US20080026457A1

US20080026457A1 - Ungulates with genetically modified immune systems

Info

Publication number: US20080026457A1
Application number: US11/789,961
Authority: US
Inventors: Kevin Wells; David Ayares
Original assignee: Revivicor Inc
Current assignee: Revivicor Inc
Priority date: 2004-10-22
Filing date: 2007-04-26
Publication date: 2008-01-31
Also published as: US9585374B2; US11085054B2; US20170183685A1; US20200109415A1; US20100077494A1

Abstract

The present invention provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which lack expression of functional endogenous immunoglobulin loci. The present invention also provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which express xenogenous, such as human, immunoglobulin loci. The present invention further provides ungulate, such as porcine genomic DNA sequence of porcine heavy and light chain immunogobulins. Such animals, tissues, organs and cells can be used in research and medical therapy. In addition, methods are provided to prepare such animals, organs, tissues, and cells.

Description

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

An antigen is an agent or substance that can be recognized by the body as ‘foreign’. Often it is only one relatively small chemical group of a larger foreign substance which acts as the antigen, for example a component of the cell wall of a bacterium. Most antigens are proteins, though carbohydrates can act as weak antigens. Bacteria, viruses and other microorganisms commonly contain many antigens, as do pollens, dust mites, molds, foods, and other substances. The body reacts to antigens by making antibodies. Antibodies (also called immunoglobulins (Igs)) are proteins that are manufactured by cells of the immune system that bind to an antigen or foreign protein. Antibodies circulate in the serum of blood to detect foreign antigens and constitute the gamma globulin part of the blood proteins. These antibodies interact chemically with the antigen in a highly specific manner, like two pieces of a jigsaw puzzle, forming an antigen/antibody complex, or immune complex. This binding neutralizes or brings about the destruction of the antigen.
When a vertebrate first encounters an antigen, it exhibits a primary humoral immune response. If the animal encounters the same antigen after a few days the immune response is more rapid and has a greater magnitude. The initial encounter causes specific immune cell (B-cell) clones to proliferate and differentiate. The progeny lymphocytes include not only effector cells (antibody producing cells) but also clones of memory cells, which retain the capacity to produce both effector and memory cells upon subsequent stimulation by the original antigen. The effector cells live for only a few days. The memory cells live for a lifetime and can be reactivated by a second stimulation with the same antigen. Thus, when an antigen is encountered a second time, its memory cells quickly produce effector cells which rapidly produce massive quantities of antibodies.
By exploiting the unique ability of antibodies to interact with antigens in a highly specific manner, antibodies have been developed as molecules that can be manufactured and used for both diagnostic and therapeutic applications. Because of their unique ability to bind to antigenic epitopes, polyclonal and monoclonal antibodies can be used to identify molecules carrying that epitope or can be directed, by themselves or in conjunction with another moiety, to a specific site for diagnosis or therapy. Polyclonal and monoclonal antibodies can be generated against practically any pathogen or biological target. The term polyclonal antibody refers to immune sera that usually contain pathogen-specific antibodies of various isotypes and specificities. In contrast, monoclonal antibodies consist of a single immunoglobulin type, representing one isotype with one specificity.
In 1890, Shibasaburo Kitazato and Emil Behring conducted the fundamental experiment that demonstrated immunity can be transmitted from one animal to another by transferring the serum from an immune animal to a non-immune animal. This landmark experiment laid the foundation for the introduction of passive immunization into clinical practice. However, wide scale serum therapy was largely abandoned in the 1940s because of the toxicity associated with the administration of heterologous sera and the introduction of effective antimicrobial chemotherapy. Currently, such polyclonal antibody therapy is indicated to treat infectious diseases in relatively few situations, such as replacement therapy in immunoglobulin-deficient patients, post-exposure prophylaxis against several viruses (e.g., rabies, measles, hepatitis A and B, varicella), and toxin neutralization (diphtheria, tetanus, and botulism). Despite the limited use of serum therapy, in the United States, more than 16 metric tons of human antibodies are required each year for intravenous antibody therapy. Comparable levels of use exist in the economies of most highly industrialized countries, and the demand can be expected to grow rapidly in developing countries. Currently, human antibody for passive immunization is obtained from the pooled serum of donors. Thus, there is an inherent limitation in the amount of human antibody available for therapeutic and prophylactic therapies.
The use of antibodies for passive immunization against biological warfare agents represents a very promising defense strategy. The final line of defense against such agents is the immune system of the exposed individual. Current defense strategies against biological weapons include such measures as enhanced epidemiologic surveillance, vaccination, and use of antimicrobial agents. Since the potential threat of biological warfare and bioterrorism is inversely proportional to the number of immune persons in the targeted population, biological agents are potential weapons only against populations with a substantial proportion of susceptible persons.
Vaccination can reduce the susceptibility of a population against specific threats; provided that a safe vaccine exists that can induce a protective response. Unfortunately, inducing a protective response by vaccination may take longer than the time between exposure and onset of disease. Moreover, many vaccines require multiple doses to achieve a protective immune response, which would limit their usefulness in an emergency to provide rapid prophylaxis after an attack. In addition, not all vaccine recipients mount a protective response, even after receiving the recommended immunization schedule.
Drugs can provide protection when administered after exposure to certain agents, but none are available against many potential agents of biological warfare. Currently, no small-molecule drugs are available that prevent disease following exposure to preformed toxins. The only currently available intervention that could provide a state of immediate immunity is passive immunization with protective antibody (Arturo Casadevall “Passive Antibody Administration (Immediate Immunity) as a Specific Defense Against Biological Weapons” from Emerging Infectious Diseases, Posted Sep. 12, 2002).
In addition to providing protective immunity, modern antibody-based therapies constitute a potentially useful option against newly emergent pathogenic bacteria, fungi, virus and parasites (A. Casadevall and M. D. Scharff, Clinical Infectious Diseases 1995; 150). Therapies of patients with malignancies and cancer (C. Botti et al, Leukemia 1997; Suppl 2:S55-59; B. Bodey, S. E. Siegel, and H. E. Kaiser, Anticancer Res 1996; 16(2):661), therapy of steroid resistant rejection of transplanted organs as well as autoimmune diseases can also be achieved through the use of monoclonal or polyclonal antibody preparations (N. Bonnefoy-Berard and J. P. Revillard, J Heart Lung Transplant 1996; 15(5):435-442; C. Colby, et al Ann Pharmacother 1996; 30(10):1164-1174; M. J. Dugan, et al, Ann Hematol 1997; 75(1-2):41 2; W. Cendrowski, Boll Ist Sieroter Milan 1997; 58(4):339-343; L. K. Kastrukoff, et al Can J Neurol Sci 1978; 5(2):175178; J. E. Walker et al J Neurol Sci 1976; 29(2-4):303309).
Recent advances in the technology of antibody production provide the means to generate human antibody reagents, while avoiding the toxicities associated with human serum therapy. The advantages of antibody-based therapies include versatility, low toxicity, pathogen specificity, enhancement of immune function, and favorable pharmacokinetics.
The clinical use of monoclonal antibody therapeutics has just recently emerged. Monoclonal antibodies have now been approved as therapies in transplantation, cancer, infectious disease, cardiovascular disease and inflammation. In many more monoclonal antibodies are in late stage clinical trials to treat a broad range of disease indications. As a result, monoclonal antibodies represent one of the largest classes of drugs currently in development.
Despite the recent popularity of monoclonal antibodies as therapeutics, there are some obstacles for their use. For example, many therapeutic applications for monoclonal antibodies require repeated administrations, especially for chronic diseases such as autoimmunity or cancer. Because mice are convenient for immunization and recognize most human antigens as foreign, monoclonal antibodies against human targets with therapeutic potential have typically been of murine origin. However, murine monoclonal antibodies have inherent disadvantages as human therapeutics. For example, they require more frequent dosing to maintain a therapeutic level of monoclonal antibodies because of a shorter circulating half-life in humans than human antibodies. More critically, repeated administration of murine immunoglobulin creates the likelihood that the human immune system will recognize the mouse protein as foreign, generating a human anti-mouse antibody response, which can cause a severe allergic reaction. This possibility of reduced efficacy and safety has lead to the development of a number of technologies for reducing the immunogenicity of murine monoclonal antibodies.
Polyclonal antibodies are highly potent against multiple antigenic targets. They have the unique ability to target and kill a plurality of “evolving targets” linked with complex diseases. Also, of all drug classes, polyclonals have the highest probability of retaining activity in the event of antigen mutation. In addition, while monoclonals have limited therapeutic activity against infectious agents, polyclonals can both neutralize toxins and direct immune responses to eliminate pathogens, as well as biological warfare agents.
The development of polyclonal and monoclonal antibody production platforms to meet future demand for production capacity represents a promising area that is currently the subject of much research. One especially promising strategy is the introduction of human immunoglobulin genes into mice or large domestic animals. An extension of this technology would include inactivation of their endogenous immunoglobulin genes. Large animals, such as sheep, pigs and cattle, are all currently used in the production of plasma derived products, such as hyperimmune serum and clotting factors, for human use. This would support the use of human polyclonal antibodies from such species on the grounds of safety and ethics. Each of these species naturally produces considerable quantities of antibody in both serum and milk.
Arrangement of Genes Encoding Immunoglobulins
Antibody molecules are assembled from combinations of variable gene elements, and the possibilities resulting from combining the many variable gene elements in the germline enable the host to synthesize antibodies to an extraordinarily large number of antigens. Each antibody molecule consists of two classes of polypeptide chains, light (L) chains (that can be either kappa (κ) L-chain or lambda (λ) L-chain) and heavy (H) chains. The heavy and light chains join together to define a binding region for the epitope. A single antibody molecule has two identical copies of the L chain and two of the H chain. Each of the chains is comprised of a variable region (V) and a constant region (C). The variable region constitutes the antigen-binding site of the molecule. To achieve diverse antigen recognition, the DNA that encodes the variable region undergoes gene rearrangement. The constant region amino acid sequence is specific for a particular isotype of the antibody, as well as the host which produces the antibody, and thus does not undergo rearrangement.
The mechanism of DNA rearrangement is similar for the variable region of both the heavy- and light-chain loci, although only one joining event is needed to generate a light-chain gene whereas two are needed to generate a complete heavy-chain gene. The most common mode of rearrangement involves the looping-out and deletion of the DNA between two gene segments. This occurs when the coding sequences of the two gene segments are in the same orientation in the DNA. A second mode of recombination can occur between two gene segments that have opposite transcriptional orientations. This mode of recombination is less common, although such rearrangements can account for up to half of all V_κ to J_κ joins; the transcriptional orientation of half of the human V_κ gene segments is opposite to that of the J_κ gene segments.
The DNA sequence encoding a complete V region is generated by the somatic recombination of separate gene segments. The V region, or V domain, of an immunoglobulin heavy or light chain is encoded by more than one gene segment. For the light chain, the V domain is encoded by two separate DNA segments. The first segment encodes the first 95-101 amino acids of the light chain and is termed a V gene segment because it encodes most of the V domain. The second segment encodes the remainder of the V domain (up to 13 amino acids) and is termed a joining or J gene segment. The joining of a V and a J gene segment creates a continuous exon that encodes the whole of the light-chain V region. To make a complete immunoglobulin light-chain messenger RNA, the V-region exon is joined to the C-region sequence by RNA splicing after transcription.
A heavy-chain V region is encoded in three gene segments. In addition to the V and J gene segments (denoted V_Hand J_Hto distinguish them from the light-chain V_Land J_L), there is a third gene segment called the diversity or D_Hgene segment, which lies between the V_Hand J_Hgene segments. The process of recombination that generates a complete heavy-chain V region occurs in two separate stages. In the first, a D_Hgene segment is joined to a J_Hgene segment; then a V_Hgene segment rearranges to DJ_Hto make a complete V_H-region exon. As with the light-chain genes, RNA splicing joins the assembled V-region sequence to the neighboring C-region gene.
Diversification of the antibody repertoire occurs in two stages: primarily by rearrangement (“V(D)J recombination”) of Ig V, D and J gene segments in precursor B cells resident in the bone marrow, and then by somatic mutation and class switch recombination of these rearranged Ig genes when mature B cells are activated. Immunoglobulin somatic mutation and class switching are central to the maturation of the immune response and the generation of a “memory” response.
The genomic loci of antibodies are very large and they are located on different chromosomes. The immunoglobulin gene segments are organized into three clusters or genetic loci: the κ, λ, and heavy-chain loci. Each is organized slightly differently. For example, in humans, immunoglobulin genes are organized as follows. The λ light-chain locus is located on chromosome 22 and a cluster of V_λ gene segments is followed by four sets of J_λ gene segments each linked to a single C_λ gene. The κ light-chain locus is on chromosome 2 and the cluster of V_κ, gene segments is followed by a cluster of J_κ gene segments, and then by a single C_κ gene. The organization of the heavy-chain locus, on chromosome 14, resembles that of the κ locus, with separate clusters of V_H, D_H, and J_Hgene segments and of C_Hgenes. The heavy-chain locus differs in one important way: instead of a single C-region, it contains a series of C regions arrayed one after the other, each of which corresponds to a different isotype. There are five immunoglobulin heavy chain isotypes: IgM, IgG, IgA, IgE and IgD. Generally, a cell expresses only one at a time, beginning with IgM. The expression of other isotypes, such as IgG, can occur through isotype switching.
The joining of various V, D and J genes is an entirely random event that results in approximately 50,000 different possible combinations for VDJ(H) and approximately 1,000 for VJ(L). Subsequent random pairing of H and L chains brings the total number of antibody specificities to about 10⁷possibilities. Diversity is further increased by the imprecise joining of different genetic segments. Rearrangements occur on both DNA strands, but only one strand is transcribed (due to allelic exclusion). Only one rearrangement occurs in the life of a B cell because of irreversible deletions in DNA. Consequently, each mature B cell maintains one immunologic specificity and is maintained in the progeny or clone. This constitutes the molecular basis of the clonal selection; i.e., each antigenic determinant triggers the response of the pre-existing clone of B lymphocytes bearing the specific receptor molecule. The primary repertoire of B cells, which is established by V(D)J recombination, is primarily controlled by two closely linked genes, recombination activating gene (RAG)-1 and RAG-2.
Over the last decade, considerable diversity among vertebrates in both Ig gene diversity and antibody repertoire development has been revealed. Rodents and humans have five heavy chain classes, IgM, IgD, IgG, IgE and IgA, and each have four subclasses of IgG and one or two subclasses of IgA, while rabbits have a single IgG heavy chain gene but 13 genes for different IgA subclasses (Burnett, R. C et al. EMBO J. 8:4047; Honjo, In Honjo, T, Alt. F. W. T. H. eds, Immunoglobulin Genes p. 123 Academic Press, New York). Swine have at least six IgG subclasses (Kacskovics, I et al. 1994 J Immunol 153:3565), but no IgD (Butler et al. 1996 Inter. Immunol 8:1897-1904). A gene encoding IgD has only been described in rodents and primates. Diversity in the mechanism of repertoire development is exemplified by contrasting the pattern seen in rodents and primates with that reported for chickens, rabbits, swine and the domesticated Bovidae. Whereas the former group have a large number of V_Hgenes belonging to seven to 10 families (Rathbun, G. In Hongo, T. Alt. F. W. and Rabbitts, T. H., eds, Immunoglobulin Genes, p. 63, Academic press New York), the V_Hgenes of each member of the latter group belong to a single V_Hgene family (Sun, J. et al. 1994 J. Immunol. 1553:56118; Dufour, V et al. 1996, J Immunol. 156:2163). With the exception of the rabbit, this family is composed of less than 25 genes. Whereas rodents and primates can utilize four to six J_Hsegments, only a single J_His available for repertoire development in the chicken (Reynaud et al. 1989 Adv. Immunol. 57:353). Similarly, Butler et al. (1996 Inter. Immunol 8:1897-1904) hypothesized that swine may resemble the chicken in having only a single J_Hgene. These species generally have fewer V, D and J genes; in the pig and cow a single VH gene family exists, consisting of less than 20 gene segments (Butler et al, Advances in Swine in Biomedical Research, eds: Tumbleson and Schook, 1996; Sinclair et al, J. Immunol. 159: 3883, 1997). Together with lower numbers of J and D gene segments, this results in significantly less diversity being generated by gene rearrangement. However, there does appear to be greater numbers of light chain genes in these species. Similar to humans and mice, these species express a single κ light chain but multiple λ light chain genes. However, these do not seem to affect the restricted diversity that is achieved by rearrangement.
Since combinatorial joining of more than 100 V_H, 20-30 D_Hand four to six J_Hgene segments is a major mechanism of generating the antibody repertoire in humans, species with fewer V_H, D_Hor J_Hsegments must either generate a smaller repertoire or use alternative mechanisms for repertoire development. Ruminants, pigs, rabbits and chickens, utilize several mechanisms to generate antibody diversity. In these species there appears to be an important secondary repertoire development, which occurs in highly specialized lymphoid tissue such as ileal Peyer's patches (Binns and Licence, Adv. Exp. Med. Biol. 186: 661, 1985). Secondary repertoire development occurs in these species by a process of somatic mutation which is a random and not fully understood process. The mechanism for this repertoire diversification appears to be templated mutation, or gene conversion (Sun et al, J. Immunol. 153: 5618, 1994) and somatic hypermutation.
Gene conversion is important for antibody diversification in some higher vertebrates, such as chickens, rabbits and cows. In mice, however, conversion events appear to be infrequent among endogenous antibody genes. Gene conversion is a distinct diversifying mechanism characterized by transfers of homologous sequences from a donor antibody V gene segment to an acceptor V gene segment. If donor and acceptor segments have numerous sequence differences then gene conversion can introduce a set of sequence changes into a V region by a single event. Depending on the species, gene conversion events can occur before and/or after antigen exposure during B cell differentiation (Tsai et al. International Immunology, Vol. 14, No. 1, 55-64, January 2002).
Somatic hypermutation achieves diversification of antibody genes in all higher vertebrate species. It is typified by the introduction of single point mutations into antibody V(D)J segments. Generally, hypermutation appears to be activated in B cells by antigenic stimulation.
Production of Animals with Humanized Immune Systems
In order to reduce the immunogenicity of antibodies generated in mice for human therapeutics, various attempts have been made to replace murine protein sequences with human protein sequences in a process now known as humanization. Transgenic mice have been constructed which have had their own immunoglobulin genes functionally replaced with human immunoglobulin genes so that they produce human antibodies upon immunization. Elimination of mouse antibody production was achieved by inactivation of mouse Ig genes in embryonic stem (ES) cells by using gene-targeting technology to delete crucial cis-acting sequences involved in the process of mouse Ig gene rearrangement and expression. B cell development in these mutant mice could be restored by the introduction of megabase-sized YACs containing a human germline-configuration H- and κ L-chain minilocus transgene. The expression of fully human antibody in these transgenic mice was predominant, at a level of several 100 μg/l of blood. This level of expression is several hundred-fold higher than that detected in wild-type mice expressing the human Ig gene, indicating the importance of inactivating the endogenous mouse Ig genes in order to enhance human antibody production by mice.
The first humanization attempts utilized molecular biology techniques to construct recombinant antibodies. For example, the complementarity determining regions (CDR) from a mouse antibody specific for a hapten were grafted onto a human antibody framework, effecting a CDR replacement. The new antibody retained the binding specificity conveyed by the CDR sequences (P. T. Jones et al. Nature 321: 522-525 (1986)). The next level of humanization involved combining an entire mouse VH region with a human constant region such as gamma₁(S. L. Morrison et al., Proc. Natl. Acad. Sci., 81, pp. 6851-6855 (1984)). However, these chimeric antibodies, which still contain greater than 30% xenogeneic sequences, are sometimes only marginally less immunogenic than totally xenogeneic antibodies (M. Bruggemann et al., J. Exp. Med., 170, pp. 2153-2157 (1989)).
Subsequently, attempts were carried out to introduce human immunoglobulin genes into the mouse, thus creating transgenic mice capable of responding to antigens with antibodies having human sequences (Bruggemann et al. Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989)). Due to the large size of human immunoglobulin genomic loci, these attempts were thought to be limited by the amount of DNA, which could be stably maintained by available cloning vehicles. As a result, many investigators concentrated on producing mini-loci containing limited numbers of V region genes and having altered spatial distances between genes as compared to the natural or germline configuration (See, for example, U.S. Pat. No. 5,569,825). These studies indicated that producing human sequence antibodies in mice was possible, but serious obstacles remained regarding obtaining sufficient diversity of binding specificities and effector functions (isotypes) from these transgenic animals to meet the growing demand for antibody therapeutics.
In order to provide additional diversity, work has been conducted to add large germline fragments of the human Ig locus into transgenic mammals. For example, a majority of the human V, D, and J region genes arranged with the same spacing found in the unrearranged germline of the human genome and the human Cμ and Cδ constant regions was introduced into mice using yeast artificial chromosome (YAC) cloning vectors (See, for example, WO 94/02602). A 22 kb DNA fragment comprising sequences encoding a human gamma-2 constant region and the upstream sequences required for class-switch recombination was latter appended to the foregoing transgene. In addition, a portion of a human kappa locus comprising Vκ, Jκ and Cκ region genes, also arranged with substantially the same spacing found in the unrearranged germline of the human genome, was introduced into mice using YACS. Gene targeting was used to inactivate the murine IgH & kappa light chain immunoglobulin gene loci and such knockout strains were bred with the above transgenic strains to generate a line of mice having the human V, D, J, Cμ, Cδ and Cγ₂constant regions as well as the human Vκ, Jκ and Cκ region genes all on an inactivated murine immunoglobulin background (See, for example, PCT patent application WO 94/02602 to Kucherlapati et al.; see also Mendez et al., Nature Genetics 15:146-156 (1997)).
Yeast artificial chromosomes as cloning vectors in combination with gene targeting of endogenous loci and breeding of transgenic mouse strains provided one solution to the problem of antibody diversity. Several advantages were obtained by this approach. One advantage was that YACs can be used to transfer hundreds of kilobases of DNA into a host cell. Therefore, use of YAC cloning vehicles allows inclusion of substantial portions of the entire human Ig heavy and light chain regions into a transgenic mouse thus approaching the level of potential diversity available in the human. Another advantage of this approach is that the large number of V genes has been shown to restore full B cell development in mice deficient in murine immunoglobulin production. This ensures that these reconstituted mice are provided with the requisite cells for mounting a robust human antibody response to any given immunogen. (See, for example, WO 94/02602; L. Green and A. Jakobovits, J. Exp. Med. 188:483-495 (1998)). A further advantage is that sequences can be deleted or inserted onto the YAC by utilizing high frequency homologous recombination in yeast. This provides for facile engineering of the YAC transgenes.
In addition, Green et al. Nature Genetics 7:13-21 (1994) describe the generation of YACs containing 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus, respectively, which contained core variable and constant region sequences. The work of Green et al. was recently extended to the introduction of greater than approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and kappa light chain loci, respectively, to produce XenoMouse™ mice. See, for example, Mendez et al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), European Patent No. EP 0 463 151 B1, PCT Publication Nos. WO 94/02602, WO 96/34096 and WO 98/24893.
Several strategies exist for the generation of mammals that produce human antibodies. In particular, there is the “minilocus” approach that is typified by work of GenPharm International, Inc. and the Medical Research Council, YAC introduction of large and substantially germline fragments of the Ig loci that is typified by work of Abgenix, Inc. (formerly Cell Genesys). The introduction of entire or substantially entire loci through the use microcell fusion as typified by work of Kirin Beer Kabushiki Kaisha.
In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V_Hgenes, one or more D_Hgenes, one or more J_Hgenes, a mu constant region, and a second constant region (such as a gamma constant region) are formed into a construct for insertion into an animal. See, for example, U.S. Pat. Nos. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, 5,643,763; European Patent No. 0 546 073; PCT Publication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884; Taylor et al. Nucleic Acids Research 20:6287-6295 (1992), Chen et al. International Immunology 5:647-656 (1993), Tuaillon et al. J. Immunol. 154:6453-6465 (1995), Choi et al. Nature Genetics 4:117-123 (1993), Lonberg et al. Nature 368:856-859 (1994), Taylor et al. International Immunology 6:579-591 (1994), Tuaillon et al. J. Immunol. 154:6453-6465 (1995), and Fishwild et al. Nature Biotech. 14:845-851 (1996).
In the microcell fusion approach, portions or whole human chromosomes can be introduced into mice (see, for example, European Patent Application No. EP 0 843 961 A1). Mice generated using this approach and containing the human Ig heavy chain locus will generally possess more than one, and potentially all, of the human constant region genes. Such mice will produce, therefore, antibodies that bind to particular antigens having a number of different constant regions.
While mice remain the most developed animal for the expression of human immunoglobulins in humans, recent technological advances have allowed for progress to begin in applying these techniques to other animals, such as cows. The general approach in mice has been to genetically modify embryonic stem cells of mice to knock-out murine immunoglobulins and then insert YACs containing human immunoglobulins into the ES cells. However, ES cells are not available for cows or other large animals such as sheep and pigs. Thus, several fundamental developments had to occur before even the possibility existed to generate large animals with immunoglobulin genes knocked-out and that express human antibody. The alternative to ES cell manipulation to create genetically modified animals is cloning using somatic cells that have been genetically modified. Cloning using genetically modified somatic cells for nuclear transfer has only recently been accomplished.
Since the announcement of Dolly's (a cloned sheep) birth from an adult somatic cell in 1997 (Wilmut, I., et al (1997) Nature 385: 810-813), ungulates, including cattle (Cibelli, J et al 1998 Science 280: 1266-1258; Kubota, C. et al. 2000 Proc. Nat'l. Acad. Sci. 97: 990-995), goats (Baguisi, A. et al., (1999) Nat. Biotechnology 17: 456-461), and pigs (Polejaeva, I. A., et al. 2000 Nature 407: 86-90; Betthauser, J. et al. 2000 Nat. Biotechnology 18: 1055-1059) have been cloned.
The next technological advance was the development of the technique to genetically modify the cells prior to nuclear transfer to produce genetically modified animals. PCT publication No. WO 00/51424 to PPL Therapeutics describes the targeted genetic modification of somatic cells for nuclear transfer.
Subsequent to these fundamental developments, single and double allele knockouts of genes and the birth of live animals with these modifications have been reported. Between 2002 and 2004, three independent groups, Immerge Biotherapeutics, Inc. in collaboration with the University of Missouri (Lai et al. (Science (2002) 295: 1089-1092) & Kolber-Simonds et al. (PNAS. (2004) 101(19):7335-40)), Alexion Pharmaceuticals (Ramsoondar et al. (Biol Reprod (2003)69: 437-445) and Revivicor, Inc. (Dai et al. (Nature Biotechnology (2002) 20: 251-255) & Phelps et al. (Science (2003) January 17; 299(5605):411-4)) produced pigs that lacked one allele or both alleles of the alpha-1,3-GT gene via nuclear transfer from somatic cells with targeted genetic deletions. In 2003, Sedai et al. (Transplantation (2003) 76:900-902) reported the targeted disruption of one allele of the alpha-1,3-GT gene in cattle, followed by the successful nuclear transfer of the nucleus of the genetically modified cell and production of transgenic fetuses.
Thus, the feasibility of knocking-out immunoglobulin genes in large animals and inserting human immunoglobulin loci into their cells is just now beginning to be explored. However, due to the complexity and species differences of immunoglobulin genes, the genomic sequences and arrangement of Ig kappa, lambda and heavy chains remain poorly understood in most species. For example, in pigs, partial genomic sequence and organization has only been described for heavy chain constant alpha, heavy chain constant mu and heavy chain constant delta (Brown and Butler Mol Immunol. 1994 June; 31(8):633-42, Butler et al Vet Immunol Immunopathol. 1994 October; 43(1-3):5-12, and Zhao et al J Immunol. 2003 Aug. 1; 171(3):1312-8).
In cows, the immunoglobulin heavy chain locus has been mapped (Zhao et al. 2003 J. Biol. Chem. 278:35024-32) and the cDNA sequence for the bovine kappa gene is known (See, for example, U.S. Patent Publication No. 2003/0037347). Further, approximately 4.6 kb of the bovine mu heavy chain locus has been sequenced and transgenic calves with decreased expression of heavy chain immunoglobulins have been created by disrupting one or both alleles of the bovine mu heavy chain. In addition, a mammalian artificial chromosome (MAC) vector containing the entire unarranged sequences of the human Ig H-chain and κ L-chain has been introduced into cows (TC cows) with the technology of microcell-mediated chromosome transfer and nuclear transfer of bovine fetal fibroblast cells (see, for example, Kuroiwa et al. 2002 Nature Biotechnology 20:889, Kuroiwa et al. 2004 Nat Genet. June 6 Epub, U.S. Patent Publication Nos. 2003/0037347, 2003/0056237, 2004/0068760 and PCT Publication No. WO 02/07648).
While significant progress has been made in the production of bovine that express human immunoglobulin, little has been accomplished in other large animals, such as sheep, goats and pigs. Although cDNA sequence information for immunoglobulin genes of sheeps, goats and pigs is readily available in Genbank, the unique nature of immunoglobulin loci, which undergo massive rearrangements, creates the need to characterize beyond sequences known to be present in mRNAs (or cDNAs). Since immunoglobulin loci are modular and the coding regions are redundant, deletion of a known coding region does not ensure altered function of the locus. For example, if one were to delete the coding region of a heavy-chain variable region, the function of the locus would not be significantly altered because hundreds of other function variable genes remain in the locus. Therefore, one must first characterize the locus to identify a potential “Achilles heel”.
Despite some advancements in expressing human antibodies in cattle, greater challenges remain for inactivation of the endogenous bovine Ig genes, increasing expression levels of the human antibodies and creating human antibody expression in other large animals, such as porcine, for which the sequence and arrangement of immunoglobulin genes are largely unknown.
It is therefore an object of the present invention to provide the arrangement of ungulate immunoglobin germline gene sequence.
It is another object of the present invention to provide novel ungulate immunoglobulin genomic sequences.
It is a further object of the present invention to provide cells, tissues and animals lacking at least one allele of a heavy and/or light chain immunoglobulin gene.
It is another object of the present invention to provide ungulates that express human immunoglobulins.
It is a still further object of the present invention to provide methods to generate cells, tissues and animals lacking at least one allele of novel ungulate immunoglobulin gene sequences and/or express human immunoglobulins.

SUMMARY OF THE INVENTION

The present invention provides for the first time ungulate immunoglobin germline gene sequence arrangement as well as novel genomic sequences thereof. In addition, novel ungulate cells, tissues and animals that lack at least one allele of a heavy or light chain immunoglobulin gene are provided. Based on this discovery, ungulates can be produced that completely lack at least one allele of a heavy and/or light chain immunoglobulin gene. In addition, these ungulates can be further modified to express xenoogenous, such as human, immunoglobulin loci or fragments thereof.
In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In another aspect of the present invention, novel genomic sequences encoding the heavy chain locus of ungulate immunoglobulin are provided. In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In another embodiment, an isolated nucleotide sequence is provided that includes at least four joining regions and at least one constant region, such as the mu constant region, of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No. 4. In a further embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No 1. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in the 3′ region of Seq ID No 4. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In one embodiment, the nucleotide sequence contains at least 17, 20, 25 or 30 contiguous nucleotides of Seq ID No 4 or residues 1-9,070 of Seq ID No 29. In another embodiment, the nucleotide sequence contains residues 9,070-11039 of Seq ID No 29. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.
In another embodiment, novel genomic sequences encoding the kappa light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate kappa light chain regions. In one embodiment, nucleic acid sequence is provided that encodes the porcine kappa light chain locus. In another embodiment, the nucleic acid sequence can contain at least one joining region, one constant region and/or one enhancer region of kappa light chain. In a further embodiment, the nucleotide sequence can include at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In a further embodiment, an isolated nucleotide sequence is provided that contains at least one, at least two, at least three, at least four or five joining regions and 3′ flanking sequence to the joining region of porcine genomic kappa light chain, for example, as represented in Seq ID No 12. In another embodiment, an isolated nucleotide sequence of porcine genomic kappa light chain is provided that contains 5′ flanking sequence to the first joining region, for example, as represented in Seq ID No 25. In a further embodiment, an isolated nucleotide sequence is provided that contains 3′ flanking sequence to the constant region and, optionally, the 5′ portion of the enhancer region, of porcine genomic kappa light chain, for example, as represented in Seq ID Nos. 15, 16 and/or 19.
In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 30, 12, 25, 15, 16 or 19 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 30, 12, 25, 15, 16 or 19, as well as, nucleotides homologous thereto.
In another embodiment, novel genomic sequences encoding the lambda light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate lambda light chain regions. In one embodiment, the porcine lambda light chain nucleotides include a concatamer of J to C units. In a specific embodiment, an isolated porcine lambda nucleotide sequence is provided, such as that depicted in Seq ID No. 28. In one embodiment, a nucleotide sequence is provided that includes 5′ flanking sequence to the first lambda J/C unit of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 32. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 200 base pairs downstream of lambda J/C, such as that represented by Seq ID No 33. Alternatively, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 34, 35, 36, 37, 38, and/or 39.
In a further embodiment, nucleic acid sequences are provided that encode bovine lambda light chain locus, which can include at least one joining region-constant region pair and/or at least one variable region, for example, as represented by Seq ID No. 31. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39, as well as, nucleotides homologous thereto.
In another embodiment, nucleic acid targeting vector constructs are also provided. The targeting vectors can be designed to accomplish homologous recombination in cells. These targeting vectors can be transformed into mammalian cells to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. In one embodiment, the targeting vectors can contain a 3′ recombination arm and a 5′ recombination arm (i.e. flanking sequence) that is homologous to the genomic sequence of ungulate heavy chain, kappa light chain or lambda light chain genomic sequence, for example, sequence represented by Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The homologous DNA sequence can include at least 15 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous to the genomic sequence.
In one embodiment, the 5′ and 3′ recombination arms of the targeting vector can be designed such that they flank the 5′ and 3′ ends of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. The targeting of a functional region can render it inactive, which results in the inability of the cell to produce functional immunoglobulin molecules. In another embodiment, the homologous DNA sequence can include one or more intron and/or exon sequences. In addition to the nucleic acid sequences, the expression vector can contain selectable marker sequences, such as, for example, enhanced Green Fluorescent Protein (eGFP) gene sequences, initiation and/or enhancer sequences, poly A-tail sequences, and/or nucleic acid sequences that provide for the expression of the construct in prokaryotic and/or eukaryotic host cells. The selectable marker can be located between the 5′ and 3′ recombination arm sequence.
In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J6 region of the porcine immunoglobulin heavy chain locus. Since the J6 region is the only functional joining region of the porcine immunoglobulin heavy chain locus, this will prevent the expression of a functional porcine heavy chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the J6 region, including J1-4, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the J6 region, including the mu constant region (a “J6 targeting construct”), see for example, FIG. 1. Further, this J6 targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 5 and FIG. 1. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the diversity region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the diversity region of the porcine heavy chain locus. In a further embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the mu constant region and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the mu constant region of the porcine heavy chain locus.
In another particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the constant region of the porcine immunoglobulin kappa light chain locus. Since the present invention discovered that there is only one constant region of the porcine immunoglobulin kappa light chain locus, this will prevent the expression of a functional porcine kappa light chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the constant region, optionally including the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the constant region, optionally including at least part of the enhancer region (a “Kappa constant targeting construct”), see for example, FIG. 2. Further, this kappa constant targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 20 and FIG. 2. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the joining region of the porcine kappa light chain locus.
In another particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J/C region of the porcine lambda light chain. See FIG. 3. Disruption of the J/C region will prevent the expression of a functional porcine kappa light chain immunoglobulin. In one embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the first J/C unit and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the last J/C unit. Further, this lambda light chain targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example FIG. 4.
In a further embodiment, more than one targeting vector can be used to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. For example, two targeting vectors can be used to target the gene of interest. A first targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 5′ flanking sequence of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. A second targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ flanking sequence at least one functional variable, joining, diversity, and/or constant region of the genomic sequence.
In a particular embodiment, the first targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 5′ flanking sequence of the first J/C unit in the J/C cluster region. See FIG. 5. According to this embodiment, a second targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ flanking sequence of the last J/C unit in the J/C cluster region. See FIG. 6.
In another embodiment, primers are provided to generate 3′ and 5′ sequences of a targeting vector. The oligonucleotide primers can be capable of hybridizing to porcine immunoglobulin genomic sequence, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. In a particular embodiment, the primers hybridize under stringent conditions to Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. Another embodiment provides oligonucleotide probes capable of hybridizing to porcine heavy chain, kappa light chain or lambda light chain nucleic acid sequences, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The polynucleotide primers or probes can have at least 14 bases, 20 bases, 30 bases, or 50 bases which hybridize to a polynucleotide of the present invention. The probe or primer can be at least 14 nucleotides in length, and in a particular embodiment, are at least 15, 20, 25, 28, or 30 nucleotides in length.
In one embodiment, primers are provided to amplify a fragment of porcine Ig heavy-chain that includes the functional joining region (the J6 region). In one non-limiting embodiment, the amplified fragment of heavy chain can be represented by Seq ID No 4 and the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 2, to produce the 5′ recombination arm and complementary to a portion of Ig heavy-chain mu constant region, such as, but not limited to Seq ID No 3, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 4) can be subcloned and assembled into a targeting vector.
In other embodiments, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the constant region. In another embodiment, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the J region. In one non-limiting embodiment, the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 21 or 10, to produce the 5′ recombination arm and complementary to genomic sequence 3′ of the constant region, such as, but not limited to Seq ID No 14, 24 or 18, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 20) can be subcloned and assembled into a targeting vector.
In another aspect of the present invention, ungulate cells lacking at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the process, sequences and/or constructs described herein are provided. These cells can be obtained as a result of homologous recombination. Particularly, by inactivating at least one allele of an ungulate heavy chain, kappa light chain or lambda light chain gene, cells can be produced which have reduced capability for expression of ungulate antibodies. In other embodiments, mammalian cells lacking both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be produced according to the process, sequences and/or constructs described herein. In a further embodiment, porcine animals are provided in which at least one allele of an ungulate heavy chain, kappa light chain and/or lambda light chain gene is inactivated via a genetic targeting event produced according to the process, sequences and/or constructs described herein. In another aspect of the present invention, porcine animals are provided in which both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene are inactivated via a genetic targeting event. The gene can be targeted via homologous recombination.
In other embodiments, the gene can be disrupted, i.e. a portion of the genetic code can be altered, thereby affecting transcription and/or translation of that segment of the gene. For example, disruption of a gene can occur through substitution, deletion (“knock-out”) or insertion (“knock-in”) techniques. Additional genes for a desired protein or regulatory sequence that modulate transcription of an existing sequence can be inserted. To achieve multiple genetic modifications of ungulate immunoglobulin genes, in one embodiment, cells can be modified sequentially to contain multiple genetic modifications. In other embodiments, animals can be bred together to produce animals that contain multiple genetic modifications of immunoglobulin genes. As an illustrative example, animals that lack expression of at least one allele of an ungulate heavy chain gene can be further genetically modified or bred with animals lacking at least one allele of a kappa light chain gene.
In embodiments of the present invention, alleles of ungulate heavy chain, kappa light chain or lambda light chain gene are rendered inactive according to the process, sequences and/or constructs described herein, such that functional ungulate immunoglobulins can no longer be produced. In one embodiment, the targeted immunoglobulin gene can be transcribed into RNA, but not translated into protein. In another embodiment, the targeted immunoglobulin gene can be transcribed in an inactive truncated form. Such a truncated RNA may either not be translated or can be translated into a nonfunctional protein. In an alternative embodiment, the targeted immunoglobulin gene can be inactivated in such a way that no transcription of the gene occurs. In a further embodiment, the targeted immunoglobulin gene can be transcribed and then translated into a nonfunctional protein.
In a further aspect of the present invention, ungulate, such as porcine or bovine, cells lacking one allele, optionally both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be used as donor cells for nuclear transfer into recipient cells to produce cloned, transgenic animals. Alternatively, ungulate heavy chain, kappa light chain and/or lambda light chain gene knockouts can be created in embryonic stem cells, which are then used to produce offspring. Offspring lacking a single allele of a functional ungulate heavy chain, kappa light chain and/or lambda light chain gene produced according to the process, sequences and/or constructs described herein can be breed to further produce offspring lacking functionality in both alleles through mendelian type inheritance.
In one aspect of the present invention, a method is provided to disrupt the expression of an ungulate immunoglobulin gene by (i) analyzing the germline configuration of the ungulate heavy chain, kappa light chain or lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end and the 3′ end of at least one functional region of the locus; and (iii) transfecting a targeting construct containing the flanking sequence into a cell wherein, upon successful homologous recombination, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene. In one embodiment, the germline configuration of the porcine heavy chain locus is provided. The porcine heavy chain locus contains at least four variable regions, two diversity regions, six joining regions and five constant regions, for example, as illustrated in FIG. 1. In a specific embodiment, only one of the six joining regions, J6, is functional. In another embodiment, the germline configuration of the porcine kappa light chain locus is provided. The porcine kappa light chain locus contains at least six variable regions, six joining regions, one constant region and one enhancer region, for example, as illustrated in FIG. 2. In a further embodiment, the germline configuration of the porcine lambda light chain locus is provided. The porcine lambda light chain locus contains a variable region and the J/C region. See FIG. 3.
In a further aspect of the present invention, a method is provided to disrupt the expression of an ungulate lambda light chain locus by (i) analyzing the germline configuration of the ungulate lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end of at least one functional region of the locus; (ii) constructing a 5′ targeting construct; (iv) determining the location of nucleotide sequences that flank the 3′ end of at least one functional region of the locus; (v) constructing a 3′ targeting construct; (vi) transfecting both the 5′ and the 3′ targeting constructs into a cell wherein, upon successful homologous recombination of each targeting construct, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene. See FIGS. 5 and 6.
In one embodiment, the germline configuration of the porcine lambda light chain locus is provided. The porcine lambda light chain locus contains a variable region and a J/C region. See FIG. 3.
In further aspects of the present invention provides ungulates and ungulate cells that lack at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the processes, sequences and/or constructs described herein, which are further modified to express at least part of a human antibody (i.e. immunoglobulin (Ig)) locus. In additional embodiments, porcine animals are provided that express xenogenous immunoglobulin. This human locus can undergo rearrangement and express a diverse population of human antibody molecules in the ungulate. These cloned, transgenic ungulates provide a replenishable, theoretically infinite supply of human antibodies (such as polyclonal antibodies), which can be used for therapeutic, diagnostic, purification, and other clinically relevant purposes. In one particular embodiment, artificial chromosomes (ACs), such as yeast or mammalian artificial chromosomes (YACS or MACS) can be used to allow expression of human immunoglobulin genes into ungulate cells and animals. All or part of human immunoglobulin genes, such as the Ig heavy chain gene (human chromosome 414), Ig kappa chain gene (human chromosome #2) and/or the Ig lambda chain gene (chromosome #22) can be inserted into the artificial chromosomes, which can then be inserted into ungulate cells. In further embodiments, ungulates and ungulate cells are provided that contain either part or all of at least one human antibody gene locus, which undergoes rearrangement and expresses a diverse population of human antibody molecules.
In additional embodiments, methods of producing xenogenous antibodies are provided, wherein the method can include: (a) administering one or more antigens of interest to an ungulate whose cells comprise one or more artificial chromosomes and lack any expression of functional endogenous immunoglobulin, each artificial chromosome comprising one or more xenogenous immunoglobulin loci that undergo rearrangement, resulting in production of xenogenous antibodies against the one or more antigens; and/or (b) recovering the xenogenous antibodies from the ungulate. In one embodiment, the immunoglobulin loci can undergo rearrangement in a B cell.
In one aspect of the present invention, an ungulate, such as a pig or a cow, can be prepared by a method in accordance with any aspect of the present invention. These cloned, transgenic ungulates (e.g., porcine and bovine animals) provide a replenishable, theoretically infinite supply of human polyclonal antibodies, which can be used as therapeutics, diagnostics and for purification purposes. For example, transgenic animals produced according to the process, sequences and/or constructs described herein that produce polyclonal human antibodies in the bloodstream can be used to produce an array of different antibodies which are specific to a desired antigen. The availability of large quantities of polyclonal antibodies can also be used for treatment and prophylaxis of infectious disease, vaccination against biological warfare agents, modulation of the immune system, removal of undesired human cells such as cancer cells, and modulation of specific human molecules.
In other embodiments, animals or cells lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can contain additional genetic modifications to eliminate the expression of xenoantigens. Such animals can be modified to eliminate the expression of at least one allele of the alpha-1,3-galactosyltransferase gene, the CMP-Neu5Ac hydroxylase gene (see, for example, U.S. Ser. No. 10/863,116), the iGb3 synthase gene (see, for example, U.S. Patent Application 60/517,524), and/or the Forssman synthase gene (see, for example, U.S. Patent Application 60/568,922). In additional embodiments, the animals discloses herein can also contain genetic modifications to express fucosyltransferase and/or sialyltransferase. To achieve these additional genetic modifications, in one embodiment, cells can be modified to contain multiple genetic modifications. In other embodiments, animals can be bred together to achieve multiple genetic modifications. In one specific embodiment, animals, such as pigs, lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can be bred with animals, such as pigs, lacking expression of alpha-1,3-galactosyl transferase (for example, as described in WO 04/028243).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the design of a targeting vector that disrupts the expression of the joining region of the porcine heavy chain immunoglobulin gene.
FIG. 2 illustrates the design of a targeting vector that disrupts the expression of the constant region of the porcine kappa light chain immunoglobulin gene.
FIG. 3 illustrates the genomic organization of the porcine lambda immunoglobulin locus, including a concatamer of J-C sequences or units as well as flanking regions that include the variable region 5′ to the JC cluster region. Bacterial artificial chromosomes (BAC1 and BAC2) represent fragments of the porcine immunoglobulin genome that can be obtained from BAC libraries.
FIG. 4 represents the design of a targeting vector that disrupts the expression of the JC cluster region of the porcine lambda light chain immunoglobulin gene. “SM” stands for a selectable marker gene, which can be used in the targeting vector.
FIG. 5 illustrates a targeting strategy to insert a site specific recombinase target or recognition site into the region 5′ of the JC cluster region of the porcine lambda immunoglobulin locus. “SM” stands for a selectable marker gene, which can be used in the targeting vector. “SSRRS” stands for a specific recombinase target or recognition site.
FIG. 6 illustrates a targeting strategy to insert a site specific recombinase target or recognition site into the region 3′ of the JC cluster region of the porcine lambda immunoglobulin locus. “SM” stands for a selectable marker gene, which can be used in the targeting vector. “SSRRS” stands for a specific recombinase target or recognition site.
FIG. 7 illustrates the site specific recombinase mediated transfer of a YAC into a host genome. “SSRRS” stands for a specific recombinase target or recognition site.

DETAILED DESCRIPTION

The present invention provides for the first time ungulate immunoglobin germline gene sequence arrangement as well as novel genomic sequences thereof. In addition, novel ungulate cells, tissues and animals that lack at least one allele of a heavy or light chain immunoglobulin gene are provided. Based on this discovery, ungulates can be produced that completely lack at least one allele of a heavy and/or light chain immunoglobulin gene. In addition, these ungulates can be further modified to express xenoogenous, such as human, immunoglobulin loci or fragments thereof.
In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
Definitions
The terms “recombinant DNA technology,” “DNA cloning,” “molecular cloning,” or “gene cloning” refer to the process of transferring a DNA sequence into a cell or organism. The transfer of a DNA fragment can be from one organism to a self-replicating genetic element (e.g., bacterial plasmid) that permits a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount. Plasmids and other types of cloning vectors such as artificial chromosomes can be used to copy genes and other pieces of chromosomes to generate enough identical material for further study. In addition to bacterial plasmids, which can carry up to 20 kb of foreign DNA, other cloning vectors include viruses, cosmids, and artificial chromosomes (e.g., bacteria artificial chromosomes (BACs) or yeast artificial chromosomes (YACs)). When the fragment of chromosomal DNA is ultimately joined with its cloning vector in the lab, it is called a “recombinant DNA molecule.” Shortly after the recombinant plasmid is introduced into suitable host cells, the newly inserted segment will be reproduced along with the host cell DNA.
“Cosmids” are artificially constructed cloning vectors that carry up to 45 kb of foreign DNA. They can be packaged in lambda phage particles for infection into E. coli cells.
As used herein, the term “mammal” (as in “genetically modified (or altered) mammal”) is meant to include any non-human mammal, including but not limited to pigs, sheep, goats, cattle (bovine), deer, mules, horses, monkeys, dogs, cats, rats, mice, birds, chickens, reptiles, fish, and insects. In one embodiment of the invention, genetically altered pigs and methods of production thereof are provided.
The term “ungulate” refers to hoofed mammals. Artiodactyls are even-toed (cloven-hooved) ungulates, including antelopes, camels, cows, deer, goats, pigs, and sheep. Perissodactyls are odd toes ungulates, which include horses, zebras, rhinoceroses, and tapirs. The term ungulate as used herein refers to an adult, embryonic or fetal ungulate animal.
As used herein, the terms “porcine”, “porcine animal”, “pig” and “swine” are generic terms referring to the same type of animal without regard to gender, size, or breed.
A “homologous DNA sequence or homologous DNA” is a DNA sequence that is at least about 80%, 85%, 90%, 95%, 98% or 99% identical with a reference DNA sequence. A homologous sequence hybridizes under stringent conditions to the target sequence, stringent hybridization conditions include those that will allow hybridization occur if there is at least 85, at least 95% or 98% identity between the sequences.
An “isogenic or substantially isogenic DNA sequence” is a DNA sequence that is identical to or nearly identical to a reference DNA sequence. The term “substantially isogenic” refers to DNA that is at least about 97-99% identical with the reference DNA sequence, or at least about 99.5-99.9% identical with the reference DNA sequence, and in certain uses 100% identical with the reference DNA sequence.
“Homologous recombination” refers to the process of DNA recombination based on sequence homology.
“Gene targeting” refers to homologous recombination between two DNA sequences, one of which is located on a chromosome and the other of which is not.
“Non-homologous or random integration” refers to any process by which DNA is integrated into the genome that does not involve homologous recombination.
A “selectable marker gene” is a gene, the expression of which allows cells containing the gene to be identified. A selectable marker can be one that allows a cell to proliferate on a medium that prevents or slows the growth of cells without the gene. Examples include antibiotic resistance genes and genes which allow an organism to grow on a selected metabolite. Alternatively, the gene can facilitate visual screening of transformants by conferring on cells a phenotype that is easily identified. Such an identifiable phenotype can be, for example, the production of luminescence or the production of a colored compound, or the production of a detectable change in the medium surrounding the cell.
The term “contiguous” is used herein in its standard meaning, i.e., without interruption, or uninterrupted.
“Stringent conditions” refers to conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C., or (2) employ during hybridization a denaturing agent such as, for example, formamide. One skilled in the art can determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. For example, stringency can generally be reduced by increasing the salt content present during hybridization and washing, reducing the temperature, or a combination thereof. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y., (1989).
I. Immunoglobulin Genes
In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
In another aspect of the present invention, a method is provided to disrupt the expression of an ungulate immunoglobulin gene by (i) analyzing the germline configuration of the ungulate heavy chain, kappa light chain or lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end and the 3′ end of at least one functional region of the locus; and (iii) transfecting a targeting construct containing the flanking sequence into a cell wherein, upon successful homologous recombination, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene.
In one embodiment, the germline configuration of the porcine heavy chain locus is provided. The porcine heavy chain locus contains at least four variable regions, two diversity regions, six joining regions and five constant regions, for example, as illustrated in FIG. 1. In a specific embodiment, only one of the six joining regions, J6, is functional.
In another embodiment, the germline configuration of the porcine kappa light chain locus is provided. The porcine kappa light chain locus contains at least six variable regions, six joining regions, one constant region and one enhancer region, for example, as illustrated in FIG. 2.
In a further embodiment, the germline configuration of the porcine lambda light chain locus is provided.
Isolated nucleotide sequences as depicted in Seq ID Nos 1-39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to any one of Seq ID Nos 1-39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of any one of Seq ID Nos 1-39 are provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1-39, as well as, nucleotides homologous thereto.
Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (see, for example, Altschul, S. F. et al (1997) Nucleic Acids Res 25:3389-3402 and Karlin et al, (1900) Proc. Natl. Acad. Sci. USA 87, 2264-2268) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. See, for example, Altschul et al., (1994) (Nature Genetics 6, 119-129). The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter (low co M'plexity) are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919), which is recommended for query sequences over 85 in length (nucleotide bases or amino acids).
Porcine Heavy Chain
In another aspect of the present invention, novel genomic sequences encoding the heavy chain locus of ungulate immunoglobulin are provided. In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In another embodiment, an isolated nucleotide sequence is provided that includes at least four joining regions and at least one constant region, such as the mu constant region, of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No. 4. In a further embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No 1. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in the 3′ region of Seq ID No 4. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.
In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In one embodiment, the nucleotide sequence contains at least 17, 20, 25 or 30 contiguous nucleotides of Seq ID No 4 or residues 1-9,070 of Seq ID No 29. In other embodiments, nucleotide sequences that contain at least 50, 100, 1,000, 2,500, 4,000, 4,500, 5,000, 7,000, 8,000, 8,500, 9,000, 10,000 or 15,000 contiguous nucleotides of Seq ID No. 29 are provided. In another embodiment, the nucleotide sequence contains residues 9,070-11039 of Seq ID No 29.
In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.

In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In Seq ID No. 29, the Diversity region of heavy chain is represented, for example, by residues 1089-1099 (D(pseudo)), the Joining region of heavy chain is represented, for example, by residues 1887-3352 (for example: J(psuedo): 1887-1931, J(psuedo): 2364-2411, J(psuedo): 2756-2804, J (functional J): 3296-3352), the recombination signals are represented, for example, by residues 3001-3261 (Nonamer), 3292-3298 (Heptamer), the Constant Region is represented by the following residues: 3353-9070 (J to C mu intron), 5522-8700 (Switch region), 9071-9388 (Mu Exon 1), 9389-9469 (Mu Intron A), 9470-9802 (Mu Exon 2), 9830-10069 (Mu Intron B), 10070-10387 (Mu Exon 3), 10388-10517 (Mu Intron C), 10815-11052 (Mu Exon 4), 11034-11039 (Poly(A) signal).


Seq ID No. 29

tctagaagacgctggagagaggccagacttcctcggaacagctcaaagag

ctctgtcaaagccagatcccatcacacgtgggcaccaataggccatgcca

gcctccaagggccgaactgggttctccacggcgcacatgaagcctgcagc

ctggcttatcctcttccgtggtgaagaggcaggcccgggactggacgagg

ggctagcagggtgtggtaggcaccttgcgccccccaccccggcaggaacc

agagaccctggggctgagagtgagcctccaaacaggatgccccacccttc

aggccacctttcaatccagctacactccacctgccattctcctctgggca

cagggcccagcccctggatcttggccttggctcgacttgcacccacgcgc

acacacacacttcctaacgtgctgtccgctcacccctccccagcgtggtc

catgggcagcacggcagtgcgcgtccggcggtagtgagtgcagaggtccc

ttcccctcccccaggagccccaggggtgtgtgcagatctgggggctcctg

tcccttacaccttcatgcccctcccctcatacccaccctccaggcgggag

gcagcgagacctttgcccagggactcagccaacgggcacacgggaggcca

gccctcagcagctggctcccaaagaggaggtgggaggtaggtccacagct

gccacagagagaaaccctgacggaccccacaggggccacgccagccggaa

ccagctccctcgtgggtgagcaatggccagggccccgccggccaccacgg

ctggccttgcgccagctgagaactcacgtccagtgcagggagactcaaga

cagcctgtgcacacagcctcggatctgctcccatttcaagcagaaaaagg

aaaccgtgcaggcagccctcagcatttcaaggattgtagcagcggccaac

tattcgtcggcagtggccgattagaatgaccgtggagaagggcggaaggg

tctctcgtgggctctgcggccaacaggccctggctccacctgcccgctgc

cagcccgaggggcttgggccgagccaggaaccacagtgctcaccgggacc

acagtgactgaccaaactcccggccagagcagccccaggccagccgggct

ctcgccctggaggactcaccatcagatgcacaagggggcgagtgtggaag

agacgtgtcgcccgggccatttgggaaggcgaagggaccttccaggtgga

caggaggtgggacgcactccaggcaagggactgggtccccaaggcctggg

gaaggggtactggcttgggggttagcctggccagggaacggggagcgggg

cggggggctgagcagggaggacctgacctcgtgggagcgaggcaagtcag

gcttcaggcagcagccgcacatcccagaccaggaggctgaggcaggaggg

gcttgcagcggggcgggggcctgcctggctccgggggctcctgggggacg

ctggctcttgtttccgtgtcccgcagcacagggccagctcgctgggccta

tgcttaccttgatgtctggggccggggcgtcagggtcgtcgtctcctcag

gggagagtcccctgaggctacgctgggg*ggggactatggcagctccacc

aggggcctggggaccaggggcctggaccaggctgcagcccggaggacggg

cagggctctggctctccagcatctggccctcggaaatggcagaacccctg

gcgggtgagcgagctgagagcgggtcagacagacaggggccggccggaaa

ggagaagttgggggcagagcccgccaggggccaggcccaaggttctgtgt

gccagggcctgggtgggcacattggtgtggccatggctacttagattcgt

ggggccagggcatcctggtcaccgtctcctcaggtgagcctggtgtctga

tgtccagctaggcgctggtgggccgcgggtgggcctgtctcaggctaggg

caggggctgggatgtgtatttgtcaaggaggggcaacagggtgcagactg

tgcccctggaaacttgaccactggggcaggggcgtcctggtcacgtctcc

tcaggtaagacggccctgtgcccctctctcgcgggactggaaaaggaatt

ttccaagattccttggtctgtgtggggccctctggggcccccgggggtgg

ctcccctcctgcccagatggggcctcggcctgtggagcacgggctgggca

cacagctcgagtctagggccacagaggcccgggctcagggctctgtgtgg

cccggcgactggcagggggctcgggtttttggacaccccctaatgggggc

cacagcactgtgaccatcttcacagctggggccgaggagtcgaggtcacc

gtctcctcaggtgagtcctcgtcagccctctctcactctctggggggttt

tgctgcattttgtgggggaaagaggatgcctgggtctcaggtctaaaggt

ctagggccagcgccggggcccaggaaggggccgaggggccaggctcggct

cggccaggagcagagcttccagacatctcgcctcctggcggctgcagtca

ggcctttggccgggggggtctcagcaccaccaggcctcttggctcccgag

gtccccggccccggctgcctcaccaggcaccgtgcgcggtgggcccgggc

tcttggtcggccaccctttcttaactgggatccgggcttagttgtcgcaa

tgtgacaacgggctcgaaagctggggccaggggaccctagtctacgacgc

ctcgggtgggtgtcccgcacccctccccactttcacggcactcggcgaga

cctggggagtcaggtgttggggacactttggaggtcaggaacgggagctg

gggagagggctctgtcagcggggtccagagatgggccgccctccaaggac

gccctgcgcggggacaagggcttcttggcctggcctggccgcttcacttg

ggcgtcagggggggcttcccggggcaggcggtcagtcgaggcgggttgga

attctgagtctgggttcggggttcggggttcggccttcatgaacagacag

cccaggcgggccgttgtttggcccctgggggcctggttggaatgcgaggt

ctcgggaagtcaggagggagcctggccagcagagggttcccagccctgcg

gccgagggacctggagacgggcagggcattggccgtcgcagggccaggcc

acaccccccaGGTTTTTGTggggcgagcctggagattgcacCACTGTGAT

TACTATGCTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTC

AGgtaagaacggccctccagggcctttaatttctgctctcgtctgtgggc

ttttctgactctgatcctcgggaggcgtctgtgccccccccggggatgag

gccggcttgccaggaggggtcagggaccaggagcctgtgggaagttctga

cgggggctgcaggcgggaagggccccaccggggggcgagccccaggccgc

tgggcggcaggagacccgtgagagtgcgccttgaggagggtgtctgcgga

accacgaacgcccgccgggaagggcttgctgcaatgcggtcttcagacgg

gaggcgtcttctgccctcaccgtctttcaagcccttgtgggtctgaaaga

gccatgtcggagagagaagggacaggcctgtcccgacctggccgagagcg

ggcagccccgggggagagcggggcgatcggcctgggctctgtgaggccag

gtccaagggaggacgtgtggtcctcgtgacaggtgcacttgcgaaacctt

agaagacggggtatgttggaagcggctcctgatgtttaagaaaagggaga

ctgtaaagtgagcagagtcctcaagtgtgttaaggttttaaaggtcaaag

tgttttaaacctttgtgactgcagttagcaagcgtgcggggagtgaatgg

ggtgccagggtggccgagaggcagtacgagggccgtgccgtcctctaatt

cagggcttagttttgcagaataaagtcggcctgttttctaaaagcattgg

tggtgctgagctggtggaggaggccgcgggcagccctggccacctgcagc

aggtggcaggaagcaggtcggccaagaggctattttaggaagccagaaaa

cacggtcgatgaatttatagcttctggtttccaggaggtggttgggcatg

gctttgcgcagcgccacagaaccgaaagtgcccactgagaaaaaacaact

cctgcttaatttgcatttttctaaaagaagaaacagaggctgacggaaac

tggaaagttcctgttttaactactcgaattgagttttcggtcttagctta

tcaactgctcacttagattcattttcaaagtaaacgtttaagagccgagg

cattcctatcctcttctaaggcgttattcctggaggctcattcaccgcca

gcacctccgctgcctgcaggcattgctgtcaccgtcaccgtgacggcgcg

cacgattttcagttggcccgcttcccctcgtgattaggacagacgcgggc

actctggcccagccgtcttggctcagtatctgcaggcgtccgtctcggga

cggagctcaggggaagagcgtgactccagttgaacgtgatagtcggtgcg

ttgagaggagacccagtcgggtgtcgagtcagaaggggcccggggcccga

ggccctgggcaggacggcccgtgccctgcatcacgggcccagcgtcctag

aggcaggactctggtggagagtgtgagggtgcctggggcccctccggagc

tggggccgtgcggtgcaggttgggctctcggcgcggtgttggctgtttct

gcgggatttggaggaattcttccagtgatgggagtcgccagtgaccgggc

accaggctggtaagagggaggccgccgtcgtggccagagcagctgggagg

gttcggtaaaaggctcgcccgtttcctttaatgaggacttttcctggagg

gcatttagtctagtcgggaccgttttcgactcgggaagagggatgcggag

gagggcatgtgcccaggagccgaaggcgccgcggggagaagcccagggct

ctcctgtccccacagaggcgacgccactgccgcagacagacagggccttt

ccctctgatgacggcaaaggcgcctcggctcttgcggggtgctggggggg

agtcgccccgaagccgctcacccagaggcctgaggggtgagactgaccga

tgcctcttggccgggcctggggccggaccgagggggactccgtggaggca

gggcgatggtggctgcgggagggaaccgaccctgggccgagcccggcttg

gcgattcccgggcgagggccctcagccgaggcgagtgggtccggcggaac

caccctttctggccagcgccacagggctctcgggactgtccggggcgacg

ctgggctgcccgtggcaggccTGGGCTGACCTGGACTTCACCAGACAGAA

CAGGGCTTTCAGGGCTGAGCTGAGCCAGGTTTAGCGAGGCCAAGTGGGGC

TGAACCAGGCTCAACTGGCCTGAGCTGGGTTGAGCTGGGCTGACCTGGGC

TGAGCTGAGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGAC

TGGCTGAGCTGAGCTGGGTTGAGCTGAGCTGAGCTGGCCTGGGTTGAGCT

GGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGTTGAGCTGGGTTGATCT

GAGCTGAGCTGGGCTGAGCTGAGCTAGGCTGGGGTGAGCTGGGCTGAGCT

GGTTTGAGTTGGGTTGAGCTGAGCTGAGCTGGGCTGTGCTGGCTGAGCTA

GGCTGAGCTAGGCTAGGTTGAGCTGGGCTGGGCTGAGGTGAGCTAGGCTG

GGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCGTTGAGCTGGCTGG

GCTGGATTGAGCTGGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGT

TGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGTTGAGCTGTCC

TGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCAGC

AGAGCTGGGTTGGGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCTGGCC

TGGGTTGAGCTGGGCTGAGCTGAGCTGGGCTGAGCTGGCCTGTGTTGAGC

TGGGCTGGGTTGAGCTGGGCTGAGCTGGATTGAGCTGGGTTGAGCTGAGC

TGGGCTGGGCTGTGCTGACTGAGCTGGGCTGAGCTAGGCTGGGGTGAGCT

GGGCTGAGCTGATCCGAGCTAGGCTGGGCTGGTTTGGGCTGAGCTGAGCT

GAGCTAGGCTGGATTGATCTGGCTGAGCTGGGTTGAGCTGAGCTGGGCTG

AGCTGGTCTGAGCTGGCCTGGGTCGAGCTGAGCTGGACTGGTTTGAGCTG

GGTCGATCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTG

AGCTGGGTTGAGCTGGGCTGAGCTGAGGGCTGGGGTGAGCTGGGCTGAAC

TAGCCTAGCTAGGTTGGGCTGAGCTGGGCTGGTTTGGGCTGAGCTGAGCT

GAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCAGGCCTGGGGTG

AGCTGGGCTAGGTGGAGCTGAGCTGGGTCGAGCTGAGTTGGGCTGAGCTG

GCCTGGGTTGAGGTAGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTG

GCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGA

GCTGGGCTCGGTTGAGCTGGGCTGAGCTGAGCCGACCTAGGCTGGGATGA

GCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGG

CTGAGCTGGGCCTGGAGCCTGGCCTGGGGTGAGCTGGGCTGAGCTGCGCT

GAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAAGCTG

GGCCGAGCTGGCCTGGGATGAGCTGGGCCGGTTTGGGCTGAGCTGAGCTG

AGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGGGTGA

GCTGGGCTGAGCTAAGCTGAGCTGGGCTGGTTTGGGCTGAGCTGGCTGAG

CTGGGTCCTGCTGAGCTGGGCTGAGCTGACCAGGGGTGAGCTGGGCTGAG

TTAGGCTGGGCTCAGCTAGGCTGGGTTGATCTGGCAGGGCTGGTTTGCGC

TGGGTCAAGCTCCCGGGAGATGGCCTGGGATGAGCTGGGCTGGTTTGGGC

TGAGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCT

GAGCTGGCCTGGGGTGAGCTGGGCTGGGTGGAGCTGAGCTGGGCTGAACT

GGGCTAAGCTGGCTGAGCTGGATCGAGCTGAGCTGGGCTGAGCTGGCCTG

GGGTTAGCTGGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCTGG

GCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGAGCTGG

GCTGGGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGGCTGGGCTGAGCTGA

GCTAGGCTGCATTGAGCTGGCTGGGATGGATTGAGCTGGCTGAGCTGGCT

GAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTG

AGCTGAGCTGGGCTGAGCTGGGCTCAGCAGAGCTGGGTTGAGCTGAGCTG

GGTTGAGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGTTGAGCTGAGCTG

GGTTGAGCTGGGCTCGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCT

GGGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTGAGCT

AGCTGGGCTCAGCTAGGCTGGGTTGAGCTGAGCTGGGCTGAACTGGGCTG

AGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCTGGGCTGAGCAGAGCTG

GGCTGAGCAGAGCTGGGTTGGTCTGAGCTGGGTTGAGCTGGGCTGAGCTG

GGCTGAGCAGAGTTGGGTTGAGCTGAGCTGGGTTCAGCTGGGCTGAGCTA

GGCTGGGTTGAGCTGGGTTGAGTTGGGCTGAGCTGGGCTGGGTTGAGCGG

AGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCGGAACTGGGTTGATCTG

AATTGAGCTGGGCTGAGCCGGGCTGAGCCGGGCTGAGCTGGGCTAGGTTG

AGCTTGGGTGAGCTTGCCTCAGCTGGTCTGAGCTAGGTTGGGTGGAGCTA

GGCTGGATTGAGCTGGGCTGAGCTGAGCTGATCTGGCCTCAGCTGGGCTG

AGGTAGGCTGAACTGGGCTGTGCTGGGCTGAGCTGAGCTGAGCCAGTTTG

AGCTGGGTTGAGCTGGGCTGAGCTGGGCTGTGTTGATCTTTCCTGAACTG

GGCTGAGCTGGGCTGAGCTGGCCTAGCTGGATTGAACGGGGGTAAGCTGG

GCCAGGCTGGACTGGGCTGAGCTGAGCTAGGCTGAGCTGAGTTGAATTGG

GTTAAGCTGGGCTGAGATGGGCTGAGCTGGGCTGAGCTGGGTTGAGCCAG

GTCGGACTGGGTTACCCTGGGCCACACTGGGCTGAGCTGGGCGGAGCTCG

attaacctggtcaggctgagtcgggtccagcagacatgcgctggccaggc

tggcttgacctggacacgttcgatgagctgccttgggatggttcacctca

gctgagccaggtggctccagctgggctgagctggtgaccctgggtgacct

cggtgaccaggttgtcctgagtccgggccaagccgaggctgcatcagact

cgccagacccaaggcctgggccccggctggcaagccaggggcggtgaagg

ctgggctggcaggactgtcccggaaggaggtgcacgtggagccgcccgga

ccccgaccggcaggacctggaaagacgcctctcactcccctttctcttct

gtcccctctcgggtcctcagAGAGCCAGTCTGCCCCGAATCTCTACCCCC

TCGTCTCCTGCGTCAGCCCCCCGTCCGATGAGAGCCTGGTGGCCCTGGGC

TGCCTGGCCCGGGACTTCCTGCCCAGCTCCGTCACCTTCTCCTGGAACTA

CAAGAACAGCAGCAAGGTCAGCAGCCAGAACATCCAGGACTTCCCGTCC

GTCCTGAGAGGCGGCAAGTACTTGGCCTCCTCCCGGGTGCTCCTACCCTCT

GTGAGCATCCCCCAGGACCCAGAGGCCTTCCTGGTGTGCGAGGTCCAGCA

CCCCAGTGGCACCAAGTCCGTGTCCATCTCTGGGCCAGgtgagctgggct

ccccctgtggctgtggcgggggcggggccgggtgccgccggcacagtgac

gccccgttcctgcctgcagTCGTAGAGGAGCAGCCCCCCGTCTTGAACATC

TTCGTCCCCACCCGGGAGTCCTTCTCCAGTACTCCCCAGCGCACGTCCAAG

CTCATCTGCCAGGCCTCAGACTTCAGCCCCAAGCAGATCTCCATGGCCTGG

TTCCGTGATGGGAAACGGGTGGTGTCTGGCGTCAGGACAGGCCCCGTGGAG

ACCCTACAGTCCAGTGGGGTGACCTACAGGCTCCACAGCATGCTGACCGTCA

CGGAGTCCGAGTGGGTCAGCCAGAGCGTCTTCACCTGCCAGGTGGAGCACAAA

GGGCTGAACTACGAGAAGAACGCGTCCTCTCTGTGCACCTCCAgtgagtgcag

cccctcgggccgggcggcggggcggcgggagccacacacacaccagctgctcc

ctgagccttggcttccgggagtggccaaggcggggaggggctgtgcagggcagc

tggagggcactgtcagctggggcccagcaccccctcaccccggcagggcccggg

ctccgaggggccccgcagtcgcaggccctgctcttgggggaagccctacttggc

cccttcagggcgctgacgctccccccacccacccccgcctagATCCCAACTCTC

CCATCACCGTCTTCGCCATCGCCCCCTCCTTCGCTGGCATCTTCCTCACCAAGT

CGGCGAAGCTTTCCTGCCTGGTCAGGGGCCTCGTCACCAGGGAGAGCGTCAACA

TCTCCTGGACCCGCCAGGACGGCGAGGTTCTGAAGACCAGTATCGTCTTCTCTG

AGATCTACGCCAACGGCACCTTCGGCGCCAGGGGCGAAGCCTCCGTCTGCGTGG

AGGACTGGGAGTCGGGCGACAGGTTCACGTGCACGGTGACCCACACGGACCTGC

CCTCGCCGCTGAAGCAGAGCGTCTCCAAGCCCAGAGgtaggccctgccctgccc

ctgcctccgcccggcctgtgccttggccgccggggcgggagccgagcctggccg

aggagcgccctcggccccccgcggtcccgacccacacccctcctgctctcctcc

ccagGGATCGCCAGGCACATGCCGTCCGTGTACGTGCTGCCGCCGGCCCCGGAG

GAGCTGAGCCTGCAGGAGTGGGCCTCGGTCACCTGCCTGGTGAAGGGCTTCTCC

CCGGCGGACGTGTTCGTGCAGTGGCTGCAGAAGGGGGAGCCCGTGTCCGCCGAC

AAGTACGTGACCAGCGGGCCGGTGCCCGAGCCCGAGCCCAAGGCCCCCGCCTCC

TACTTCGTGCAGAGCGTCCTGACGGTGAGCGCCAAGGACTGGAGCGACGGGGAG

ACCTACACCTGCGTCGTGGGCCACGAGGCCCTGCCCCACACGGTGACCGAGAGG

ACCGTGGACAAGTCCACCGGTAAACCCACCCTGTACAACGTCTCCCTGGTCCTG

TCCGACACGGCCAGCACCTGCTACTGACCCCCTGGCTGCCCGCCGCGGCCGGGG

CCAGAGCCCCCGGGCGACCATCGCTCTGTGTGGGCCTGTGTGCAACCCGACCC

TGTCGGGGTGAGCGGTCGCATTTCTGAAAATTAGAaataaaAGATCTCGTGC

CG

Seq ID No. 1

TCTAgAAGACGCTGGAGAGAGGCCagACTTCCTGGGAACAGCTCAAAGAG

CTCTGTCAAAGCCAGATCCCATCACACGTGGGCACCAATAGGCCATGCCA

GCCTCCAAGGGCCGAACTGGGTTCTCCACGGCGCACATGAAGCCTGCAGC

CTGGCTTATCCTCTTCCGTGGTGAAGAGGCAGGCCCGGGACTGGACGAGG

GGCTAGCAGGGTGTGGTAGGCACCTTGCGCCCCCCACCCCGGCAGGAACC

AGAGACCCTGGGGCTGAGAGTGAGCCTCCAAACAGGATGCCCCACCCTTC

AGGCCACCTTTCAATCCAGCTACACTCCACCTGCCATTCTCCTCTGGGCA

CAGGGCCCAGCCCCTGGATCTTGGCCTTGGCTCGACTTGCACCGACGCGC

ACACACACACTTCCTAACGTGCTGTCCGCTCACCCCTCCCCAGCGTGGTC

CATGGGCAGCACGGCAGTGCGCGTCCGGCGGTAGTGAGTGCAGAGGTCCC

TTCCCCTCCCCCAGGAGCCCCAGGGGTGTGTGCAGATCTGGGGGCTCCTG

TCCCTTACACCTTCATGCCCCTCCCCTCATACCCACCCTCCAGGCGGGAG

GCAGCGAGACCTTTGCCCAGGGACTCAGCCAACGGGCACACGGGAGGCC

A GCCCTCAGCAGCTGGG

Seq ID No. 4

GGCCAGACTTCCTCGGAACAGCTCAAAGAGCTCTGTCAAAGCCAGATCCC
0
ATCACACGTGGGCACCAATAGGCCATGCCAGCCTCCAAGGGCCGAACTGG

GTTCTCCACGGCGCACATGAAGCCTGCAGCCTGGCTTATCCTCTTCCGTG

GTGAAGAGGCAGGCCCGGGACTGGACGAGGGGCTAGCAGGGTGTGGTAG

GCACCTTGCGCCCCCCACCCCGGCAGGAACCAGAGACCCTGGGGCTGAGA

G

TGAGCCTCCAAACAGGATGCCCCACCCTTCAGGCCACCTTTCAATCCAGC

TACACTCCACCTGCCATTCTCCTCTGGGCACAGGGCCCAGCCCCTGGATC

TTGGCCTTGGCTCGACTTGCACCCACGCGCACACACACACTTCCTAACGT

GCTGTCCGCTCACCCCTCCCCAGCGTGGTCCATGGGCAGCACGGCAGTGC

GCGTCCGGCGGTAGTGAGTGCAGAGGTCCCTTCCCCTCCCCCAGGAGCCC

CAGGGGTGTGTGCAGATCTGGGGGCTCCTGTCCCTTACACCTTCATGCCC

CTCCCCTCATACCCACCCTCCAGGCGGGAGGCAGCGAGACCTTTGCCCAG

GGACTCAGCCAACGGGCACACGGGAGGCCAGCCCTCAGCAGCTGGCTCCC

AAAGAGGAGGTGGGAGGTAGGTCCACAGCTGCCACAGAGAGAAACCCTG

ACGGACCCCACAGGGGCCACGCCAGCCGGAACCAGCTCCCTCGTGGGTGA

GCAATGGCCAGGGCCCCGCCGGCCACCACGGCTGGCCTTGCGCCAGCTGA

G

AACTCACGTCCAGTGCAGGGAGACTCAAGACAGCCTGTGCACACAGCCTC

GGATCTGCTCCCATTTCAAGCAGAAAAAGGAAACCGTGCAGGCAGCCCTC

AGCATTTCAAGGATTGTAGCAGCGGCCAACTATTCGTCGGCAGTGGCCGA

TTAGAATGACCGTGGAGAAGGGCGGAAGGGTCTCTCGTGGGCTCTGCGGC

CAACAGGCCCTGGCTCCACCTGCCCGCTGCCAGCCCGAGGGGCTTGGGCC

GAGCCAGGAACCACAGTGCTCACCGGGACCACAGTGACTGACCAAACTCC

CGGCCAGAGCAGCCCCAGGCCAGCCGGGCTCTCGCCCTGGAGGACTCACC

ATCAGATGCACAAGGGGGCGAGTGTGGAAGAGACGTGTCGCCCGGGCCA

T

TTGGGAAGGCGAAGGGACCTTCCAGGTGGACAGGAGGTGGGACGCACTC

C

AGGCAAGGGACTGGGTCCCCAAGGCCTGGGGAAGGGGTACTGGCTTGGG

G

GTTAGCCTGGCCAGGGAACGGGGAGCGGGGCGGGGGGCTGAGCAGGGAG

G

ACCTGACCTCGTGGGAGCGAGGCAAGTCAGGCTTCAGGCAGCAGCCGCAC

ATCCCAGACCAGGAGGCTGAGGCAGGAGGGGCTTGCAGCGGGGCGGGGG

C

CTGCCTGGCTCCGGGGGCTCCTGGGGGACGCTGGCTCTTGTTTCCGTGTC

CCGCAGCACAGGGCCAGCTCGCTGGGCCTATGCTTACCTTGATGTCTGGG

GCCGGGGCGTCAGGGTCGTCGTCTCCTCAGGGGAGAGTCCCCTGAGGCTA

CGCTGGGG*GGGGACTATGGCAGCTCCACCAGGGGCCTGGGGACCAGGG

G

CCTGGACCAGGCTGCAGCCCGGAGGACGGGCAGGGCTCTGGCTCTCCAGC

ATCTGGCCCTCGGAAATGGCAGAACCCCTGGCGGGTGAGCGAGCTGAGA

G

CGGGTCAGACAGACAGGGGCCGGCCGGAAAGGAGAAGTTGGGGGCAGAG

C

CCGCCAGGGGCCAGGCCCAAGGTTCTGTGTGCCAGGGCCTGGGTGGGCAC

ATTGGTGTGGCCATGGCTACTTAGATTCGTGGGGCCAGGGCATCCTGGTC

ACCGTCTCCTCAGGTGAGCCTGGTGTCTGATGTCCAGCTAGGCGCTGGTG

GGCCGCGGGTGGGCCTGTCTCAGGCTAGGGCAGGGGCTGGGATGTGTATT

TGTCAAGGAGGGGCAACAGGGTGCAGACTGTGCCCCTGGAAACTTGACCA

CTGGGGCAGGGGCGTCCTGGTCACGTCTCCTCAGGTAAGACGGCCCTGTG

CCCCTCTCTCGCGGGACTGGAAAAGGAATTTTCCAAGATTCCTTGGTCTG

TGTGGGGCCCTCTGGGGCCCCCGGGGGTGGCTCCCCTCCTGCCCAGATGG

GGCCTCGGCCTGTGGAGCACGGGCTGGGCACACAGCTCGAGTCTAGGGCC

ACAGAGGCCCGGGCTCAGGGCTCTGTGTGGCCCGGCGACTGGCAGGGGG

C

TCGGGTTTTTGGACACCCCCTAATGGGGGCCACAGCACTGTGACCATCTT

CACAGCTGGGGCCGAGGAGTCGAGGTCACCGTCTCCTCAGGTGAGTCCTC

GTCAGCCCTCTCTCACTCTCTGGGGGGTTTTGCTGCATTTTGTGGGGGAA

AGAGGATGCCTGGGTCTCAGGTCTAAAGGTCTAGGGCCAGCGCCGGGGCC

CAGGAAGGGGCCGAGGGGCCAGGCTCGGCTCGGCCAGGAGCAGAGCTTC

C

AGACATCTCGCCTCCTGGCGGGTGCAGTCAGGCCTTTGGCCGGGGGGGTC

TCAGCACCACGAGGCCTCTTGGCTCCCGAGGTGGCCGGCCCCGGCTGCCT

CACCAGGCACCGTGCGCGGTGGGCCCGGGCTCTTGGTCGGCCACCCTTTC

TTAACTGGGATCCGGGCTTAGTTGTCGCAATGTGACAACGGGCTCGAAAG

CTGGGGCCAGGGGACCCTAGT*TAGGACGCCTCGGGTGGGTGTCCCGCAC

CCCTCCCCACTTTCACGGCACTCGGCGAGACCTGGGGAGTCAGGTGTTGG

GGACACTTTGGAGGTCAGGAACGGGAGCTGGGGAGAGGGCTCTGTCAGC

G

GGGTCCAGAGATGGGCCGCCCTCCAAGGACGCCCTGCGCGGGGACAAGG

G

CTTCTTGGCCTGGCCTGGCCGCTTCACTTGGGCGTCAGGGGGGGCTTCCC

GGGGCAGGCGGTCAGTCGAGGCGGGTTGGAATTCTGAGTCTGGGTTCGGG

GTTCGGGGTTCGGCCTTCATGAACAGACAGCCCAGGCGGGCCGTTGTTTG

GCCCCTGGGGGCCTGGTTGGAATGCGAGGTCTCGGGAAGTCAGGAGGGA

G

CCTGGCCAGCAGAGGGTTCCCAGCCCTGCGGCCGAGGGACCTGGAGACG

G

GCAGGGCATTGGCCGTCGCAGGGCCAGGCCACACCCCCCAGGTTTTTGTG

GGGCGAGCCTGGAGATTGCACCACTGTGATTACTATGCTATGGATCTCTG

GGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAGGTAAGAACGGCCCTCCAG

GGCCTTTAATTTCTGCTCTCGTCTGTGGGCTTTTCTGACTCTGATCCTCG

GGAGGCGTCTGTGCCCCCCCCGGGGATGAGGCCGGCTTGCCAGGAGGGGT

CAGGGACCAGGAGCCTGTGGGAAGTTCTGACGGGGGCTGCAGGCGGGAA

G

GGCCCCACCGGGGGGCGAGCCCCAGGCCGCTGGGCGGCAGGAGACCCGT

G

AGAGTGCGCCTTGAGGAGGGTGTCTGCGGAACCACGAACGCCCGCCGGG

A

AGGGCTTGCTGCAATGCGGTCTTCAGACGGGAGGCGTCTTCTGCCCTCAC

CGTCTTTCAAGCCCTTGTGGGTCTGAAAGAGCCATGTCGGAGAGAGAAGG

GACAGGCCTGTCCCGACCTGGCCGAGAGCGGGCAGCCCCGGGGGAGAGC

G

GGGCGATCGGCCTGGGCTCTGTGAGGCCAGGTCCAAGGGAGGACGTGTG

G

TCCTCGTGACAGGTGCACTTGCGAAACCTTAGAAGACGGGGTATGTTGGA

AGCGGCTCCTGATGTTTAAGAAAAGGGAGACTGTAAAGTGAGCAGAGTCC

TCAAGTGTGTTAAGGTTTTAAAGGTCAAAGTGTTTTAAACCTTTGTGACT

GCAGTTAGCAAGCGTGCGGGGAGTGAATGGGGTGCCAGGGTGGCCGAGA

G

GCAGTACGAGGGCCGTGCCGTCCTCTAATTCAGGGCTTAGTTTTGCAGAA

TAAAGTCGGCCTGTTTTCTAAAAGCATTGGTGGTGCTGAGCTGGTGGAGG

AGGCCGCGGGCAGCCCTGGCCACCTGCAGCAGGTGGCAGGAAGCAGGTC

G

GCCAAGAGGCTATTTTAGGAAGCCAGAAAACACGGTCGATGAATTTATAG

CTTCTGGTTTCCAGGAGGTGGTTGGGCATGGCTTTGCGCAGCGCCACAGA

ACCGAAAGTGCCCACTGAGAAAAAACAACTCCTGCTTAATTTGCATTTTT

CTAAAAGAAGAAACAGAGGCTGACGGAAACTGGAAAGTTCCTGTTTTAAC

TACTCGAATTGAGTTTTCGGTCTTAGCTTATCAACTGCTCACTTAGATTC

ATTTTCAAAGTAAACGTTTAAGAGCCGAGGCATTCCTATCCTCTTCTAAG

GCGTTATTCCTGGAGGCTCATTCACCGCCAGCACCTCCGCTGCCTGCAGG

CATTGCTGTCACCGTCACCGTGACGGCGCGCACGATTTTCAGTTGGCCCG

CTTCCCCTCGTGATTAGGACAGACGCGGGCACTCTGGCCCAGCCGTCTTG

GCTCAGTATCTGCAGGCGTCCGTCTCGGGACGGAGCTCAGGGGAAGAGCG

TGACTCCAGTTGAACGTGATAGTCGGTGCGTTGAGAGGAGACCCAGTCGG

GTGTCGAGTCAGAAGGGGCCCGGGGCCCGAGGCCCTGGGCAGGACGGCC

C

GTGCCCTGCATCACGGGCCCAGCGTCCTAGAGGCAGGACTCTGGTGGAGA

GTGTGAGGGTGCCTGGGGCCCCTCCGGAGCTGGGGCCGTGCGGTGCAGGT

TGGGCTCTCGGCGCGGTGTTGGCTGTTTCTGCGGGATTTGGAGGAATTCT

TCCAGTGATGGGAGTCGCCAGTGACCGGGCACCAGGGTGGTAAGAGGGA

G

GCGGCCGTCGTGGCCAGAGCAGCTGGGAGGGTTCGGTAAAAGGCTCGCCC

GTTTCCTTTAATGAGGACTTTTCCTGGAGGGCATTTAGTCTAGTCGGGAC

CGTTTTCGACTCGGGAAGAGGGATGCGGAGGAGGGCATGTGCCCAGGAG

C

CGAAGGCGCCGCGGGGAGAAGCCCAGGGCTCTCCTGTCCCCACAGAGGC

G

ACGCCACTGCCGCAGACAGACAGGGCCTTTCCCTCTGATGACGGCAAAGG

CGCCTCGGCTCTTGCGGGGTGCTGGGGGGGAGTCGCCCCGAAGCCGCTCA

CCCAGAGGCCTGAGGGGTGAGACTGACCGATGCCTCTTGGCCGGGCCTGG

GGCCGGACCGAGGGGGACTCCGTGGAGGCAGGGCGATGGTGGCTGCGGG

A

GGGAACCGACCCTGGGCCGAGCCCGGCTTGGCGATTCCCGGGCGAGGGCC

CTCAGCCGAGGCGAGTGGGTCCGGCGGAACCACCCTTTCTGGCCAGCGCC

ACAGGGCTCTCGGGACTGTCCGGGGCGACGCTGGGCTGCCCGTGGCAGGC

CTGGGCTGACCTGGACTTCACCAGACAGAACAGGGCTTTCAGGGCTGAGC

TGAGCCAGGTTTAGCGAGGCCAAGTGGGGCTGAACCAGGCTCAACTGGCC

TGAGCTGGGTTGAGCTGGGCTGACCTGGGCTGAGCTGAGCTGGGCTGGGC

TGGGCTGGGGTGGGCTGGGCTGGGCTGGACTGGCTGAGCTGAGCTGGGTT

GAGCTGAGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCT

GGGTTGAGCTGGGTTGAGCTGGGTTGATCTGAGCTGAGCTGGGCTGAGCT

GAGCTAGGCTGGGGTGAGCTGGGCTGAGCTGGTTTGAGTTGGGTTGAGCT

GAGCTGAGCTGGGCTGTGCTGGCTGAGCTAGGCTGAGCTAGGCTAGGTTG

AGCTGGGCTGGGCTGAGCTGAGCTAGGCTGGGCTGATTTGGGCTGAGCTG

AGCTGAGCTAGGCTGCGTTGAGCTGGGTGGGCTGGATTGAGCTGGCTGAG

CTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGAGCTGGACTGGTT

TGAGCTGGGTCGATCTGGGTTGAGCTGTCCTGGGTTGAGCTGGGCTGGGT

TGAGCTGAGCTGGGTTGAGCTGGGCTCAGCAGAGCTGGGTTGGGCTGAGC

TGGGTTGAGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGAGC

TGAGCTGGGCTGAGCTGGCCTGTGTTGAGCTGGGCTGGGTTGAGCTGGGC

TGAGCTGGATTGAGCTGGGTTGAGCTGAGCTGGGCTGGGCTGTGCTGACT

GAGCTGGGCTGAGCTAGGCTGGGGTGAGCTGGGCTGAGCTGATCCGAGCT

AGGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGGATTGATCT

GGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCTGGTCTGAGCTGGGCTG

GGTCGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGCTGAGCTG

GCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTG

AGCTGAGGGCTGGGGTGAGCTGGGCTGAACTAGCCTAGCTAGGTTGGGCT

GAGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCA

GGCTGAGCTGGGCTGAGCAGGCCTGGGGTGAGCTGGGCTAGGTGGAGCT

G

AGCTGGGTCGAGCTGAGTTGGGCTGAGCTGGCCTGGGTTGAGGTAGGCTG

AGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGG

GTCAAGCTGGGCCGAGCTGOCCTGGGTTGAGCTGGGCTCGGTTGAGCTGG

GCTGAGCTGAGCCGACCTAGGCTGGGATGAGCTGGGCTGATTTGGGCTGA

GCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCCTGGAGCCT

GGCCTGGGGTGAGCTGGGCTGAGCTGCGCTGAGCTAGGCTGGGTTGAGCT

GGCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGATG

AGCTGGGCCGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAG

GCTGAGCTGGGCTGAGCTGGCCTGGGGTGAGCTGGGCTGAGCTAAGCTGA

GCTGGGCTGGTTTGGGCTGAGCTGGCTGAGCTGGGTCCTGCTGAGCTGGG

CTGAGCTGACCAGGGGTGAGCTGGGCTGAGTTAGGCTGGGCTCAGCTAGG

CTGGGTTGATCTGGCAGGGCTGGTTTGCGCTGGGTCAAGCTCCCGGGAGA

TGGCCTGGGATGAGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTGAGC

TAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGGGTGAGCT

GGGCTGGGTGGAGCTGAGCTGGGCTGAACTGGGCTAAGCTGGCTGAGCTG

GATCGAGCTGAGCTGGGCTGAGCTGGCCTGGGGTTAGCTGGGCTGAGCTG

AGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAA

GCTGGGCCGAGCTGGCCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAG

GCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAGGCTGCATTGAGCTGG

CTGGGATGGATTGAGCTGGCTGAGCTGGCTGAGCTGGCTGAGCTGGGCTG

AGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGCTGAGCTG

GGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGGTGAGCTG

GGCTGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCGAGCA

GAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCAGCAGAGCTGGGTT

GAGCTGAGCTGGGTTGAGCTGGGCTGAGCTAGCTGGGCTCAGCTAGGCTG

GGTTGAGCTGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAACTGGGCTG

AGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGCTGAGCAGAGCTGGGTT

G

GTCTGAGCTGGGTTGAGCTGGGCTGAGCTGGGCTGAGCAGAGTTGGGTTG

AGCTGAGCTGGGTTCAGCTGGGCTGAGCTAGGCTGGGTTGAGCTGGGTTG

AGTTGGGCTGAGCTGGGCTGGGTTGAGCGGAGCTGGGCTGAACTGGGCTG

AGCTGGGCTGAGCGGAACTGGGTTGATCTGAATTGAGCTGGGCTGAGCCG

GGCTGAGCCGGGCTGAGCTGGGCTAGGTTGAGCTTGGGTGAGCTTGCCTC

AGCTGGTCTGAGCTAGGTTGGGTGGAGCTAGGCTGGATTGAGCTGGGCTG

AGCTGAGCTGATCTGGCCTCAGCTGGGCTGAGGTAGGCTGAACTGGGCTG

TGCTGGGCTGAGCTGAGCTGAGCCAGTTTGAGCTGGGTTGAGCTGGGCTG

AGCTGGGCTGTGTTGATCTTTCCTGAACTGGGCTGAGCTGGGCTGAGCTG

GCCTAGCTGGATTGAACGGGGGTAAGCTGGGCCAGGCTGGACTGGGCTGA

GCTGAGCTAGGCTGAGCTGAGTTGAATTGGGTTAAGCTGGGCTGAGATGG

GCTGAGCTGGGCTGAGCTGGGTTGAGCCAGGTCGGACTGGGTTACCCTGG

GCCACACTGGGCTGAGCTGGGCGGAGCTCGATTAACCTGGTCAGGCTGAG

TCGGGTCCAGCAGACATGCGCTGGCCAGGCTGGCTTGACCTGGACACGTT

CGATGAGCTGCCTTGGGATGGTTCACCTCAGCTGAGCCAGGTGGCTCCAG

CTGGGCTGAGCTGGTGACCCTGGGTGACCTCGGTGACCAGGTTGTCCTGA

GTCCGGGCCAAGCCGAGGCTGCATCAGACTCGCCAGACCCAAGGCCTGGG

CCCCGGCTGGCAAGCCAGGGGCGGTGAAGGCTGGGCTGGCAGGACTGTC

CCGGAAGGAGGTGCACGTGGAGCCGCCCGGACCCCGACCGGCAGGACCT

GGAAAGACGCCTCTCACTCCCCTTTCTCTTCTGTCCCCTCTCGGGTCCTCA

GAGAGCCAGTCTGCCCCGAATCTCTACCCCCTCGTCTCCTGCGTCAGCCCC

CCGTCCGATGAGAGCCTGGTGGCCCTGGGCTGCCTGGCCCGGGACTTCCT

GCCCAGCTCCGTCACCTTCTCCTGGAA

Porcine Kappa Light Chain

In another embodiment, novel genomic sequences encoding the kappa light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate kappa light chain regions. In one embodiment, nucleic acid sequence is provided that encodes the porcine kappa light chain locus. In another embodiment, the nucleic acid sequence can contain at least one joining region, one constant region and/or one enhancer region of kappa light chain. In a further embodiment, the nucleotide sequence can include at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In a further embodiment, an isolated nucleotide sequence is provided that contains at least one, at least two, at least three, at least four or five joining regions and 3′ flanking sequence to the joining region of porcine genomic kappa light chain, for example, as represented in Seq ID No 12. In another embodiment, an isolated nucleotide sequence of porcine genomic kappa light chain is provided that contains 5′ flanking sequence to the first joining region, for example, as represented in Seq ID No. 25. In a further embodiment, an isolated nucleotide sequence is provided that contains 3′ flanking sequence to the constant region and, optionally, the 5′ portion of the enhancer region, of porcine genomic kappa light chain, for example, as represented in Seq ID Nos. 15, 16 and/or 19.
In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 30, 12, 25, 15, 16 or 19 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In other embodiments, nucleotide sequences that contain at least 50, 100, 1,000, 2,500, 5,000, 7,000, 8,000, 8,500, 9,000, 10,000 or 15,000 contiguous nucleotides of Seq ID No. 30 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 30, 12, 25, 15, 16 or 19, as well as, nucleotides homologous thereto.

In one embodiment, an isolated nucleotide sequence encoding kappa light chain is provided that includes at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In Seq ID No. 30, the coding region of kappa light chain is represented, for example by residues 1-549 and 10026-10549, whereas the intronic sequence is represented, for example, by residues 550-10025, the Joining region of kappa light chain is represented, for example, by residues 5822-7207 (for example, J1:5822-5859, J2:6180-6218, J3:6486-6523, J4:6826-6863, J5:7170-7207), the Constant Region is represented by the following residues: 10026-10549 (C exon) and 10026-10354 (C coding), 10524-10529 (Poly(A) signal) and 11160-11264 (SINE element).


Seq ID No 30

GCGTCCGAAGTCAAAAATATCTGCAGCCTTCATGTATTCATAGAAACAAG

GAATGTCTACATTTTCCAAAGTGGGACCAGAATCTTGGGTGATGTCTAAG

GCATGTGCATTTGCACATGGTAGGCAAAGGACTTTGCTTCTCCCAGCACA

TCTTTCTGCAGAGATCCATGGAAACAAGACTCAACTCCAAAGCAGCAAAG

AAGCAGCAAGTTCTCAAGTGATCTCCTCTGACTCCCTCCTCCCAGGCTAA

TGAAGCCATGTTGCCCCTGGGGGATTAAGGGCAGGTGTCCATTGTGGCAC

CCAGCCCGAAGACAAGCAATTTGATCAGGTTCTGAGCACTCCTGAATGTG

GACTCTGGAATTTTCTCCTCACCTTGTGGCATATCAGCTTAAGTCAAGTA

CAAGTGACAAACAACATAATCCTAAGAAGAGAGGAATCAAGCTGAAGTC

A

AAGGATCACTGCCTTGGATTCTACTGTGAATGATGACCTGGAAAATATCC

TGAACAACAGCTTCAGGGTGATCATCAGAGACAAAAGTTCCAGAGCCAGg

tagggaaaccctcaagccttgcaaagagcaaaatcatgccattgggttct

taacctgctgagtgatttactatatgttactgtgggaggcaaagcgctca

aatagcctgggtaagtatgtcaaataaaaagcaaaagtggtgtttcttga

aatgttagacctgaggaaggaatattgataacttaccaataattttcaga

atgatttatagatgtgcacttagtcagtgtctctccaccccgcacctgac

aagcagtttagaatttattctaagaatctaggtttgctgggggctacatg

ggaatcagcttcagtgaagagtttgttggaatgattcactaaattttcta

tttccagcataaatccaagaacctctcagactagtttattgacactgctt

ttcctccataatccatctcatctccgtccatcatggacactttgtagaat

gacaggtcctggcagagactcacagatgcttctgaaacatcctttgcctt

caaagaatgaacagcacacatactaaggatctcagtgatccacaaattag

tttttgccacaatggttcttatgataaaagtctttcattaacagcaaatt

gttttataatagttgttctgctttataataattgcatgcttcactttctt

ttcttttctttttttttctttttttgctttttagtgccgcaggtgcagca

tatgaaatttcccaggctaggggtcaaatcagaactacacctactggcct

acgccacagccacagcaactcaggatctaagccatgtcggtgacctacac

tacagctcatggcaatgccagatccttaacccaatgagcgaggccaggga

tcgaacccatgtcctcatggatactagtcaggctcattatccgctgagcc

ataacaggaactcccgagtttgctttttatcaaaattggtacagccttat

tgtttctgaaaaccacaaaatgaatgtattcacataattttaaaaggtta

aataatttatgatatacaagacaatagaaagagaaaacgtcattgcctct

ttcttccacgacaacacgcctccttaattgatttgaagaaataactactg

agcatggtttagtgtacttctttcagcaattagcctgtattcatagccat

acatattcaattaaaatgagatcatgatatcacacaatacataccataca

gcctatagggatttttacaatcatcttccacatgactacataaaaaccta

cctaaaaaaaaaaaaaaccctacttcatcctcctattggctgctttgtgc

tccattaaaaagctctatcataattaggttatgatgaggatttccatttt

ctacctttcaagcaacatttcaatgcacagtcttatatacacatttgagc

ctacttttctttttctttctttttttggtttttttttttttttttttttt

ggtctttttgtcttttctaaggctgcatatggaggttcccaggctagctg

tctaatcagaactatagctgctggcctacgccacatccacagcaatacaa

gatctgagccatgtctgcaacttacaccacagctcacagcaacggtggat

ccttaaaccactgagcaaggccagggatcaaacccataacttcatggctc

ctagttggatttgttaaccactgagccatgatggcaactcctgagcctac

ttttctaatcatttccaaccctaggacacttttttaagtttcatttttct

ccccccaccccctgttttctgaagtgtgtttgcttccactgggtgacttc

actcccaggatctcatctgcaggatactgcagctaagtgtatgagctctg

aatttgaatcccaactctgccactcaaagggataggagtttccgatgtgg

cccaatgggatcagtggcatctctgcagtgccaggacgcaggttccatcc

ctggcccagcacagtgggttaagaatctggcattgctgcagctgaggcat

agatttcaattgtgcctcagatctgatccttggcccaaggactgcatatg

cctcagggcaaccaaaaaagagaaaaggggggtgatagcattagtttcta

gatttgggggataattaaataaagtgatccatgtacaatgtatggcattt

tgtaaatgctcaacaaatttcaactattatggagttcccatcatggctca

gtggaagggaatctgattagcatccatgaggacacaggtccaaccccgac

cttgctcagtgggcattgctgtgagctgtggcatgggttacagacgaagc

tcggatctggcattgctgtggctgtggtgtaagccagcaactacagctct

cattcagcccctagcctgggaacctccatatgcctaaaagacaaaaaata

aaatttaaattaaaaataaagaaatgttaactattatgattggtactgct

tgcattactgcaaagaaagtcactttctatactctttaatatcttagttg

actgtgtgctcagtgaactattttggacacttaatttccactctcttcta

tctccaacttgacaactctctttcctctcttctggtgagatccactgctg

actttgctctttaaggcaactagaaaagtgctcagtgacaaaatcaaaga

aagttaccttaatcttcagaattacaatcttaagttctcttgtaaagctt

actatttcagtggttagtattattccttggtcccttacaacttatcagct

ctgatctattgctgattttcaactatttattgttggagttttttcctttt

ttccctgttcattctgcaaatgtttgctgagcatftgtcaagtgaagata

ctggactgggccttccaaatataagacaatgaaacatcgggagttctcat

tatggtgcagcagaaacgaatccaactaggaaatgtgaggttgcaggttc

gatccctgcccttgctcagtgggttaaggatccagcattaccgtgagctg

tggtgtaggttgcagacgtggctcagatcctgcgttgctgtggctgtggc

ataggctggcagctctagctctgattcgaccgctagcctgggaacctcca

tgcgccccgagtgcagcccttaaaaagcaaaaaaaaaagaaagaaagaaa

aagacaatgaaacatcaaacagctaacaatccagtagggtagaaagaatc

tggcaacagataagagcgattaaatgttctaggtccagtgaccttgcctc

tgtgctctacacagtcgtgccacttgctgagggagaaggtctctcttgag

ttgagtcctgaaagacattagttgttcacaaactaatgccagtgagtgaa

ggtgtttccaagcagagggagagtttggtaaaaagctggaagtcacagaa

agactctaaagagtttaggatggtgggagcaacatacgctgagatggggc

tggaaggttaagagggaaacaactatagtaagtgaagctggactcacagc

aaagtgaggacctcagcatccttgatggggttaccatggaaacaccaagg

cacaccttgatttccaaaacagcaggcacctgattcagcccaatgtgaca

tggtgggtacccctctagctctacctgttctgtgacaactgacaaccaac

gaagttaagtctggattttctactctgctgatccttgtttttgtttcaca

cgtcatctatagcttcatgccaaaatagagttcaaggtaagacgcgggcc

ttggtttgatatacatgtagtctatcttgtttgagacaatatggtggcaa

ggaagaggttcaaacaggaaaatactctctaattatgattaactgagaaa

agctaaagagtcccataatgacactgaatgaagttcatcatttgcaaaag

ccttcccccccccccaggagactataaaaaagtgcaattttttaaatgaa

cttatttacaaaacagaaatagactcacagacataggaaacgaacagatg

gttaccaagggtgaaagggagtaggagggataaataaggagtctggggtt

agcagatacaccccagtgtacacaaaataaacaacagggacctactatat

agcacagggaactatatgcagtagcttacaataacctataatggaaaaga

atgtgaaaaagaatatatgtatgcgtgtgtgtgtaactgaatcactttgc

tgtaacctgaatctaacataacattgtaaatcaactacagtttttttttt

ttttaagtgcagggttttggtgttttttttttttcatttttgtttttgtt

tttgttttttgctttttagggccacacccagacatatgggggttcccagg

ctaggggtctaattagagctacagttgccggcttgcaccacagccacagc

aacatcagatccgagccgcacttgcgacttacaccacagctcatggcaat

accagatccttaacccactgagcaaggcccagggatcgtacccgcaacct

catggttcctagtcagattcatttctgctgcgctacaatgggaactccaa

gtgcagttttttgtaatgtgcttgtctttctttgtaattcatattcatcc

tacttcccaataaataaataaatacataaataataaacataccattgtaa

atcaactacaattttttttaaatgcagggtttttgttttttgttttttgt

tttgtctttttgccttttctagggccgctcccatggcatatggaggttcc

caggctaggggtcgaatcggagctgtagccaccggcctacgccagagcca

cagcaacgcgggatccgagccgcgtctgcaacctacaccacagctcacgg

caacgccggatcgttaacccactgagcaagggcagggatcgaacctgcaa

cctcatggttcctagtcagattcgttaactactgagccacaacggaaact

cctaaagtgcagtttttaaatgtgcttgtctttctttgtaatttacactc

aacctacttcccaataaataaataaataaacaaataaatcatagacatgg

ttgaattctaaaggaagggaccatcaggccttagacagaaatacgtcatc

ttctagtattttaaaacacactaaagaagacaaacatgctctgccagaga

agcccagggcctccacagctgcttgcaaagggagttaggcttcagtagct

gacccaaggctctgttcctcttcagggaaaagggtttttgttcagtgaga

cagcagacagctgtcactgtgGTGGACGTTCGGCCAAGGAACCAAGCTGG

AACTCAAACgtaagtcaatccaaacgttccttccttggctgtctgtgtct

tacggtctctgtggctctgaaatgattcatgtgctgactctctgaaacca

gactgacattctccagggcaaaactaaagcctgtcatcaaactggaaaac

tgagggcacattttctgggcagaactaagagtcaggcactgggtgaggaa

aaacttgttagaatgatagtttcagaaacttactgggaagcaaagcccat

gttctgaacagagctctgctcaagggtcaggaggggaaccagtttttgta

caggagggaagttgagacgaacccctgtgTATATGGTTTCGGCGCGGGGA

CCAAGCTGGAGCTCAAACgtaagtggctttttccgactgattctttgctg

tttctaattgttggttggctttttgtccatttttcagtgttttcatcgaa

ttagttgtcagggaccaaacaaattgccttcccagattaggtaccaggga

ggggacattgctgcatgggagaccagagggtggctaatttttaacgtttc

caagccaaaataactggggaagggggcttgctgtcctgtgagggtaggtt

tttatagaagtggaagttaaggggaaatcgctatgGTTGACTTTTGGCTC

GGGGACCAAAGTGGAGCCCAAAAttgagtacattttccatcaattatttg

tgagatttttgtcctgttgtgtcatttgtgcaagtttttgacattttggt

tgaatgagccattcccagggacccaaaaggatgagaccgaaaagtagaaa

agagccaacttttaagctgagcagacagaccgaattgttgagtttgtgag

gagagtagggtttgtagggagaaaggggaacagatcgctggctttttctc

tgaattagcctttctcatgggactggcttcagagggggtttttgatgagg

gaagtgttctagagccttaactgtgGGTTGTGTTCGGTAGCGGGACCAAG

CTGGAAATCAAACgtaagtgcacttttctactcctttttctttcttatac

gggtgtgaaattggggacttttcatgtttggagtatgagttgaggtcagt

tctgaagagagtgggactcatccaaaaatctgaggagtaagggtcagaac

agagttgtctcatggaagaacaaagacctagttagttgatgaggcagcta

aatgagtcagttgacttgggatccaaatggccagacttcgtctgtaacca

acaatctaatgagatgtagcagcaaaaagagatttccattgaggggaaag

taaaattgttaatattgtgGATCACCTTTGGTGAAGGGACATCCGTGGAG

ATTGAACgtaagtattttttctctactaccttctgaaatttgtctaaatg

ccagtgttgacttttagaggcttaagtgtcagttttgtgaaaaatgggta

aacaagagcatttcatatttattatcagtttcaaaagttaaactcagctc

caaaaatgaatttgtagacaaaaagattaatttaagccaaattgaatgat

tcaaaggaaaaaaaaattagtgtagatgaaaaaggaattcttacagctcc

aaagagcaaaagcgaattaattttctttgaactttgccaaatcttgtaaa

tgatttttgttctttacaatttaaaaaggttagagaaatgtatttcttag

tctgttttctctcttctgtctgataaattattatatgagataaaaatgaa

aattaataggatgtgctaaaaaatcagtaagaagttagaaaaatatatgt

ttatgttaaagttgccacttaattgagaatcagaagcaatgttattttta

aagtctaaaatgagagataaactgtcaatacttaaattctgcagagattc

tatatcttgacagatatctcctttttcaaaaatccaatttctatggtaga

ctaaatttgaaatgatcttcctcataatggagggaaaagatggactgacc

ccaaaagctcagatttaaagaaatctgtttaagtgaaagaaaataaaaga

actgcattttttaaaggcccatgaatttgtagaaaaataggaaatatttt

aataagtgtattcttttattttcctgttattacttgatggtgtttttata

ccgccaaggaggccgtggcaccgtcagtgtgatctgtagaccccatggcg

gccttttttcgcgattgaatgaccttggcggtgggtccccagggctctgg

tggcagcgcaccagccgctaaaagccgctaaaaactgccgctaaaggcca

cagcaaccccgcgaccgcccgttcaactgtgctgacacagtgatacagat

aatgtcgctaacagaggagaatagaaatatgacgggcacacgctaatgtg

gggaaaagagggagaagcctgatttttattttttagagattctagagata

aaattcccagtattatatccttttaataaaaaatttctattaggagatta

taaagaatttaaagctatttttttaagtggggtgtaattctttcagtagt

ctcttgtcaaatggatttaagtaatagaggcttaatccaaatgagagaaa

tagacgcataaccctttcaaggcaaaagctacaagagcaaaaattgaaca

cagcagccagccatctagccactcagattttgatcagttttactgagttt

gaagtaaatatcatgaaggtataattgctgataaaaaaataagatacagg

tgtgacacatctttaagtttcagaaatttaatggcttcagtaggattata

tttcacgtatacaaagtatctaagcagataaaaatgccattaatggaaac

ttaatagaaatatatttttaaattccttcattctgtgacagaaattttct

aatctgggtcttttaatcacctaccctttgaaagagtttagtaatttgct

atttgccatcgctgtttactccagctaatttcaaaagtgatacttgagaa

agattatttttggtttgcaaccacctggcaggactattttagggccattt

taaaactcttttcaaactaagtattftaaactgttctaaaccatttaggg

ccttttaaaaatcttttcatgaatttcaaacttcgttaaaagttattaag

gtgtctggcaagaacttccttatcaaatatgctaatagtttaatctgtta

atgcaggatataaaattaaagtgatcaaggcttgacccaaacaggagtat

cttcatagcatatttcccctcctttttttctagaattcatatgattttgc

tgccaaggctattttatataatctctggaaaaaaaatagtaatgaaggtt

aaaagagaagaaaatatcagaacattaagaattcggtattttactaactg

cttggttaacatgaaggtttttattttattaaggtttctatctttataaa

aatctgttcccttttctgctgatttctccaagcaaaagattcttgatttg

ttttttaactcttactctcccacccaagggcctgaatgcccacaaagggg

acttccaggaggccatctggcagctgctcaccgtcagaagtgaagccagc

cagttcctcctgggcaggtggccaaaattacagttgacccctcctggtct

ggctgaaccttgccccatatggtgacagccatctggccagggcccaggtc

tccctctgaagcctttgggaggagagggagagtggctggcccgatcacag

atgcggaaggggctgactcctcaaccggggtgcagactctgcagggtggg

tctgggcccaacacacccaaagcacgcccaggaaggaaaggcagcttggt

atcactgcccagagctaggagaggcaccgggaaaatgatctgtccaagac

ccgttcttgcttctaaactccgagggggtcagatgaagtggttttgtttc

ttggcctgaagcatcgtgttccctgcaagaagcggggaacacagaggaag

gagagaaaagatgaactgaacaaagcatgcaaggcaaaaaaggccttagg

atggctgcaggaagttagttcttctgcattggctccttactggctcgtcg

atcgcccacaaacaacgcacccagtggagaacttccctgttacttaaaca

ccattctctgtgcttgcttcctcagGGGCTGATGCCAAGCCATCCGTCTT

CATCTTCCCGCCATCGAAGGAGCAGTTAGCGACCCCAACTGTCTCTGTGG

TGTGCTTGATCAATAACTTCTTCCCCAGAGAAATCAGTGTCAAGTGGAAA

GTGGATGGGGTGGTCCAAAGCAGTGGTCATCCGGATAGTGTCACAGAGCA

GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTCTCGCTGCCCA

CGTCACAGTACCTAAGTCATAATTTATATTCCTGTGAGGTCACCCACAAG

ACCCTGGCCTCCCCTCTGGTCACAAGCTTCAACAGGAACGAGTGTGAGGC

TtagAGGCCCACAGGCCCCTGGCCTGCCCCCAGCCCCAGCCCCCCTCCCC

ACCTGAAGCCTCAGGCCCTTGCCCCAGAGGATCCTTGGCAATCCCCCAGC

CCCTCTTCCCTCCTCATCCCCTCCCCCTCTTTGGCTTTAACCGTGTTAAT

ACTGGGGGGTGGGGGAATGAATAaataaaGTGAACCTTTGCACCTGTGAt

ttctctctcctgtctgattttaaggttgttaaatgttgttttccccatta

tagttaatcttttaaggaactacatactgagttgctaaaaactacaccat

cacttataaaattcacgccttctcagttctcccctcccctcctgtcctcc

gtaagacaggcctccgtgaaacccataagcacttctctttacaccctctc

ctgggccggggtaggagactttttgatgtcccctcttcagcaagcctcag

aaccattttgagggggacagttcttacagtcacat*tcctgtgatctaat

gactttagttaccgaaaagccagtctctcaaaaagaagggaacggctaga

aaccaagtcatagaaatatatatgtataaaatatatatatatccatatat

gtaaaataacaaaataatgataacagcataggtcaacaggcaacagggaa

tgttgaagtccattctggcacttcaatttaagggaataggatgccttcat

tacattttaaatacaatacacatggagagcttcctatctgccaaagacca

tcctgaatgccttccacactcactacaaggttaaaagcattcattacaat

gttgatcgaggagttcccgttgtggctcagcaggttaagaacgtgactgg

tatccaggaggatgcgggtttggtccccagcctcgctcagtggattaagg

atccagtgttgctgcaagatcacgggctcagatcccgtgttctatggcta

tggtgtaggctggtagctgcatgcagccctaatttgacccctagcctggg

aactgccatatgccacatgtgaggcccttaaaacctaaaagaaaaaaaaa

gaaaagaaatatcttacacccaatttatagataagagagaagctaaggtg

gcaggcccaggatcaaagccctacctgcctatcttgacacctgatacaaa

ttctgtcttctagggtttccaacactgcatagaacagagggtcaaacatg

ctaccctcccagggactcctcccttcaaatgacataaattttgttgccca

tctctgggggcaaaactcaacaatcaatggcatctctagtaccaagcaag

gctcttctcatgaagcaaaactctgaagccagatccatcatgacccaagg

aagtaaagacaggtgttactggttgaactgtatccttcaattcaatatgc

tcaatttccaactcccagtccccgtaaatacaaccccctttgggaagaga

gtccttgcagatgtagccacgttaaaaagagattatacagaaaggctagt

gaggatgcagtgaaacgggatctttcatacattgctggtggaaatgtaaa

atgctgcaggcactctagaaaataatttgccagttttttgaaaagctaaa

caaaatagtttagttgcattctgggttatttatcccccagaaattaaaaa

ttatgtccgcacaaaaacgtgtacataatcattcataacagccttgtac

Seq ID No. 12

caaggaaccaagctggaactcaaacgtaagtcaatccaaacgttccttcc

ttggctgtctgtgtcttacggtctctgtggctctgaaatgattcatgtgc

tgactctctgaaaccagactgacattctccagggcaaaactaaagcctgt

catcaaactggaaaactgagggcacattttctgggcagaactaagagtca

ggcactgggtgaggaaaaacttgttagaatgatagtttcagaaacttact

gggaagcaaagcccatgttctgaacagagctctgctcaagggtcaggagg

ggaaccagtttttgtacaggagggaagttgagacgaacccctgtgtatat

ggtttcggcgcggggaccaagctggagctcaaacgtaagtggctttttcc

gactgattctttgctgtttctaattgttggttggctttttgtccattttt

cagtgttttcatcgaattagttgtcagggaccaaacaaattgccttccca

gattaggtaccagggaggggacattgctgcatgggagaccagagggtggc

taatttttaacgtttccaagccaaaataactggggaagggggcttgctgt

cctgtgagggtaggtttttatagaagtggaagttaaggggaaatcgctat

ggttcacttttggctcggggaccaaagtggagcccaaaattgagtacatt

ttccatcaattatttgtgagatttttgtcctgttgtgtcatttgtgcaag

tttttgacattttggttgaatgagccattcccagggacccaaaaggatga

gaccgaaaagtagaaaagagccaacttttaagctgagcagacagaccgaa

ttgttgagtttgtgaggagagtagggtttgtagggagaaaggggaacaga

tcgctggctttttctctgaattagcctttctcatgggactggcttcagag

ggggtttttgatgagggaagtgttctagagccttaactgtgggttgtgtt

cggtagcgggaccaagctggaaatcaaacgtaagtgcacttttctactcc

tttttctttcttatacgggtgtgaaattggggacttttcatgtttggagt

atgagttgaggtcagttctgaagagagtgggactcatccaaaaatctgag

gagtaagggtcagaacagagttgtctcatggaagaacaaagacctagtta

gttgatgaggcagctaaatgagtcagttgacttgggatccaaatggccag

acttcgtctgtaaccaacaatctaatgagatgtagcagcaaaaagagatt

tccattgaggggaaagtaaaattgttaatattgtggatcacctttggtga

agggacatccgtggagattgaacgtaagtattttttctctactaccttct

gaaatttgtctaaatgccagtgttgacttttagaggcttaagtgtcagtt

ttgtgaaaaatgggtaaacaagagcatttcatatttattatcagtttcaa

aagttaaactcagctccaaaaatgaatttgtagacaaaaagattaattta

agccaaattgaatgattcaaaggaaaaaaaaattagtgtagatgaaaaag

gaattcttacagctccaaagagcaaaagcgaattaattttctttgaactt

tgccaaatcttgtaaatgatttttgttctttacaatttaaaaaggttaga

gaaatgtatttcttagtctgttttctctcttctgtctgataaattattat

atgagataaaaatgaaaattaataggatgtgctaaaaaatcagtaagaag

ttagaaaaatatatgtttatgttaaagttgccacttaattgagaatcaga

agcaatgttatttttaaagtctaaaatgagagataaactgtcaatactta

aattctgcagagattctatatcttgacagatatctcctttttcaaaaatc

caatttctatggtagactaaatttgaaatgatcttcctcataatggaggg

aaaagatggactgaccccaaaagctcagattt*aagaaaacctgtttaag

*gaaagaaaataaaagaactgcattttttaaaggcccatgaatttgtaga

aaaataggaaatattttaataagtgtattcttttattttcctgttattac

ttgatggtgtttttataccgccaaggaggccgtggcaccgtcagtgtgat

ctgtagaccccatggcggccttttttcgcgattgaatgaccttggcggtg

ggtccccagggctctggtggcagcgcaccagccgctaaaagccgctaaaa

actgccgctaaaggccacagcaaccccgcgaccgcccgttcaactgtgct

gacacagtgatacagataatgtcgctaacagaggagaatagaaatatgac

gggcacacgctaatgtggggaaaagagggagaagcctgatttttattttt

tagagattctagagataaaattcccagtattatatccttttaataaaaaa

tttctattaggagattataaagaatttaaagctatttttttaagtggggt

gtaattctttcagtagtctcttgtcaaatggatttaagtaatagaggctt

aatccaaatgagagaaatagacgcataaccctttcaaggcaaaagctaca

agagcaaaaattgaacacagcagccagccatctagccactcagattttga

tcagttttactgagtttgaagtaaatatcatgaaggtataattgctgata

aaaaaataagatacaggtgtgacacatctttaagtttcagaaatttaatg

gcttcagtaggattatatttcacgtatacaaagtatctaagcagataaaa

atgccattaatggaaacttaatagaaatatatttttaaattccttcattc

tgtgacagaaattttctaatctgggtcttttaatcacctaccctttgaaa

gagtttagtaatttgctatttgccatcgctgtttactccagctaatttca

aaagtgatacttgagaaagattatttttggtttgcaaccacctggcagga

ctattttagggccattttaaaactcttttcaaactaagtattttaaactg

ttctaaaccatttagggccttttaaaaatcttttcatgaatttcaaactt

cgttaaaagttattaaggtgtctggcaagaacttccttatcaaatatgct

aatagtttaatctgttaatgcaggatataaaattaaagtgatcaaggctt

gacccaaacaggagtatcttcatagcatatttcccctcctttttttctag

aattcatatgattttgctgccaaggctattttatataatctctggaaaaa

aaatagtaatgaaggttaaaagagaagaaaatatcagaacattaagaatt

cggtattttactaactgcttggttaacatgaaggtttttattttattaag

gtttctatctttataaaaatctgttcccttttctgctgatttctccaagc

aaaagattcttgatttgttttttaactcttactctcccacccaagggcct

gaatgcccacaaaggggacttccaggaggccatctggcagctgctcaccg

tcagaagtgaagccagccagttcctcctgggcaggtggccaaaattacag

ttgacccctcctggtctggctgaaccttgccccatatggtgacagccatc

tggccagggcccaggtctccctctgaagcctttgggaggagagggagagt

ggctggcccgatcacagatgcggaaggggctgactcctcaaccggggtgc

agactctgcagggtgggtctgggcccaacacacccaaagcacgcccagga

aggaaaggcagcttggtatcactgcccagagctaggagaggcaccgggaa

aatgatctgtccaagacccgttcttgcttctaaactccgagggggtcaga

tgaagtggttttgtttcttggcctgaagcatcgtgttccctgcaagaagc

ggggaacacagaggaaggagagaaaagatgaactgaacaaagcatgcaag

gcaaaaaaggccttaggatggctgcaggaagttagttcttctgcattggc

tccttactggctcgtcgatcgcccacaaacaacgcacccagtggagaact

tccctgttacttaaacaccattctctgtgcttgcttcctcaggggctgat

gccaagccatccgtcttcatcttcccgccatcgaaggagcagttagcgac

cccaactgtctctgtggtgtgcttgatca

Seq ID No. 15

gatgccaagccatccgtcttcatcttcccgccatcgaaggagcagttagc

gaccccaactgtctctgtggtgtgcttgatcaataacttcttccccagag

aaatcagtgtcaagtggaaagtggatggggtggtccaaagcagtggtcat

ccggatagtgtcacagagcaggacagcaaggacagcacctacagcctcag

cagcaccctctcgctgcccacgtcacagtacctaagtcataatttatatt

cctgtgaggtcacccacaagaccctggcctcccctctggtcacAAGCTTC

AACAGGAACGAGTGTGAGGCTTAGAGGCCCACAGGCCCCTGGCCTGCCCC

CAGCCCCAGCCCCGCTCCCCACCTCAAGCCTCAGGCCCTTGCCCCAGAGG

ATCCTTGGCAATCCCCCAGCCCCTCTTCCCTCCTCATCCCCTCCCCCTCT

TTGGCTTTAACCGTGTTAATACTGGGGGGTGGGGGAATGAATAAATAAAG

TGAACCTTTGCACCTGTGATTTCTCTCTCCTGTCTGATTTTAAGGTTGTT

AAATGTTGTTTTCCCCATTATAGTTAATCTTTTAAGGAACTACATACTGA

GTTGCTAAAAACTACACCATCACTTATAAAATTCAcgCCTTCTCAGTTCT

CCCCTCCCCTCCTGTCCTCCGTAAGACAGGCCTCCGTGAAACCCATAAGC

ACTTCTCTTTACACCCTCTCCTGGGCCGGGGTAGGAGACTTTTTGATGTC

CCCTcTTCAGCAAGCCTCAGAACCATTTTGAGGGGGACAGTTCTTACAGT

CACAT*TCCtGtGATCTAATGACTTTAGTTaCCGAAAAGCCAGTGTCTCA

AAAAGAAGGGAACGGCTAGAAACCAAGTCATAGAAATATATATGTATAAA

ATATATATATATCCATATATGTAAAATAACAAAATAATGATAACAGCATA

GGTCAACAGGCAACAGGGAATGTTGAAGTCCATTCTGGCACTTCAATTTA

AGGGAATAGGATGCCTTCATTACATTTTAAATACAATACACATGGAGAGC

TTCCTATCTGCCAAAGACCATCCTGAATGCCTTCCACACTCACTACAAGG

TTAAAAGCATTCATTACAATGTTGATCGAGGAGTTCCCGTTGTGGCTCAG

CAGGTTAAGAACGTGACTGGTATCCAGGAGGATGCGGGTTTGGTCCCCAG

CCTCGCTCAGTGGATTAAGGATCCAGTGTTGCTGCAAGATCACGGGCTCA

GATCCCGTGTTCTATGGCTATGGTGTAGGCTGGTAGCTGCATGCAGCCCT

AATTTGACCCCTAGCCTGGGAACTGCCATAtGCCACATGTGAGGCCCTTA

AAACCTAAAAGAAAAAaAAAGAAAAGAAATATCTTACACCCAATTTATAG

ATAAGAGAGAAGCTAAGGTGGCAGGCCCAGGATCAAAGCCCTACCTGCCT

ATCTTGACACCTGAtACAAATTCTGTCTTCTAGGGtTTCCAACACTGCAT

AGAACAGAGGGTCAAACATGCTACCCTCCCAGGGACTCCTCCCTTCAAAT

GACATAAATTTTGTTGCCCATCTCTGGGGGCAAAACTCAACAATCAATGG

CATCTCTAGTACCAAGCAAGGCTCTTCTCATGAAGCAAAACTCTGAAGCC

AGATCCATCATGACCCAAGGAAGTAAAGACAGGTGTTACTGGTTGAACTG

TATCCTTCAATTCAATATGCTCAATTTCCAACTCCCAGTCCCCGTAAATA

CAACCCCCTTTGGGAAGAGAGTCCTTGCAGATGTAGCCACGTTAAAAAGA

GATTATACAGAAAGGCTAGTGAGGATGCAGTGAAACGGGATCTTTCATAC

ATTGCTGGTGGAAATGTAAAATGCTGCAGGCACTCTAGAAAATAATTTGC

CAGTTTTTTGAAAAGCTAAACAAAATAGTTTAGTTGCATTCTGGGTTATT

TATCCCCCAGAAATTAAAAATTATGTCCGCACAAAAACGTGTACATAATC

ATTCATAACAGCCTTGTACGAAAAGCTT

Seq ID No. 16

GGATCCTTAACCCACTAATCGAGGATCAAACACGCATCCTCATGGACAAT

ATGTTGGGTTCTTAGCCTGCTGAGACACAACAGGAACTCCCCTGGCACCA

CTTTAGAGGCCAGAGAAACAGCACAGATAAAATTCCCTGCCCTCATGAAG

CTTATAGTCTAGCTGGGGAGATATCATAGGCAAGATAAACACATACAAAT

ACATCATCTTAGGTAATAATATATACTAAGGAGAAAATTACAGGGGAGAA

AGAGGACAGGAATTGCTAGGGTAGGATTATAAGTTCAGATAGTTCATCAG

GAACACTGTfGCTGAGAAGATAACATTTAGGTAAAGACCGAAGTAGTAAG

GAAATGGACCGTGTGCCTAAGTGGGTAAGACCATTCTAGGCAGCAGGAAC

AGCGATGAAAGCACTGAGGTGGGTGTTCACTGCACAGAGTTGTTCACTGC

ACAGAGTTGTGTGGGGAGGGGTAGGTCTTGCAGGGTCTTATGGTCACAGG

AAGAATTGTTTTACTCCCACCGAGATGAAGGTTGGTGGATTTTGAGCAGA

AGAATAATTCTGCCTGGTTTATATATAACAGGATTTCCCTGGGTGCTCTG

ATGAGAATAATCTGTCAGGGGTGGGATAGGGAGAGATATGGCAATAGGAG

CCTTGGCTAGGAGCCCACGACAATAATTCCAAGTGAGAGGTGGTGCTGCA

TTGAAAGCAGGACTAACAAGACCTGCTGACAGTGTGGATGTAGAAAAAGA

TAGAGGAGACGAAGGTGCATCTAGGGTTTTCTGCCTGAGGAATTAGAAAG

ATAAAGCTAAAGCTTATAGAAGATGCAGCGCTCTGGGGAGAAAGACCAGC

AGCTCAGTTTTGATCCATCTGGAATTAATTTTGGCATAAAGTATGAGGTA

TGTGGGTTAACATTATTTGTTTTTTTTTTTTCCATGTAGCTATCCAACTG

TCCCAGCATCATTTATTTTAAAAGACTTTCCTTTCCCCTATTGGATTGTT

TTGGCACCTTCACTGAAGATCAACTGAGCATAAAATTGGGTCTATTTCTA

AGCTCTTGATTCCATTCCATGACCTATTTGTTCATCTTTACCCCAGTAGA

CACTGCCTTGATGATTAAAGCCCCTGTTACCATGTCTGTTTTGGACATGG

TAAATCTGAGATGCCTATTAGCCAACCAAGCAAGCACGGCCCTTAGAGAG

CTAGATATGAGAGCCTGGAATTCAGACGAGAAAGGTCAGTCCTAGAGACA

TACATGTAGTGCCATCACCATGCGGATGGTGTTAAAAGCCATCAGACTGC

AACAGACTGTGAGAGGGTACCAAGCTAGAGAGCATGGATAGAGAAACCCA

AGCACTGAGCTGGGAGGTGCTCCTACATTAAGAGATTAGTGAGATGAAGG

ACTGAGAAGATTGATCAGAGAAGAAGGAaAATCAGGAAAATGGTGCTGTC

cTGAAAATCCAAGGGAAGAGATGTTCCAAAGAGGAGAaAACTGATCAGTT

GTCAGCTAGCGTCAATTGGGATGAAAATGGACCATTGGACAGAGGGATGT

AGTGGGTCATGGGTGAATAGATAAGAGCAGCTTCTATAGAATGGCAGGGG

CAAAATTCTCATCTGATCGGCATGGGTTcTAAAGAAAACGGGAAGAAAAA

ATTGAGTGCATGACCAGTCCCTTCAAGTAGAGAGGTgGAAAAGGGAAGGA

GGAAAATGAGGCCACGACAACATGAGAGAAATGACAGCATTTTTAAAAAT

TTTTTATTTTATTTtATTTATTTATTTTTGCTTTTTAGGGCTGCCCCTGC

AAcatatggaggttcccaggttaggggtctaatcagagctatagctgcca

gcctacaccacagccatagcaatgccagatctacatgacctacaccacag

ctcacagcaacgccggatccttaacccactgagtgaggccagagatcaaa

cccatatccttatggatactagtcaggttcattaccactgagccaaaatg

ggaaATCCTGAGTAATGACAGCATTTTTTAATGTGCCAGGAAGCAAAACT

TGCCACCCCGAAATGTCTCTCAGGCATGTGGATTATTTTGAGCTGAAAAC

GATTAAGGCCCAAAAAACACAAGAAGAAATGTGGACCTTCCCCCAACAGC

CTAAAAAATTTAGATTGAGGGCCTGTTCCCAGAATAGAGCTATTGCCAGA

CTTGTCTACAGAGGCTAAGGGCTAGGTGTGGTGGGGAAACCCTCAGAGAT

CAGAGGGACGTTTATGTACCAAGCATTGACATTTCCATCTCCATGCGAAT

GGCCTTCTTCCCCTCTGTAGCCCCAAACCACCACCCCCAAAATCTTCTTC

TGTCTTTAGCTGAAGATGGTGTTGAAGGTGATAGTTTCAGCCACTTTGGC

GAGTTCCTCAGTTGTTCTGGGTCTTTCCTCCGGATCCACATTATTCGACT

GTGTTTGATTTTCTCCTGTTTATCTGTCTCATTGGCACCCATTTCATTCT

TAGACCAGCCCAAAGAACCTAGAAGAGTGAAGGAAAATTTCTTCCACCCT

GACAAATGCTAAATGAGAATCACCgCAGTAGAGGAAAATGATCTGGTgCT

GCGGGAGATAGAAGAGAAAATcGCTGGAGAGATGTCACTGAGTAGGTGAG

ATGGGAAAGGGGGGGCACAGGTGGAGGTGTTGCCCTCAGCTAGGAAGACA

GACAGTTcacagaagagaagcgggtgtccgtGGACATCTTGCCTCATGGA

TGAGGAAACCGAGGCTAAGAAAGACTGCAAAAGAAAGGTAAGGATTGCAG

AGAGGTCGATCCATGACTAAAATCACAGTAACCAACCCCAAACCACCATG

TTTTCTCCTAGTCTGGCACGTGGCAGGTACTGTGTAGGTTTTCAATATTA

TTGGTTTGTAACAGTACCTATTAGGCCTCCATCcCCTCCTCTAATACTAA

CAAAAGTGTGAGACTGGTCAGTGAAAAATGGTCTTCTTTCTCTATGCAAT

CTTTCTCAAGAAGATACATAACTTTTTATTTTATCATaGGCTTGAAGAGC

AAATGAGAAACAgCCTCCAACCTATGACACCGTAACAAAGTGTTTATGAT

CAGTGAAGGGCAAGAAACAAAACATACACaGTAAAGACCCTCCATAATAT

TGtGGGCTGGCCCAaCACAGGCCAGGTTGTAAAAGCTTTTTATTCTTTGA

TAGAGGAATGGATAGTAATGTTTCAACCTGGACAGAGAT*CATGTTCACT

GAATCCTTCCAAAAATTCATGGGTAGTTTGAAtTATAAGGAAAATAAGAC

TTAGGATAAATACTTTgTCCA*GATCCCAGAGTTAATgCCAAAATCAGTT

TTCAGACTCCAGGCAGCCTGATCAAGAGCCTAAACTTTAAAGACACAGTC

CCTTAATAACTACTATTCACAGTTGCACTTTCAgGGCGCAAAGACTCATT

GAATCCTACAATAGAATGAGTTTAGATATCAAATCTCTCAGTAATAGATG

AGGAGACTAAATAGCGGGCATGACCTGGTCACTTAAAGACAGAATTGAGA

TTCAAGGCTAGTGTTCTTTCTACCTGTTTTGTTTCTACAAGATGTAGCAA

TGCGCTAATTACAGACCTCTCAGGGAAGGAATTCACAACCCTCAGCAAAA

ACCAAAGACAAATCTAAGACAACTAAGAGTGTTGGTTTAATTTGGAAAAA

TAACTCACTAACCAAACGCCCCTCTTAGCACCCCAATGTCTTCCACCATC

ACAGTGCTCAGGCCTCAACCATGCCCCAATCACCCCAGCCCCAGACTGGT

TATTACCAAGTTTCATGATGACTGGCCTGAGAAGATCAAAAAAGCAATGA

CATCTTACAGGGGACTACCCCGAGGACCAAGATAGCAACTGTCATAGCAA

CCGTCACACTGCTTTGGTCA

Seq ID No. 19

ggatcaaacacgcatcctcatggacaatatgttgggttcttagcctgctg

agacacaacaggaactcccctggcaccactttagaggccagagaaacagc

acagataaaattccctgccctcatgaagcttatagtctagctggggagat

atcataggcaagataaacacatacaaatacatcatcttaggtaataatat

atactaaggagaaaattacaggggagaaagaggacaggaattgctagggt

aggattataagttcagatagttcatcaggaacactgttgctgagaagata

acatttaggtaaagaccgaagtagtaaggaaatggaccgtgtgcctaagt

gggtaagaccattctaggcagcaggaacagcgatgaaagcactgaggtgg

gtgttcactgcacagagttgttcactgcacagagttgtgtggggaggggt

aggtcttgcaggctcttatggtcacaggaagaattgttttactcccaccg

agatgaaggttggtggattttgagcagaagaataattctgcctggtttat

atataacaggatttccctgggtgctctgatgagaataatctgtcaggggt

gggatagggagagatatggcaataggagccttggctaggagcccacgaca

ataattccaagtgagaggtggtgctgcattgaaagcaggactaacaagac

ctgctgacagtgtggatgtagaaaaagatagaggagacgaaggtgcatct

agggttttctgcctgaggaattagaaagataaagctaaagcttatagaag

atgcagcgctctggggagaaagaccagcagctcagttttgatccatctgg

aattaattttggcataaagtatgaggtatgtgggttaacattatttgttt

tttttttttccatgtagctatccaactgtcccagcatcatttattttaaa

agactttcctttcccctattggattgttttggcaccttcactgaagatca

actgagcataaaattgggtctatttctaagctcttgattccattccatga

cctatttgttcatctttaccccagtagacactgccttgatgattaaagcc

cctgttaccatgtctgttttggacatggtaaatctgagatgcctattagc

caaccaagcaagcacggcccttagagagctagatatgagagcctggaatt

cagacgagaaaggtcagtcctagagacatacatgtagtgccatcaccatg

cggatggtgttaaaagccatcagactgcaacagactgtgagagggtacca

agctagagagcatggatagagaaacccaagcactgagctgggaggtgctc

ctacattaagagattagtgagatgaaggactgagaagattgatcagagaa

gaaggaaaatcaggaaaatggtgctgtcctgaaaatccaagggaagagat

gttccaaagaggagaaaactgatcagttgtcagctagcgtcaattgggat

gaaaatggaccattggacagagggatgtagtgggtcatgggtgaatagat

aagagcagcttctatagaatggcaggggcaaaattctcatctgatcggca

tgggttctaaagaaaacgggaagaaaaaattgagtgcatgaccagtccct

tcaagtagagaggtggaaaagggaaggaggaaaatgaggccacgacaaca

tgagagaaatgacagcatttttaaaaattttttattttattttatttatt

tatttttgctttttagggctgcccctgcaacatatggaggttcccaggtt

aggggtctaatcagagctatagctgccagcctacaccacagccatagcaa

tgccagatctacatgacctacaccacagctcacagcaacgccggatcctt

aacccactgagtgaggccagagatcaaacccatatccttatggatactag

tcaggttcattaccactgagccaaaatgggaaatcctgagtaatgacagc

attttttaatgtgccaggaagcaaaacttgccaccccgaaatgtctctca

ggcatgtggattattttgagctgaaaacgattaaggcccaaaaaacacaa

gaagaaatgtggaccttcccccaacagcctaaaaaatttagattgagggc

ctgttcccagaatagagctattgccagacttgtctacagaggctaagggc

taggtgtggtggggaaaccctcagagatcagagggacgtttatgtaccaa

gcattgacatttccatctccatgcgaatggccttcttcccctctgtagcc

ccaaaccaccacccccaaaatcttcttctgtctttagctgaagatggtgt

tgaaggtgatagtttcagccactttggcgagttcctcagttgttctgggt

ctttcctccTgatccacattattcgactgtgtttgattttctcctgttta

tctgtctcattggcacccatttcattcttagaccagcccaaagaacctag

aagagtgaaggaaaatttcttccaccctgacaaatgctaaatgagaatca

ccgcagtagaggaaaatgatctggtgctgcgggagatagaagagaaaatc

gctggagagatgtcactgagtaggtgagatgggaaaggggtgacacaggt

ggaggtgttgccctcagctaggaagacagacagttcacagaagagaagcg

ggtgtccgtggacatcttgcctcatggatgaggaaaccgaggctaagaaa

gactgcaaaagaaaggtaaggattgcagagaggtcgatccatgactaaaa

tcacagtaaccaaccccaaaccaccatgttttctcctagtctggcacgtg

gcaggtactgtgtaggttttcaatattattggtttgtaacagtacctatt

aggcctccatcccctcctctaatactaacaaaagtgtgagactggtcagt

gaaaaatggtcttctttctctatgaatctttctcaagaagatacataact

ttttattttatcataggcttgaagagcaaatgagaaacagcctccaacct

atgacaccgtaacaaaatgtttatgatcagtgaagggcaagaaacaaaac

atacacagtaaagaccctccataatattgtgggtggcccaacacaggcca

ggttgtaaaagctttttattctttgatagaggaatggatagtaatgtttc

aacctggacagagatcatgttcactgaatccttccaaaaattcatgggta

gtttgaattataaggaaaataagacttaggataaatactttgtccaagat

cccagagttaatgccaaaatcagttttcagactccaggcagcctgatcaa

gagcctaaactttaaagacacagtcccttaataactactattcacagttg

cactttcagggcgcaaagactcattgaatcctacaatagaatgagtttag

atatcaaatctctcagtaatagatgaggagactaaatagcgggcatgacc

tggtcacttaaagacagaattgagattcaaggctagtgttctttctacct

gttttgtttctacaagatgtagcaatgcgctaattacagacctctcaggg

aaggaattcacaaccctcagcaaaaaccaaagacaaatctaagacaacta

agagtgttggtttaatttggaaaaataactcactaaccaaacgcccctct

tagcaccccaatgtcttccaccatcacagtgctcaggcctcaaccatgcc

ccaatcacc

Seq ID No. 25

GCACATGGTAGGCAAAGGACTTTGCTTCTCCCAGCACATCTTTCTGCAGA

GATCCATGGAAACAAGACTCAACTCCAAAGCAGCAAAGAAGCAGCAAGTT

CTCAAGTGATCTCCTCTGACTCCCTCCTCCCAGGCTAATGAAGCCATGTT

GCCCCTGGGGGATTAAGGGCAGGTGTCCATTGTGGGACCCAGCCCGAAGA

CAAGCAATTTGATCAGGTTCTGAGCACTCCTGAATGTGGACTCTGGAATT

TTCTCCTCACCTTGTGGCATATCAGCTTAAGTCAAGTACAAGTGACAAAC

AACATAATCCTAAGAAGAGAGGAATCAAGCTGAAGTCAAAGGATCACTGC

CTTGGATTCTACTGTGAATGATGACCTGGAAAATATCCTGAACAACAGCT

TCAGGGTGATCATCAGAGACAAAAGTTCCAGAGCCAGGTAGGGAAACCCT

CAAGCCTTGCAAAGAGCAAAATCATGCCATTGGGTTCTTAACCTGCTGAG

TGATTTACTATATGTTACTGTGGGAGGCAAAGCGCTCAAATAGCCTGGGT

AAGTATGTCAAATAAAAAGCAAAAGTGGTGTTTCTTGAAATGTTAGACCT

GAGGAAGGAATATTGATAACTTACCAATAATTTTCAGAATGATTTATAGA

TGTGCACTTAGTCAGTGTCTCTCCACCCCGCACCTGACAAGCAGTTTAGA

ATTTATTCTAAGAATCTAGGTTTGCTGGGGGCTACATGGGAATCAGCTTC

AGTGAAGAGTTTGTTGGAATGATTCACTAAATTTTCTATTTCCAGCATAA

ATCCAAGAACCTCTCAGACTAGTTTATTGACACTGCTTTTCCTCCATAAT

CCATCTCATCTCCGTCCATCATGGACACTTTGTAGAATGACAGGTCCTGG

CAgAGACTCaCAGATGCTTCTGAAACATCCTTTGCCTTCAAAGAATGAAC

AGCACACATACTAAGGATCTCAGTGATCCACAAATTAGTTTTTGCCACAA

TGGTTCTTATGATAAAAGTCTTTCATTAACAGCAAATTGTTTTATAATAG

TTGTTCTGCTTTATAATAATTGCATGCTrCACTTTCTTTTCTTTTCTTTT

TTTTTCTTTTTTTGCTTTTTAGTGCCGCAGGTgcagcatatgaaatttcc

caggctaggggtcaaatcagaactacacctactggcctacgccacagcca

cagcaactcaggatctaagccatgtcggtgacctacactacagctcatgg

caatgccagatccttaacccaatgagcgaggccagggatcgaacccatgt

cctcatggatactagtcaggctcattatccgctgagccataacaggaact

cccGAGTTTGCTTTTTATCAAAATTGGTACAGCCTTATTGTTTCTGAAAA

CCACAAAATGAATGTATTCACATAATTTTAAAAGGTTAAATAATTTATGA

TATACAAGACAATAGAAAGAGAAAACGTCATTGCCTCTTTCTTCCACGAC

AACACGCCTCCTTAATTGATTTGAAGAAATAACTACTGAGCATGGTTTAG

TGTACTTCTTTCAGCAATTAGCCTGTATTCATAGCCATACATATTCAATT

AAAATGAGATCATGATATCACACAATACATACCATACAGCCTATAGGGAT

TTTTACAATCATCTTCCACATGACTACATAAAAACCTACCTAAAAAAAAA

AAAAACCCTACTTCATCCTCCTATTGGCTGCTTTGTGCACCATTAAAAAG

CTCTATCATAATTAGGTTATGATGAGGATTTCCATTTTCTACCTTTCAAG

CAACATTTCAATGCACAGTCTTATATACACATTTGAGCCTACTTTTCTTT

TTCTTTCTTTTTTTGGTTTTTTTTTTTTTTTTTTTTTTGGTCTTTTTGTC

TTTTCTAAGgctgcatatggaggttcccaggctagctgtctaatcagaac

tatagctgctggcctacgccacatccacagcaatacaagatctgagccat

gtctgcaacttacaccacagctcacagcaacggtggatccttaaaccact

gagcaaggccagggatcaaacccatAACTTCATGGCTCCTAGTTGGATTT

GTTAACCACTGAGCCATGATGGCAACTCCTGAGCCTACTTTTCTAATCAT

TTCCAACCCTAGGACACTTTTTTAAGTTTCATTTTTCTCCCCCCACCCCC

TGTTTTCTGAAGtGTGTTTGCTTCCACTGGGTGACTTCACtCCCAGGATC

TCATCTGCAGGATACTGCAGCTAAGTGTATGAGCTCTGAATTTGAATCCC

AACTCTGCCACTCAAAGGGATAGGAGTTTCCGATGTGGCCCAATGGGATC

AGTGGCATCTCTGCAGTGCCAGGACGCaggttccatccctggcccagcac

agtgggttaagaatctggCATTGCTGCAGCTGAGGCATAGATTTCAATTG

TGCCTCAgATCTGATCCTTGGCCCAAGGACTGCATATGCCTCAGGGCAAC

CAAAAAAGAGAAAAGGGGGGTGATAGCATTAGTTTCTAGATTTGGGGGAT

AATTAAATAAAGTGATCCATGTACAATGTATGGCATTTTGTAAATGCTCA

ACAAATTTCAACTATTATggagttcccatcatggctcagtggaagggaat

ctgattagcatccatgaggacacaggtCCAACCCCGACCTTGCTCAGTGG

GCATTGCTGTGAGCTGTGGCATGGGTTACAGACGAAGCTCGGATCTGGCA

TTGCTGTGGCTGTGGTGTAAGCCAgCAActacagctctcattcagcccct

agcctgggaacctccatatgccTAAAAGACAAAAAATAAAATTTAAATTA

AAAATAAAGAAATGTTAACTATTATGATTGgTACTGCTTGCATTACTGCA

AAGAAAGTCACTTTCTATACTCTTTAATATCTTAGTTGACTGTGTGCTCA

GTGAACTATTTTGGACACTTAATTTCCACTCTCTTCTATCTCCAACTTGA

CAACTCTCTTTCCTCTCTTCTGGTGAGATCCACTGCTGACTTTGCTCTTT

AAGGCAACTAGAAAAGTGCTCAGTGACAAAATCAAAGAAAGTTACCTTAA

TCTTCAGAATTACAATCTTAAGTTCTCTTGTAAAGCTTACTATTTCAGTG

GTTAGTATTATTCCTTGGTCCCTTACAACTTATCAGCTCTGATCTATTGC

TGATTTTCAACTATTTATTGTTGGAGTTTTTTCCTTTTTTCCCTGTTCAT

TCTGCAAATGTTTGCTGAGCATTTGTCAAGTGAAGATACTGGACTGGGCC

TTCCAAATATAAGACAATGAAACATCGGGAGTTCTCATTATGGTGCAGCA

GAaacgaatccaactaggaaatgtgaggttgcaggttcgatccctgccct

tgctcagtgggttaaggatccagcattaccgtgagctgtggtgtaggttg

cagacgtggctcagatcctgcgttgctgtggctgtggcataggctggcag

ctctagctctgattcgaccgctagcctgggaacctccatGCGCCCCGAGT

GCAGCCCTTAAAAAGCAAAAAAAAAAGAAAGAAAGAAAAAGACAATGAAA

CATCAAACAGCTAACAATCCAGTAGGGTAGAAAGAATCTGGGAACAGATA

AGAGCGATTAAATGTTCTAGGTCCAGTGACCTTGCCTCTGTGCTCTACAC

AGTCGTGCCACTTGCTGAGGGAGAAGGTCTCTCTTGAGTTGAGTCCTGAA

AGACATTAGTTGTTCACAAACTAATGCCAGTGAGTGAAGGTGTTTCCAAG

CAGAGGGAGAGTTTGGTAAAAAGCTGGAAGTCACAGAAAGACTCTAAAGA

GTTTAGGATGGTGGGAGCAACATACGCTGAGATGGGGCTGGAAGGTTAAG

AGGGAAACAACTATAGTAAGTGAAGCTGGACTCACAGCAAAGTGAGGACC

TCAGCATCCTTGATGGGGTTACCATGGAAACACCAAGGCACACCTTGATT

TCCAAAACAGCAGGCACCTGATTCAGCCCAATGTGACATGGTGGGTACCC

CTCTAGCTCTACCTGTTCTGTGACAACTGACAACCAACGAAGTTAAGTCT

GGATTTTCTACTCTGCTGATCCTTGTTTTTGTTTCACACGTCATCTATAG

CTTCATGCCAAAATAGAGTTCAAGGTAAGACGCGGGCCTTGGTTTGATAT

ACATGTAGTCTATCTTGTTTGAGACAATATGGTGGCAAGGAAGAGGTTCA

AACAGGAAAATACTCTCTAATTATGATTAAGTGAGAAAAGCTAAAGAGTC

CCATAATGACACTGAATGAAGTTCATCATTTGCAAAAGCCTTCCCCCCCC

CCCAGGAGACTATAAAAAAGTGCAATTTTTTAAATGAACTTATTTACAAA

ACAGAAATAGAGTCACAGACATAGGAAACGAACAGATGGTTACCAAGGGT

GAAAGGGAGTAGGAGGGATAAATAAGGAGTCTGGGGTTAGCAGATACACC

CCAGTGTACACAAAATAAACAACAGGGACCTACTATATAGCACAGGGAAC

TATATGCAGTAGCTTACAATAACCTATAATGGAAAAGAATGTGAAAAAGA

ATATATGTATGCGTGTGTGTGTAACTGAATCACTTTGCTGTAACCTGAAT

CTAACATAACATTGTAAATCAACTACAGTTTTTTTTTTTTTTAAGTGCAG

GGTTTTGGTGTTTTTTTTTTTTCATTTTTGTTTTTGTTTTTGTTTTTTGC

TTTTTAGGGCCACACCCAGACATATGGGGGTTCCCAGGctAGGGGTcTAa

TTAGAGcTACAGtTGCCGGCTTGCAccacagccacagcaacatcagatcc

gagccgcacttgcgacttacaccacagctcatggcaataccagatcctta

acccactgagcaaggcccagggatcgtacccgcaacctcatggttcctag

tcagattcattTCTGCTGCGCTACAATGGGAACTCCAAGTGCAGTTTTTT

GTAATGTGCTtGTCTTTCTTTGTAATTCATATTCATCCTACTTCCCAATA

AATAAATAAATACATAAATAATAAACATACCATTGTAAATCAACTACAAT

TTTTTTTAAATGCAGGGTTTTTGTTTTTTGTTTTTTGTTTTGTCTTTTTG

CCTTTTGTAgggccgctcccatggcatatggaggttcccaggctaggggt

cgaatcggagctgtagccaccggcctacgccagagccacagcaacgcggg

atccgagccgcgtctgcaacctacaccacagctcacggcaacgccggatc

gttaacccactgagcaagggcagggatcgaacctgcaacctcatggttcc

tagtcagattcgttaactactgagccacaacggaaacTCCTAAAGTGCAG

TTTTTAAATGTGCTTGTCTTTCTTTGTAATTTACACTCAACCTACTTCCC

AATAAATAAATAAATAAACAAATAAATCATAGACATGGTTGAATTCTAAA

GGAAGGGACCATCAGGCCTTAGACAGAAATACGTCATCTTCTAGTATTTT

AAAACACACTAAAGAAGACAAACATGCTCTGCCAGAGAAGCCCAGGGCCT

CCACAGCTGCTTGCAAAGGGAGTTAGGCTTCAGTAGCTGACCCAAGGCTC

TGTTCCTCTTCAGGGAAAAGGGTTTTTGTTCAGTGAGACAGCAGACAGCT

GTCACTGTGgtggacgttcggccaaggaaccaagctggaactcaaacGTA

AGTCAATCCAAACGTTCCTTCCTTGGCTGTCTGTGTCTTACGGTCTCTGT

GGCTCTGAAATGATTCATGTGCTGACTCTCTGAAACCAGACTGACATTCT

CCAGGGCAAAACTAAAGCCTGTCATGAAACcGGAAAACTGAGGGCACATT

TTCTGGGCAGAACTAAGAGTCAGGCACTGGGTGAGGAAAAACTTGTTAGA

ATGATAGTTTCAGAAACTTACTGGGAAGCAAAGCCCATGTTCTGAACAGA

GCTCTGCTCAAGGGTCAGGAGGGGAACCAGTTTTTGTACAGGAGGGAAGT

TGAGACGAACCCCTGTGTAtatggtttcggcgcggggaccaagctggagc

tcaaacGTAAGTGGCTTTTTCCGACTGATTCTTTGCTGTTTCTAATTGTT

GGTTGGCTTTTTGTCCATTTTTCAGTGTTTTCATCGAATTAGTTGTCAGG

GACCAAACAAATTGCCTTCCCAGATTAGGTACCAGGGAGGGGACATTGCT

GCATGGGAGACCAGAGGGTGGCTAATTTTTAACGTTTCCAAGCCAAAATA

ACTGGGGAAGGGGGCTTGCTGTCCTGTGAGGGTAGGTTTTTATAGAAGTG

GAAGTTAAGGGGAAATCGCTATGGTtcacttttggctcggggaccaaagt

ggagcccaaaattgaGTACATTTTCCATCAATTATTTGTGAGATTTTTGT

CCTGTTGTGTCATTTGTGCAAGTTTTTGACATTTTGGTTGAATGAGCCAT

TCCCAGGGACCCAAAAGGATGAGACCGAAAAGTAGAAAAGAGCCAACTTT

TAAGCTGAGCAGACAGACCGAATTGTTGAGTTTGTGAGGAGAGTAGGGTT

TGTAGGGAGAAAGGGGAACAGATCGCTGGCTTTTTCTCTGAATTAGCCTT

TCTCATGGGACTGGCTTCAGAGGGGGTTTTTGATGAGGGAAGTGTTCTAG

AGCCTTAACTGTGGgttgtgttcggtagcgggaccaagctggaaatcaaa

CGTAAGTGCACTTTTCTACTCC

Porcine Lambda Light Chain

In another embodiment, novel genomic sequences encoding the lambda light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate lambda light chain regions. In one embodiment, the porcine lambda light chain nucleotides include a concatamer of J to C units. In a specific embodiment, an isolated porcine lambda nucleotide sequence is provided, such as that depicted in Seq ID No. 28. See FIG. 3 for a diagram of the organization of the porcine lamba immunoglobulin locus.
In one embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first lambda J/C region of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 32.
Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 200 base pairs downstream of lambda J/C, such as that represented by Seq ID No 33. Alternatively, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 11.8 kb downstream of the J/C cluster, near the enhancer (such as that represented by Seq ID No. 34), approximately 12 Kb downstream of lambda, including the enhancer region (such as that represented by Seq ID No. 35), approximately 17.6 Kb downstream of lambda (such as that represented by Seq ID No. 36, approximately 19.1 Kb downstream of lambda (such as that represented by Seq ID No. 37), approximately 21.3 Kb downstream of lambda (such as that represented by Seq ID No. 38), and/or approximately 27 Kb downstream of lambda (such as that represented by Seq ID No. 39).

In still further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 500 or 1,000 contiguous nucleotides of Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39, as well as, nucleotides homologous thereto.


Seq ID No. 28

CCTTCCTCCTGCACCTGTCAACTCCCAATAAACCGTCCTCCTTGTCATTC

AGAAATCATGCTCTCCGCTCACTTGTGTCTACCCATTTTCGGGCTTGCAT

GGGGTCATCCTCGAAGGTGGAGAGAGTCCCCCTTGGCCTTGGGGAAGTCG

AGGGGGGCGGGGGGAGGCCTGAGGCATGTGCCAGCGAGGGGGGTCACCTC

CACGCCCCTGAGGACCTTCTAGAACCAGGGGCGTGGGGCCACCGCCTGAG

TGGAAGGCTGTCCACTTTTCCCCCGGGCCCCCAGGCTCCCTCCTCCGTGT

GGACCTTGTCCACCTCTGACTGGCCCAGCCACTCATGCATTGTTTCCCCG

AAACCCCAGGACGATAGCTCAGCACGCGACAGTGTCCCCCTCTGAGGGCC

TCTGTCCATTTCAGGACGACCCGCATGTACAGCGTGACCACTCTGCTCAC

GCCCACTCACCACGTCCTAGAGCCCCACCCCCAGCCCCATCCTTAGGGGC

ACAGCCAGcTCCGACCGCCCCGGGGACACCACCCTCTGCCCCTTcCCCAG

GCCCTCCCTGTCACACGCACCACAGGGCCCTCCGTCCCGAGACCCTGCTC

CCTCATCCCTCGGTCCCCTCAGGTAGCCTTCCACCCGCGTGTGTCCCGAG

GTCCCAGATGCAGCAAGGCCCCTGGGACAACGCCAGATCTCTGCTCTcCC

CGACCCCTCAGAAGCCAGCCCACGCCTGGCCCCACCACCACTGCCTAACg

TCCAAGTGTCCATAGGCCTCGGGACCTCCAAGTCCAGGTTCTGCCTCTGG

GATTCCGCCATGGGTCTGCCTGGGAAATGATGCACTTGGAGGAGCTCAGC

ATGGGATGCGGGACCTTGTCTCTAGGCGCTcCCTCAGGATCCCACAGCTG

CCCTGTGAGACACACACACACACACACACACACACACACACACACACACA

CACACAAACACGCATGCACGCACGCCGGCACACACGCTATTGCAGAGATG

GCCACGGTAGCTGTGCCTCGAGGCCGAGTGGAGTGTCTAGAACTCTCGGG

GGTCCCCTCTGCAGACGACACTGCTCCATCCCCCCCGTGCCCTGAAGGGC

TCCTCACTCTCCCATCAGGATCTCTCCAAGCTGCTGACCTGGAGAGGAAG

GGGCCTGGGACAGGCGGGGACACTCAGACCTCCCTGCTGCCCCTCCTCTG

CCTGGGCTTGGACGGCTCCCCCCTTCCCACGGGTGAAGGTGCAGGTGGGG

AGAGGGCACCCCCCTCAGCCTCCCAGACCCAGACCAGCCCCCGTGGCAGG

GGCAGCCTGTGAGCCTCCAGCCAGATGCAGGTGGCCTGGGGTGGGGGGTG

GAGGGGGCGGGAGGTTTATGTTTGAGGCTGTATCACTGTGTAATATTTTC

GGCGGTGGGACCCATCTGACCGTCCTCGGTGAGTCTCCCCTTTTCTCTCC

TCCTTGGGGATCCGAGTGAAATCTGGGTCGATCTTCTCTCCGTTCTCCTC

CGACTGGGGCTGAGGTCTGAACCTCGGTGGGGTCCGAAGAGGAGGCCCCT

AGGCCAGGCTCCTCAGCCCCTCCAGCCCGACcgGCCCTCTTGACACAGGG

TCCAGCTAAGGGCAGACATGGAGGCTGCTAGTCCAGGGCCAGGCTCTGAG

ACCCAAGGGCGCTGCCCAAGGAACCCTTGCCCCAGGGACCCTGGGAGCAA

AGCTCCTCACTCAGAGCCTGCAGCCCTGGGGTCTGAGGACAAGGAGGGAC

TGAGGACTGGGCGTGGGGAGTTCAGGCGGGGACACCAGGTCCAGGGAGGT

GACAAAGGCGCTGGGAGGGGGCGGACGGTGCCGGGGACTCCTCCTGGGCC

CTGTGGGCTCGGGGTCCTTGTGAGGACCCTGAGGGACTGAGGGGCCCCTG

GGCCTAGGGACTTGCAgTgAGGGAGGCAGGGAGTGTCCCTTGAGAACGTG

GCCTCCGCGGGCTGGGTCCCCCTGCTGCTCCCAGCC*GGGAGGACACCCC

AGAGCAAGCGCCCCAGGTGGGCGGGGAGGGTCTCCTCACAGGGGCAGCTG

ACAGATAGAGGCCCCCGCCAGGCAGATGCTTGATCCTGGCAgTTATACTG

GGTTC**GCACAACTTTCCCTGAACAAGGGGCCCTCCGAACAGACACAGA

CGCAACCCAGTCGACCcaggCTCAGCACAgAAAATGCACTGACACCCAAA

ACCCTCATCTggggGCCTGGCCGGcAtCCCGCCCCAGGACCCAAGGCCCC

TGCCCCCTGGCAGCCCTGGACACGGTCCTCTGTGGGCGGTGGGGTCgGGG

CTGTGGTGACGGTGGCATCGGGGAGCCTGTGCCCCCTCCCTGAAAGGGCG

GAGAGGCTCAAGAGGGGAGAGAAATGTCCTCCCCTAGGAAGACGTCGGAC

GGGGGCGGGGGGGTGGTCTCCGACAGACAGATGCCCGGGACCGACAGACC

TGCCGAGGGAAGAGGGCACCTCGGTCGGGTTAGGCTCCAGGCAGCACGAG

GGAGCGAGGCTGGGAGGGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAG

ACTTCAGCAGGCCCCCAGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACG

CAAGGTGAGTGACCCCACCTGTGGCTGACCTGACCTCAgGGgGACAAGGC

TCAGCCTGGGAGTCTGTGTCCCCATCGCCTGcACAGGGGATTCCCCTGAT

GGACACTGAGCCAACGACCTCCCGTCTGTCCCCGACCCCCAGGTCAGCCC

AAgGCCaCTCCCACGGTCAACCTCTTCCCGCCCTCCTCTGAGGAGCTCGG

CACCAACAAGGCCACCCTGGTGTGTCTAATAAGTGACTTCTACCCGGGCG

CCGTGACGGTGACCTGGAAGGCAGGCGGCACCACCGTCACCCAGGGCGTG

GAGACCACCAAGCCCTCGAAACAGAGCAACAACAAGTACGCGGCCAGCAG

CTACCTGGCCCTGTCCGCCAGTGACTGGAAATCTTCCAGCGGCTTCACCT

GCCAGGTCACCCACGAGGGGACCATTGTGGAGAAGACAGTGACGCCCTCC

GAGTGCGCCTAGGTCCCTGGGCCCCCACCCTCAGGGGCCTGGAGCCACAG

GACCCCCGCGAGGGTCTCCCCGCGACCCTGGTCCAGCCCAGCCCTTCCTC

CTGCACCTGTCAACTCCCAATAAACCGTCCTCCTTGTCATTCAGAAATCA

TGCTCTCCGCTCACTTGTGTCTACCCATTTTCGGGCTTGCATGGGGTCAT

CCTCGAAGGTGGAGAGAGTCCCCCTTGGCCTTGGGgAAATCGAGGGGGGC

GGGGGGAGGCCTGAGGCATGTGCCAGCGAGGGGGGTCACCTCCACGCCCC

TGAGGACCTTCTAGAACCAGGGGCGTGGGGCCACCGCCAGAGTGGAAGGC

TGTCCACTTTTCCCCCGGGCCCCCAGGCTCCCTCCTCCGTGTGGACCTTG

TCCACCTCTGACTGGCCCAGCCACTCATGCATTGTTTCCCCGAAACCCCA

GGACGATAGCTCAGCACGCGACAGTGTCCCCCTCTGAGGGCCTCTGTCCA

TTTCAGGACGACCCGCATGTACAGCGTGACCACTCTGCTCACGCCCACTC

ACCACGTCCTAGAGCCCCACCCCCAGCCCCATCCTTAGGGGCACAGCCAG

CTCCGACCGCCCCGGGGACACCACCCTCTGCCCCTTCCCCAGGCCCTCCC

TGTCACACGCACCACAGGGCCCTCCGTCCCGAGACCCTGCTCCCTCATCC

CTCGGTCCCCTCAGGTAGCCTTCCACCCGCGTGTGTCCCGAGGTCCCAGA

TGCAGCAAGGCCCCTGGGACAACGCCAGATCTCTGCTCTCCCCGACCCTC

AGAAGCCAGCCCACGCCTGGCCCACCACCACTGCCTAACGTCCAAGTGTC

CATAGGCTCGGGAcCTCcAaGTCCAGGTTCTGCCTCTGGGATTCCGCCAT

GGGTCTGCCTGGAATGATGCACTTGGAGgAgCTCAGcATGGGATGcGGAA

CTTGTCTAGcGCTCCTCAGATCCAcAGcTGCCTGtGAgAcacacacacac

acacacacacaccAAAcaCGcATGCACGCACGCCGGCACACACGCTATTA

CAGAGATGGCCACGGTAGCTGTGCCTCGAGGCCGAGTGGAGTGTCTAGAA

CTCTCGGGGGTCCCCTCTGCAGACGACACTGCTCCATCCCCCCCGTGCCC

TGAAGGGCTCCTCACTCTCCCATCAGGATCTCTCCAAGCTGCTGACCTGG

AGAGGAAGGGGCCTGGGACAGGCGGGGACACTCAGACCTCCCTGCTGCCC

CTGCTCTGCCTGGGCTTGGACGGCTCCCCCCTTCCCACGGGTGAAGGTGC

AGGTGGGGAGAGGGCACCCCCCTCACCCTCCCAGACCCAGACCAGCCCCC

GTGGCAGGGGCAGCCTGTGAGCCTCCAGCCAGATGCAGGTGGCCTGGGGT

GGGGGGTGGAGGGGGCGGGAGGTTTATGTTTGAGGCTGTATTCATCTGTG

TAATATttTCGGCGGTGGGACCCATCTGACCGTCCTCGGTGAGTCTCCCC

TtttctttcctccttggggatccgagtgaaATcTGGGTCGATCTTCTCTC

CGTTCTCCTCCGACTGGGGCTGAGGTCTGAACCTCGGTgGGGTCCGAAGA

GGAGGCCCCTAGGCC*GGCTCcTCAGCCCCTCCAGGCCGACCCGCCCTCT

TGACACAGGGTCCAGCTAAGGGCAGACAT***GGCTGCTAGTCCAGGGCC

AGGCTcTGAGACCCAAGGGCGCTGCCCAAGGAACCCTTGCCCCAGGGACC

CTGGGAGCAAAGCTCCTCACTCAGAGCCTGCAGCCCTGGgGTCTGAGGAC

AAGGAGGGACTGAGGACTGGGCGTGGGGAGTTCAGGCgGGGACACCGGGT

CCAGGGAGGTGACAAAGGCGCTGGGAGGGGGCGGACGGTGCCGGAGACTC

CTCCTGGGCCCTGTGGGCTCGTGGTCCTTGTGAGGACCCTGAGGG*CTGA

GGGGCCCCTGGGCCTAGGGACTTGCAGTGAGGGAGGCAGGGAGTGTCCCT

TGAGAACGTGGCCTCCGCGGGCTGGGTCCCCCTCGTGCTCCCAGCAGGGA

GGACACCCCAGAGCAAGCGCCCCAGGTGGGCGGGGAGGGTCTCCTCACAG

GGGCAGCTGACAGATAGAC*GgccCCCGCCAGACAGATGCTTGATCCTGG

TCag***TACTGGGTTCGCcACTTCCCTGAACAGGGGCCCTCCGAACAGA

CACAGACGCAGACCaggCTCAGCACAgAAAATGCACTGACACCCAAAACC

CTCATCTGggGGCCTGGCCGGCATCCCGCCCCAGGACCCAAGGCCCCTGC

CCCCTGGCAGCCCTGGACACGGTCCTCTGTGGGCGGTGGGGTCgGGGCTG

TGGTGACGGTGGCATCGGGGAGCCTGTGCCCCCTCCCTGAAAGGGCGGAG

AGGCTCAAGAGGGGACAGAAATGTCCTCCCCTAGGAAGACCTCGGACGGG

GGCGGGGGGGTGGTCTCCGACAGACAGATGCCCGGGACCGACAGACCTGC

CGAGGGAAGAGGGCACCTCGGTCGGGTTAGGCTCCAGGCAGCACGAGGGA

GCGAGGCTGGGAGGGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAGACT

TCAGCAGGCCCCCAGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACGCAA

GGTGAGTGACCCCACCTGTGGCTGACCTGACCTGACCtCAGGGGGACAAG

GCTGAGCCTGGGACTCTgTGTCCCCATCGCCTGCACAGGGGATTCCCCTG

ATGGACACTGAGCCAACGACCTCCCGTCTCTCCCCGACCCCCAGGTCAGC

CCAAGGCCACTCCCACGGTCAACCTGTTCCCGCCCTCCTCTGAGGAGCTC

GGCACCAACAAGGCCACCCTGGTGTGTCTA

Seq ID No. 32

GCCACGCCCACTCCATCATGCGGGGAGGGGATGGGCAGACCCTCCAGAAA

GAAGCTCCCTGGGGTGCAGGTTAACAGCTTTCCCAGACACAGCCAGTACT

AGAGTGAGGTGAATAAGACATCCTCCTTGCTTGTGAAATTTAGGAAGTGC

CCCCAAACATCAGTCATTAAGATAAATAATATTGAATGCACTTTTTTTTT

TTTATTTTTTTTTTTTGCTTTTTAGGGCCTAATCTGCAGCatatggaagt

tcccaggctacaagtcgaaccagagctgcagctgccagcctacatcacag

ccacagcaacaccagatccgagccacatctgtgactaacactgcagttca

cagcaacgccagatccttaacccattgagtgaggccagggatcaaaccca

catcctcatggatactagtctggttcgtaaaccactgagccaCAAGGGGA

ACTCCTGAATGCAATATTTTTGAAAATTGAAATTAAATCTGTCACTCTTT

CACTTAAGAGTCCCCTTAGATTGGGGAAAATTTAAATATCTGTCATCTTA

GTGCATCTTTGCTCATATGATGTGAATAAAATCCCAAAATCCATATGAAT

GAAGCATCAAAATGTACATGAAGTCAGCCTGACCCTGCACTGCCCTCACT

TGCCTCATGTACCCCCCACCTCAAAGGAAGATGCAGAAAGGAGTCCAGCC

CCTACACCGCCACCTGCCCCCACCACTGGAGCCCCTCAGGTCTCCCACCT

CCTTTTCTGAGCTTCAGTCTTCCTGTGGCATTGCCTACCTCTACAGCTGC

CCCCTACTAGGCCCTCCCCCTGGGGCTGAGCTCCAGGCACTGGACTGGGA

AAGTTAGAGGTTAAAGCATGGAAAATTCCCAAAGCCACCAGTTCCAGGCT

GCCCCCCACCCCACCGCCACGTCCAAAAAGGGGCATCTTCCCAGATCTCT

GGCTGGTATTGGTAGGACCCAGGACATAGTCTTTATACCAATTCTGCTGT

GTGTCTTAGGAAAGAaactctccctctctgtgcttcagtttcctcatcaa

taaaAGGAGCAGGCCAGGTTGGAGGGTCTGTGACGTCTGCTGAAGCAGCA

GGATTCTCTCTCCTTTTGCTGGAGGAGAACTGATCCTTCACCCCCAGGAT

CAACAGAGAAGCCAAGGTCTTCAGCCTTCCTGGGGACCCCTCAGAGGGAA

CTCAGGGCCACAGAGCCAGACCCTGATGCCAGAACCTTTGTCATATGCCC

AGACGGAGACTTCATCCCCCTCCTCCTCAGACCCTCCAGGCCCCAACAGT

GAGATGCTGAAGATATTAAGAGAAGGGCAAGTCAGcTTAAGTTTGGGGGT

AGAGGGGAACAGGGAGTGAGGAGATCTGGCCTGAGAGATAGGAGCCCTGG

TGGCCACAGGAGGACTCTTTGGGTCCTGTCGGATGGACACAGGGCGGCCC

GGGGGCATGTTGGAGCCCGGCTGGTTCTTACCAGAGGCAGGGGGCACCCT

CTGACACGGGAGCAGGGCATGTTCCATACATGACACACCCCTCTGCTCCA

GGGCAGGTGGGTGGCGGCACAGAGGAGCCAGGGACTCTGAGCAAGGGGTC

CACCAGTGGGGCAGTTGGATCCAGACTTCTCTGGGCCAGCGAGAGTCTAG

CCCTCAGCCGTTCTCTGTCCAGGAGGGGGGTGGGGCAGGCCTGGGCGGCC

AGAGCTCATCCCTCAAGGGTTCCCAGGGTCCTGCCAGACCCAGATTTCCG

ACCGCAGCCACCACAAGAGGATGTGGTCTGCTGTGGCAGCTGCCAAGACC

TTGCAGCAGGTGCAGGGTGGGGGGGTGGGGGCACCTGGGGGCAGCTGGGG

TCACTGAGTTCAGGGAAAACCCCTTTTTTCCCCTAAACCTGGGGCCATCC

CTAGGGGAAACCACAACTTCTGAGCCCTGGGCAGTGGCTGCTGGGAGGGA

AGAGCTTCATCCTGGACCCTGGGGGGGAACCCAGCTCCAAAGGTGCAAGG

GGCCCAGGTCCAAGGCTAGAGTGGGCCAAGCACCGCAATGGCCAGGGAGT

GGGGGAGGTGGAGCTGGACTGGATCAGGGCCTCCTTGGGACTCCCTACAC

CCTGTGTGACATGTTAGGGTACCCACACCCCATCACCAGTCAGGGCCTGG

CCCATCTCCAGGGCCAGGGATGTGCATGTAAGTGTGTGTGAGTGTGTGTG

TGTGGTGTAGTACACCCCTTGGCATCCGGTTCCGAGGCCTTGGGTTCCTC

CAAAGTTGCTCTCTGAATTAGGTCAAACTGTGAGGTCCTGATCGCCATCA

TCAACTTCGTTCTCCCCACCTCCCATCATTATCAAGAGCTGGGGAGGGTC

TGGGATTTCTTCCCACCCACAAGCCAAAAGATAAGCCTGCTGGTGATGGC

AGAAGACACAGGATCCTGGGTCAGAGACAAAGGCCAGTGTGTCACAGCGA

GAGAGGCAGCCGGACTATCAGCTGTCACAGAGAGGCCTTAGTCCGCTGAA

CTCAGGCCCCAGTGACTCCTGTTCCACTGGGCACTGGCCCCCCTCCACAG

CGCCCCCAGGCCCCAGGGAGAGGCGTCACAGCTTAGAGATGGCCCTGCTG

AACAGGGAACAAGAACAGGTGTGCCCCATCCAGCGCCCCAGGGGTGGGAC

AGGTGGGCTGGATTTGGTGTGAAGCCCTTGAGCCCTGgAACCCAAcCACA

GCAgGGCAGTTGGTAGATGCCATTTGGGGAGAGGCCCCAGGAGTAAGGGC

CATGGGCCCTTGAGGGGGCCAGGAGCTGAGGACAGGGACAGAGACGGCCC

AGGCAGAGGACAGGGCCATGAGGGGTGCACTGAGATGGCCACTGCCAGCA

GGGGCAGCTGCCAACCCGTCCAGGGAACTTATTCAGCAGTCAGCTGGAGG

TGCCATTGACGCTGAGGGCAGATGAAGCCCAGGCCAGGCTAGGTGGGCTG

TGAAGACCCCAGGGGACAGAGCTCTGTCCCTGGGCAGCACTGGCCTCTCA

TTCTGCAGGGCTTGACGGGATCCCAAGGCCTGCTGCCCCTGATGGTAGTG

GCAGTACCGCCCAGAGCAGGACCCCAGCATGGAAACCCCAACGGGACGCA

GCCTGCGGAGCCCACAAAACCAGTAAGGAGCCGAAGCAGTCATGGCACGG

GGAGTGTGGACTTCCCTTTGATGGGGCCCAGGCATGAAGGACAGAATGGG

ACAGCGGCCATGAGCAGAAAATCAGCCGGAGGGGATGGGCCTAGGCAGAC

GCTGGCTTTATTTGAAGTGTTGGCATTTTGTCTGGTGTGTATTGTTGGTA

TTGATTTTATTTTAGTATGTCAGTGACATACTGACATATTATGTAACGAC

ATATTATTATGTGTTTTAAGAAGCACTCCAAGGGAACAGGCTGTCTGTAA

TGTGTCCAGAGAAGAGAGCAAGAGCTTGGCTCAGTCTCCCCCAAGGAGGT

CAGTTCCTCAACAGGGGTCCTAAATGTTTCCTGGAGCCAGGCCTGAATCA

AGGGGgTCATATCTACACGTGGGGCAGACCCATGGACCATTTTCGGAGCA

ATAAGATGGCAGGGAGGATACCAAGCTGGTCTTACAGATCCAGGGCTTTG

ACCTGTGACGCGGGCGCTCCTCCAGGCAAAGGGAGAAGCCAGCAGGAAGC

TTTCAGAACTGGGGAGAACAGGGTGCAGACCTCCAGGGTCTTGTACAACG

CACCCTTTATCCTGGGGTCCAGGAGGGGTCACTGAGGGATTTAAGTGGGG

GACCATCAGAACCAGGTTTGTGTTTTGGAAAAATGGCTCCAAAGCAGAGA

CCAGTGTGAGGCCAGATTAGATGATGAAGAAGAGGCAGTGGAAAGTCGAT

GGGTGGCCAGGTAGCAAGAGGGCCTATGGAGTTGGCAAGTGAATTTAAAG

TGGTGGCACCAGAGGGCAGATGGGGAGGAGCAGGCACTGTCATGGACTGT

CTATAGAAATCTAAAATGTATACCCTTTTTAGCAATATGCAGTGAGTCAT

AAAAGAACACATATATATTTAAATTGTGTAATTCCACTTCTAAGGATTCA

TCCCAAGGGGGGAAAATAATCAAAGATGTAACCAAAGGTTTACAAACAAG

AACTCATCATTAATCTTCCTTGTTGTTATTTCAACGATATTATTATTATT

ACTATTATTATTATTATTATTttgtctttttgcattttctagggccactc

ccacggcatagagaggttcccaggctaggggtcaaatcggagctacagct

gccggcctacgccagagccacagcaacgcaggatctgagccacagcaatg

caggatctacaccacagctcatggtaacgctggatccttaacccaatgag

tgaggccagggatcgaacctgtaacttcatggttcctagtcggattcatt

aaccactgagccacgacaggaactccAACATTATTAATGATGGGAGAAAA

CTGGAAGTAACCTAAATATCCAGCAGAAAGGGTGTGGCCAAATACAGCAT

GGAGTAGCCATCATAAGGAATCTTACACAAGCCTCCAAAATTGTGTTTCT

GAAATTGGGTTTAAAGTACGTTTGCATTTTAAAAAGCCTGCCAGAAAATA

CAGAAAAATGTCTGTGATATGTCTCTGGCTGATAGGATTTTGCTTAGTTT

TAATTTTGGCTTTATAATTTTCTATAGTTATGAAAATGTTCACAAGAAGA

TATATTTCATTTTAGCTTCTAAAATAATTATAACACAGAAGTAATTTGTG

CTTTAAAAAAATATTCAACACAGAAGTATATAAAGTAAAAATTGaggagt

tcccatcgtggctcagtgattaacaaacccaactagtatccatgaggata

tggatttgatccctggccttgctcagtgggttgaggatccagtgttgctg

tgagctgtggtgtaggttgcagacacagcactctggcgttgctgtgactc

tggcgtaggccggcagctacagctccatttggacccttagcctgggaacc

tccatatgcctgagatacggcccTAAAAAGTCAAAAGCCAAAAAAATAGT

AAAAATTGAGTGTTTCTACTTACCACCCCTGCCCACATCTTATGCTAAAA

CCCGTTCTCCAGAGACAAACATCGTCAGGTGGGTCTATATATTTCCAGCC

CTCCTCCTGTGTGTGTATGTCCGTAAAACACACACACACACACACACACG

CACACACACACACACGTATCTAATTAGCATTGGTATTAGTTTTTCAAAAG

GGAGGTCATGCTCTACCTTTTAGGCGGCAAATAGATTATTTAAACAAATC

TGTTGACATTTTCTATATCAACCCATAAGATCTCCCATGTTCTTGGAAAG

GCTTTGTAAGACATCAACATCTGGGTAAACCAGCATGGTTTTTAGGGGGT

TGTGTGGATTTTTTTCATATTTTTTAGGGCACACCTGCAgcatatggagg

ttcccaggctaggggttgaatcagagctgtagctgccggcctacaccaca

gccacagcaacgccagatccttaacccactgagaaaggccagggattgaa

cctgcatcctcatggATGCTGGTCAGATTTATTTCTGCTGAGCCACAACA

GGAACTCCCTGAACCAGAATGCTTTTAACCATTCCACTTTGCATGGACAT

TTAGATTGTTTCCATTTAAAAATACAAATTACAaggagttcccgtcgtgg

ctcagtggtaacgaattggactaggaaccatgaggtttcgggttcgatcc

ctggccttgctcggtgggttaaggatccagcattgatgtgagatatggtg

taggtcgcagacgtggctcggatcccacgttgctgtggctctggcgtagg

ccggcaacaacagctccgattcgacccctagccTGggaacctccatgtgc

cacaggagcagccctaGAAAAGGCAAAAAGACAAAAAAATAAAAAATTAA

AATGAAAAAATAAAATAAAAATACAAATTACAAGAGACGGCTACAAGGAA

ATCCCCAAGTGTGTGCAAATGCCATATATGTATAAAATGTACTAGTGTCT

CCTCGCGGGAAAGTTGCCTAAAAGTGGGTTGGCTGGACAGAGAGGACAGG

CTTTGACATTCTCATAGGTAGTAGCAATGGGCTTCTCAAAATGCTGTTCC

AGTTTACACTCACCATAGCAAATGACAGTGCCTCTTCCTCTCCACCCTTG

CCAATAATGTGACAGGTGGATCTTTTTCTATTTTGTGTATCTGACAAGCA

AAAAATGAGAACAggagttcctgtcgtggtgcagtggagacaaatctgac

taggaaccatgaaatttcgggttcaatccctggcctcactcagtaggtaa

aggatccagggttgcagtgagctgtggggtaggtcgcagacacagtgcaa

atttggccctgttgtggctgtggtgtaggccggcagctatagctccaatt

ggacccctagcctgggaacctccttatgccgtgggtgaggccctAAAAAA

AAGAGTGCAAAAAAAAAAAATAAGAACAAAAATGATCATCGTTTAATTCT

TTATTTGATCATTGGTGAAACTTATTTTCCTTTTATATTTTTATTGACTG

ATTTTATTTCTCCTATGAATTTACCGGTCATAGTTTTGCCTGGGTGTTTT

TACTCCGGTTTTAGTTTTGGTTGGTTGTATTTTCTTAGAGAGCTATAGAA

ACTCTTCATCTATTTGGAATAGTAATTCCTCATTAAGTATTTGTGCTGCA

AAAAATTTTCCCTGATCTGTTTTATGCTTTTGTTTGTGGGGTCTTTCACG

AGAAAGCCTTTTTAGTTTTTACACCTCAGCTTGGTTGTTTTTCTTGATTG

TGTCTGTAATCTGCGGCCAACATAGGAAACACATTTTTACTTTAGTGTTT

TTTTCCTATTTTCTTCAAGTACGTCCATTGTTTTGGTGTCTGATTTTACT

TTGCCTGGGGTTTGTTTTTGTGTGGCAGGAATATAAACTTATGTATTTTC

CAAATGGAGAGCCAATGGTTGTATATTTGTTGAATTCAAATGCAACTTTA

TCAAACACCAAATCATCGATTTATCACAACTCTTCTCTGGTTTATTGATC

TAATGATCAATTCCTGTTCCACGCTGTTTTAATTATTTTAGCTTTGTGGA

TTTTGGTGCCTGGTAGAGAACAAAGCCTCCATTATTTTCATTCAAAATAG

TCCCGTCTATTATCTGCCATTGTTGTAGTATTAGACTTTAAAATCAATTT

ACTGATTTTCAAAAGTTATTCCTTTGGTGATGTGGAATACTTTATACTTC

ATAAGGTACATGGATTCATTTGTGGGGAATTGATGTCTTTGCTATTGTGG

CCATTTGTCAAGTTGTGTAATATTTTACCCATGCCAACTTTGCATATTGT

ATGTGAGTTTATTCCCAGGGTTTTTAATAGGATGTTTATTGAAGTTGTCA

GTGTTTCCACAATTTCATCGCCTCAGTGCTTACTGTTTGCATAAAAGGAA

ACCTACTCACTTTTGCCTATTGCTCTTGTATTCAATCATTTTAGTTAACT

CTTGTGTTAATTTTGAGAGTTTTTCAGCTGACTGTCTGGGGTTTTCTTTA

ATAGACTAGCCCTTTGTCTGTAAAGAATAATTTTATCGAATTTTTCTTAA

CACTCACACTCTCCCCACCCCCACCCCCGCTGATCTCCTTTCATTGGGTC

AAATCTGTAGAATACAATAAAAGTAAGAGTGGGAACCTTAGCCTTTAAGT

CGATTTTGCCTTTAAATGTGAATGTTGCTATGTTTCGGGACATTCTCTTT

ATCAAGTTGCGGATGTTTCCTTAGATAATTAACTTAATAAAAGACTGGAT

GTTTGCTTTCTTCAAATCAGAATTGTGTTGAATTTATATTGCTATTCTGT

TTAATTTTGTTTCAAAAAATTTACATGCACACCTTAAAGATAACCATGAC

CAAATAGTCCTCCTGCTGAGAGAAAATGTTGGCCCCAATGCCACAGGTTA

CCTCCCGACTCAGATAAACTACAATGGGAGATAAAATCAGATTTGGCAAA

GCCTGTGGATTCTTGCCATAACTCTCAGAGCATGACTTGGGTGTTTTTTC

CTTTTCTAAGTATTTTAATGGTATTTTTGTGTTACAATAGGAAATCTAGG

ACACAGAGAGTGATTCAATGAGGGGAACGCATTCTGGGATGACTCTAGGC

CTCTGGTTTGGGGAGAGCTCTATTGAAGTAAAGACAATGAGAGGAAGCAA

GTTTGCAGGGAACTGTGAGGAATTTAGATGGGGAATGTTGGGTTTGAGGT

TTCTATAGGGCACGCAAGCAGAGATGCACTCAGGAGGAAGAAGGAGCATA

AATCTAGAGGCAAAAAGAGAGGTCAGGACTGGAAATAGAGATGCGAGACA

CCAGGGTGGCAGTCAGAGAGCACAGTGTGGGTCAGAAGACAGTGGAAGAA

CACAAGGGACAGAGAGGGATCTCCAACTTCACTGGGATGAGGGCCTTGTT

GGCCTTGACCTGAGAGATTTCCAGGAGTTGAGGGTGGGAAGGAGAGGGCT

CCTGCACATGTCCTGACATGAAACGGTGCCCAGCATATGGGTGCTTGGAA

GACATTGTTGGACAGATGGATGGATGATGGATGATGGATGAATGGATGGA

TGGAAGATGATGGATAAATGGATGATGGATGGATGGACAGAAGGACAAAG

AGATGGACAGAAAGACAGTGATCTGAGAGAGCAGAGAAGGCTTCATGAAA

GGACAGGAACTGAACTGTCTCAGTGGGTGGAGACAATGGTGTAGGGGGTT

TCCACATGGAGGCACCAGGGGTCAGGAATAATCTAGTGTCCACAGGCCCA

GGAAGGAAGCTGTCTGCAGGAAATTGTGGGGAAGAACCTCAGAGTCCTTA

AATGAGGTCAGGAGTGGTCAGGAGGGTCTGATCAGGTAAGGACTCATGTC

CATCATCACATGGTCACCTAAGGGCATGTAGCTCTCAGCATCTCCATCAG

GACAGTCTCAGAATGGGGGCGGGGTCACACACTGGGTGACTCAAGGCGTG

GGTCATGCCTGCCTCGGACGTGGGCCTGGGCATGGGGACACCTCCAGACC

ATGGGCCCGCCCAGGGCTGCACTGGcctctggtgggctagctacccgtcc

aagcaacacaggacacagccctacctgctgcaaccctgtgcccgaaacgc

ccatctggttcctgctccagcccggccccagggaacaggactcaggtgct

agcccaatggggttttgttcgagcctcagtcagcgtggTATTTCTCCGGC

AGCGAGACTCAGTTCACCGCCTTAGGttaagtggttctcatgaatttcct

agcagtcctgcactctgctatgccgggaaagtcacttttgtcgctggggg

ctgtttccccgtgcccttggagaatcaaggattgcccaactttctctgtg

ggggaggtggctggtcttggggtgaccagcaggaagggccccaaaagcag

gagcagctgcctccagAATACAACTGTCGGCTACAGCTCAAACAGGAGGC

CTGGACTGGGGTTTAACCACCAGGGCGGCACGAAGGAGCGAGGCTGGGAG

GGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAGACTTCAGCAGGCCCCC

AGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACGCAAGGTGAGTGACCCC

ACCTGTGGCTGACCTGACCTCAGGGGGACAAGGCTCAGCCTGAGACTCTG

TGTCCCCATCGCCTGCACAGgggattcccctgatggacactgagccaacg

acctcccgtctctccccgacccccaggtcagcccaaggccgcccccacgg

tcaacctcttcccgccctcctctgaggagctcggcaccaacaaggccacc

ctggtgtgtctaataagtgacttctacccgAAGGGCGAATTCCAGCACAC

TGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGATGCATAG

CTTGAGTATCTA

Seq ID No. 33

agatctttaaaccaccgagcaaggccagggatcgaacccgcatcctcatg

aatcctagttgggttcgttaaccgctgaaccacaatgggaactcctGTCT

TTCACATTTAATTCACAACCTCTCCAGGATTCTGGGGGTGGGTGGGGAAT

CCTAGGTACCCACTGGGAAAGTAATCCAAGGGGAGAGGCTCACGGACTcT

AGGGATCGGCGGAGGAGGGAAGGTATCTCCCAGGAAACTGGCCAGGACAC

ATTGGTCCTCCGCCCTCCCCTTCCTCCCACTCCTCCTCCAGACAGGACTG

TGCCCACCCCCTGCCACCTTTCTGGCCAGAACTGTCCATGGCAGGTGACC

TTCACATGAGCCCTTCCTCCCTGCCTGCCCTAGTGGGACCCTCCATACCT

CCCCCTGGACCCCGTTGTCCTTTCTTTCCAGTGTGGCCCTGAGCATAACT

GATGCCATCATGGGCTGCTGACCCACCCGGGACTGTGTTGTGCAGTGAGT

CACTTCTCTGTCATCAGGGCTTTGTAATTGATAGATAGTGTTTCATCATC

ATTAGGACCGGGTGGCCTCTATGCTCTGTTAGTCTCCAAACACTGATGAA

AACCTTCGTTGGCATAGTCCCAGCTTCCTGTTGCCCATCCATAAATCTTG

ACTTAGGGATGCACATCCTGTCTCCAAGCAACCACCCCTCCCCTAGGCTA

ACTATAAAACTGTCCCAATGGCCCTTGTGTGGTGCAGAGTTCATGCTTCC

AGATCATTTCTGTGCTAGATCCATATCTCACCTTGTAAGTCATCCTATAA

TAAACTGATCCATTGATTATTTGCTTCTGTTTTTTCCATCTCAAAACAGC

TTCTCAGTTCAGTTCGAATTTTTTATTCCCTCCATCCACCCATACTTTCC

TCAGCCTGGGGAACCCTTGCCCCCAGTCCCATGCCCTTCCTCCCTCTCTG

CCCAGCTCAGCACCTGCCCACCCTCACCCTTCCTGTGACTCCCTAGGACT

GGACCATCCACTGGGGCCAGGACACTCCAGCAGCCTTGGCTTCATGGGCT

CTGAAATCCATGGCCCATCTCTATTCCTCACTGGATGGCAGGTTCAGAGA

TGTGAAAGGTCTAGGAGGAAGCCAGGAAGGAAACTGTTGCATGAAAGGCC

GGCCTGATGGTTCAGTACTTAAATAATATGAGCTCTGAGCTCCCCAGGAA

CCAAAGCATGGAGGGAGTATGTGCCTCAGAATCTCTCTGAGATTCAGCAA

AGCCTTTGCTAGAGGGAAAATAGTGGCTCAACCTTGAGGGCCAGCATCTT

GCACCACAGTTAAAAGTGGGTATTTGTTTTACCTGAGGCCTCAGCATTAT

GGGAACCGGGCTCTGACACAAACACAGGTGCAGCCCGGCAGCCTCAGAAC

ACAGCAACGACCACAAGCTGGGACAGCTGCCCCTGAACGGGGAGTCCACC

ATGCTTCTGTCTCGGGTACCACCAGGTCACCATCCCTGGGGGAGGTAGTT

CCATAGCAGTAGTCCCCTGATTTCGCCCCTCGGGCGTGTAGCCAGGCAAG

CTCCTGCCTCTGGACCCAGGGTGGACCCTTGCTCCCCACTACCCTGCACA

TGCCAGACAGTCAAGACCACTCCCACCTCTGTCTGAGGCCCCCTTGGGTG

TCCCAGGGCCCCCGAGCTGTCCTCTACTCATGGTTCTTCCACCTGGGTAC

AAAAGAGGCGAGGGACACTTTTCTCAGGTTTGCGGCTCAGAAAGGTACCT

TCCTAGGGTTTGTCCACTGGGAGTCACCTCCCTTGCATCTCAATGTCAGT

GGGGAAAACTGGGTCCCATGGGGGGATTAGTGCCACTGTGAGGCCCCTGA

AGTCTGGGGCCTCTAGACACTATGATGATGAGGGATGTGGTGAAAAACCC

CACCCCAGCCCTTCTTGCCGGGACCCTGGGCTGTGGCTCCCCCATTGCAC

TTGGGGTCAGAGGGGTGGATGGTGGCTATGGTGAGGCATGTTTCCCATGA

GCTGGGGGCACCCTGGGTGACTTTCTCCTGTGAATCCTGAATTAGCAGCT

ATAACAAATTGCCCAAACTCTTAGGCTTAAAACAACACACATTTATTCCT

CTGGGTCCCAGGGTCAGAAGTCCAAAATGAGTCCTATAGGCTAAATTTGA

GGTGTCTCTGGGTTGAGCTCCTCCTGGAAGCCTTTTCCAGCCTCTAGAGT

CCCAAGTCCTTGGCTCTGGGCCCCTCCCTCAAGCTTCAAAGCCACAGAAG

CTTCTAATCTCTCTCCCTTCCCCTCTGACCTCTGCTCCCATCCTCATACC

CTGTCCCCTCACTCTGACCCTCCTGCCTCCCTCTTTCCCTTATAAAGACC

CTGCATGGGGCCACGGAGATAATCCAGGGTAATCGCCCCTCTTCCAGCCC

TTAACTCCATCCCATCTGCAAAATCCCTGTCACCCCATAATGGACCTACT

GATGGTCTGGGGGTTAGGACGTGGACAACTTGGGGCCTTATTCATCTGAT

CACAACTCCAGTTCCCAGACCCCCAGACCCCCGGGCATTAGGGAAACTTC

TCCCAGTTCCTCTCCCTCTGTGTCCTGCCCAGTCTCCAGGATGGGCCACT

CCCGAGGGCCCTTCAGCTCAGGCTCCCCCTCCTTTCTCCCTGGCCTCTTG

TGGCCCCATCTCCTCCTCCGCTCACAGGGAGAGAACTTTGATTTCAGCTT

TGGCTCTGGGGCTTTGCTTCCTTCTGGCCATTGGCTGAAGGGCGGGTTTC

TCCAGGTCTTACCTGTCAGTCATCAAACCGCCCTTGGAGGAAGACCCTAA

TATGATCCTTACCCTACAGATGGAGACTCGAGGCCCAGAGATCCTGAGTG

ACCTGCTCACATTCACAGCAGGGACTGAACCCCAGTCACCTACCCAACTC

CAGGGCTCAGCGCTTTTTTTTTTTTTTTTCTTTTTgccttttcgagggcc

gctcccgcaacatatggagatttccaggctaggggtctaattggagcagt

cgacactggcctaagccaaagccacagcaacaagggcaagccgcttctgc

agcctataccacagctcacggcaatgccggatccttaacccactgagcaa

agccagggattgaacctgcaacctcatgtttcctagtcaaatttgttaac

cactgacccatgacgggaactcccAGGGCTCAGCTCTTGACTCCAGGTTC

GCAGCTGCCCTGAAAGCAATGCAACCCTGGCTGGCCCCGCCTCATGCATC

CGGCCTCCTGCCCAAAGAGCTCTGAGCCCACCTGGGCCTAGGTCCTCCTC

CCTGGGACTCATGGCCTAAGGGTACAGAGTTACTGGGGCTGATGAAGGGA

CCAATGGGGACAGGGGCCTCAAATCAAAGTGGCTGTCTCTCTCATGTCCC

TTCCTCTCCTCAGGGTCCAAAATCAGGGTCAGGGCCCCAGGGCAGGGGCT

GAGAGGGCCTCTTTCTGAAGGCCCTGTCTCAGTGCAGGTTATGGGGGTCT

GGGGGAGGGTCAATGCAGGGCTCACCCTTCAGTGCCCCAAAGCCTAGAGA

GTGAGTGCCTGCCAGTGGCTTCCCAGGCCCAATCCCTTGACTGCCTGGGA

ATGCTCAAATGCAGGAACTGTCACAACACCTTCAGTCAGGGGCTGCTCTG

GGAGGAAAAACACTCAGAATTGGGGGTTCAGGGAAGGCCCAGTGCCAAGC

ATAGCAGGAGCTCAGGTGGCTGCAGATGGTGTGAACCCCAGGAGCAGGAT

GGCCGGCACTCCCCCCAGACCCTCCAGAGCCCCAGGTTGGCTGCCCTCTT

CACTGCCGACACCCCTGGGTCCACTTCTGCCCTTTCCCACCTAAAACCTT

TAGGGCTCCCACTTTCTCCCAAATGTGAGACATCACCACGGCTCCCAGGG

AGTGTCCAGAAGGGCATCTGGCTGAGAGGTCCTGACATCTGGGAGCCTCA

GGCCCCACAATGGACAGACGCCCTGCCAGGATGCTGCTGCAGGGCTGTTA

GCTAGGCGGGGTGGAGATGGGGTACTTTGCCTCTCAGAGGCCCCGGCCCC

ACCATGAAACCTCAGTGACACCCCATTTCCCTGAGTTCACATACCTGTAT

CCTACTCCAGTCACCTTCCCCACGAACCCCTGGGAGCCCAGGATGATGCT

GGGGCTGGAGCCACGACCAGCCCACGAGTGATCCAGCTCTGCCAATCAGC

AGTCATTTCCCAAGTGTTCCAGCCCTGCCAGGTCCCACTACAGCAGTAAT

GGAGGCCCCAGACACCAGTCCAGCAGTTAGAGGGCTGGACTAGCACCAGC

TTTCAAGCCTCAGCATCTCAAGGTGAATGGCCAGTGCCCCTCCCCGTGGC

CATCACAGGATCGCAGATATGACCCTAGGGGAAGAAATATCCTGGGAGTA

AGGAAGTGCCCATACTCAAGGATGGCCCCTCTGTGACCTAACCTGTCCCT

GAGGATTGTACTTCCAGGCGTTAAAACAGTAGAACGCCTGCCTGTGAACC

CCCGCCAAGGGACTGCTTGGGGAGGCCCCCTAAACCAGAACACAGGCACT

CCAGCAGGACCTCTGAACTCTGACCACCCTCAGCAAGTGGCACCCCCCGC

AGCTTCCAAGGCAC

Seq ID No. 34

AACAAGATGCTACCCCACCAACAAAATTCACCGGAGAAGACAAGGACAGG

GGGTTCCTGGGGTCCTGACAGGGTCACCAAAGAGGGTTCTGGGGCAGCAG

CAACTCCAGCCGCCTCAGAACAGAGCCTGGAAGCTGTACCCTCAGAGCAG

AGGCGGAGAGAGAAAGGGCCTCTTGGTGGGTCAGCAGGAGCAGAGGCTCA

GAGGTGGGGGTTGCAGCCCCCCCTTCAACAGGCCAACACAGTGAAGCAGC

TGACCCCTCCACCTTGGAGACCCCAGACTCCTGTCTCCCACGCCACCTTG

GTTTTTAAGGTAATTTTTATTTTATATCAGAGTATGGTTGACTTACAATG

TTGTGTTGGTTTCAGGTGTACAGCAGAGTGATTCACTTCTACATAGACTC

ATATCTATTCTTTCTCAGATTCTTTTCCCATATAGGTTATTACAGAATAT

TGAGTAGATCCCTGCTGATTACCCATTTTTATAATTGTATATGTTAATCC

CAAACTCCTAATTTATCCCTCCCCAGACTATGATTCTTTATATCTCTATC

TGTTTCCTAATCTGTCTCCTCTAAGTCACCCTAGGAGAGCAGAGGGGTCA

CGTCTGTCCTGTCCTGGCCCAGCCACCTCTCTCCACCCAGGAATCCCTTG

CATTTGGTGCCAAGGGCCCGGCCCCGCCCTAAAGAGAAAGGAGAACGGGA

TGTGGACAGGACACCGGGCAGAGAGGGACAAGCAGAGGATGCCAGGGTAG

GGAGGTCTCCAGGGTGGATGGTGGTCTGTCCGGAGGCAGGATGAGGCAGG

AAGGGTGTGGATGTACTCGGTGAGGCTGGCGCATGGCCTGGAGTGTCCTG

AGCCCTGGGAGGCCTCAGCCCTGGATCAGATCTGTGATTCCAAAGGGCCA

CTGCATCCAGAGACCGTTGAGTGGCCCATTGTCCTGAACCATTTATAGAA

CACAGGACAAGCGGTACCTGACTAAGCTGCTCACAGATTCCATGAGGCTG

ATGCCAGGGTTGTCACCCCATCTCACAGGCAGGGAAACTGATGCATATAC

TGCAGAGCCAGGCAGAGGCCCTCCCAGTGCCCCCTCCCAGCCTGTGGCCC

CCCTCCAGTGGCTGGACACTGAGGCCACACTGGGGCACCCTGTGGAGATC

t

Seq ID No. 35

AGATCTGGCCAGGCCAGAGAAGCCCATGTGGTGACCTCCCTCCATCACTC

CACGCCCTGACCTGCCAGGGAGCAGAAAGTAGGCCCAGGGTGGACCCGGT

GGCCACCTGCCACCCCATGGCTGGGAGAAGGGAGGGCCTGGGCAAAGGGC

CTGGGAAGCCTGTGGTGGGACCCCAGACCCCAGGGTGGACAGGGAGGGTC

CCACACCCACAGCCATTTGCTTCCCTCTGTGGGTTCAGTGTCCTCATCTC

ATCTGTGGGGAGGGGGCTGATAATGAATCTCCCCCATTGGGGTGGGCTTG

GGGATTAAAGGGCCAGTGTCTGTGATATGCCTGGACCATAGTGACCCTCA

CCCTCCCCAGCCATTGCTGTCACCTTCCGGGCTCTTGCCCAGGCCTGCCT

GACATGCTGTGTGACCCTGGGCAAGATGATCCCCCTTTCTGGGCCCCAGC

CTTCCTCTCTGCTCCGGAAGTGCTTCCTGGGGAAACCTGTGGGCTGGATC

CTATAGGAAACCTGTCCAATTCCTGGATGCACAGAGGGGCAGGGAGGCCC

TGGGCCTGGAGGGGCAGGGAGGCTCGAGGTGGGAGCAGGGTAGGGGCCAG

TCCAGGGCAAGGAGGTGGGTGGGTAGGGTG

Seq ID No. 36:

GATCTGTGTTCCATCTCAGAGCTATCTTAGCAGAGAGGTGCAGGGGCCTC

CAGGGCCACCAAAGTCCAGGCTCAGCCAGAGGCAATGGGGTATCGATGAG

CTACAGGACACAGGCGTCAGCCCAGTGTCAGGGAGAATCACCTTGTTTGT

TTTCTGAGTTCCTCTTAAAATAGAGTTAATTGGTCTTGGCCTTACGGTTT

ACAATAACAACTGCACCCTGTAAACAACGTGAAGAGTACAGAACAACAAA

TGGGGGAAAACATATTTCACCTGAAAGAGCCACCGCTCATATTTTGATGG

ATTTCCTTCTAGTTTAATCCTGTTTTAATTGTAAACTGTTAAAACAAACA

TAAATAAAGAAAATGCATCTGTAAAGTTTAAAAGTCATATCTATGGTGAT

GGTTGCAAAACACTGTGAATGTTCACTTTGAAATCGTGAACTCTACGTGA

TATGCATGTCCCGTTAATTAACCTCACAGGCTCAGAATGTGGTTCATTAT

TTCTTTAATTTTCCTTTAATTTTATGTCCTCTGTGTGTGCCCTTAAACCA

ACTACTTTTCAGCTCTGCCTGTTTTTGACCTTCACATAGATGACATTTGT

GAGTGTTTTCTTTCTCAACACTGGGTCTGATACCCACCCACGCTGTCTGC

TGTCACTGCGGACGTGGAGGGCCACCACCCAGCTATGGCCCCAGCCAGGC

CAACACTGGATGAATCTGCCCCCAGAGCAGGGCCACCAACACTGGAGGTG

CAGAGAGGGTTTCTTCAGGGCCATCATTATCCAAGGCATTGTTTCTACTG

TAAGCTTTCAAAATGCTTCCCCTGATTATTAAAAGAAATAATAAGATGGG

GGGAAAGTACAAGAAGGGAAGTTTCCAGCCCAGCCTGAAGATCGTGCTGG

TTGTATCTGGAGCCTGTCTTCCTGACAGGCCTCTATTCCCAGAGTTA

Seq ID No. 37:

GGATCCTAGGGAAGGGAGGGCGGGGGCCTGGACAAAGGGGGCCTAAAGGA

CATTCTCACCTATCCCACTGGACCcctgctgtgctctgagggagggagca

gagagggggtctgaggccttttcccagCTCCTCTGAGTCCCTCCTCCGAG

CACCTGGACGGAAGCCCCTCCTCAGGGAGTCCTCAGACCCCTCCCCTCCA

GCCAGGTTGGCCTGTGTGGAGTCCCCAGTAAGAATAGAATGCTCAGGGCT

TCGAGCTGAGCCCTGGCTACTTGGGGGGGTGCTGGGGATTGGGGGTGCTG

GGCGGGGAGCTGGGGTGTCACTAGATGCCAGTAGGCTGTGGGCTCGGGTC

TGGGGGGTCTGCACATGTGCAGCTGTGGGAAGGCCCTATTGGTGGTACCC

TCAGACACATATGGCCCCTCAATTTCTGAGACCAGAGAGCCCAGTCTGGC

CTTCCCAGAACAGCTGCCCCTGGTGGGGGAGATGTAGGGGGGCCTTCAGC

CCAGGACCCCCAACGGCAGGGCCTGAGGCCCCCATCCCCTTGTCCTGGGC

CCAGAGCCTCAGCTATCAGGCCTATCAGAGATCCTGGCTGCCCAGCTCAG

GTTCCCCAGGAGCCAGAGGGAGGCCAGGGGTTACTAGGAAATCCGGAAAG

GGTCTTTGAGGCTGGGCCCCACCCTCTCAGCTTTCACAGGAGAAACAGAG

GCCCACAGGGGGCAAAGGACTTGCCAGACTCACAATGAGCCCAGCAGCTG

GACTCAAGGCCCAGTGTTCGGCCCCACAACAGCACTCACGTGCCCTTGAT

CGTGAGGGGCCCCCTCTCAGCCAGGCATTCAGACCTGTGACCTGCATCTA

AGATTCAGCATCAGCCATTCTGAGCTGAAGAGCCCTCAGGGTCTGCAGTC

AAGGCCAGAGGGCCAGACCTCCAACGGCCAGACATCCCAGCCAGATTCCT

TTCTGGTCAATGGGCCCCAGTCTGGCTTGGCTCCTGCAGGCCCAGTGCCG

CCTTCTTCCCCTGGGCCTGTGGAGTCCAGCCTTTCAGTTTCCCACCCACA

TCCTCAGCCACAATCCAGGCTCAGAGGCAATGTCCGTGGGCAGCCCCTGT

GTGACCCCTCTGTGGGTGATCCTCAGTCCTACCCTTAGCAGACAGCGCAT

GAGGGGCCCTCTTGAACCTGAGGGATACTCCATGTCGGAGGGGAGAAGCT

GGCCTTCCCCACCCCCACTTCCAGGCCTTGGGGAGCAGAGAAAGACCCCA

GACCTGGGTCCCTTCTAACAGGCCAGGCCCCAGCCCAGCTCTCCACCAGC

CCCAGGGGCCTGGGGTCCACGCCTGGGGACTGGAGGGTGGGCCTGTCAGG

CGCTGACCCAGAGGCAGGACAGCCAAGTTCAGGATCCCAGCCAGGTGGTC

CCCGTGCACCATGCAGGGGTGTCACCCACACAGGGGTGTTGCCACCCTCA

CCTGACTGTCCTCATGGGCCACATGGAGGTATCCTGGGTTCATTACTGGT

CAACATACCCGTGTCCCTGCAGTGCCCCCTCTGGcgcacgcgtgcacgcg

cacacgcacacactcatacaGAGGCTCCAGCCAAGAGTGCCCTGTAGTAG

GCACTGCTGTCACTTCTCTAAAAGGTCGCAATCATACTTGTAAAGACCCA

AGATTGTTCAGAAATCCCAGATGGAGAAGTCTGGAAAGATCtTTTTCTCC

TTTCACGGGCTGGGGAAATGTGACCTGGCCAAGGTCACACAGCAAGTGGT

GGAACCCTGGCCCCTGATTCCAGCTCATTCCAGTTCCCAAGGCCCTGCCA

GAGCCCAGAGGCTGGGCCCTCTGGGGCAGAGGAGCTGGGGTCCTCCCCCC

TACACAGAGCACACAGCCCCGCAAGAGAGAAGAGACACCTTGGGGAGAGG

AATCTCCAGACCAGAGATCCCAGTATGGGTCTCCTCTATGCTGACGGGAT

GGGATGTCAAGAGGGGAGGGGGCTGGGCTTTAGGGAAACACACAAAAATC

GCTGAGAACACTGACAGGTGCGACACACCCACCCCTAATGCTAACCTGTG

GCCCATTACTCAgatct

Seq ID No. 38

GATCTTCTCCTAAGACCAAGGAAAACTGGTCATACCAGGTCCACTTGTCC

CCTGTGGCCATTGTCCCTCCTTCCCCAGAAGAAACAAGCACTTTCCACTC

CACAAGTAGCTCCTGATCAGCTTGGAAGCCCGGTGCTGCTCTGGGCCCTG

GGGACACGGCAGGGGCATCAGAGACCAAATCCTGGAACAAAGTTCCAGTG

GGTGAGGCAGGCCGGACAAGCAACACGTTATACCATAATATGAGGCAAAA

TATAATGTGAGTTCTTTATGAAAGGAAGGGGTTGCAGGTGCAACTGTTGG

CTTAGGTGGATGGTCACCCCTGAATGGAGGAGGGGGTTCCCAGGGCATGT

GCCTGGGGAGAAGGGCTCCTGGCAGGAGGGACAGCAAGTGCAAGGGCCCT

GTGATCAAATGTGCCTGGCAAGTTGCAGGAACAGCTAGAAGGCCAGCAAG

GTTGGAACCAAGGAAGGGGTGAGGGGAGGGGCAGGGCCCTCAGGGCCTTG

CCCAGCAGCCTGAGCATCTGGAGATTTGTCCAAAGTTTCAAATGTACCTG

GGCAACCTCATGCCCATATACCATTCCTAACTTCTGCACTTAACATCTCT

AGGACTGGGACCCAGCCAGTCAAGCGGGGGGACCCAGAGAGCTCCGGTGT

GAACACCGAGGTGCTGGTGGGTCTGCGTGTGTGGACATAGGGCAGTCCCG

GTCCTTCCTTCACTAACACGGCCCGGGAAGCCCTGTGCCTCCCTGGTGCG

CGGGTCGGCGCTTCCGGAGGGTACAGGCCCACCTGGAGCCCGGGCACAGT

GCATGCAAGTCGGGTTCACGGCAACCTGAGCTGGCTCTGCAGGGCAGTGG

GACTCACAGCCAGGGGTACAGGGCAGACCGGTCCTGCCTCTGCGCCCCTC

CCTGGCCTGTGGCCCCTGGACGTGATCCCCAACAGTTAGCATGCCCCGCC

GGTGCTGAGAACCTGGACGAGGTCCGCAGGCGTCACTGGGCGGTCACTGA

GCCCGCCCCAGGCCCCCTCTGCCCCTTCCTGGGGTGACCGTGGACTCCTG

GATGACCCTGGACCCTAGACTTCCCAGGGTGTCTCGCGGAGGTTCCTCAG

CCAGGATCTCTGCGTCTCCTCCTTCCATAGAGGGGACGGCGCCCCCTTGT

GGCCAAGGAGGGGACGGTGGGTCCCGGAGCTGGGGCGGAGAACACAGGGA

GCCCCTCCCAGACCCCGCTCTGGGCAGAACCTGGGAAGGGATGTGGCCAT

CGGGGGATCCCTCCAGGCCATCTCCTCAGATGGGGGCTGGTCGACTAGCT

TCTGAGTCCTCCAAGGAACCGGGTCCTTCTAGTCATGACTCTGCCCAGAT

GAAGAAGGAGAGCACTTCTCTCCATCAGGAGGATCTGAGCTTCTCTTAAT

TAGAATCAGCTCCTTGGCTTCTACCCCTTAAAAAAAGGTACAGAAACTTT

GCACCTTGATCCAGTATCAGGGGAATTTATCAATCAATGTGGGAGAAATT

GGCATCTTTACCACACTGAATCTTTCAATCCATGAATATCCTCTCTCTCT

TCCATGCATAGGTTTTAATAATTCTCAATGGAGTTTAATGTAAGTTTTCC

TCATAGACAATTGCCTTTGGACATCTCTTTAGAGTCATCTCTAGTAAACT

GATATTCTTAATGCAATTATAAAATGTATCCTGCTTAATGTTATTTTCTA

TTCATTTGCTGTTATATAGAGATACAATGAGTTTCCACATTTGAAACTGG

ATCTGGTAAATTGGCTACCCTTTTTTTATAGATTCTATTAATTTTTATAC

ATTCTGTGGGACTTGCTACATACTTAATCATGTCACCTGTGAAGAATGAC

AATTTGGTTGCTACCCTCCCAATTCTTATATGTCTCATTTCTTTCCCTCT

GCTGGTACTCTGGCAGCAGCAGGGAAGATAATGGGCCTCCTTATCTTGTC

ACAAAAGGATGTTTTTAAAGATTTCGTTATAAAACATAACGCTTTCTGGT

TTTCTTTAAAGATTCTCTCACCAGCTTAAGAAAATTTTCTTATACTCTGT

ATGATAAATGGGTTTTTGACAATCATTTGTTGCATTTTACCTAGTGTTTT

CTCTGCATCTTTATATGCTTTTTCTCCTTTAATCCTGAAAATTGTTTCGA

TTTTTCTAACATTGAACCAATCTTACATTCCTGGAATGGATGGACCAGAC

TAGTCCACATGTTTATTCTGCCCAATGGCTAGATTTTGTGTTCaatattt

tgttcagaatgtttgcatctatattcttGAGTGAGACAGAGCTGCCCTTG

TTAGGTTTCACAACCGAGGTTGTGTTAGCTTCATAAAATGAGACGTTTAT

TCTCTAAAAGAATTGTTTCGCTTCTCTGGATGAATTTGTGTAAGGTTAGA

ATTGCTTACCAGTGAagatctCGGGgCCAGTTCTTCTTTAGGGGAAGATT

TTCAACAATTAAGCTCAATGCCTTTAGAAGAACTGAGAGTTTCTATTATT

TCTTGAGTTAAATATATGTATTTAATTAGACTTTCTAGGAATAGTCTCAT

TTCATCTCAAATAATTGACATATGCTATTAAAGCAGATTCTCATGAACCA

TTGTAGGTATTCCAGGTCTAGAAAAATGTTCCCCTTTGCATCCCTAATGT

GTTTAATTTTCACCTTCTTTCTTTTGTTCTTGAGAAATTCACCAAATCAT

TTTCAATTTCAGTCATATCCCAAAGCAACCAACTCTCTACCTTCTTGTTT

TATCATCCCTGCTGGATTTTTGTTATCTACTTCTTCAGTATTTGTTCTTC

CCTTTCTTCTATTCCTCATTCCATTTTTCCCTTGTTTTCTAACTTTCTGA

GATATATGCTTAGTTCCTTCATTTGAAGCCTTTTTATTTTCTTTTTTTTT

TTTTGGTCTTTTTGTCTTTtGTTGTTGTTGTTGTGCTATTtCTTGGGCCG

CTCCCGCGGCATATGGAGGTTCCCAGGCTAGGAGTCGAATCGGAGCTGTA

GCCACCGGCCTACGCCAGAGCCACAGCAATGCGGGATCCGAGCCGCGTCT

GCAACCTACACCACAGCTCATGGCAACGCCGGATCGTTAACCCACTGAGC

AAGGGCAGGAACCGAACCCGCAACCTCATGGTTCCTAGTCGGATTCGTAA

CCACTGTGCCACAACAGGAACTCCGCCTTTTTATTTTCTATAAAAATTTC

TATGTACATTTTAAGGTTATAGGTTTCCTTCTATGTACCCCATTGGCTGT

ATCCTCAGGGTTCTGTGGAGTGATTTCATTATTGTTCAAGTTCAATATGT

CTTCTGATTTTCCAATTTGAATACCTCTCTAAATCAGTAGGTGAATATTT

CTTTTTCTTTTTCTTTTCTTTTCTTCTTTTTTTTTTTCTTTCAGCCAGGT

CCATGGCATGCAGAAATTCCCAGGCCAGGAATCAAACTCTCACCATGGCA

GTGACAATGTCGGATCCTTTACCCACTAGGCCACCAGGGAACTCTGGGAG

CATATGTTTTTATTTCCCGACATCTGAGGATGCCTAGTATGTCTTCATTA

TTGATTTCTAGTTTGCCACTGATTTCTAGTATTTTGCTCATAGAGTGTAT

GCTCAATGGTTTTGGTCATTTGAAATGTATTTAGTCCTGCTTTATGACCC

AGTATGTGGTCAGTTTTGTCAATGTTCCTTTTCTGCTTGAAGAGAACCTA

CATGCTGTAACTCTGGGTGCATGTTCTGTATATAAGTCTATAGGCTGAGC

CGGGGGAGCCTTCTAATCTGCCGTTATCTTCTTCGAGTTATTCTAGGTAC

TATTTCTTAGCCATAAACCTTTAAATTCTGATATCAATATAATGACCCCA

GCCCGCTTAGGGTCGGCACTTCATGTTATCTTTTTCCATCCATTTAATGC

CTCCCCACTGTTTTGGCCACACCCGTGGGATATGGGAGTTCCTGGGCCAA

GGATCaGATCTGAGCCGCAGCTGCCACCTATGCCACAGCAgcagcaatga

tggatctttaacccactgcaccacactggggattgaacccaagcctcagc

agcaacccaagctactgcagagacaacaccagatccttaacctgctgtgc

catagcgggaaTTTCCATCCATTTACTTTCAAGCCAGCTGAATAACCTAG

CCCACCATGGCTGGACATGGGTGCTCTGCTTCAAATGATTTTGTTCAGTC

AGCATCCATCTCTGAAATGTGTGCCAAGCATTTATATGCATGCAAGAGTC

ATGTTGGCACTTCTATCATTTCCAACAGTTCAGTAGCCTTTGTATCATGA

CATTTCTTGGCCTTTTCTCTACAATATTTGAGGCTGAGCAGACTGGCCGT

GCCCCTGTCCATGCTTCCAGAGCCTGTGTGCAGACTTCTGCTCTAGACAG

AGACAGCTAACCATCCTGCAGTGCCCAGAAAACCCAACTCAAAGACCCTC

AAGTAAGGAAGGATTTATTGGCTCACGTAATCTGGAATCCAGGCATGGGG

TATTCAGGGCGACCTGAACCAGAGGCCCTGGCCCTGTTCTCTAAGCTTCT

TCCTGCCCTGCCCTCGTTCTGGAAGTGACCCTGAAGGACAGCAATGAAGG

GCAGCTCCCCCAGGGACAGATGACTGAGAGGTCCATTTCAAGTCCAACTT

GGCCTAGATTGAGAGGCAGCAAGAAATATGGACCTACAGTGAGTCACAGG

ATTTACCAGTGGTTTGGCTGGGTTGTCAGTGTTACAGGCTAAACATTTGG

GTCCCTCCAAAATTAACATGTTGCCACTCTAACCACCAAAATCatggtat

ttgggggtggggcccttggaggtaattaggtttagaaAGAATGAAGAGGG

GGCCCTTGTGATGGGACTAGTGCCTTTATAGAGAGAGAAGAGAGAGGG

Seq ID No. 39

CACCTCATCCCCAACCACCTGGATGGTGGCAAGTGGCAGGCTGAGAGGCT

GCATATGAGCTCATCAAGAGGGTCCCCACCCCACAGAGGCTGACCCAGCT

GCCACTGCCACCTAGTGGCTGATCGGCCAAGAGCAGGAGCCCCAGGGGCA

GCTCCATTCCCTGGGGCGGCCAGGGAACCACCTGGTGGTAGGACAATTCC

ATTGCACCTCATCCATCAGGAAAAGGTTTGCCTTCCCTGGCAGTAATGCA

TCTTCCCATAACATGGTCCCTGGCCTCTTGGAATGGCTTGGCCACCGTCA

TGGCCTCACCCACAAAGCCTTGTGTCTCAGCAAGGAACTTATTCCACAGC

AAAGGACTTGCAGCCTGGAATGAACTGGTCTGACTACATACCCCATTGCC

CAGAAGTAGGTGGTCTATTGCAAAGTGGAGTGGCTTACCCAAGACTCAGT

TGTGCCCAAGTTGAGAGATAGCATCCTAAAATATGGGCTTATGTCTCACT

GGCTGAGGTTTATTCTTTGAATCAAAGACAATTATATGGTGTGGTCCCCC

CAGAGATAGAATACATGAGTCTGGGAATCAAGGGATAGAAGTAAGAAGAG

ATTTTGTCACCATTAATCCCAATAACTCGCCCAAAGAATATTTGCTTTCT

GTCCTGGCAGCTCTGCTGCTTTGGCAATAACTTCCTAGAATATAATGTCT

CCACCAGGGGACTCCACAACGGTTCCATTGATTTGAAGCCAATGGGCAGA

GGAGGGGCTGCCTTACTGGTCGGACTGGTCAGCCCTGATTACTAAGGAGA

AATCAGGCAACTTCAACAAAACTAAGGCAGGGGGGACTTTGTCTAGAACC

CAAAGCACTAAGCATCTTAGTACTTTTTAGTTCTCAGAGCCTCCAAGAAC

AAAGATTTAGCCCCTCAGCACCACCAGGTAAAGAACAGGTAAATCCAGCT

GAGGACAAGAGAAATATTGAATGGATAGAGGAAGAAAGAAATTATAGATA

TCAACTATGGCCTCATGACTAGAGTCTCCAGATTAAGCGGAATAAAAATA

CAGATGATTaGATCTGAACATCAGGCCAAACAACGAACAACAGTTTAAGT

GCGACCTAGGCAATATTTGGGACATACTTATACTAAAATTTTTTCGCTAT

TTGAGCATCCTGTATTTTATCTGGCAACTTTATTCATCCCTAGCGAAAAA

GGAACTGTGGTAACTTAGTGTATTTTTACTTTGCTCATTATTGTGTATAT

ACCTACTTGTATTTATCAATCATATTTACTCTGTTCTCAGTATTACTTTA

TATAGCAGTTGGTGGTGATGGTTAGCAACATATTCAGTGGAACTGTGACT

GAATTTGAGGAGAAATTAACAGAGTTGGCTGTGGCTACAATAACCCTTCG

GGACATGTGTCCCCTCATTTTGGGGAGATGGTTagatctCTGGGTAAATG

TTAGGGCATCTGAGCCAGAAACCAAGATTTTGCCAGCTGGTGCAATGTCA

GATTTTACCAGCAGAGGGTGCCAGAGGAATGCGGCAAAACCCGAGTGCCA

GAAAGCACCTCCCTGTTTTCCAGCTTTTCTTCCTTTTTATTTATTTTATT

TACGGCCCAGGAGTCCGTAATAGCGCTGAGGATGGCCCAGGCTCTTCTCA

GCAGCCCTGACTGACTAGTTCAGCAATGCGCTCAGGCCCCATCTGGCCAC

CGGGCAGCCTCTTCTGTGGTAGCTCCAGCCTCAGCCAGTGCAAAAGGCTA

CCCTACACTGGCGCCACTTCTACAATCAGCACTGGCCACACCCTCCACGC

CATCCGGCACGGAGCCAGGTGATCTGCCGGCCAGATTGCAGTTCGTGCTG

CCTGAGTCCAGGTGATTACACTGGCTGCATCTTTTCTTTCTGGACCAtTC

attccattttttt

Bovine Lambda Light Chain

In a further embodiment, nucleic acid sequences are provided that encode bovine lambda light chain locus, which can include at least one joining region-constant region pair and/or at least one variable region, for example, as represented by Seq ID No. 31. In Seq ID No 31, bovine lambda C can be found at residues 993-1333, a J to C pair can be found at the complement of residues 33848-35628 where C is the complement of 33848-34328 and J is the complement of 35599-35628, V regions can be found at (or in the complement of) residues 10676-10728, 11092-11446, 15088-15381, 25239-25528, 29784-30228, and 51718-52357. Seq ID No. 31 can be found in Genbank ACCESSION No. AC117274. Further provided are vectors and/or targeting constructs that contain all or part of Seq ID No. 31, for example at least 100, 250, 500, 1000, 2000, 5000, 10000, 20000, 500000, 75000 or 100000 contiguous nucleotides of Seq ID No. 31, as well as cells and animals that contain a disrupted bovine lambda gene.


Seq ID No	1	tgggttctat gccacccagc ttggtctctg
31		atggtcactt gaggccccca tctcatggca

	61	aagagggaac tggattgcag atgagggacc
		gtgggcagac atcagaggga cacagaaccc

	121	tcaaggctgg ggaccagagt cagagggcca
		ggaagggctg gggaccttgg gtctagggat

	181	ccgggtcagg gactcggcaa aggtggaggg
		ctccccaagg cctccatggg gcggacctgc

	241	agatcctggg ccggccaggg acccagggaa
		agtgcaaggg gaagacgggg gaggagaagg

	301	tgctgaactc agaactgggg aaagagatag
		gaggtcagga tgcaggggac acggactcct

	361	gagtctgcag gacacactcc tcagaagcag
		gagtccctga agaagcagag agacaggtac

	421	cagggcagga aacctccaga cccaagaaga
		ctcagagagg aacctgagct cagatctgcg

	481	gatgggggga ccgaggacag gcagacaggc
		tccccctcga ccagcacaga ggctccaagg

	541	gacacagact tggagaccaa cggacgcctt
		cgggcaaagg ctcgaacaca catgtcagct

	601	caaaatatac ctggactgac tcacaggagg
		ccagggaggc cacatcatcc actcagggga

	661	cagactgcca gccccaggca gaccccatca
		accgtcagac gggcaggcaa ggagagtgag

	721	ggtcagatgt ctgtgtggga aaccaagaac
		cagggagtct caggacagcg ctggcagggg

	781	tccaggctca ggctttccca ggaagatggg
		gaggtgcctg agaaaacccc acccaccttc

	841	cctggcacag gccctctggc tcacagtggt
		gcctggactc ggggtcctgc tgggctctca

	901	aaggatcctg tgtccccctg tgacacagac
		tcaggggctc ccatgacggg caccagacct

	961	ctgattgtgg tcttcttccc ctcgcccact
		ttgcaggtca gcccaagtcc acaccctcgg

	1021	tcaccctgtt cccgccctcc aaggaggagc
		tcagcaccaa caaggccacc ctggtgtgtc

	1081	tcatcagcga cttctacccg ggtagcgtga
		ccgtggtcta gaaggcagac ggcagcacca

	1141	tcacccgcaa cgtggagacc acccgggcct
		ccaaacagag caacagcaag tacgcggcca

	1201	gcagctacct gagcctgatg ggcagcgact
		ggaaatcgaa aggcagttac agctgcgagg

	1261	tcacgcacga ggggagcacc gtgacgaaga
		cagtgaagcc tcagagtgtt cttagggccc

	1321	tgggccccca ccccggaaag ttctaccctc
		ccaccctggt tccccctagc ccttcctcct

	1381	gcacacaatc agctcttaat aaaatgtcct
		cattgtcatt cagaaatgaa tgctctctgc

	1441	tcatttttgt tgatacattt ggtgccctga
		gctcagttat cttcaaagga aacaaatcct

	1501	cttagccttt gggaatcagg agagagggtg
		gaagcttggg ggtttgggga gggatgattt

	1561	cactgtcatc cagaatcccc cagagaacat
		tctggaacag gggatggggc cactgcagga

	1621	gtggaagtct gtccaccctc cccatcagcc
		gccatgcttc ctcctctgtg tggaccgtgt

	1681	ccagctctga tggtcacggc aacacactct
		ggttgccacg ggcccagggc agtatctcgg

	1741	ctccctccac tgggtgctca gcaatcacat
		ctggaagctg ctcctgctca agcggccctc

	1801	tgtccactta gatgatgacc cccctgaagt
		catgcgtgtt ttggctgaaa ccccaccctg

	1861	gtgattccca gtcgtcacag ccaagactcc
		ccccgactcg acctttccaa gggcactacc

	1921	ctctgcccct cccccagggc tccccctcac
		agtcttcagg ggaccggcaa gcccccaacc

	1981	ctggtcactc atctcacagt tcccccaggt
		cgccctcctc ccacttgcat ggcaggaggg

	2041	tcccagctga cttcgaggtc tctgaccagc
		ccagctctgc tctgcgaccc cttaaaactc

	2101	agcccaccac ggagcccagc accatctcag
		gtccaagtgg ccgttttggt tgatgggttc

	2161	cgtgagctca agcccagaat caggttaggg
		aggtcgtggc gtggtcatct ctgaccttgg

	2221	gtggtttctt aggagctcag aatgggagct
		gatacacgga taggctgtgc taggcactcc

	2281	cacgggacca cacgtgagca ccgttagaca
		cacacacaca cacacacaca cacacacaca

	2341	cacacacgag tcactacaaa cacggccatg
		ttggttggac gcatctctag gaccagaggc

	2401	gcttccagaa tccgccatgg cctcactctg
		cggagaccac agctccatcc cctccgggct

	2461	gaaaaccgtc tcctcaccct cccaccgggg
		tgacccccaa agctgctcac gaggagcccc

	2521	cacctcctcc aggagaagtt ccctgggacc
		cggtgtgaca cccagccgtc cctcctgccc

	2581	ctcccccgcc tggagatggc cggcgcccca
		tttcccaggg gtgaactcac aggacgggag

	2641	gggtcgctcc cctcacccgc ccggagggtc
		aaccagcccc tttgaccagg aggggggcgg

	2701	acctggggct ccgagtgcag ctgcaggcgg
		gcccccgggg gtggcggggc tggcggcagg

	2761	gtttatgctg gaggctgtgt cactgtgcgt
		gtttgctcgg tggagggacc cagctggcca

	2821	tccggggtga gtctcccctt tccagctttc
		cggagtcagg agtgacaaat gggtagattc

	2881	ttgtgttttt cttacccatc tggggctgag
		gtctccgtca ccctaggcct gtaaccctcc

	2941	cccttttagc ctgttccctc tgggcttctt
		cacgtttcct tgagggacag tttcactgtc

	3001	acccagcaaa gcccagagaa tatccagatg
		gggcaggcaa tatgggacgg caagctagtc

	3061	caccctctta ccttgggctc cccgcggcct
		ccggataatg tctgagctgc ctccctggat

	3121	gcttcacctt ctgagactgt gaggcaagaa
		accccctccc caaaagggag gagacccgac

	3181	cccagtgcag atgaacgtgc tgtgagggga
		ccctgggagt aagtggggtc tggcggggac

	3241	cgtgatcatt gcagactgat gccccaggca
		gggtgagagg tcatggccgc cgacaccagc

	3301	agctgcaggg agcacaggcc gggggcaagt
		catgcagaca ggacaggacg tgtgaccctg

	3361	aagagtcaga gtgacacgcg gggggggggc
		ccggagctcc cgagattagg gcttgggtcc

	3421	taacgggatc caggagggtc cacgggccca
		ccccagccct ctccctgcac ccaatcaact

	3481	tgcaataaaa cgtcctctat tgtcttacaa
		aaaccctgct ctctgctcat gtttttcctt

	3541	gccccgcatt taatcgtcaa cctctccagg
		attctggaac tggggtgggg nnnnnnnnnn

	3601	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnmnnnnnnn nnnnnnnnnn

	3661	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		agcttatgtg gtgggcaggg gggtagtaag

	3721	atcaaaagtg cttaaattaa taaagccggc
		atgatatacg agtttggata aaaaatagat

	3781	ggaaaagtaa gaaaggacag gaggggggtg
		aggcggaaga aagggggaag aaggaaaaaa

	3841	aaataagaga gaggaacaaa gaaagggagg
		ggggccggtg atgggggtgg gatagaatat

	3901	aataattgga gtaaagagta gcgggtggct
		gttaattccg ggggggaata gagaaaaaaa

	3961	aaaaaaaatg tgcgggtggg cggtaagtat
		ggagatttta taaatattat gtgtggaata

	4021	atgagcgggg gtggacgggc aaggcgagag
		taaaaagggg cgagagaaaa aaattaggat

	4081	ggaatatatg gggtaaattt taaatagagg
		gtgatatatg ttagattgag caagatataa

	4141	atatagatgg tgggggaaaa gagacaaggg
		tgagcgccaa aacgccctcc cgtatcattt

	4201	gccttccttc ctttaccacc tcgttcaaac
		tctttttcga gaaccctgaa gcggtcaggc

	4261	ccggggctgg gggtgggata cccggggagg
		ggctgcgcct cctcctttgc agagggggtc

	4321	gaggagtggg agctgaggca ggagactggc
		aggctggaga gatggctgtt gacttcctgc

	4381	ctgtttgaac tcacagtcac agtgccagac
		ccactgaatt gggctaaata ccatattttt

	4441	ctggggagag agtgtagagc gagcgactga
		ggcgagctca tgtcatctac agggccgcca

	4501	gctgcaggga ctttgtgtgt gtcgtgctcg
		ttgctcagtt gtgtccgact ctttatgact

	4561	tcatggactg taacctgcca ggctcctctg
		tccgtggaat tctccaggca agaatactgg

	4621	agtgggtagc cattctcatc tccgggggat
		cttcctgacc caagaatcaa acctgagtct

	4681	cccgcattgc aggcagcttc tttcttgtct
		gagccaccag ggaagcccct taagtggagg

	4741	atctaaatag agtgtttagg agtataagag
		aaaggaagga cgtctataca agatccttcg

	4801	gttcctgtaa ctacgactcg agttaacaag
		ccctgtgtga gtgagttgcc agtaattatt

	4861	gctaacctgt ttctttcact cactgagcca
		ggtatcctgt gagacggcat acttacctcc

	4921	tcttctgcat tcctcgggat ggagctgtgc
		ggtggcctct aggactacca catcgaccag

	4981	gtcagaccca gggacagagg attgctgaga
		tgcactgaga agtttgtcag cctaggtctt

	5041	cacccacaca gactgtgctg tcgtctacca
		cgtaattctt cctgtccaaa gaactggtta

	5101	aacgctcctg aagcgtattc tggtctgctt
		caaaaagtgc ctctttcctt tataagttcc

	5161	gccaatcctg gactttgtcc caggccagtc
		tactttattt gtgggaaagg tttttttggt

	5221	cttttttgtt ttaaactctg cagaaattgc
		ttacactttt ggtgtgcaat ggctcactct

	5281	tacggttcta gctgtattca aaggggttgc
		ttttctttgt ttttaaagct ttttgaacgt

	5341	ggaccatttt taaagtcttt attaaacgtc
		taacatcgtt tctggtttat tttctggtgg

	5401	tctggccatg aggcctacgg gtcttagctc
		ccctaccagg gtccaaccca catcccttgc

	5461	actggacggc aaggtcttaa cctttgaacc
		accagagagc ttctgaaagg ggctgctttt

	5521	ctccaatcct ctttgctccc tgcctgctgg
		tagggattca gcacccctgc aatagccctg

	5581	tctgttctta ggggctcagt agcctttctg
		cctgggtgtg gagctggggt tgtaagagag

	5641	cttcatggat ttggacacga cctacgactc
		agaggtaaga ctccatctta gcgctgtaat

	5701	gacctctttc caacaaccac ccccaccacc
		ctggaccact gatcaggaga gatgattctc

	5761	tctcttatca tcaacgtggt cagtcccaaa
		cttgcacccg gcctgtcata gatgtagcag

	5821	gtaagcaata aatatttgtt gaatgttaag
		tgaattgaaa taacataagt gaaaaagaaa

	5881	acacttaaaa acatgtgttt ttataattac
		acagtaaaca tataatcatt gtagaaaaaa

	5941	atcgaaagag tggcgggggc caagtgaaaa
		ccaccatccc tggtatgtcc acccgcccgg

	6001	gtagccccag gtaagaggtg cggacacgga
		tggccctgta gacacagaga cacacgctca

	6061	tatgctgggt cttgtcttgt gacctcttgg
		ggatgatgtt attttcacga tgccattcaa

	6121	accttctacc acaccatttt tagagggtcg
		ttcatcgtaa atcagttcac tgctttgttt

	6181	tctgattttg aaagtgtcac attcttcgag
		aaatgagaag gaacaggcgc gcataaggaa

	6241	gaaagtaaac acgtggcctt gcttccaggg
		ggcactcagc gtgttggtgt gcacgctggc

	6301	agtcttttct ctgtgacagt catggccttt
		tcccaaaggt gggctcagat aagaccgcct

	6361	cccatcccct gtccctgtcc ccgtccccta
		cggtggaacc cacccacggc acgtctccga

	6421	ggccctttgg ggctgtggac gttaggctgt
		gtggacatgc tgctggtggg gacccagggc

	6481	tgggcagcac gttgtccctg ggtcccgggc
		cagtgaggag ctcccaagga gcagggctgc

	6541	tgggccaaag ggcagtgcgt cccgaggcca
		tggacaaggg gatacatttc ctgctgaagg

	6601	gctggactgc gtctccctgg ggccccttgg
		agtcatgggc agtggggagg cctctgctca

	6661	ccccgttgcc cacccatggc tcagtctgca
		gccaggagcg cctggggctg ggacgccgag

	6721	gccggagccc ctccctgctg tgctgacggg
		ctcggtgacc ctgccgcccc ctccctgggg

	6781	ccctgctgac cgcgggggcc accccggcca
		gttctgagat tcccctgggg tccagccctc

	6841	caggatccca ggacccagga tggcaaggat
		gttgaggagg cagctagggg gcagcatcag

	6901	gcccagaccg gggctgggca ggggctgggc
		gcaggcgggt gggggggtct gcacnccccc

	6961	acctgcnagc tgdncnnncn tttgntnncg
		tcctccctgn tcctggtctg tcccgcccgg

	7021	ggggcccccc ctggtcttgt ttgttccccc
		tccccgtccc ttcccccctt tttccgtcct

	7081	cctcccttct tttattcgcc ccttgtggtc
		gttttttttc cgtccctctt ttgttttttt

	7141	gtctttttct ttttccccct cttctccctt
		gctctctttt tcattcgtcg gtttttctgc

	7201	tcccttccct ctcccccccg ctttttttcc
		ctgtctgctt tttgtgttct ccctctctac

	7261	cccccctgca gcctattttt tttatatatc
		catttccccc tagtatttgg cccccgctta

	7321	cttctcccta atttttattt tcctttcttt
		aactaaaatc accgtgtggt tataagtttt

	7381	aacctttttt gcaccgccca caatgcaatc
		ttcacgcacg ccccccccgt cagcctcctt

	7441	aaataccttt gcctactgcc cccctccttg
		tataataacg cgtcacgtgg tcaaccatta

	7501	tcacctctcc accaccttac cacattttcc
		ttcnnnnnnn nnnnnnnnnn nnnnnnnnnn

	7561	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	7621	nnnnnnnnnn nnntgaaaaa agaaaaggct
		gggcaggttt taatatgggg gggttggagt

	7681	ggaatgaaaa tgcattggag tggttgcaac
		aaatggaaag gtctcaggag cgctcctccc

	7741	ccatcaggag ctggaaagaa gtggaagcaa
		agcaaggaat tcgtgtgatg gccagaggtc

	7801	aggggcaggg agctgcaaag actgccggct
		gtttgtgact gnccgtctcc gggtgcattt

	7861	gttagcaggg aggcattaca ctcatgtctt
		ggtttgctaa ctaattctta ctattgttta

	7921	gttgcaaggt catgtctgac tctttgcaac
		ccagggactg cagcccgcca ggctcctctg

	7981	tccatgggat ttcgcaggca agaatactgg
		aggtggtagc cattttcttc accatgggat

	8041	cttcccgagc cagaaatgga acccgagtcg
		cctcctgtgc atggggtctg ctgcctaaca

	8101	ggcagatatt tgacgtctga gccaacaggg
		aggacagacg gtaattatac caaccattga

	8161	aagaggaatt acacactaat ctttatcaaa
		atctttcaaa cagtagagga gaaaggatac

	8221	tctctagttt attccataaa gttggaatta
		cgcttatcaa taaagacatt acaagaaaag

	8281	aaagtgaagc cccaaatgcc ttataaatat
		acaagaaaaa atcttttaag atattagcca

	8341	acttaatcaa caaaaaatgt atcaaaagtc
		caagtaacat tcaccccagg aatgcaagtg

	8401	tggttcagcc taagacaatc agtcatgagt
		ataccacgga aacaaattaa agagaaaaga

	8461	cattaaatct cacaaatggt gcagaaaaag
		atttggcaat atcgaacatc ttttcatgac

	8521	caaaggaaaa aaaagaaaca aaacaccaga
		aaattctgtg tagaaagaat atatctcaac

	8581	ccaatgaagg gcatttatga aaaacccaca
		gcatacatca cactccatga gaaagactga

	8641	aagctttccc cactgccatt gaactctgtc
		ctggaaattc tagtcacagc gacagaacaa

	8701	gagaaagaaa taacggccgt ctaaactggt
		aggaagaaat caaagcgtct ctattctctg

	8761	ggcgcataat acaatataga caaatttcta
		aagtccacaa aaattcctag agctcataat

	8821	gaatccagaa atgcgtcagg gctcaagatt
		cagatgcaaa aatcgtctgg gttttgatgc

	8881	accaacaaac aattccatta acaataatac
		caaggaatta atttaactta gaagagaaaa

	8941	gacctgttta cagagagtta taaaacattt
		ggtgatgaaa ttaaataaga gtaaatcata

	9001	tagaaacacc gttcgtgttt tggagaccta
		atgtcataaa cgtggcaaca cagagacgcc

	9061	tcacggggaa ccctgagcct ccttctccaa
		acaggcctgc tcatcatttc acaggtaacc

	9121	tgagacccta aagcttgact ctgaggcact
		ttgagggcat gaagagagca gtagctcctc

	9181	ccatgggacc gacagtcaag gcccagggaa
		tgaccacctg gacagatgac ttcccggcct

	9241	catcagcagt cggtgcagag tggccaccag
		ggggcagcag agagtcgctc aacactgcac

	9301	ctggagatga ggcaacctgg gcatcaggtg
		cccatgcagg ggctggatac ccacacctca

	9361	cacctgagga caggggccgg ctttctgtgg
		tgtcgccctc tcaggatgca cagactccac

	9421	cctcttcgct tgcattgaca gcctctgtcc
		ttcctggagg acaagctcca ccttccccat

	9481	ctctccccag ggggctgggg ccaacagtgt
		tctctcttgt ccactccagg aacacagagc

	9541	caagagattt atttgtctta attagaaaaa
		ctatttgtat tcctgcattt ccccagtaac

	9601	tgaaggcaac tttaaaaaat gtatttcctg
		gacttccctg gtgggccagt ggctagactc

	9661	tgagctccca gtgcatgggg cctgggttca
		atccctgctc aggaaactac atcccacagg

	9721	ctgcaaataa gatcctgcat gccacccgat
		gcaggcaaag aaacaagtgt tcggtatgca

	9781	tgtatttcac gtgaggtgtt tctataattt
		acagccagta ttctgtctta cacttagtca

	9841	ttcctttgag cacatgatcg gtcgatggcc
		cagaccacac acaggaatac tgaggcccag

	9901	cacccaccgg ctgcccagaa cctcatggcc
		aagggtggac acttacagga cctcagggga

	9961	cctttaagaa cgccccgtgc tcttggcagc
		ggagcagtgt taagcatggc tctgtccctc

	10021	gggagctgtg tctgggctgc gtgcatcacc
		tgtggtgtgg gcctggtgag ggtcaccgtc

	10081	caggggccct cgagggtcag aagaaccttc
		ccttaaaagt tctagaggtg gagctagaac

	10141	cagacccaca tgtgaactgc acccaaaaac
		agtgaaggat gagacacttc aaagtcctgg

	10201	gtgaaattaa gggccttccc ctgaaccagg
		atggagcaga ggaaggactt ggcttccagg

	10261	aaaccctgac gtctccaccg tgactctggc
		cggggtcatg gcagggccca ggatcctttg

	10321	gtgcaaagga ctcagggttc ctggaaaata
		cagtctccac ctctgagccc tcagtgagaa

	10381	gggcttctct cccaggagtg gggcaaggac
		ccagattggg gtggagctgt ccccccagac

	10441	cctgagacca gcaggtgcag gagcagcccc
		gggctgaggg gagtgtgagg gacgttcccc

	10501	ccgctctcaa ccgctgtagc cctgggctga
		gcctctccga ccacggctgc aggcagcccc

	10561	caccccaccc cccgaccctg gctcggactg
		atttgtatcc ccagcagcaa ggggataaga

	10621	caggcctggg aggagccctg cccagcctgg
		gtttggcgag cagactcagg gcgcctccac

	10681	catggcctgg accccctcct cctcggcctc
		ctggctcact gcacaggtga gccccagggt

	10741	ccacccaccc cagcccagaa ctcggggaca
		ggcctggccc tgactctgag ctcagtggga

	10801	tctgcccgtg agggcaggag gctcctgggg
		ctgctgcagg gtgggcagct ggaggggctg

	10861	aaatccccct ctgtgctcac tgctaggtca
		gccctgaggg ctgtgcctgc cagggaaagg

	10921	ggggtctcct ttactcagag actccatcca
		ccaggcacat gagccggggg tgctgagact

	10981	gacggggagg gtgtccctgg gggccagaga
		atctttggca cttaatctgc atcaggcagg

	11041	gggcttctgt tcctaggttc ttcacgtcca
		gctacctctc ctttcctctc ctgcaggcgc

	11101	tgtgtcctcc tacgagctga ctcagtcacc
		cccggcatcg atgtccccag gacagacggc

	11161	caggatcacg tgttgggggc ccagcgttgg
		aggtganaat gttgagtggc accagcagaa

	11221	gccaggccag gcctgtgcgc tggtctccta
		tggtgacgat aaccgaccca cgggggtccc

	11281	tgaccagttc tctggcgcca actcagggaa
		catggccacc ctgcccatca gcggggcccg

	11341	ggccaaggat gaggccgact attactgtca
		gctgtgggac agcagcagta acaatcctca

	11401	cagtgacaca ggcagacggg aagggagatg
		caaaccccct gcctggcccg cgcggcccag

	11461	cctcctcgga gcagctgcag gtcccgctga
		ggcccggtgc cctctgtgct cagggcctct

	11521	gttcatcttg ctgagcagcg gcaagtgggc
		attggttcca agtcctgggg gcatatcagc

	11581	acccttgagc cagagggtta ggggttaggg
		ttagggttag gctgtcctga gtcctaggac

	11641	agccgtgtcc cctgtccatg ctcagcttct
		ctcaggactg gtgggaagat tccagaacca

	11701	ggcaggaaac cgtcagtcgc ttgtggccgc
		tgagtcaggc agccattctg gtcagcctac

	11761	cggatcgtcc agcactgaga cccggggcct
		ccctggaggg caggaggtgg gactgcagcc

	11821	cggcccccac accgtcaccc caaaccctcg
		gagaaccgcg ctccccagga cgcctgcccc

	11881	tttgcaacct gacatccgaa cattttcatc
		agaacttctg caaaatattc acaccgctcc

	11941	tttatgcaca ttcctcagaa gctaaaagtt
		atcatggctt gctaaccact ctccttaaat

	12001	attcttctct aacgtccatc ttccctgctc
		cttagacgcg ttttcattcc acatgtctta

	12061	ctgcctttgg tctgctcgtg tattttcttt
		tttttttttt ttttattgga atatatttgc

	12121	gttacaatgt tgaatttgaa ttggtttctg
		ttgtacaaca atgtgaatta gttatacatg

	12181	tcctgaggag gggcggctgc gtgggtgcag
		gagggccgag aggagctact ccacgttcaa

	12241	ggtcaggagg ggcggccgtg aggagatacc
		cctcgtccaa ggtaagagaa acccaagtaa

	12301	gacggtaggt gttgcgagag ggcatcagag
		ggcagacaca ctgaaaccat aatcacagaa

	12361	actagccaat gtgatcacac ggaccacagc
		ctggtctaac tcagtgaaac taagccatgc

	12421	ccatggggcc aaccaagatg ggcgggtcat
		gtgcccatgg ggccaaccaa gatgggcggg

	12481	tcatggtgaa gaggtctgat ggaatgtggt
		ccactggaga agggaaaggc aaaccacttc

	12541	agtattcttg ccttgagagc cccatgaaca
		gtatgaaaag gcaaaatgat aggatactga

	12601	aagaggaact ccccaggtca gtaggtgccc
		aatatgctac tggagatcag tggagaaata

	12661	actccagaaa gaatgaaggg atggagccaa
		agcaaaaaca atacccagtt gtggatgtga

	12721	ctggtgatag aagcaagggc caatgatgta
		aagagcaata ttgcatagga acctggaatg

	12781	ttaagtccaa gannnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	12841	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnagaatttt

	12901	gagcattact ttactagcgt gtgagacgag
		tgcaattgtg cggtagtttg agcattcttt

	12961	ggcattgcct ttctttggga ttggaatgaa
		aactgacctg ttccaggcct gtggccactg

	13021	ctgagttttc caaatttgct ggcgtattga
		gtgcatcact ttaacagcat catcttttag

	13081	gatttgaaat agctcaactg gaattctatc
		actttagcta attccattca ttagctttgt

	13141	ttgtagtgat gcttcctaag gcccccctgg
		ctttatcttc ctggatgtct ggctctggtg

	13201	agtgatcaca ccgctgtgat tatctgggtc
		atgaaggtct ttttgtatag ttcttcttag

	13261	gaacagatat tatgatctcc atccttgcat
		ctcgttatat ctagagaagc actgactccc

	13321	ttcatggtga cgtcagatcc tcatgactaa
		caaatggcct tttgtaagat gagtgcctca

	13381	tggtattgag ctcccccgtc accaagacct
		tatgactgac ctcccccact gccccaggtg

	13441	cctctcgaag cgtctgagat gccgcctccc
		aggctgcact cctcattttg cccccaataa

	13501	aacttaactt gcagctctcc agctgtgcat
		ctgtgtttag ttgacagtac aaatataatg

	13561	gaaaatttaa attaaatata atctatgggg
		agaaatccaa acatcttatg agggagagag

	13621	agggagagaa aggaaagaag aagaagcagg
		aggaggagga gagtagagaa acagggggag

	13681	ggcggcaggg agacagaggg gaggacaccg
		aggggaaagg gaggaaggcg agtgcagtga

	13741	gagagaggcc agagttcatc agagtctgga
		ctcgcagccc aatcccacgg gtgtgtcccg

	13801	aagcagggga gagcctgagc caggcggaga
		cagagctgtg tctccagtcc tcgtggccgt

	13861	gacctggagc tgtgtggtca gcccccctga
		ccccagcctg gccctgctgg tggtcggagg

	13921	cagtgatcct ggacacagtg tctgagcgtc
		tgtctgaaat ccctgtggag gcgccactca

	13981	ggacggacct cgcctggccc cacctggatc
		tgcaggtcca ggcccgagtg gggcttcctg

	14041	cctggaactg agcagctgga ggggcgtctg
		caccccagca gtggagcggc cccaggggcg

	14101	ctcagagctg ccggggggac acagagcttg
		tctgagaccc agggctcgtc tccgaggggt

	14161	cccctaaggt gtcttctggc cagggtcaga
		gccgggatga gcacaggtct gagtcagact

	14221	ttcagagctg gtggctgcat ccctggggac
		agagggctgg gtcctaacct gggggtcaga

	14281	gggcaggacg ggagcccagc tgacccctgg
		ggactggcct cctctgtggt ctcccctggg

	14341	cagtcacagc ttccccggac gtggactctg
		aggaggacag ctggggcctg gctgtcagga

	14401	gggggttcga gaggccacac tcagaggagg
		agaccctggc ctgcttgggt tgtgactgag

	14461	tttttggggt cctctaggag actctggccc
		tgcaggccct gcaaggtcat ctctagtgga

	14521	gcaggactcc acaagattga tgaactgaat
		cctctaggag aggtgtggtt gtgagggggc

	14581	agcattctag aaccaacagc gtgtgcaggt
		agctggcacc gggtctagtg gcggcgggca

	14641	gggcactcag ggccgactag gggtctgggg
		gattcaatgg tgcccacagc actgggtctt

	14701	ccatcagaat cccagacttc acaaggcagt
		ttcggggatt aggtcaggac gtgagggcca

	14761	cagagaggtg gtgatggcct agacaagtcc
		ttcacagaga gagctccagg ggccatgata

	14821	agatggatgg gtctgtattg tcagtttccc
		cacatcaaca ccgtggtccc gccagcccat

	14881	aatgctctgt ggatgcccct gtgcagagcc
		tacctggagg cccgggaggc ggggccgcct

	14941	gggggctcag ctccggggta accgggccag
		gcctgtccct gctgtgtcca cagtcctccc

	15001	ggggttggag gagagtgtga gcaggacagg
		agggtttgtg tctcacttcc ctggctgtct

	15061	gtgtcactgg gaacattgta actgccactg
		gcccacgaca gacagtaata gtcggcttca

	15121	tcctcggcac ggaccccact gatggtcaag
		atggctgttt tgccggagct ggagccagag

	15181	aactggtcag ggatccctga gcgccgctta
		ctgtctttat aaatgaccag cttaggggcc

	15241	tggcccggct tctgctggta ccactgagta
		tattgttcat ccagcagctc ccccgagcag

	15301	gtgatcttgg ccgtctgtcc caaggccact
		gacactgaag tcaactgtgt cagttcatag

	15361	gagaccacgg agcctggaag agaggaggga
		gaggggatga gaaggaagga ctccttcccc

	15421	aagtgagaag ggcgcctccc ctgaggttgt
		gtctgggctg agctctgggt ttgaggcagg

	15481	ctcagtcctg agtgctgggg gaccagggcc
		ggggtgcagt gctggggggc cgcacctgtg

	15541	cagagagtga ggaggggcag caggagaggg
		gtccaggcca tggtggacgt gccccgagct

	15601	ctgcctctga gcccccagca gtgctgggct
		ctctgagacc ctttattccc tctcagagct

	15661	ttgcaggggc cagtgagggt ttgggtttat
		gcaaattcac cccccggggg cccctcactc

	15721	agaggcgggg tcaccacacc atcagccctg
		tctgtcccca gcttcctcct cggcttctca

	15781	cgtctgcaca tcagacttgt cctcagggac
		tgaggtcact gtcaccttcc ctgtgtctga

	15841	ccacatgacc actgtcccaa gcccccctgc
		ctgtggtcct gggctcccca gtggggcggt

	15901	cagcttggca gcgtcctggc cgtggactgc
		ggcatggtgt cctggggttc actgtgtatg

	15961	tgaccctcag aggtggtcac tagttctgag
		gggatggcct gtccagtcct gacttcctgc

	16021	caagcgctgc tccctggaca cctgtggacg
		cacagggctg gttcccctga agccccgctt

	16081	gggcagccca gcctctgacc tgctgctcct
		ggccgcgctc tgctgccccc tgctggctac

	16141	cccatgtgct gcctctagca gagctgtgat
		ttctcagcat aactgattac tgtctccagt

	16201	actttcatgt ccctgtgacg ggctgagtta
		gcatttctca cactagagaa ccacagtcct

	16261	cctgtgtaaa gtgatcacac tcctctctgt
		gggacttttg taaaagattc tgcagccagg

	16321	agtcatgggt ggtcttagct gagaaatgct
		ggatcagaga gacctgataa ccgatgtgaa

	16381	gaggggaacc tggaagatct tcagttcagt
		tcatttcagt cattcagttg tgtccgactg

	16441	tttgggatcc catggactgc cacacgccag
		tcctccctgt ccatcaccaa cttctgaagc

	16501	ttgttcaaac tcatgtccat caagttggag
		atgcctttca accatctcat cctctgtcat

	16561	ccccttctcc tcccgccttc aatcttccct
		agcattaggg tcttttccgt gagtcagttc

	16621	ttcgcatcag gtggccaagt tttggagttt
		cagtttcagc atcagtcctt tcaatgaata

	16681	gtaaggactg atttccttta ggatggactg
		gtttgatatc cttgcagttc aagggactct

	16741	caagagtctt ctccaacact gcagttaaaa
		gccatcaatt cttcggtgct cagctttctt

	16801	tttggtacaa ctctcacatt catacatgac
		taccgaaaat acattagtcg tgtagaacca

	16861	gtttggggct tcccacgtgg ctctagtggt
		aaagaatatg cctgccaact cagaagatgt

	16921	aagagatgcg gttcaatctc tgggtcggga
		agatcccctg gagaagggca tgacaaccca

	16981	ctccagtatt tttgcctgga gaatcccatg
		gacagagaag cctggtggac tgcagtccat

	17041	ggagtctcac agagtcagac acgactgaag
		caacttagct acttggaaaa gagcatgcac

	17101	gaagctgtct aaaaaacagg tcaagaagtc
		ttgtgttttg aaggtttact gagaaagttg

	17161	atgcactgct ccaacacttc ctctcagttg
		aaaagatcag aagcgttaga tcaaatggtg

	17221	gtcaatacct tggatgcgct ccaacaggtt
		atatctgcag atggaaatga aggcagttta

	17281	tggggtaact ggaggacaag atgagatcat
		acacttggaa cactgtctgg catcaaaggc

	17341	gtgtacagta aacattagct gttattagca
		aaataaattc agcttgaatc acccaaatca

	17401	gatggcattc ttaaagccac tgagtggtaa
		aatcaggggt gtgcagccaa aacgtccatt

	17461	ttgactcatt atgatttcca tgtcacaaga
		ctagaaagtc actttctcct cagcagaaga

	17521	gaaggtagaa cattttaacc tttttttgga
		gtgtcaaggg aattttgttt acactgtaaa

	17581	gtcagtgaaa atattgaagc ttttcatttg
		tggaaaatat taaatatgta aaattgaaat

	17641	tttaaaattt attcctgggt agttttgttt
		ttccagtagt catgcatgga tgtgagagtt

	17701	ggactataaa gaaagctgag cgctgaagaa
		ttaatgcttt tgaactgtgg cactggagaa

	17761	gactcttgag agtcccttgg tctgcaagga
		gatcaaacca gtccatccta aaggaaatca

	17821	gtcctgaata ttcactggaa ggactgatgc
		tgaagctgaa actccaatac tttggccacc

	17881	tgatgtgaag aactgactca tatgaaaaga
		ctcagatgct gggaaagatt gaaggtggga

	17941	ggagaagggg acgacagagg atgagatggc
		tgaatggcat caccgactcg atggacatga

	18001	gtctgaataa gctctgggag ttgttgatgg
		acagggaggc cctggagtgc tgcagtccat

	18061	gggattgcaa agagttggac atgactgagt
		gactgaactg aactgagttt ggtaacagat

	18121	atgagaatta tataatttaa atctaaactc
		ttggtatttc tttctttggc ggttccaaaa

	18181	gagctgtccc ttctgttaac tatataaatc
		ctttttgaga attactaaat tgataatgtt

	18241	cacaagttat ccaatttctc attactctta
		gttgtcagta taagaaatcc catttgattt

	18301	atcatgttat agtatctgca actctaatag
		ttcagttctg acaaattttt attttattta

	18361	aaaatattgg catacagtaa aatttcaaac
		aatatacaat tctccctttc agtttaaaaa

	18421	acaaaacaaa acaaaagtaa tattagttaa
		aaaaatccgg gaagaatcca agcatttaaa

	18481	attgcatcac atttctatgc tagacaagct
		gatataaagt tataattaat aaaggattgg

	18541	actattaaac tctttacata tgaggtaaca
		tggctctcta gcaaaacatt taaaaatatg

	18601	ttgtgggtaa attattgttg tccttaaaga
		aataaaaaga cataagcgta agcaattggn

	18661	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	18721	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnna aaatggataa ggggggagga

	18781	catgggtagg ggagcgcgat ggaggaagta
		aggtggtcga gggagttggg gggggaataa

	18841	gtgggtaaaa gggaagcggg cggaaggagg
		gggaagcagg agagaggggt gggcgtcaga

	18901	tcggggggag gggtatgagg gagagggaat
		ggtagacggg gggtgggaag cataaaggaa

	18961	aagatagggg ggggaaaagt tagaagaaga
		atgaggggat aggcggaaag ggaagagaaa

	19021	tgggagaaga acagaaaaat agggggaggg
		ggggcgtaaa gagggggggg gagggcaggt

	19081	gtggagatga cagatacggg gaatgccccg
		gtataaaaga gtatatggcg tggggcgaga

	19141	aggctgtcat cctgtgggag gggggacgcg
		gagaaccctt cgggctatag ggaggattcg

	19201	gggggatcgt tcgggaaggc agtcagcaca
		gcacccacca agggtgcagg gatggatctg

	19261	gggtcccaaa gaagaggccc aatcccgcgt
		cttggcagca aggagccctg gagactggga

	19321	agtgtccagg acactgaccc aggggttcga
		ggaacccaga agtgtgtctg tgaagatgtg

	19381	ttttgtgggg ggacaggtcc agagctttga
		gcagaaaagc ggccatggcc tgtggagggc

	19441	caaccacgct gatctttttt aaaaggtttt
		tgttttgatg tggaccattt ttaaagtctt

	19501	cattgaattt gctacaatat tgtttctggt
		ttatgctctg gtttcttcgg ctgcaaggtt

	19561	tgtgtgatcg tatctcctca accaggactg
		aacccacagc ccctgcactg gaaggcgaag

	19621	tcttaaccca gatcgccagg aacgtccctc
		ccctcactga tctaatccaa gaccctcatt

	19681	aaggaaaaac cgagattcaa agctccccca
		ggaggactcg gtggggagga gagagccaag

	19741	cactcagcac tcagtccagc acggcgccct
		ccctgtccag ggcgagggct cggccgaagg

	19801	accaccggag accctgtcgg attcaccagt
		aggattgtga ggaatttcaa cttacttttt

	19861	aaatctgtct ctcaaggctg ttacaagcgg
		actttaccag taacttaaaa gttgaaaggg

	19921	acttcccagg cggcacttgc ggtgaagaac
		ccgccggctg gttttaggag acataagaga

	19981	tgtgggttag atccctggtt caggaggatt
		cccctggaga aggaaatggc aacccactcc

	20041	agtattcttg cctggaaagc ctcacggaca
		gaggaggctg gcgggctaca gtccacgggg

	20101	tcgcacacga ctgaatcgac ttagcttcaa
		gttgagacag gaagaggcag tgactggtgg

	20161	caaaacaccg cacccatgct cccaggggac
		ctgcagcgct ctggttcatg agctgtgcta

	20221	acaaaaatca acccaacgag aggcccagac
		agagggaagc tgagttcatc aaacacgggc

	20281	atgatgtgga ggagataatc caggaaggga
		cctgccaagc ccatgacaga ccggtgtcct

	20341	gtctgagggc cgtcctggca gagcagtgca
		gggccctccg agaccgcccg agctccagac

	20401	ccggctgggg gctacagggt ggggctgagc
		tgcaaggact ctgctgtgag ccccacgtca

	20461	gggaggatca ccttgtttgt tttctgagtt
		tctcttaaaa tagcctttat gggtcctggt

	20521	ctttggtttt aaaataacaa ctgttctccg
		taaacaacgt gaaaaaaaac aaacaggagg

	20581	aaaacaacgc agcccgggca tttcacccgg
		aagagccgcc tctaacactt tgacgggttg

	20641	ccttctattt taaccctgtt ttcattgtaa
		actgtaaaaa ccacatcata aataaattaa

	20701	aggtctctgt gaagtttaaa aagtaagcat
		ggcggtggcg atggctgtgc cacaccgtga

	20761	acgctcgttt caaaacggta aattctaggg
		accccctggt ggtccagtgg gtgagatttt

	20821	gcttccattg caggagccgt gggtttgatc
		cctggttggg gaactaagat cccacatgct

	20881	gtatggagtg gccaaaaaga attttttgta
		aatggtgagt tttaggtgac gtgaatttcc

	20941	cattgatgca cttcacaggc tcagatgcag
		ccaggccctc aggaagcccg agtccaccgg

	21001	tcctttactt ttccttagag ttttatggct
		tctgtttctg cccttaaacc caccatgttt

	21061	caacctcatc tgattttgga ctttataata
		aagttaggct gtgtttcagg aaactttgct

	21121	cagtattctg taataatcta aatggaaaga
		atttgaaaaa agagcagaca cttgtacatg

	21181	cataactgaa tcactttggt gtacacctga
		aactcgagtg cagccgctca gtcgtgtccg

	21241	accctgcgac cccacggact gcagcacgcg
		ggcttccctg cccatcacca actcccggag

	21301	ttcactcaaa cacatgtccg tcgactcggt
		gatgccgtcc aaccgtctca tcctctgtcg

	21361	tccccttctc ctcccgcctt caatcttttc
		cagcatcagg gtcttttcaa atgagtcagt

	21421	tcttcacacc aggtggccag agtattggag
		tttcagcttc agcatcagcc cttccaacga

	21481	ccccccatac ctgaagctaa cacagtgcta
		atccactgtg ctgcaacatg aaagaaaaac

	21541	acatttttta agtttaggct gtgtgtgtct
		tccttctctc aacactgcgt ctgaccccac

	21601	ccacactgcc cagcactgca ttccccgtgg
		acaggaggcc ccctgcccca cagctgcgtg

	21661	ccggccggtc actgccgagc agacctgccc
		gcccagagtg gggcccctgg cactggggac

	21721	aaggcagggg cctctccagg gccggtcact
		gtccactgtt cctactggtt ttgttttcaa

	21781	aagtggaggc agcgtaatat ttccctgatt
		ataaaaagaa gtacacaggt tctccacaaa

	21841	taaaacaggg gaaaagtata aagaatggaa
		gttcccagca cagcctggag atcacgccgg

	21901	gtgcacctgg ggtgtccttc caggctggac
		ctcacatttc acgcagacat cagaaggctg

	21961	cgagatctac ccagaaggct gggtagatgg
		gggataggtc agtgacaaac agtagacaga

	22021	gagatataca gacagatgat ggatagacag
		acgctaagac accgagcgag gggacagacg

	22081	gatggaagac accatccttt gtcactgacc
		acacacccac atgggtgtgg tgagccggct

	22141	gtcatacttg tgaacctgct gctctcacaa
		caccagctgg gtccctccag ccccagcgtc

	22201	ccacacagca gactcccggc tccatcccca
		ggcaggaatc ccaccaccaa ctggggtgga

	22261	ccctccccgc aggaaggtcg tgctgtctaa
		ggccttgaga gcaagttaca gacctacttc

	22321	tgggaagaca gcgcacaacc gcctaccccg
		cagagcccag gaggacccct gagtcctagg

	22381	gaagggacca cgcggcctgg acggggagcg
		gccccaggac gctgccccca acctgtccca

	22441	cctcactcct gctctgctct gaggcggggc
		gcagagaggg gccctgaggc ctcttcccag

	22501	ttcttgggag cacccactgg gcctgaacca
		ggccagaagc cccctcctca aggtgtcccc

	22561	agaccactcc cctccacctc cggttgctct
		gtctcctggc agcagggagc cccagtgaga

	22621	agagacagct ccaggctgtg atcttggccc
		ctggctgctc tggcagtgtg gggggtgggg

	22681	gtcgctggga ggccatgagt gctgggggtc
		ggggctgtga aagcacctcg aggtcagtgg

	22741	gctgttggtc gggctctgcg aggtccgcac
		gggtagagct gtgccaggac acaggaggcc

	22801	tggtcagtgg tcccaagagt cagggccaaa
		ggaaggggtt cgggcccctc tggttcctca

	22861	gcttctgagg ccggggaccc cagtctggcc
		ttggtagggg ggcgattgga gggtacaacg

	22921	atccaaaaga aaacacacat ctacgaggga
		agagtcctga ggaggagaga gctacacaga

	22981	gggtctgcac actgcggaca ctgcttggag
		tctgagagct cgagtgcggg gcacagtgag

	23041	cgaagggagg acggaacctc caaggacacc
		ggacgccgat ggccagagac acacgcacgt

	23101	cccatgaggg ccggctgctc agacgcaggg
		gagctcctca ttaaggcctc tcgctgaata

	23161	gtgaggagaa ctggccccgt gtgtggggaa
		acttagccca gaagaaacgc tgccctggcc

	23221	ccaaggatca nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	23281	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn tgccctttgc

	23341	ctccagggag ggaggaagcg tggatcttgg
		gtttgccttg ggtttaaagg atccacccac

	23401	tcccttttta gccactccct gtgctggcaa
		tttcttaaga ctggaggtcg caaagagttg

	23461	gacacactga gcgagtgaac tgcactgagc
		ctaagaaaag tctttgaatt cctccaaaca

	23521	aaacacactt gtcttgggta ctttccttgg
		ttttgttaca aatgtctggt ccctctgttc

	23581	tcctggccag ctcctgggtg tcattttgac
		ctgacgaagt caaagggagc ctggaccctc

	23641	aaaatctgta ggacccagca cccctccatt
		acacctctgt tcccccgcga acgggcacgt

	23701	gtttcgccgt ctggcgtaat gtgtaagcga
		cggtgtgata ctcgggagtc ttactctgtt

	23761	tctttttctt ctggggtgac accaccatcc
		gcacgactct gtctgaatgt gaacatttgg

	23821	gtgatttgat gtggcccaga ctcccccaac
		gaatgtacct tcaggttggt tttcttcttt

	23881	tatattttgc ttttgtgaat agacacagga
		tcccatcagt tgtatgtagt gagaaagtaa

	23941	aaacccactc agccttagct ggatggagat
		ctagtagtaa gatagcacgt tagccggaaa

	24001	tggaaatttc agccagaatc tgaaaagcgt
		gtcctggaag gagaagaggg actcaggccc

	24061	gagcacactg ctccacgctg gagcctcagg
		ctctgacagc tgtacctgcc ggggtcttca

	24121	tgggacaggc catgcaggcc acgatcccgt
		tgagaagttt cttgcctttc catcacattg

	24181	gcaattgcac gctttgctct tgcttctaca
		tggagtttta cttttatccc agacagtttg

	24241	gtttcttctc tgattttcgc caattgtaca
		gatcgttaca gtatttctta accacataga

	24301	attcggcagg gggggtgggg ggacagggta
		gggtggggtg agagtgaggg gagggggctg

	24361	caccgagcag catctggggt cgtagctccc
		tgacggggat agacctcgtg cccctgcagt

	24421	gacagcacag agtcctcctc tctgaactgc
		cagggacgct cctgcaattg acttaatgaa

	24481	aggcatctaa ttaggaattt tggggtgaca
		ttttacattt aagtgtgtga gcagtgatta

	24541	tagttcatat cattttatag tttcgtgatt
		ttactagctt aaagggtttt tggggtttct

	24601	ttttgtttta aaagctaaaa tctgtttttt
		aattccatgg aatacaaaaa aaaaaagtct

	24661	gtagaatatt ttaaagagtg aaggctttgt
		tcggaatgtg agcgctttgc tccactgaac

	24721	cgaacggtaa taacatttgt agaagagacg
		cagagtgaaa ggtacctctt tttattgagt

	24781	gacatgacag cacccatcgc gtgagttatt
		ggctggagtt tagagacagg ccatgttggg

	24841	ctaaactcct tattgctgtt ctcagccttt
		gagtaataat cagaagcttt ctctgaagag

	24901	agtggggtca gctgtcagac tcctaggtgt
		ctacctgcag cagggctggg attaaatgca

	24961	gcagccagta gatacgggat ggggcaagag
		gtcaccttgt ccctttgttg ctgctgggag

	25021	agaggcttgt cctggtgcca gtggggccaa
		agctgtgact ttgtgaccac aggatgtctc

	25081	tgaccctgcc ttgggttccc tgagggtgga
		gggacagcag ggtctccccg gttccttggc

	25141	cggagaagga ccccccaccc cttgctctct
		gacatccccc caggacttgc cccggagtag

	25201	gttcttcagg atgggcatcc gggccccacc
		ctgactcctg gagctggccg gctagagctt

	25261	gctgcagaat gaggccttgg ccattgcggc
		cctgaaggag ctgcccgtca agctcttccc

	25321	gaggctgttt acggcggcct ttgccaggag
		gcacacccat gccgtgaagg cgatggtgca

	25381	ggcctggccc ttcccctacc tcccgatggg
		ggccctgatg aaggactacc agcctcatct

	25441	ggagaccttc caggctgtac ttgatggcct
		ggacctcctg cttgctgagg aggtccgccg

	25501	taggtaaggt cgacctggca gactggtggg
		gcctggggtg tgagcaagat gcagccaggc

	25561	caggaagatg aggggtcacc tgggaacagg
		cgttgggtgt acaggactgg ttgaggctca

	25621	gaggggacaa aaggcacgtg ggcctccccc
		ccagtgtccc ttaaagtggg aaccaagggg

	25681	gccccggaag ccggaggagc tgtggtgtgt
		ggagtgcaga gccctcgcgg ggtcctgatg

	25741	cccgtcggac tctgcacagc tcagcgtgtg
		ccccgcggcc cggtaggcgg tggaagctgc

	25801	aggtgctgga cttgcgccgg aacgcccacc
		agggacttct ggaccttgtg gtccggcatc

	25861	aaggccagcg tgtgctcact gctggagccc
		gagtcagccc agcccatgca gaagaggagc

	25921	agggtagagg gttccagggg tgggggctga
		agcctgtgcc gggccctttg gaggtgctgg

	25981	tcgacctgtg cctcaaggag gacacgctgg
		acgagaccct ctgctacctg ctgaagaagg

	26041	ccaagcagag gaggagcctg ctgcacctgc
		gctgccagaa gctgaggatc ttcgccatgc

	26101	ccatgcagag catcaggagg atcctgaggc
		tggtgcagct ggactccatc caggacctgg

	26161	aggtgaactg cacctggaag ctggctgggc
		cggatgggca acctgcgcgg ctgctgctgt

	26221	cgtgcatgcg cctgttgccg cgcaccgccc
		ccgaccggga ggagcactgc gttggccagc

	26281	tcaccgccca gttcctgagc ctgccccacc
		tgcaggagct ctacctggac tccatctcct

	26341	tcctcaaggg cccgctgcac caggtgctca
		ggtgaggcgt ggcgccagct ccaaagacca

	26401	gagcaggcct ctcttgtttc gtgcccgctg
		gggacattgc cagggtgccc ggccactcgg

	26461	aagtcctcac gatgccaccg ctctgaccct
		gggcatcttg tcaggtcact tccctggtta

	26521	gggtcagagg cgtggcctag gttaaatgct
		gtcaaagggg actcctttct gggagtccgc

	26581	atagtggggg cttggtgtga tgcccttggg
		aattctttcc gagagagtga tgtcttagct

	26641	gagataatga cagataacta agcgagaagg
		acggtccatc aggtgtgagg tttgaagtcc

	26701	aaagctctgt ctctccctcc cacctgcccc
		ttctgtcctg agctgtttta ggctccaggt

	26761	gagctgtggg aagtgggtga ttctggagat
		gacaagaagg gatcaggagg ggaaaattgt

	26821	ggctcctaag cagtccagag aagagaaaaa
		gtcaaataag cattattgtt aaagtggctc

	26881	cagtctcttt aagtccaaat tataattata
		attttcctct aagacttctg aatacatagg

	26941	aaatcctcag taacaggtta ttgctctgcc
		ttgaacacag tgataaaagc tgggaggatg

	27001	cagcctaatc tgtctgtgtg aatgagttgt
		attgattccc tttttggcag ctgcaaactc

	27061	caagcattag gaataaatat gttcactgag
		aaccccgaag aaagaaagaa agaaaaaaaa

	27121	aaagaattgt aggtgttgat ggacggtttg
		tggcccctga atatctgggg gatgttcacc

	27181	cagggatcac gtgtaactgc tgggaccccc
		agccccatgt ccactgcatc cagcctgctg

	27241	ttgaattccg cggatcnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	27301	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnncaat

	27361	tcgagctcgg taccccaaag gtccgtctag
		tcaaggctat ggtttttcca gtggtcatgt

	27421	atggatgtga gagttggact gtgaagaaag
		ctgagtgcca aagaattatt cttttgtact

	27481	gggtgttgga gaagactctt gagagtccct
		tgaactgcaa ggagatccaa ccagtccgtt

	27541	ctaaaggaga tcagtcctga atgttcattg
		gaaggactga tgctgaagct gaaactccaa

	27601	tactttggcc acctgacgtg aagagttgac
		tcattggaaa agaccatgat gctgagagga

	27661	attgggggca ggaggagaag gggacgacag
		aggatgagat ggctggatgg catcaccaac

	27721	tcgatgngac atgagtttgg ttaaactcca
		ggagttggtg atggacttgg aggcctggtg

	27781	tgctgggatt catggggtcg cagagtcgga
		catgactgag cgactgaact gaactgaact

	27841	gagctgaaga gctcacctgt accagagctc
		ctcaggtcct cctgcaggcc tggctgtaat

	27901	ggcccccagg tcaccgtcct gcctccttca
		tcccatcctt tcacgacagg ctgggagtgg

	27961	ggtgaggtga gttgtcttgt atctagaatt
		tctgcatgcg accctcagag tgcaatttag

	28021	ctccagagaa ctgagctcca agagttcatt
		ttttcctttt cttctttatg atactaccct

	28081	cttctgagca gagacctcat gtcagggaga
		aggggactct gccttcctca gccttttgtt

	28141	cctccaagac ccacacgggg agggtcgcct
		gcttcactga gccggaaggt tcaattgctc

	28201	atgtcctcca gaaacacccc cccccccaga
		gacccccaga aataagtgga acagcacctt

	28261	gtttcccaga caagtgggac acacgttatg
		aaccacctca gtgattaaaa tagtaacctc

	28321	tgtgtatgtg tatttactgg agaaggaaac
		ggcaacctac tccactattc ctgcctagaa

	28381	aattccatgg gagagaagcc aggcaggcta
		cagtccacgg ggtcacagag actgaacata

	28441	cacaagcaca tggaagtgta ttttgcagta
		tttttaaatt tgttcagttc aacatggagt

	28501	acaagaattc aaatcgtgaa gtcaattgac
		caagaaacca gaagaaatca ctgtgttgtg

	28561	atctctgtgg aggtaacatg ggtacctgtg
		ctctgaccct cacagcctct ggctctctct

	28621	ctacatgtac atacacatat atttccatgt
		atgtatgtat tcggaagatt tcacatacgt

	28681	ctcaccagtc cacagccccc gcgttccctg
		atgcccagaa catctgtgat agctgtgagt

	28741	attgtcacca gataagatct tccaggttcc
		tgcactcaca ttggttatca ggtctctctg

	28801	atccagcatt tctcagctaa gattccttgt
		gactcctggc tgcagaatct tctgcaaaag

	28861	tcccacagag aggagtgtga tcactgtaca
		caggagggcc gtggttctct agtgtgagaa

	28921	aagctaactc agcccgtcac agggacgtga
		atgtacctga gacagtaatc agttatgctg

	28981	agaaatcaca gctctgctag aggcagcaca
		tggggtagcc agcagggggc agcagagcac

	29041	ggccaggagc cgcaggtcag aggctgggct
		gcccaagcgg ggcttcaggg gaaccagccc

	29101	tgcgggtcca caggtgtcca gggagcagcg
		cttggcagga agtcaggacc ggacaggcca

	29161	tcccctcagg actagtgacc acctctgagg
		gtcacatcca cagtgaaccc cagagcacca

	29221	tgcctcagtc cacggccagg acgctgccag
		gctgaccgcc ccactgggga gtccagggga

	29281	gaccacaggc cggggggctt gggacagtga
		tcatgtggtc agacacagag aaggtgacag

	29341	tgacctcagt ccctgaggac aagtctgatg
		tgcagacgtg agaagccgag gaggaagctg

	29401	gggacagaca gggctgatgg tgtggtgacc
		ccgcctctca gtgaggggcc cccgggggtg

	29461	aatttgcata aacccaagcc ctcactgccc
		ccacaaagct ctgagaggga ataaaggggc

	29521	tcggagagcc cagcactgct gcgggctcag
		aggcagagct cggggcgcgt ccaccatggc

	29581	ctgggcccct ctcgtactgc ccctcctcac
		tctctgcgca ggtgcggccc cccagcctcg

	29641	gtccccaagt gaccaggcct caggctggcc
		tgtcagctca gcacaggggc tgctgcaggg

	29701	aatcggggcc gctgggagga gacgctcttc
		ccacactccc cttcctctcc tctcttctag

	29761	gtcacctggc ttcttctcag ctgactcagc
		cgcctgcggt gtccgtgtcc ttgggacaga

	29821	cggccagcat cacctgccag ggagacgact
		tagaaagcta ttatgctcac tggtaccagc

	29881	agaagccaag ccaggccccc tgtgctggtc
		atttatgagt ctagtgagag accctcaggg

	29941	atccctgacc ggttctctgg ctccagctca
		gggaacacgg ccaccctgac catcagcggg

	30001	gcccagactg aggacgaggc cgactattac
		tgtcagtcat atgacagcag cggtgatcct

	30061	cacagtgaca cagacagacg gggaagtgag
		acacaaacct tccagtcctg ctcacgctct

	30121	cctccagccc cgggaggact gtgggcacag
		cagggacagg cctggcccgg ttcccccgga

	30181	gctgagcccc caggcggccc cgcctcccgg
		ccctccaggc aggctctgca caggggcgtt

	30241	agcagtggac gatgggctgg caggccctgc
		tgtgtcgggg tctgggctgt ggagtgacct

	30301	ggagaacgga ggcctggatg aggactaaca
		gagggacaga gactcagtgc taatggcccc

	30361	tgggtgtcca tgtgatgctg gctggaccct
		cagcagccaa aatctcctgg attgacccca

	30421	gaacttccca gatccagatc cacgtggctt
		tagaaaggct taggaggtga acaagtgggg

	30481	tgagggctac catggtgacc tggaccagaa
		ctcctgagac ccatggcacc ccactccagt

	30541	actcttccct ggaaaatccc atggacggag
		gagcctggaa ggcttcagcc catggggtcg

	30601	ctaagagtca gacacgactg agcgacgtca
		ctttcccttt tcactttcat gcattggaga

	30661	aggaaatggc aacccagtcc agtgttcctg
		cctggaaaat cccagggaca ggggagcctg

	30721	gtgggctgcc atccatgggg ccacacagag
		tcagacacga ctgaagcaac ttagcagcag

	30781	cagcagcagc ccaataaaac tcagcttaag
		taatggcatc taaatggacc ctattgccaa

	30841	ataaggtcca ctcgcgtgca ctctgtttag
		gacttcagtt cctgattgtg gagggttccc

	30901	acaagacgtg tgtgtatatt ggtgttgccg
		gaaaacagtg tcaatgtgag catcccagac

	30961	tcatcaccct cctactccca ctattccatt
		gtctctgcag gtattaagca taaaggttaa

	31021	gggtcttatt agatggaaga ggagtgaata
		ctcgtctgtg cttaacacat accaagtacc

	31081	atcaaggtcc ttcctattta ttaacgtgtg
		ttttaatcag aaatatgcta tgtagaagca

	31141	tccggacgat agcccatgtt acagacgggg
		aagctgaggc atgaagttct cagcaccttg

	31201	tttcacgtca gacctgaaac ggggcagagc
		cggcagcaaa caaggttcct cttcccaagc

	31261	gcccgctctt cacccgcttc ctatggcttc
		tcactgtgct tcctaaacta agctctcccc

	31321	aaccctgtgg agacaggatt agagacttta
		ggagaaaaga ccaggaacat cccacacccg

	31381	acccgagtga gccactaaga caaggctttg
		taaggacaga accagcaggt gtcctcagcg

	31441	agccagggag agacctcgca ccaaaaacaa
		tattgtagca tcctgaccct ggacttctga

	31501	cctccagaaa tgtgaaaaag aaacgtgtgg
		ggtttaatca actcaccggt gttatttggt

	31561	tatgactgcc tgagttaaga aggagttggg
		aacacttgag tgtaggtgtt tatggaacat

	31621	aagtcttgtt tctctgaaat aaattcccaa
		gggtataatt cctaggttgt agggtaactg

	31681	ccacaaatct aggcagctta ttaaaaaaca
		aagatatcac tttgccagca aaggttcata

	31741	tagtcaaatt atggttttta tagtagtcat
		gtatggatgt aaaagttgga tcataaagaa

	31801	ggctgagcac cagagaattg atcccttcaa
		atcgtggtgc tggagaagac tcttgagagt

	31861	cccttggaca gcaaggagat ccaaccagtc
		aatcctaaag gaaatgaact gtgaatattc

	31921	actggaagga ctgatgctga agctgaagat
		ccaatacttt ggccacctga tgcgaagagt

	31981	tgactcattg gaaaagaccc tgatgctgga
		aagcttgagg gcaggaggag aagagggcgg

	32041	cagaggatga gacggttgga tggcatcact
		gactcaatgg acatgagttt gagccaactc

	32101	tgggagacag tgaaggatag ggaaggctgg
		cgtggtacag tgcatgcggt cacaaagagt

	32161	ctgacacatc ttagtgactc aacaacgaca
		gcaacacagg catcacacgc ttagtgtgat

	32221	aagcggcaga actgttttcc aggggtccgn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	32281	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	32341	nnnnnnnnng tacgattcga gctcggaccc
		tgacattgtg agtcacgtca tgagcagctg

	32401	ttttccggtc ttcagggatt gtggacgatt
		tctgtttggg tttgctcatg ataatttagt

	32461	tacagcttag gttctttctt tccaggccac
		gagcgacatg ttttcaggtg agatgacgtg

	32521	gtgggggatg ggcggccaag cccccactgg
		ggggggaggg attctgttgt gggcaggagt

	32581	tggcagcatc cctgaactga tgacctgcga
		tccaggtgac aagaaccggg ggatattatt

	32641	cctctgcctt ctcatgtcat gtcctcggtt
		cttcatgatg aaaacatatg acaatacagg

	32701	ggagttagat ttgggcgggc acaactctgg
		gtgggggacc cggtggcatt gtgcccagca

	32761	gggccatcaa gatgagggcg acctgggtgg
		tccccttctc ccctggggtc ttagttttcc

	32821	cctcatggaa atgggatcag gcagcagcca
		tggaacaccg cgaccgtggc ttctctcacc

	32881	tcctcgtctg tgattttggg tcgggatacc
		aggcatgaag acctggggcg gggggacatc

	32941	actcctctgc agcagggagg ccgcagagtc
		ctccgtccat gaggacttcg tccctgggct

	33001	gaccctgcgg actgctggag gctgaagctg
		gaggcacagg cgggctgcga ggccagggtc

	33061	ctgaggacga cagagccagt ggggctgcag
		ctctgagcag atggcccctc gccccgggcc

	33121	ctgagcttgt gtgtccagct gcaggttcgc
		tcaggtgagc cactacgtta tgggggaggc

	33181	gccctgggca gggatcgggg gtgctgactc
		ctccgagatt ccgaccttct gggagcactc

	33241	tggccacact ctaagcctgg caagagctgg
		gttcatcagt ctaactctcc tcctgaagtc

	33301	caatggactc tctccatgcg gcagtcactg
		gatggcctct ttatccccga tggtgtcctt

	33361	ttccgctgac ctggctctcc tgaccacctc
		ccagcccccc accatacagg aagatggcac

	33421	ctggtccctg cagagctaag tccacccctg
		gcctggcttc agatgcctac agtcctcctg

	33481	cgggaggccc cgctccccac taggccccaa
		gcctgccgtg tgagtctcag tctcacctgg

	33541	aaccctcctc atttctcccc agtcctcagc
		tcccaacccc agaggtatcc cctgcccctt

	33601	tcaaggccct tgtcccttcc tggggggatg
		gggtgtatgg gagggcaagc ctgatccccc

	33661	gagcctgtgc cgctgacaat gtccgtctct
		ggatcatcgc tcccctggct ctcagagctc

	33721	cctggtccct ggggatgggt tgcggtgatg
		acaagtggat ggactctcag gtcacacctg

	33781	tcccttccct aaggaactga cccttaaccc
		cgacactcgg ccagacccag aaagcacttc

	33841	agacatgtcg gctgataaat gagaaggtct
		ttattcagga gaaacaggaa cagggaggga

	33901	ggagaggccc ctggtgtgag gcgacctggg
		taggggctca ggggtccatg gagaggtggg

	33961	ggagggggtg tgggccagag ggcccccgag
		ggtgggggtc cagggcccta agaacacgct

	34021	gaggtcttca ctgtcttcgt cacggtgctc
		ccctcgtgcg tgacctcgca gctgtaactg

	34081	cctttcgatt tccagtcgct gcccgtcagg
		ctcagtagct gctggccgcg tatttgctgt

	34141	tgctctgttt ggaggcccgg gtggtctcca
		cgttgcgggt gatggtgctg ccgtctgcct

	34201	tccaggccac ggtcacgcta cccgggtaga
		agtcgctgat gagacacacc agggtggcct

	34261	tgttggcgct gagctcctcg gtggggggcg
		ggaacagggt gaccgagggt gcggacttgg

	34321	gctgacccgt gtggacagag gagagggtgt
		aagacgccgg ggaggttctg accttgtccc

	34381	cacggtagcc ctgtttgcct tctctgtgcc
		ctccgaccct tgccctcagc ccctgggcgg

	34441	cagacagccc ctcagaagcc attgcaatcc
		actctccaag tgaccagcca aacgtggcct

	34501	cagagtcccc ggctgcgacc agggctgctc
		tcctccgtcc tcctggcccc gggagtctgt

	34561	gtctgctctt ggcactgacc ccttgagccc
		tcagcccctg ccagacccct ccgtgacctt

	34621	ccgctcatgc agcccaggtg cctcctccgt
		gaacccgggt ccccccgccc acctgccagg

	34681	acggtcctga tgggagatgt ggggacaagc
		gtgctagggt catgtgcgga gccgggcccg

	34741	ggcctccctc tcctcgccca gcccagcctc
		agctctcctg gccaaagccc ggggctcctc

	34801	tgaggtcctg cctgtctacc gtccgccctg
		cctgagtgca gggcccctcg cctcacctgc

	34861	cttcagggga cggtgccccc acacagcacc
		tccaaagacc ccgattctgt gggagtcaga

	34921	gccctgttca tatctcctaa gtccaatgct
		cgcttcgagg ccagcggagg ccgaccctcg

	34981	gacaggtgtg acccctgggt cccaggggat
		caggtctccc agactgacga gtttctgccc

	35041	catgggaccc gctcctttct gaccgctgtc
		ctgagatcct ctggtcagct tgccccgtct

	35101	cagctgtgtc cacccggccc ctcagcccag
		agcgggcgag acccctctct ctctgccctc

	35161	cagggccttc cctcaggctg ccctctgtgt
		tcctggggcc tggtcatagc ccccgccgag

	35221	cccccaagct cctgtctggc ctcccggctg
		gggcatggag ctcacagcac agagcccggg

	35281	gcttggagat gcccctagtc agcaccagcc
		tctggcccgc accccagcgt ctgccctgca

	35341	agaggggaac aagtccctgc attcctggac
		caaacaccag ccccggcgcc ccgactggcc

	35401	ccattggacg gtcggccact ggatgctcct
		gctggttacc ccaagaccaa cccgcctccc

	35461	ctcccggccc cacggagaaa ggtggggatc
		ggcccttaag gccgggggga cagagaggaa

	35521	gctgccccca gagcaagaga agtgactttc
		ccgagagagc agagggtgag agaggctggg

	35581	gtagggtgag agccacttac ccaggacggt
		gacccaggtc ccgccgccta agacaaaata

	35641	cagagactaa gtctcggacc aaaacccgcc
		gggacagcgc ctggggcctg tcccccgggg

	35701	gggctgggcc gagcgggaac ctgctgggcg
		tgacgggcgc agggctgcag ccggtggggc

	35761	tgtgtcctcc gctgaggggt gttgtggagc
		cagccttcca gaggccaggg gaccttgtgt

	35821	cctggaggtg ccctgtgccc agccccctgg
		ccgaggcagc agccacacac gcccttgggg

	35881	tcacccagtg ccccctcact cggaggctgt
		cctggccacc actgacgcct tagcgctgag

	35941	ggagacgtgg agcgccgcgt ctgtgcgggg
		cggcagagga gtaccggcct ggcttggacc

	36001	tgcccagccg ctcctggcct cactgtaagg
		cctctgggtg ttccttcccc acagtcctca

	36061	cagtccagcc aggcagcttc cttcctgggg
		ctgtggacac cgggctattc ctcaggcccc

	36121	aagtggggaa ccctgccctt tttctccacc
		cacggagatg cagttcagtt tgttctcttc

	36181	aatgaacatt ctctgctgtc agatcactgt
		ctttctgtac atctgtttgt ccatccatcg

	36241	atccaacatc catccatcca tccatcaccc
		agccatccat ctgtcatcca acatccatcc

	36301	ttccatccat tgtccatcca tctgtccatc
		ttgcatctgt ctgtccaaca gtggccatca

	36361	agcacccgtc tgccaagccc tgtgtcacac
		gctgggactt ggtgggggga gccctcgccc

	36421	tcccaccctc ccatctctcc tgaaacttct
		ggggtcaagt ctaacaaggt cccatcccgt

	36481	ctagtctgag gtccccccgc agcctcctct
		tccactctct ctgcttctga cccacactgt

	36541	gcactcggac gaccacccag ggcccttgca
		tccctgtttc cttcctgacc tctttttttt

	36601	ggctctggat ttatacacat tctgcctcct
		ggaggcgtct cagcttgagt gtcccacaga

	36661	cgcctcagac tcagcatctt ccatcgaaac
		tgctcccagg tccttgcaga cctggtcccc

	36721	cacattgttc tcaattcggt agatttctcc
		acaagccaga ggcctggact catcccataa

	36781	tgcctgcccc tcattgagtc agcctctgtg
		tcctaccata accaaacatc cccttaaaaa

	36841	tctcagaaga acaaaaaaag cacccagatg
		gcactgtcag agtttatgat gacaagaatc

	36901	ctcagttcag ttcagtcact cagtcgtgtc
		cgactctttg cgaccccatg aatcgcagca

	36961	cgccaggcct ccctgtccat caccaactcc
		cggagttcac tcagactcac gtccattgag

	37021	tcagtgatgc catccagcca tctcatcctc
		tctcgtcccc ttctcctcct gcccccaatc

	37081	cctcccagca tcagagtttt ttccaatgag
		tcaactcttc gcgtgaggtg accaaagtac

	37141	tggagtttca gcttcagcat cattccttcc
		aaagaaatcc cagggctgat ctccttcaga

	37201	atggactggt tggatctcct tacagtccaa
		gggactctca agagtcttct ccaacaccac

	37261	agttcaaaag cctcaattct ttggcgctca
		gccttcttca cagtccaact ctcacatcca

	37321	tacatgacca caggaaaaac cataaccttg
		actagatgga cctttgttgg caaagtaatg

	37381	tctctgcttt ttaatatgct atctaggttg
		ctcataactt tccttccaag aagtaagtgt

	37441	cttttaattt catggctgca atcaacatct
		gcagtgattt tggagcccca aaaaataaag

	37501	tctgccactg tttccactgt ttccccatct
		atttcccatg aagtgatggg accagatgcc

	37561	atgatctttg ttttctgaat gttgagcttt
		aagccaactt ttcactctcc actttcactt

	37621	tcatcaagag gctttttagt tcctcttcac
		tttctgccat aagggtggtg tcatctgcat

	37681	atctgaggtt attgatattt ctcctggcaa
		tcttgattcc agtttgtgtt tcttccagtc

	37741	cagtgtttct catgatgtac tctgcatata
		agttaaataa gcagggtgat aatatacagc

	37801	cttgacgtac tccttttcct atttggaacc
		agtctgttgt tccatgtcca gttctaactg

	37861	ttgcttcctg acctgcatac agatttctca
		agaggcaggt caggtggtct ggtattccca

	37921	tctctttcag aattttccac agttgattgt
		gatccacaca gtcaaaggct ttggcatagt

	37981	caataaagca gaaatagatg tttttctgaa
		actctcttgc tttttccatg atccagcaga

	38041	tgttggcaat ttgatctctg gttcctctgc
		cttttctaaa accagcttga acatcaggaa

	38101	gttcacggtt catgtattgc tgaagcctgg
		cttggagaat tttgagcatt cctttgctag

	38161	cgtgtgagat gagtgcaatt gtgcggcagt
		ttgagcattc tttggcattg cctttctttg

	38221	ggattggaat gaaaactgac ctgttccagg
		cctgtggcca ctgttgagtt ttcccaattt

	38281	gctggcatat tgagtgcagc actttcacag
		catcatcttt caggatttga aatcgctcca

	38341	ctggaattcc atcacctcca ctagctttgt
		ttgtagtgat gctctctaag gcccacttga

	38401	cttcacattc caggatgtct ggctctagat
		gagtgatcac accatcgtga ttatctgggt

	38461	cgtgaagatc ttttttgtac agttcttctg
		tgtattcttg ccacctcttc ttaatatctt

	38521	ctgcttctgt taggcccata ccgtttctgt
		cctcgcctat cgagccctcg cctccctacg

	38581	tagagactct aagcaggaag gtgacccgtg
		ctgcactggg tccagcatgc ttttaattca

	38641	gcagtggaac ttctgggtca tgattgtgtt
		taagggatgc gcatacgatt tttgaagcaa

	38701	aatttaacag gacagcagtg taaagtcagt
		acttatttct gattaaagaa agcaaatatc

	38761	cagcctgtta ctaagttaat taactaaaga
		aacatcttca acttaataaa cagtatctcc

	38821	tgaaacttac agcatgcttc acatttaaag
		gcaaaaccat tttagaggcc agggttccca

	38881	cgcttacgtt tattatttaa tatatgctac
		agattcaagc ccatgacaca aaatgggggg

	38941	aagagtgtga gtgttaggaa aaatgagata
		aaattggttt ttgcaggtga tgggctagtt

	39001	tactttaaaa aaaaaaacaa aacaagctca
		agatgaactg aaggactatt agaactggta

	39061	caagagttaa cctgtgatcg aatacaagca
		ggctgggcaa aactcagcag gttttcttct

	39121	atacaggcag taatgattga gaatacgaaa
		cggcggaagc gcttacaacc tcgataacag

	39181	ttctattaaa agccctagga atgaacttaa
		cacggnnnnn nnnnnnnnnn nnnnnnnnnn

	39241	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	39301	nnnnnnnnnn nnnnngctcc ccccaccctc
		ccctcctccc cccccaccac cagtgcccca

	39361	ggtctcgtgc ccagagagct gaagatgcca
		gcaggcccgc tgcctgcctc gctcgcgtgg

	39421	cccgggctcg ctgccggtct gcctgcccag
		cacacagatg cagccccagc tctcgctgcc

	39481	acccgcctcc cccaggcagg actctcccac
		aacaccaagg gcgtctctgg gttcaggatg

	39541	gccctcgttg aggtgtaaag tgcttcccgg
		ggctgagacg aatgggccgg agatccaaac

	39601	gaggccaagg ccgccacggc gcctggcgca
		gggcacccat ggtgcagagc ggcccagctc

	39661	cctccctccc tccctccctc cctgcttctt
		tatgctcccg gctatgtcta tttttactct

	39721	gcaatttaga aatgataccg aaggacaaac
		accgttcccc ctgtgtgtct gctctaaacc

	39781	ctttatctac ttatctatta gcgtgtccaa
		gttttgctgc taagtgaatg aaggaacact

	39841	acccacaagc agcaacgtcc ccacgaccct
		cgcctgttca actgggaatg taaatgtgct

	39901	ttcaaaggac ctaagtttct atgttcaaaa
		ccgttgtgtg tttcttttgg gagtgaacct

	39961	aggccactcg ttgttctgcc tttcaaagca
		ttcttaacaa ctctccagaa cccagggctt

	40021	ggcttacgtt tccagaaatt ccaaagacag
		acacttggaa acctgatgaa gaaggcctgt

	40081	gagcacagca ggggccgggg tacctgaggt
		aggtgggggg ctcggtgctg atggacacgg

	40141	ccttgtactt ctcatcgttg ccgtccagga
		tctcctccac ctcggaggct ttcagcaggg

	40201	tcacgctggt ggccagggtc gtgtatccat
		gatctgcaac cagagacggg gctgcggtca

	40261	gcccgcgggc gggcagcagg caggagcagc
		caggagacgc agcacaccga ggtcctcaca

	40321	tgcaggaggt gggggaagcg gctgtggacc
		tcacgactgc ccgatgtggg cctcttccaa

	40381	agggccggcc tggaccctgg ctttctccag
		aggccctgct gggccgtccg cacaggctcc

	40441	agccacaggg cctcttggga caggagggct
		ccagagtgag ccggccggcg ggaagaggtc

	40501	tgacaccgct gcagtccaca acacgaagcg
		aggtggagat gggatgaggg atgagaaaca

	40561	cttttctttt aaaacaagag cccagagagt
		tggaaagagc tgctgcacac gcaacatgaa

	40621	ctcctggccc cggtgccagc ggcgctggga
		gcccgagttc tcggcaatcc gaccacagct

	40681	tgcctaggga gccgggtgga gacggagggt
		taggggaagg cggctcccca gggagcgcga

	40741	ggcccggggt cgccaaggct cgccaggggc
		aagcgcagct aggggcgcag ggttagtgac

	40801	cggcactgca cccggcgcag gagggccagg
		gaggggctga aaggtcacag cagtgtgtgg

	40861	acaagaggct ccggctcctg cgttaaaaga
		acgcggtgga cagaccacga cagcgccacg

	40921	gacacactca taccggacgg actgcggagt
		gcacgcgcgc gcacacacac acacacacca

	40981	cacacacaca cacacggccc gggacacact
		cataccggac ggactgcgga gtgcacgcgc

	41041	acacacacac ccaccacaca cacacccacc
		acacacacac ccaccacaca cacacacaca

	41101	cacacacacc cccacacaca cccacacaca
		cccacacaca cccacacaca cacacccaca

	41161	cacacacaca cacacacaca cacacacacg
		gcccggtggc cccaggcgca cacagcacgg

	41221	agcaaacatg cacagagcac agagcgagcg
		ctagcggacc ggctgccaga ccaggcgcca

	41281	cgcgatggat tgggggcggg gacggggagg
		ggcgggagca aacggnnnnn nnnnnnnnnn

	41341	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	41401	nnnnnnnnnn nnnnnnnnnn nnnnngtatt
		aaagaagccg ggagcgagaa tatgacggca

	41461	agaggatgta ggtgggggcg gggcaagagt
		aaagagagcg gacggtagag gggatgcgat

	41521	tgtgatgcgg aagcgagacg aggagtgatg
		ccgtattaga ttgatagcaa gaggaacagt

	41581	aggagggggg ggggagagga gggggaggtg
		gggggtggtg ggtgggaagg gaactttaaa

	41641	aaaaagaggg gagagttgga ggggggaata
		aacgggcggt aaaaaagaac aatttgaaat

	41701	taccagggtg gggcggccag gggggtgatt
		cattcttgga gggggcaaca tatggggggt

	41761	ggctgtcgcg gattaggaga aaataaatat
		caggggtgat taagtgtttg gcgttgggga

	41821	ataatgaagt aagaatcaaa tatgaatcgc
		gttggcatcg ttagccatcg ggggaaacat

	41881	ttcccatgca aggaacaagg atgtgagaat
		gcgtccgtct gaaccaccgt cccggggtcc

	41941	cagtaggact cgccgagctg atagttgccg
		gagcaacagt taagggagca gaagctgcta

	42001	caaaaccacc acctgccaaa gtagggtctc
		caattacgga gtgcgcctcc tgggtgtcgg

	42061	tccaaacctt tggaaaggac ctggaaataa
		gtgctaccca ccagatatta atataaaccc

	42121	acctggccag gagaggcagg cgctgctggc
		acaggaagtg tccccagact cagtcatcaa

	42181	ggtaaataat attttgggac ctccctggaa
		atccagtggt taggactctg cggttcaatc

	42241	cctggtcggg gaactaagat cccacaagtc
		acaagacatg gccaaattta aaaaagaaaa

	42301	aaagagagag aaatatttag tgcaataggt
		tttagaattg aaattaagct cctgcccacc

	42361	cccacccccc aatctggatg aataaagcat
		tgaaatagta agtgaagtca ggctctgaca

	42421	tgcactgatg tgactcacct taagcaaccc
		ccaccctagg actggtcggg gttccaggag

	42481	tttcaggggt gccaggaaga tggagtccag
		cccctgccct ctccccccac cacgtcctcc

	42541	actggagccg cctaccccac ctcccacccc
		tccgcaccct gctacccccc acccctgccc

	42601	ccaggtctcc cctgtcctgt gtctgagctc
		cacactttct gggcagtgtc tccctctaca

	42661	gctggtttct gctgcccgct accgggcccg
		tcccctctgt tcagttcagt tcagtcgctc

	42721	agtcatgtct gactctttgt gaccccatgg
		actgcagcac accaggcctc cctggccatc

	42781	accaaccccc agaacttact caaactcatg
		tccatcgagc cagtgatgcc atccaaccat

	42841	ctcatcctct gtcgacccct tctcctggcc
		tcaatctttc ccagcatcag ggtcttttcc

	42901	aatgagtcag ttctttgcat caggtagcca
		aagtattgga gtttcagctt cagcatcatt

	42961	tcttccaatg aatattcagg actcatttcc
		tttgggatga actggttgga tctccttgca

	43021	gtccaaggga ctctcaagag tcttctccaa
		caccacagtt caaaagcatc aattcttcag

	43081	tgctcagctc tctttatagt ccaactctca
		catccatacg tgaccactgg aaaaaccata

	43141	gcctcgacta gatggaactt tgtgggcaaa
		gtaatgtctc tgcttttgaa tatgctgtct

	43201	aggttggtca taacttttct tccaaggagc
		aagcgtcttt taatttcatg gctgcagtca

	43261	ccatctgcag tgatttttgg agcccaagaa
		aataaagtct gtcactgttt ccactgtttc

	43321	cccgtctatt taacggaggg aaatttccca
		gagcccccag gttccaggct gggccccacc

	43381	ccactcccat gtcccagaga gcctggtcct
		cccaggctcc cggctggcgc tggtaagtcc

	43441	caggatatag tctttacatc aagttgctgt
		gtgtcttagg aaagaaactc tccctctctg

	43501	tgcctctgtt ccctcatccg cagaagtgac
		tgccaggtcg gggagtctgt gacgtctcca

	43561	gaagccggag gattttctcc ccatttgctg
		aaagagagct cggggtgggg gaagcttctg

	43621	cacccctagg atcaccagag gagccagggt
		cttcagggtt cccggggacc cctcagtggg

	43681	ggctcaggaa ccacagagcc agaccctgat
		tccaaaaacc tggtcacacc tccagatgac

	43741	cctttgtccc ttggctccgc ctcaaatgct
		ccaagcccca acagtgaagc gcttaagaga

	43801	aggatccacc aggcttgagt ttggggagga
		gggaagtggg gagctggggg agggcctggg

	43861	cctgggagac aggaatccac catggcttca
		ggcagggtct ctggggcctg cggggtggag

	43921	agcgggcagg agcagacaga ggtgactgga
		cacgacacac ccctccactc caagggaggt

	43981	gggcaggggc ggggcacaga ggaacaagag
		accctgagaa ggggtccacc gagcagactg

	44041	ctggacccag acatctctga gccagctgga
		atccagctct aagccatgct cagcccaggc

	44101	agggtatagg gcaggactga gtggagtggc
		cagagctgca gctgcatggg ctgggaaggc

	44161	cctgcccgtc ccctgagggt cccccagggt
		ctagccagac tccaatttcc gaccgcagca

	44221	cacacaggag gaagtggtcg gggtggagtt
		ggcccagagg tctgggcagg tgcagggtgg

	44281	gggaaggggg gcagctggag tcacccgctg
		aattcaggga cagtcccttt ttctccctga

	44341	aacctggggc tgtcccgggg gccaccgcag
		cctccaggca gcggggggac ccagccccca

	44401	atatgtgaga agagcaggtc ccaggctgga
		gagagcgaag caccatggtg gggagaagtt

	44461	agactggatc ggggccccta ggggctcccc
		cggacctgca cggcagccgt cagggcaccc

	44521	gcaccccatt gctgttcagt gctggccagt
		gtccaaggcc agggatgtgt gtgtgtgtgt

	44581	gtgcgtgcgt gcgtgcgtgt gtgtgtgcgt
		gtgtgcgcgt gcgtgcgtgt gtgtgtgtgt

	44641	gcgtgcgtgt gcgtgcgtag acgtgtgcgt
		gcgtgcgtgc gtgcgtgcgt gtgtgtgcgc

	44701	acgcgcgcag cccagcctca gcactggacc
		aggcagcctg ggattcctcc aaaactgcct

	44761	tgtgagtttg gtcaaaccgt gaggctctga
		tcaccgccat ccattcgccc cctcctgccc

	44821	ccctcatcac cgtggttgtt gtcattatcg
		agagctgtgg agggtctggg aggtcatccc

	44881	acctgccagc taaaccgtga ggctgccgca
		atcgcactga tgcgggcaga cccgagacgc

	44941	tgtgccggag acgaaggcca gcttgtcacc
		ccgccagagc ggcagtcggg ccacaagcat

	45001	catccaagca gtggttctct gagcccgacg
		gggtgatgca aaggagccag gagacacctg

	45061	cgcgtccaag ctgggggacc ccaggtctgt
		tatgccggac agtaaacacg ttcagctccg

	45121	gagggagagg gttcccctac cttccagggt
		ttctcattcc acaaacatcc aaagacaatc

	45181	cataccgaag gcgatccgtg cctttgctcc
		tgagacgtgc ggaagcacag agatccacag

	45241	acactgtctc ccaggatcct atgtatgtaa
		aggaaccgaa gtcccaggct gtgtgtctgg

	45301	taccacatcc cacggaacag gctggactga
		ttttcaccaa atgtagcaga aacgttaagg

	45361	agtatcagct tcaaaatatg agggccagac
		atgtctgaga agtcccttcc agaaaagtcc

	45421	ctttggggtc cttccccaga gttgctgaaa
		cagagaaccg gaagggctgc agagctgaac

	45481	ttaaacaact ggatcgcaaa ggtccgtctc
		atcagagcga tggtttttcc agtggtcatg

	45541	tatggatgag agagttggac cataaagaaa
		gctgagcgcc gaagaatcga tgcttttgaa

	45601	ctctggtgtt ggagaagact cttgagagtc
		ccttggactg caaggagatc caaccagtca

	45661	atcctaaagg aaatcaatcc tgaatattca
		tgggaaggac tgatgctgaa gctgaaactc

	45721	caatactttg gccacttgat gcaaagaact
		gactcactgg aaaaaccctg atgctgggaa

	45781	aggttgaagg caggaggaga aggggtcgac
		agaggatgag atggttgggt ggcatcaccc

	45841	acccatggac tcaatggaca tgggtttgag
		taaactctgg gagttggtga tggacagaga

	45901	atcctggcat gctgcggtcc atggggtcat
		agagagtcag acacaactga gcgactgaca

	45961	gaactgaagc aactggcaag ccggagggta
		ggtgccggct gcgatgagcg ggaacgtgca

	46021	acctgccacg tggagctctt cctacaccca
		gagtcctgac ggcactggga ccctagccct

	46081	ccacggcctc tccagggcca cgagacaccc
		tcacagagca gagaagcgga acagagctgg

	46141	tgtgcagaac caggccccgg gggtggggcg
		gggctggtgg gcaggcttta gtgagaagcc

	46201	cttgagccct ggaaccagag cagagcagaa
		cagttggcag aggcccccct gggagaggcc

	46261	ccccgcccag agtaccggcc ctgggccctg
		ggggagaggg cggtgctggg ggcagggaca

	46321	gaaggcccag gcagaggatg ggccccgtgg
		gacggggcgc accaaaacag cccctgccag

	46381	caaggggaag ctggggcact ttcgaccccc
		tccaaggagg agcccacacc agcgcatctg

	46441	cccaaggtgc ccttggccct gggggcacat
		gaggcccagg ccaggccagg gggcccatga

	46501	ggcccccagg ggtcagtgca gtgtccccag
		gcagccctgg cctctcatcc tgctgggcct

	46561	ggcctcttat cccgtgggcg cccacggcct
		gctgcccccg acagcggcgc ctcagagcac

	46621	agccccccgc atggaagccc cgtcaggaaa
		gagcccttgg agcctgcagg acaggtaagg

	46681	gccgagggag tcatggtgca gggaagtggg
		gcttcccttc gatgggaccc aggggtgaat

	46741	gaccgcaggg gcggggaacg agaagggaaa
		ccagctggag agaaggagcc tgggcagacg

	46801	tggctgcacg cacagcgctg accctgggcc
		cagtgtgcct ttgtgttggg ttttattttt

	46861	aattttgtat tgagatgcta tttatctcgt
		ggagcttttg ccgccctgag attttgtacc

	46921	cgtggctggt gtccctcttg cctcaccccg
		gcctctgtag cagggcagac acggcgcaac

	46981	ggggcagggc gtgcccagga ggcactgtca
		ttttgggggc agcggcccca caaggcaggt

	47041	ctgccttcct cccctcttac aggcagcgac
		agaggtccag agaggtgagg caagctgccc

	47101	aatgtcacac agcacacggg cgcagtccca
		ggactgtaga aatcccggga ctagacaggc

	47161	accagagtgt cctgtgtttt taaaaaaacg
		gcccaagaga agaggcaagt ctgcaaggcg

	47221	tcccgggaag gcagcagggg cttggctcgg
		tctcccccaa ggaggccagc tcctcagcga

	47281	ggttcctaag tgtctaacgg agccaagcct
		gaaccaaggg ggtcacgtgc agctatggga

	47341	cactgacctg ggatggggga gctccaggca
		aagggagtag ggaggccaag gaggagagag

	47401	gggtgcacag gcctgcaggg agcttccaga
		gctggggaaa acggggttca gaccacgggg

	47461	tcatgtccac ccctccttta tcctgggatc
		cggggcaggt attgagggat ttatgtgcgg

	47521	ggctgtcagg gtccagttcg tgctgtggaa
		aaattgtttc agatcagaga ccagcgtgag

	47581	gtcaggttag aggatggaga agaagctgtg
		aaaaggtgat ggagagcggg gggacggtcc

	47641	tcggtgatca ggcaccgaga tcgcccatgg
		aatccgcagg cgaatttaca gtgacgtcgt

	47701	cagagggctg tcggggagga acaggcactg
		tcatgaactg gctacaaaaa tctaaaatgt

	47761	gcaccctttt cggcaatatg cagcaagtca
		taaaagaaaa cgcatttctt taaaattgcg

	47821	taattccgct tttaggaatt catctggggg
		cgggggaaca atcaaaaaga tgtgaccaaa

	47881	ggtttacaag ccaggaagtc aactcgttaa
		tgatgggaga aaaccggaaa taacctgaat

	47941	atccaacaga aagggtgtga tgaagcgcag
		catggcacat ccaccgcaag gaatcctaac

	48001	acaaacttcc aaaacaatat ttctgacgtt
		gggtttttaa agcatgcgtg cactttcaaa

	48061	agcttgtcag aaaacataga aatatgccaa
		taatgtgtct ctagccaaat tttttaattt

	48121	ttgctttata attttataaa gttataattg
		tatgaaatat aatgataaaa ttataaacta

	48181	taaaaaagtt atgaaaatgt tcacaagaag
		atatacatgt aattttatct tctacaatac

	48241	tttttaatac cagaataacg tgcttttaaa
		aaagattgag cacagaagcg tataaagtaa

	48301	aaattgagag tttctgctca ccaaccacac
		gtcttacctt aaaacccatt ctccagcgag

	48361	agacagtgtc atgtgggtct gtacacttct
		ggcctttctc ctaggcatgt atgtccctga

	48421	aaactcacac acacggctaa tggtgctggg
		attttagttt tcaaaacgga ctcatactct

	48481	gcctatgagc ctgcaactat ttattcagtc
		tgttgagatt ttctatatca gcccacatgg

	48541	atcccgcatg ttctctgaat ggctctgtat
		gaattcaaag tttggaagaa gcagcgtgtc

	48601	tttaatcatt cgcctattaa tggacgtttg
		gggtgtttcc actacaaaan nnnnnnnnnn

	48661	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	48721	nnnnnnnnnn nnnnnnnnnn nnnnnnnnng
		atacaattcg agctcggtac cctggcttga

	48781	actatatgaa cagagaacga tgagaacagt
		ttctcaaact tggaacagtt aacattttgg

	48841	gctaaatgat tcttttttgt gtggagttgg
		cctatgaata gaggatatta gcagcatcat

	48901	ttaaccttta ctcactacat acctgtagca
		actacatcct ctccatttgt gtcaatcaaa

	48961	actgtctccg gacatggaca agtgtgcccc
		tgggatgggt ggaatgacct tttgttaaga

	49021	accactgggt cagagattca tagatttttg
		tcttgttgac tttttaaaaa tacatcttgg

	49081	tttttatttt attggtttct gctcttatct
		ttatgattac cttcctttta cttggggctt

	49141	ccctgataga ttttcccttc tggctcagct
		ggtaaagaat ctgcctgcaa tgcaggagac

	49201	ctgggttcag tccctgggtt gggaggatcc
		cctggagagg agaagggcta cccaccccag

	49261	tattctggcc tggaggattc catggagtgt
		atagtccatg gggtcgcaga gtcggacatg

	49321	actgagtgac tttcacacac acatatgtcc
		ctggtagctc agctagtaaa gaatcccacc

	49381	cgcaatgcag gagaccccgg tccaattcct
		gggtccggaa gattcccttt tgtttactcc

	49441	ataagatctt atctggggac aaaactaaca
		gctatgccag accttctgga catcagggaa

	49501	cgtgaggggt gtggactgga cagatgtgtg
		tgttctccca aacacaaaca tacatctgta

	49561	tacatgtaca tggagagagg gggagggagg
		ctgtgagtct ccaggggacc gtgcaaccat

	49621	gtgacattca tggaggcgtt tgcgggtgat
		cactacacag tttcttcttc tggtttcttg

	49681	gtcaattgac ttcacaattc caattcctat
		acttcatttt agactgaggg aattttacac

	49741	tattgtaaga catatgtata catgagttat
		gttcagcgcc atgagggctc attttgtgtg

	49801	tccactttgc ctggaaacaa agttggactg
		atttacttct aggggtgcct gggggtgttt

	49861	ctggaggaca ggagcatttg aacccaaggg
		ctcggtgaag catgagcctc tctgcaggtg

	49921	gacccaggag gaacgcaagg ccgaggaagg
		cagactctcc tcctccctaa cccgaggtct

	49981	ctgctcagaa aagggacaat ataatgacta
		gaagaaaaga aagaacatca gctgtgggag

	50041	gtttgttctc tggagcagat tcacacgttg
		aggctcatgt gcaggaattc taggtgaaac

	50101	agagcagtca cccatgtgtg ttggaaaatt
		ttaaattaca tttgcagtta cgactttgtt

	50161	taagccagac agggtagcac agcaaagtca
		ccatgtggtc acctgtgttt tgtaaaggag

	50221	agagaacttg ctggcacatt caggaaaggc
		cgtgtctcag ctttggaggc acactgagag

	50281	gccacaagca gatggtgagg accagggtct
		cgggcagagg gatcaattca ctgctcttca

	50341	cttttgccac atctgtgtgc tgtccatcct
		ggccagagta gttcagtctt cagatgctgg

	50401	agttcccatt ggtagaaatc caatctgggt
		catttttaaa cctctcttgg ttctacttaa

	50461	tggttttaaa atctctttgg ctcaagaaaa
		aaaataaaca taattttaaa gggtggtttg

	50521	gggccttgac tataaagtac attatctggg
		ccatttcaga gcatggttga attaatacat

	50581	ttcgtgctta ctatagctcc tattttcttg
		attctttaca ggtaattttt gttaggaatc

	50641	gggtactgtg aatattttct tgttgaatac
		gggatctttg tattttttcc taattttttt

	50701	ttttttttca tttttggttt taccttcagg
		aaagtcacta ggactcagga aagtcctttg

	50761	tccgcctgtt atttcagtct cttacctggg
		gccagggcag cgtttcctct gggctaagtt

	50821	tccccacaac cggggccagt tctcctcact
		cttcaccctg aggccttaat gaggagctcc

	50881	cctgcgtctg agcagccggc cctcctgtga
		cgtgcgtgtg tctctggcca tcggcgtccg

	50941	gtgtccttgg aggttccgtc ctcccttcgc
		tcactgtgcc ccgcactcga gctctcaggc

	51001	tccaagcagt gtccgcagtg tgcagaccct
		ctgtgtagct ctctcctcct caggactctt

	51061	ccctctagat gtgtgttttc ttttggctcc
		ttggacctcc gctctgaacg caggcctggt

	51121	gctgagtgtg atctctggag ggaagcctgg
		gaggctggac gggtccgccc tgcggtgtgg

	51181	tgacaggtgt gggctcgggg cggggcctgc
		acgtcgtcct gacccgagcc gggactgggc

	51241	tccgggcctc aggcatcact gactgaatct
		ccctcacaga ggggtcaggg cctgggcggg

	51301	ggaaccgtct ctgcaatgac agcccctccc
		agggagggca cagcggggag ctgccgaggc

	51361	tccagcccta gtgggaggtc ggggagccca
		ggggagcggc ctgacggccc cacaccggcc

	51421	cagggctggt tcgttctgtt tctcgagctc
		aacagaagct ccgaggagct gggcagttct

	51481	ctgaattcgt cccggagttt tggctgctga
		gtgtcctgtc agcaccgtat ggacatccag

	51541	agtccattag cagtggtctc tgtccctctg
		tctgtccttc atcaggctct ttgtccaggt

	51601	caccacacgg ccaacaccag gacagtctgg
		tcccgccagc ccatcgtccc tgcggacgcc

	51661	cctgtgcagc ctgccgaagg gccgggaggc
		cgggggaacc gggccaggcc tgtccctgct

	51721	gtgtccacag tcctcccggg gctggaggag
		agcgtgagca ggacgggagg gtttgtgtct

	51781	cacttccccg tctgtctgtg tcactgtgag
		gattatcact gctgtcagct gactgacagt

	51841	aatagtcggc ctcgtcctcg gtctgggccc
		cgctgatggt cagcgtggct gttttgcctg

	51901	agctggagcc agagaaccgg tcagagatcc
		ctgagggccg ctcactatct ttataaatga

	51961	ccctcacagg gccctggccc ggcttctgct
		ggtaccactg agtatattgt tcatccagca

	52021	ggtcccccga gcaggtgatc ttggccgtct
		gtcccaaggc cactgacact gaagtcggct

	52081	gggtcagttc ataggagacc acggagccgg
		aagagaggag ggagagggga tgagaaagaa

	52141	ggaccccttc cccgggcatc ccaccctgag
		gcggtgcctg gagtgcactc tgggttcggg

	52201	gcaggcccca gcccagggtc ctgtgtggcc
		ggagcctgcg ggcagggccg gggggccgca

	52261	cctgtgcaga gagtgaggag gggcagcagg
		agaggggtcc aggccatggt ggatgcgccc

	52321	cgagctctgc ctctgagccc gcagcagcac
		tgggctctct gagacccttt attccctctc

	52381	agagctttgc aggggccagt gagggtttgg
		gtttatgcaa attcaccccc gggggcccct

	52441	cactgagagg cggggtcacc acaccatcag
		ccctgtctgt ccccagcttc ctcctcggct

	52501	tctcacgtct gcacatcaga cttgtcctca
		gggactgagg tcactgtcac cttccccgtc

	52561	tctgaccaca tgaccactgt cccaagcccc
		ccggcctgtg gtctcccctg gactccccag

	52621	tggggcggtc agcctggcag catcctggcc
		gtggactgag gcatggtgct ctggggttca

	52681	ctgtggatgt gaccctcaga ggtggtcact
		agtcctgagg ggatggcctg tccagtcctg

	52741	acttcctgcc aagcgctgct ccttggacag
		ctgtggaccc gcagggctgc ttcccctgaa

	52801	gctccccttg ggcagcccag cctctgacct
		gctgctcctg gccacgctct gctgccccct

	52861	gctggtggag gacgatcagg gcagcggctc
		ccctcccgca ggtcacccca aggcccctgt

	52921	cagcagagag ggtgtggacc tgggagtcca
		gccctgcctg gcccagcact agaggccgcc

	52981	tgcaccggga agttgctgtg ctgtgaccct
		gtctcagggc ggagatgacc gcgccgtccc

	53041	tttggtttgt tagtggagtg gagggtccgg
		gatgactcta gccgtaaact gccaggctcc

	53101	gtagcaacct gtgcgatgcc cccggggacc
		cagggctcct tgtgctggtg taccaaggtt

	53161	ggcactagtc ccaccccagg agggcacttc
		gctgatggtg ttcctggcag ttgagtgcat

	53221	ttgagaactt acatcatttt catcatcaca
		tcttcatcac cagtatcatc accaccatca

	53281	ccattccatc atctcttctc tctttttctt
		ttatgtcatc tcacaatctc acacccctca

	53341	agagtttgca ttggtagcat atttacttta
		gcacagtgtg cctcttttta ggaaactggg

	53401	ggtctcctgc tgatacccct gggaacccat
		ccagaaattg tactgatggc tgaacccctg

	53461	cgtttggatt cttgccgagg agaccctagg
		gcctcaaagt tctctgaatc actcccatag

	53521	ttaacaacac tcattgggcc tttttatact
		ttaatttgga aaaatatcct tgaagttagt

	53581	acctacctcc acattttaca gcaggtaaag
		ctgcttcgca tttgagagca agtccccaga

	53641	tcaataaaga gaatgggatg aacccaggat
		ggggcccagg ggtcctggat tcagactcca

	53701	gccgtttagg acagaacttg actaggtacg
		aagtgagcgg ggtggggggg caatctgggg

	53761	ggaactgtgg cacccccagg gctcggggcc
		atccccacca catcctggct ttcatcagta

	53821	gccccctcag cctgcgtgtg gaggaggcca
		gggaagctat ggtccaggtc atgctggaga

	53881	atatgtgggg ctggggtgct gctgggtcct
		aggggtctgg ccaggtcctg ctgcctctgc

	53941	tgggcagtga taattggtcc tcatcctcct
		gagaagtcac gagtgacagg tgtctcatgg

	54001	ccaagctatt ggaggaggca gtgagcactc
		ccacccctgc agacatctct ggaggcatca

	54061	gtggtcctgt aggtggtcct ggggcttggg
		ccgggggacc tgagattcag ccattgactc

	54121	tcagaggggc cagctgtggg tgcagcggca
		gggctgggcg gtggaggata cctcaccaga

	54181	gccaaaataa gagatcaccc aacggataga
		aattgactca caccctttgg tctggcacat

	54241	tctgtcttga aatttcttgt ggacaggaca
		cagtccctgg ataaagggat ttctatcttg

	54301	cgtgtgcaat agagctgtcg acacgcttgg
		ctgggacatg taatcctttg aacatggtat

	54361	taaattctgt tcactaacat ctgaaaggat
		ttttgcatca ataaacctaa ggtatattgc

	54421	cctgtcattt ccttgtcttg tagtgtctct
		gagtaggctg gaaggggtaa ccagcttcac

	54481	aaatcgagtt aggaaattcc cttattcttc
		cactgtctaa tagactttca taagattagt

	54541	gttaattcct ctttaaatcg ctgctataat
		catcactgtg gccaccggta ctgaattttt

	54601	tgttaggatg atttttaaac aagcatttta
		atgatttttc cttttatttt cggctgtgct

	54661	gggtctcgtt gctgtgtgcc ggcgttctct
		cgctgtggcc agtgggggcg ctgctctcgc

	54721	gttgcgaagc tcgggcttct gactgcagtg
		gcttctctcg ttgcagagcg cgggctccag

	54781	ggcgctcagg ctcgcgtggc tgcggcacgt
		gggctcagta gtcctggggc acaggtgcag

	54841	cagcctctca ggacgttttg ttcccagatg
		gtgggtcggt cgaaccggtg tcccctgcgt

	54901	tgcaaggtgg attcttcacc gctggaccac
		cagcgacgtt ccctggaggt ttttaattat

	54961	ggatttaagc tctcattaga tgtctcctca
		catttcctat ttctttttga gtcagtttga

	55021	tactttgttt gtgtctgtaa gtttgtccat
		tttatccaag tcatctaatg tgttgataga

	55081	caattattgg ttagtcatct aattgttggt
		ttacaatttt gagagcattg tcctgcaatt

	55141	ccttctatct gcaagattgg taataatatc
		tcccaagagg agtcacaaac tgaaatgaga

	55201	ttanatacag gctttttttt taaaagaatg
		aacttatgtt gttgcctttc tcatagatct

	55261	tacttcttag catgactgta cttactgact
		ggggcgtttt catgtctgtg tggagagcta

	55321	ccattagtac ttcttatcgc ccaaagacat
		cgggctcctg ggcacagtga aaacactcct

	55381	ttctgtggct attttgcaaa atatggccta
		gcctagcgtc ataagggatc acagctgaca

	55441	actgctggaa cagagggaca tgcgaagcaa
		cgtgagggct ggaacctgga gggtcctctc

	55501	tggggacagt ttaaccagct ataatggaca
		ttccagcatc tgggacatgg agctgtgaac

	55561	tggaccaatg actgtcattt ttggaagaga
		aatcccagga gagaagggtc caggggaatc

	55621	tgaggccgca tgcagtgcct caggacaggg
		gacaccttct ccagcagagc aggggggccc

	55681	gcccaggccg cctgcagtga ttccaccagg
		aggagatgca tccctgcaga cctctgacag

	55741	cacggccctc tcctgagaca cagggtcaca
		cccggggccc tggaaccctt tgagacccta

	55801	aacctttcct ttcctgacca ccctgacagc
		agtctagctc agaacagaca tcttcatttt

	55861	cagcaggaaa atccttttcc tcgtttgagg
		gagcgactgg caccggagga gctgagtctt

	55921	ttaaacacag gctgcctgaa cctcagggat
		gacctgcagc tgctcagagg aggctggagt

	55981	gtgatagctc actctaatgt tactaaaagg
		aacatattgg acaccccctc tctgaaaaat

	56041	ttccctcctg cctctcatct cttagtccac
		tttatcgccg ttttactgct tttctattta

	56101	ctactcttaa cgccaaccta tcttatttcc
		cctcccagtt taacacggtt ttccctccac

	56161	ccgctctctt taatctcaga agattctgcc
		tattcctcta ttatcacacg cccctacttt

	56221	ttattttttt tcttacccgc cttttattcc
		ctcccctcct cactctctat ttaattacat

	56281	cttaactaca ccgcctgcgc tatcttcgaa
		tgtatccaaa tatttttccc ttatataaca

	56341	ctccaggccg agcggctaac ttattataat
		ttctttatag cgcctaccta atttcccttt

	56401	atttctaatt atctatatat acccatgcaa
		tttcgnnnnn nnnnnnnnnn nnnnnnnnnn

	56461	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	56521	nnnnnnnnnn nnnnntgggt gtacgttata
		gagtaaacgc gcatgaagaa gtgggtcaat

	56581	ctatggctgt gagaggcaga aaataatatt
		atcatatata atttatgtta taacacactg

	56641	aggtggtggg ctcgtagaat agtgcggacg
		gggagaaagg tgggaaggag aagacacaag

	56701	agagagatgt tcgcctcgcg ggatggatgg
		gcggagggat agaagaataa aaagaggaga

	56761	ggtatagagg ggggcggggg gcataacgtg
		tggtggggta aatagtaggc ggtaattatg

	56821	aaaaaaagaa agacgggggg ggcggtaaca
		tagaatacgc aaaaaagtca tatactgaac

	56881	ggggattagg gagaagaggt ggggggcgtg
		gggtgcgggg gaaagaggtg tgtgtataat

	56941	tggtatggag tgttatttga atatatatta
		atgtaatagg gagtgtaatt agtgaaattg

	57001	tgggagtatt atattggggt gtgggggaca
		tggcaaagtg atgatcggga taaaaaaagt

	57061	aaagcaagag gggaggggaa aataaggggg
		gggagaaggt cgaagaaaat aagaggaaga

	57121	agaaagaacg ggggtggcgg gcgggggggg
		cgccgctctt gtatctggct tttttgttgt

	57181	gtcggtggtt gttcgcgtct tgttgggtcc
		ggggcgggtg tgcggaaaaa aaaaaaggcg

	57241	ggaggcccgg ggcccggtca cgcggcaccc
		ccgcgggtcc ctggcttctc cttcggcagc

	57301	tccgggggtc ggtgagcctg cgccctccgg
		gccgccggcc cgagctgtgt gcgccctgga

	57361	gaatcggagc cgctgtggca gcacgcggag
		ggcgcgcgca agggccacgg gacggacctt

	57421	caaaggccgc ggcggagcgc ggcaagccga
		accgagggcg gtctggcgat cggccgagcc

	57481	ctgctccccc ctcccgcgtg gccccagggt
		cgcgggtgga ctggggcggg tacaaagcac

	57541	tcacccccgt cccgccccca gaaagcctcc
		caggactctc acagagcacc cgccaggagg

	57601	catccggttc ccccctcggc tcagttcagt
		tgctcagtcg tgtccaactc tttgcgaccc

	57661	catggactgc agcaccccaa gcttccctgt
		ccatcaccaa ctcccggagt ttactcaaac

	57721	tcatctattg agtcagtgat gccatccaac
		cgtctcatcc tctgttgtcc ccttctcctc

	57781	ccactttcaa tctttcccag catcagggtc
		ttttcttatg agccagttct tcacatcagg

	57841	tggtcagagt attggagttt cagcttcagc
		atcagtcctt ccaatgaaca ctcaggactg

	57901	atttccttta ggatggactg gctggatgca
		gcgccagaca ccgaccgcgt ttaccccgtg

	57961	tgtcctttcc aatggctgtc ccctgcgggc
		ctaggggcat tggtgcgggt ttgaatcctg

	58021	tggccttgaa ttttacgcct tagttccagg
		tccagggcag ggccatccgg attcaggatg

	58081	cttcccagcc cttcaggaat ggcaggtttt
		catggtcctt tctgagtgag ttctgagtgg

	58141	tcatattggt gcccttggca gggagggctc
		ctgactttcc tatcttcaca tcactgtccc

	58201	caacccccaa gagaggcctc ttggcccagg
		gactgcaggg aggatgaagt caggagcaga

	58261	agcatggggt agggggctca ggtgggcaga
		ggaggcccct ctgtgaggag gaacggcaag

	58321	cgaggaggga acaggggcac cggcagtgcc
		tggcaagctg ggtgatgtca cgactacgtc

	58381	ccgaccacac agtcctctca gccagcccga
		gaagcagggc cctcccctga cccccatctg

	58441	ggcctgggct tcagttttct cctccctgca
		atggggtgac tgtttgcctc caggagaggg

	58501	gagcatgtaa aggtggccac tctcttctgg
		cagacatgcc aggcctgggc cagcctccac

	58561	ccctttgctc ctgcagcccc tgctgacctg
		ctcctgtttg ccacaccggc ccctcctggg

	58621	ctgatcaggg cccccctcct gcaggaagcc
		ctctgggaca agcccagctt gctgtaactg

	58681	tggctttcca ctgtgacctg caacgtggga
		ggctgttact taaaactccc atgactggtg

	58741	gattgccggt ccccagaaca aggccacgca
		tccctggagg ccctcgagac catttaaggt

	58801	agttaaacat ttttacttta tgcattttca
		tgtgtatcag aaagaaaaaa aatgtatcat

	58861	cagttcatca aatccatgat ttcttgacca
		atattgctaa gatgaggctg aaataggcat

	58921	ttccattttt aaaaaactga atcactctga
		agaaacagat ggcaggcttc cctggtggtc

	58981	cggtggttaa cagtccatgc ttccagtgct
		gggggcatgg gttcgatccc tgaaaatttt

	59041	aaaaaggaag aaaaagatgg ctcccccgtc
		cctgggattc tccaggcaag aacactggag

	59101	tgggttgcca tttccttctc cagtgcatga
		aagggaaaag ggaaagtgaa gtcgctcagt

	59161	cgtgtgcgac tcttagcaac cccatggact
		gcagcctacc agactcctcc gtccatggga

	59221	ttttccaggc aagagtactg gagtggggtg
		ccattgcctt ctccaggcaa acggcctgct

	59281	actgctactg ctgctaaatc gcttcagtcg
		tgtccaactc tgtgcgaccc catagacggc

	59341	agcccaccag gctcccccgt ccctgggatt
		ctccaggcaa gaacactgga gtggggtgcc

	59401	attgccttca gcctgctgct gctgctgcta
		agtcgcttca gtcgtgtccg actctgtgtg

	59461	accgcataga cggcagccca ccaggctccc
		ccgtccctgg gattctccag gcaagaacac

	59521	tggagtgggt tgccatttcc ttctccaatg
		catgaaagtg aaaagttaaa gtgaaattgc

	59581	tcagtcgtgt ccgactctta gtgacccaat
		ggactgcagc ctaccagggt cctccatcca

	59641	tgggattttc caggcaagag tactggagtg
		gggtgccatt cggcctaggg agtgagaaat

	59701	cacggctgtc ttccctcttc tcgccctcta
		ggggtctctg tggagcctcc ctggagaggc

	59761	cgcggcggct ccggggactg gagggggagg
		gggggttgag tcagccggtg gccctcccct

	59821	cgctgcccgt ctcctccctt tttaggcaca
		agctgggcgc cctttttagg cgcagcctca

	59881	ccctgcgggc cactgcccgt gtttcggctc
		cccggagata aaacagattg cctgcacccc

	59941	gggtcatcac aaggattgta tgaccgtttc
		ccagtgtgct caccaccctc cctctgattc

	60001	tcagagacgc gccctcgcct caggaggctg
		ctcatcccag gccaaggggc ggcgtggggt

	60061	ccccagcgcc ccgcacagac actgccttct
		gaccacctcc tcccaacagc ttacctgcca

	60121	agaaggcctc ctgacccctc atcctgcccg
		gtggtttgga gaaagcctca tctggcccct

	60181	ccttctcggg gcctcagttt ccccctctgt
		gaactggcgg attctgccaa gctgacgtcc

	60241	tggccagccg cctccccgtg gccagtgtcc
		cccgggacac agctgaatgt ccctgctcgg

	60301	gatgcacctt cccaagttgg cctgtcagga
		ggcgggggcg agcagggaaa cccgactcct

	60361	ctcagacggc ccatcgcatt ggggacgctg
		aggcccggag cagcggcacc ctcctggcca

	60421	gggtcattct cccgccccgc cccgtccctc
		cgggcctccg agaccgcagc ccggcccgcc

	60481	ccgggaagga ccggatccgc gggccgggcc
		accccccttc cctggccgcg ggcgcggggc

	60541	gagtgcagaa caaaagcggg gggcggggcc
		ggggcggggg cggggcggag gatataaggg

	60601	gcggcggccg gcggcacccc agcaggccct
		gcacccccgg gggggatggc tcgggccgcc

	60661	ggcctccgcg gggcggcctc gcgcgccttt
		ttgtttttgg tgagggtgat gggggcggtc

	60721	gcggggtact attttttcat ttataattgg
		gtattagcta gcgagtggaa ccacaccctt

	60781	attccactat agccaatttt tgcgggggca
		tcttacatta cagactcgcc cgcctcttat

	60841	ttcggtacag catatcagat cgtctcttta
		ctcagacact agtgattatt gtctatagta

	60901	cacaaaaaga acggttgtgt cggcgtaatg
		gttgcatttt ccctcctcgt ttctcctgac

	60961	cacctcaatt acaccaacac tctactattt
		aaatcacgta ttgtacgcca ccctccgccc

	61021	gcgaactaaa agaatgtgca gatattctga
		agataaaatc gttcattgtt acgccccgcg

	61081	cgcttcgcgt atattactct tagaacttct
		tattcgcccg agcagttatt caccccccgc

	61141	aactagatgt cgccttaata tttgttctaa
		ccgttttgga ttctaacgat aggcgggaaa

	61201	ggtagacatt cgaccgctac gacaactaaa
		atcgacgagc acaggctatt tatatcgcga

	61261	ccacacgcgc gcggtataca naccgtaaaa
		ttatctaaca tcgagagtaa gggcacagag

	61321	cgaaatacaa gcggcgtggt gggaggtgtg
		tctgtagtga attcgcacct cgcgccgccg

	61381	cctctgtgcg tcgnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	61441	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnngatataa

	61501	tattaataaa cagcggatag atgtgtgtaa
		gggaggaggt gcataagaga ttaaagagag

	61561	gcgggcggag agaaatagag tagaggagga
		tgagagaaaa aagaaagcaa gcgtaggtac

	61621	aacggcgggt gggtagtatg ataaagtgag
		tgtatatatt tgagtaaagg aagggtagat

	61681	ggagtataaa gaagtaagga gaggagaggg
		cggcggagag agagagtgca aagaaaataa

	61741	gtgggcaaag gcggggtggg tgagaagcag
		tagaagagaa gatagagaag ggggaaaaag

	61801	aggaaaatga ggattagaac aagtaggaca
		ggatagatgt gaaaaatgag atcaggtcaa

	61861	ggtggagaaa aagtagaaac tggggcgtga
		ttgtaaaaaa gggaggccgc gatggggcag

	61921	caccataagc gaagagatga attaatgaaa
		gcaaggcagg gagaatcaaa tgagttgggt

	61981	ggaggaagga ggctgtgact tccttcgctg
		ccggaaagag aactagaata gcctcgggct

	62041	gtggggggag gtaaagataa agtgacttct
		gggccctggg ggaggcccag gagtttctac

	62101	cgagctgagc tgggtgcctc tcccaaatgc
		ccaaccccct gagagtcgac gggagagcac

	62161	agcctggcca aacctgggca gggcacacgt
		gtccttcacc ccacagtggt cacgagccca

	62221	gcgtggtccc tgcgtctggc gggaaacaca
		gaccctcaca ccccacacaa gggtccggcc

	62281	gctttcaaat aacagcagcc gtgccctctg
		ggccggtgac ccggacacag agagatgaag

	62341	tccgcatctc tcagagtgcg ctgtcctccg
		cccggtcagg cccgggtccc ctgcttctct

	62401	gaggtcacca ggagggattg catgtgggtc
		tcagggacac aggttcagtg atgtgacaga

	62461	gggtagtggg tcccagcagg gccggtcttt
		ggacccgttt ttctgaaaag ccagttggcg

	62521	acctggggtc acagcaaagc tgatcctgtt
		tggccaggag tctcccagtg acggcctccc

	62581	ccagaacatc gggcccagtg ggggctccag
		ggggtagact tgcctcccag ctcacgcccg

	62641	tgtcttgaca agtccatgat ttggtaaaat
		taatttgtgt tggatggagt tgatttagtg

	62701	gtgtgtgagt ttctgtggcg cagcaaagtc
		aatcagttac gcatacacat gtatccagct

	62761	cttcctacga ttctgttccc atataggtca
		ttatggggtg tcaggtagag cttcctgtgc

	62821	tacgcagtac ggccttattc agttcagctc
		agtcgtgtcc gactccttgt gaccccatgg

	62881	actgcagcac gccaggctcc cctgtccatc
		accaactcct ggagcttatt caaactcatg

	62941	tccatcgagc cggtgatgcc atccaaccat
		ctcatcctct gtcgttccct ctcctcctgc

	63001	cttcagtctt tcccagcacc ccctagagaa
		gggaatggca aaccacttcg gtattcttgc

	63061	cctgagaacc ccatgaacag tacggaaagt
		ccttattagt tttctatttt atatatagca

	63121	gtgcacacgt gtcagcccca atctcgcaat
		ttatcacccc cctccgccgc cgattggtag

	63181	tcatgtttgt tttctacatc tgcgactcta
		tttctgtttt gtaaacaagt tcatttacac

	63241	cactttttta gattctgcac atacgtggca
		agcccacagc aaacatgctc aatggtgaaa

	63301	gactgaaagc atttcctcta agatcaaaaa
		caagacgagg atgtccactc actccgtttt

	63361	tactcaacac agccctgaac gtcctagcca
		tggcaatcag agaagagaaa gaaattaagg

	63421	aatccaaatt ggaaaagaag aagtaaaact
		cactctttgc aaatgacatg acacttatac

	63481	ccagaaaatc ctagagatgc taccagataa
		ctattagagc tcatcagtga atttgttgca

	63541	ggatacaaaa ttaatacaca gaaatctcct
		gcattcctat agactgacaa caaaagatct

	63601	gagagagaaa ttaaggaaac catcccacgg
		catgaaaaag agtaaaatac ctaggaataa

	63661	agctacctaa agaggcaaaa gacctgtact
		cagaaaacta taaaatactg acaaaggaaa

	63721	tcagacgaca cagagagaga gagataccac
		gctcttggat gagaagaatc gatagtgtga

	63781	caatgactat actacccaga gaaacataca
		gattcagtac aacccctatc aaattcccaa

	63841	tggcattttt cacagaatca gaattagaac
		aaaaagtttt acaagtttca gggaaacaag

	63901	aaagatccta aagagccaga gcaatcttga
		gaaagaaaaa tggagctgga agagtcaggc

	63961	tccctgagtt ctgactgtgt atacaaagct
		ggcatgattt ttaacagcag gggtgtaaat

	64021	gaacttgttc acaaaacaga tggtggggtg
		ggcttccctg gtggctcagc tggtaaagaa

	64081	tcctcctgca acgcaggaga cctgggttcg
		atccctaggc tgggaagatc ccctggagaa

	64141	gggaaaggct acccactcca gtattctggc
		ctggaaaatt ccaaggacca tatagtccat

	64201	gggtttgcaa agagtcggac acgactgagc
		gacttccaat cctggaaacg tcccattgtg

	64261	gacggtgaac tggggttgtc caagctcagg
		gtaaccgttt gctgagtgac tgacactcct

	64321	tctcatgggt taaaatgtgg ggcccaaggc
		caggaccaga ccccgcagtc agccaggcag

	64381	accctgtgca gccccagcga gtgtgtggcc
		gccgtggagt tcctggcccc catgggcctc

	64441	gactggagcc cctggagtga gcccattccc
		tcccagcccg tgagaggctg ggtgcagccc

	64501	taaccatttc ccacccagtg acagatccgc
		ctgtgtggaa acctgctctt gtccccaggg

	64561	aacctggcag gactcaggga gaatgtctca
		gggcggccac agatcagggg ctgggggggc

	64621	agggctgggt ccagcagagg ccctgtgccc
		actccccgga aagagcagct gatggtcagc

	64681	atgacccacc agggcaccga cgcgtgcttg
		cacacaggcc gccccctcat ggtgacactc

	64741	ttttcctgtg gccacatctc gccccctcag
		gtccctcctg ctccccagct cctggcctgg

	64801	gaacctcttc cccgccccgg ggacgtcagg
		gctggtgtcc actgagcatc ccatgcccgg

	64861	gactgtgctg atcaccagca cctgcacccc
		ctctcgggtc tcaccaggat gggcaactcc

	64921	tgcccatcca gcacccagcc tcctgggtac
		acatcggggg aggagggaga agcctgggcc

	64981	agacccccag tgggctccct aaggaggaca
		gaaaggctgc cgtgggccag ccgagagcag

	65041	ctctctgaga gacgtgggac cccagaccac
		ctgtgagcca cccgcagtgt ctctgctcac

	65101	acgggccacc agcccagcac tagtgtggac
		gagggtgagt gggtgaggcc caggtgcacc

	65161	agggcaagtg ggtgaggccc gagtggacag
		ggtgagtggg tgaggcccag gtagaccagg

	65221	gcccatgtgg gtgaggcccg ggtggaccag
		agtgagcggg tgaggcccag gtggacaggg

	65281	cgagcgggtg aggcccaggt ggacagggcg
		agcgggtgag gcccgggtgg acagggcgag

	65341	cgggtgaggc ccgggtggac agggcgagcg
		ggtgaggccc gggtggacag ggcgagtggg

	65401	tgaggcccgg gtggaccagg gcgagtgggt
		gaggcccggg tggacagggc gagtgggtga

	65461	ggcccgggtg gaccagggcg agtgggtgag
		gcccaggtgg acagggtgag tgggtgaggc

	65521	ccaggtagac cagggcccag agcaaagccc
		cggctcagca gtgatttcct gagcgcccac

	65581	tgcttgcagg gacctcagcg atggtaaggc
		agccctgttg ggggctcccg actggggaca

	65641	gcatgcagag agcgagtggt cccctggaga
		aacagccagg gcatggccgg gcgccctgcc

	65701	aggctgcccc aggggccaca gctgagcccc
		gaggcggcca ggggccggga cagccctgat

	65761	tctgggttgg gggctggggg ccagagtgcc
		ctctgtgcag ctgggccggt gacagtggcg

	65821	cctcgctccc tgggggcccg ggagggacgg
		tcaggtggaa aatggacgtt tgcgggtctc

	65881	tggggttgac agttgtcgcc attggcactg
		ggctgttggg gcccagcagc ctcaggccag

	65941	cacccccggg gctccccacg ggccccgcac
		cctcacccca cgcagctggc ctggcgaaac

	66001	caagaggccc tgacgcccga aatagccagg
		aaaccccgac cgaccgccca gccctggcag

	66061	caggtgcctc cctctccccg gggtgggggg
		aggggttgct ccagttctgg aagcttccac

	66121	cagcccagct ggagaaaggc ccacatccca
		gcacccaggc cgcccaggcc cctgtgtcca

	66181	ggcctggccg cctgagacca cgtccgtcag
		aagcggcatc tcttatccca cgatcctgtg

	66241	tctgggatcc tggaggtcat ggcccctctc
		ggggccccag gagcccatct aagtgccagg

	66301	ctcagagctg aggctgccgc gggacacaga
		ggagctgggg ctggcctagg gcaccgcggt

	66361	cacacttccc ctgccgcccc tcacttggga
		ctctttgcgg ggagggactg agccaagtat

	66421	ggggatgggg agaaaaatgg ggaccctcac
		gatcactgcc ctgggagccc tggtgcgtct

	66481	ggagtaacaa tgcggtgact cgaagcacag
		ctgttcccca cgaggcctca cagggtcctt

	66541	ctccagggga cgggacctca gatggccagt
		cactcatcca ttccccacga ggcctcacag

	66601	ggtccttctc caggggacgg gacctcagat
		ggccagtcac tcatccattc cccatgaggt

	66661	ctcacagggt ccttctccag gggacgggac
		ctcagatggc cagtcactca tccattcccc

	66721	acgaggcctc acagggtcct tctccagggg
		acgggacccc agatgggcca gtcactcatc

	66781	catccgtctg tgcacccatc cgtccaacca
		tcacccttcc ctccatccat ctgaaagctt

	66841	ccctgaggcc tccccgggga cccagcctgc
		atgcggccct cagctgctca tcccaggcca

	66901	gtcaggcccg gcacagtcaa ggccaaagtc
		agacctggaa ggtgcctgct tcaccacggg

	66961	aggagggggg ctgtggacac agggcgcccc
		atgccctgcc cagcctgccc cccgtgctcg

	67021	gccgagatgc tgagggcaac gggggggcag
		gaggtgggac agacaggcca gcgtgggggg

	67081	ccagctgccg cctggctgcg ggtgagcaga
		ctgcccccct caccccaggt acaggtctcc

	67141	ctgatgtccc ctgccctccc tgcctccctg
		tccggctcca atcagagagg tcccggcatt

	67201	ccagggctcc gtggtcctca tgggaataaa
		aggtggggaa caagtacccg gcacgctctc

	67261	ctgagcccac ccccaaacac acacaaaaaa
		atccctccac cggtgggact tcaccagctc

	67321	gttctcaggg gagctgccag ggggtccccc
		agccccagga agccaggggc caggcctgca

	67381	agtccacagc cataacacca tgtcagctga
		cacagagaga cagtgtctgg tggacaggtg

	67441	cccccacctg cgagcctgga gagtgtggcc
		ctcgcctgcc ccagccgcgg tcagtcggct

	67501	cagcaaccgc tgtccactcc cagcgccctg
		gcctcccctg tgggcccagg tcaagtcctg

	67561	ggggtgaagc taagtcaggg agcctcatcc
		atgcccagcc cggagcccac agcgccatca

	67621	agaaatgctt cttccctcca tcaggaaaca
		ttagtgggaa agacaagagc tggggggttc

	67681	tggggtcctg ggggatcaga tgaaggggtc
		tgggagcagc agcagcctca ggcaccccaa

	67741	aacaaggccc aggagctgga ctcccagggc
		tgaggggcag agggaaggaa ggcctcctgg

	67801	ggggttggca tgagcaaagg cacccaggtg
		ggggctgagc acccctcggc tggcacacac

	67861	aggcccccac tgcagtacct tccccctcgg
		agaccctggg ctcccgtctc ccgcctggcc

	67921	tgccatcctg ctcaccaccc agaaatccct
		gagtgcggtg ccatgtgact gggccctgcc

	67981	ctggggagga aggagattca gacagacagg
		atgccagggc agagaggggc gagcagagga

	68041	tgctgggagg gggcccgggg aggcctgggg
		ggcagggggg caggagttct ccagggtgga

	68101	cggcgctgtg ctatgctcgg tgagcacaga
		ggccccgggt gtcccaggcc tgggaaccca

	68161	gcagaggggc agggacgggg ctcaaaggac
		ccaaaggccg agccctgacc agacctgtgg

	68221	gtccagaagg cagctgcgcc ctgaggccac
		tgagtggccc cgtgtcccga accaccgctg

	68281	aaacatggga cacacgttcc caggcggagc
		cactcctgcc ttccgggagg ctcccagcgg

	68341	gctcatcgct ccatcccaca gggagggaaa
		ccgaggccca gatgacgaac atcccggcga

	68401	gcaggtcaaa gccagcccct ggggtcccct
		ctcccggcct ggggcctccc ctctgcaggg

	68461	tgggaaaccg aggccacaca ggggctccat
		ggggctgccc tctgccaggc cctggacacc

	68521	ccgcgggtga cccccgcctc tatcatccca
		gccctgccag gccctggaca ccccgtggat

	68581	gacccccgcc tctatcatcc cagccctggg
		ggacagatgg gaggcccaag cgtggacccc

	68641	ctggccaccc cctaccccac agccgggagg
		agccgggagc tggtggccaa gggcctagag

	68701	gagccagann nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	68761	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnca atatagaggg

	68821	ggtgggataa agggtaatat gatgtttagg
		tagttagagt taaattagaa gggtttggat

	68881	aaagattaat aaaattacaa gcgtacatat
		cgtgtgagtg tgggtgataa tatttgtgta

	68941	tgtggggaat agaagtgagt gtgagtagta
		ttcaagatgt aagtgtgcga atacaggtct

	69001	gagcgatttg aatggaagtg aaaaaaagcg
		tgtgtgtgga ggaggcggga gaggaagata

	69061	gtgtggggga agaaaagaag gctagtgggt
		aaagaaatat cagtaggcgg ttgacgaaag

	69121	aagaactagg aagaattaat ataaaaataa
		agggaggatt aaaaaataaa gagggaggag

	69181	gtaacggaaa tagttagtta agaaaagaat
		ggagagtgga ggtaagataa ataagggagt

	69241	aatgggagtg aggaggaata aataaaaaaa
		tggtgaggga aaatagagta gaatgagaac

	69301	aagaatgaaa aagggagtga agggggtgaa
		aaaaagtgaa gttgaaaaaa gaggaaaaaa

	69361	aaggagaaga taaaaaaata aaataaaaaa
		aggaaaaaaa agaaaaaaag aaagaagggt

	69421	taaaggacga aaagaaggga agagaaaaaa
		aatagtttaa gtgggggagg gtaaaaaaga

	69481	attaataaag taaatatggt tgtggtcgaa
		aaaaaaaaaa aaattgttgt gttgatgaga

	69541	agaaaagaaa aaagaagaaa gggaaaagca
		aaaagaaagg agagaaaaag acaaccccac

	69601	cgcccgggcg catggagggt gaggatggcg
		cacgcccgcg gatggcacag catcacagca

	69661	atcctaaaac gttttcagac cggtgcatct
		tcaccgcgcg cgcgccccgc ccggccctcc

	69721	tcccgccctg accgcggacc cccacccgca
		ccggggagcc tacccccacc ccggggacgc

	69781	tccgccacgc taaggtcagg actgccgtga
		agacgcgccg gggtgaaaac gttttatctt

	69841	catgacataa gcgagtggtt ttgaaacagg
		tttacaaacc ctcgtgaaga cgcaccctta

	69901	gcgttaggtt ttgttttttt accatgtgac
		gatgcaacta ttttcttcct ctcttccaca

	69961	gtggctagtc gcctccagag cgaggggtat
		ctcttgtaca gagaccctcg gaacatccgg

	70021	aggtagtttc ccacctaggg gtaaagcgag
		aaggctcatt acgagggccg gggctcctcg

	70081	gggaagggca gggccctggc gcagaggctc
		tgccacctca gtgacacgca gaccacgcgc

	70141	ggcctgcagg cgccgggctc tgaaagcagg
		caaagcccga tctgctgaca tcaggggttc

	70201	cgcagcagcg aaggtctggc ccgcacctgg
		cccactggca gggggtaagc tctgcctccc

	70261	gacgacagca ccaagttcag gaagggccac
		gcagacactg gtgagacacg gcccccccgg

	70321	agctgcccga gaagctctga ctttgcacta
		aagatctctg gcgcggtcca aaaatgtaag

	70381	gcctctcttc cttttatctt aagactttga
		tatttttacg atgtaataaa taccaagaag

	70441	ggcttttaat ttcagacaga tgtaggataa
		tttcccccgt agcccttgct gctttgttta

	70501	gtaacgaaac tcaaaccaga aataccaaag
		gaattttcca aagagtttca aaagcgctta

	70561	tcagcaatca ctagactgct gcatacatca
		tcactgcccc aaacaatagc ctgcctgtgc

	70621	cagttactca aagtactact tacttgacga
		aaacaaatct agtcctaacg tttttacaaa

	70681	gaaactccac tcttccgcca acttttcaga
		aacaaccact cgatcacgtg gcaggggacc

	70741	gtggctggac tgggtgctgg ctccttctgt
		gaccaggcaa cactgccccc ttctcggcct

	70801	ccctacgcct cttgacaaat gttcatcagc
		tgtaaagttc accccacgag ggacccactt

	70861	ctgctatttc ccacgtacct accccattat
		aggagttttc tttgtgacag tttctgcatt

	70921	tttcatggat ttagaggttt acataatcag
		ggctgctgaa cagcatgaga gacgtggcca

	70981	caaggtccct cctgcacctt gccgcagggg
		cagggcgagt tatctggctt gagcgtggtt

	71041	accatcaggg ggtaaacaca gtttccagga
		cgtttttgac aagacactga cccggatgcc

	71101	cccactacca ccgtgcaggt cctgcaggcc
		tcccagcctc ccaggccctt cccgaggtcc

	71161	cttcggaact taggggactc ggtctgcccc
		cctgggtttt ccctgcacca gcttttgccc

	71221	cctctggacc caggtttccc aaatggaaaa
		cgaaggtgtg ggtatggaag ctccctgggc

	71281	tcctctcagc tgtgcctctg catggtgatg
		acggctgccc atcggggggg gcaggactgg

	71341	ggcagctgcg gacaccctcc caaggctgct
		acccccgagt ggtgtggggc gctgtgggca

	71401	cgctctgctc agcgcacctc ctggaaacca
		gcgcctgccg tctgcccggg gcaaccggcc

	71461	cgggagccaa gcaccactgc cgtcagagga
		gctgctggct gtgagtggac gccagtctag

	71521	ctctgaaccc tgcccaggcc tcctgaggtc
		tgaacattgt aaaatcaggc cccggacggc

	71581	aactgcctct ccctcctgcc gtctggtctc
		cataaactgc atctcaggac aaatcttctc

	71641	actcaccagg gctgaaacag aagactgcag
		ctatctttct caaatctaag gtgtgctaca

	71701	gggcaagtcg cagaaactgt ctggcctaag
		catctcatca gatgcctgag acaagagctg

	71761	tggacgccaa gctggagcca gagctcctcg
		cgttctgccc acctggcacc gcgttccacc

	71821	cagtaaacgc aggcttgatt ttcaaaagta
		ccaccgactc agagccaatg ctaaaccgac

	71881	cacttttcct gcccattaga ttgggtgaag
		gtttctttaa tcaatctgcc agtcaccaca

	71941	tgccgcctct gtgcccacag gctggcgaag
		acctttctga gctacggcat gtggcaggca

	72001	gcggcacctc tcttcagtac ggccagctgt
		caaggggagc gtttctgtga tgatgtgaaa

	72061	atacattgca tccggccccg tgtttcatga
		acacgggtga ggaaaggaaa cacacaaagt

	72121	tctgatgcga ctgacagcac gggtctcata
		actcaataca agtcagacaa accacaggga

	72181	gtcacaggga atcccaatag cctcatctag
		tgtgaccatc atgaggctta atttattcag

	72241	tgtattcaat cataaagagg gggaaaaatt
		gtaaaaaaaa aaaaaaagaa agagtgaaat

	72301	gtgtaatact gaaaactgtt gctaggagaa
		gcaagcattg gcgtttgtaa ctgctttgac

	72361	tccccaagac ccacactcgc ctcgctacaa
		aagggaggca ctgctgctca gtacttgcac

	72421	acccgaactg cggatttgta atttaaaaat
		gtgtgtgtgg acacagcaca agccagagac

	72481	tgccaaaggt tgagggacac tggaagaact
		taatatactt ggtgcatgct gccagtgaca

	72541	gtcagtcacc agctgattca atagagtgcc
		gaaaggtcac cttttaggta aggatgaagg

	72601	ggttctgggc tcgtttactt gcactaactc
		agagttagtc cgagatatcc gaagtgccag

	72661	gtgcctccca tttgctgatg gatctagctc
		agggacggct gggccctagc catccaaaaa

	72721	tcaagcattg ttctcccaac ctgtcttctc
		gctgataatg gaaggtcaga acgcccaccc

	72781	gcccacctca aagtcaaaga acaccaagcg
		ggtgagtccc cactaagctc ggtgtttcca

	72841	atcagcggtt tcaggattcc agctggggca
		atgagggagg gagcgtgcga gggatccaac

	72901	acctcgcccc gtgcgcagca agggataacc
		caacaccccg tttctgtacg tccggctgga

	72961	gttgtggaac tcagcgcgga cccggggcca
		ccgcgacccc cgggaccctg gccgcgcggc

	73021	gcatccccgc tgccgggaca cgggtaagcg
		tccccaaact gccggacgcg gggcggggcc

	73081	ttctccgcca cgccccgata ggccacgccc
		aaggacaagg atggtcgtgc ccagacggcc

	73141	ggggcgggnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	73201	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnncg gagggggggg

	73261	ggcggggcgg gggctgccgc cgcgcgtata
		ggacggtggt cgcccggcct ggggtccggc

	73321	cgggaatgac cccgcctctc cccgcatccc
		gcagccgccc cgccgcgccc tctgccgcgc

	73381	acccgcctgc gcacccgccg ccctcggccg
		cggccccggc ccccgccccg tcgggccagc

	73441	ccggcctgat ggcgcagatg gcgaccaccg
		ccgccggagt ggccgtgggc tcggctgtgg

	73501	gccacgtcgt gggcagcgct ctgaccggag
		ccttcagtgg ggggagctca gagcccgccc

	73561	agcctgcggc ccagcaggtg agcaagggct
		caggggaaac tgaggcccga cacagagccg

	73621	cagcaagaag gatcctactg gtcactcggc
		tgttggcctg gggtcatcac aggcgggctc

	73681	tcccaaccca tcccctgagg ccaaggtccc
		tagaaccccg tgggcagaca ccaaccagcc

	73741	ctttaaatat ggggaaacca aggtgcttag
		gggtcagaga tagccctagg tcgcccaacc

	73801	ctagtagaag ggagggctgt tggagttcct
		gagtgcccgc tctcccaccc cccgggaggc

	73861	cccttcctga gcccaagggt gactggtagt
		cagtgacttt gggcctgccg acctgtaccc

	73921	cactgggcac cccaccagtc ctgagccaca
		tttgggctta gtgacggggt cagggatcat

	73981	gaggatcaat gtggctgagc caggaaggtg
		ttagaacctg tcggcctgga gttcatacca

	74041	gcactgccct gggcttttct agacccatgt
		cccgcctcct gccccacctg cccctgttcc

	74101	cgcaccccac cagcagcggc aggggcttcg
		agagggctgt gggctcaccc tatttcaggg

	74161	atggagccgc taagacctgg ggcacactgc
		ccgctaggga cccctgaggc accagggccg

	74221	ggggctctgc ggaggggcag ccgccacccc
		cagctttgga gtcctctccc gggtgcccag

	74281	cccgagctga tccggctgcc tcccacgctg
		tgccccaggg cccggagcgc gccgccccgc

	74341	agcccctgca gatggggccc tgtgcctatg
		agatcaggca gttcctggac tgctccacca

	74401	cccagagcga cctgaccctg tgtgagggct
		tcagcgaggc cctgaagcag tgcaagtaca

	74461	accacggtga gcggctgctg cccgactggc
		gccagggtgg gaagggcggt ccacggctcc

	74521	cactccttcg gggtgctccc gctattccca
		ggtgctcctg cacttcccat gtgctcccga

	74581	ttctccctgg tgctccctct cctcctggct
		gctcctttgc ctcccaggtg ctcccacttc

	74641	tccctggtgc tcctgctcct cccggcggct
		cctgtacctt cggcctgacc tcctccctct

	74701	acaggtctga gctccctgcc ctaagagacc
		agagcagatt gggtggccag ccctgcaccc

	74761	acctgcaccc ccctcccacc gacagccgga
		ccatgacgtc agattgtacc caccgagctg

	74821	ggacccagag tgaggagggg gtccctcacc
		ccacagatga cctgagatga aaacgtgcaa

	74881	ttaaaagcct ttattttagc cgaacctgct
		gtgtctcctc ttgttggact gtctgcgggg

	74941	ggcggggggg agggagatgg aagtcccact
		gcggggtggg gtgccacccc ttcagctgct

	75001	gccccctgtg gggagggtga ccttgtcatc
		ctgcgtaatc cgacgggcag cgcagaccgg

	75061	atggtgaggc actaactgct gacctcaagc
		ctcaagggcg tccgactccg gccagctgga

	75121	gaccctggag gagcgtgccg cctccttctc
		gtctctgggg gcccctcggt ggcctcacgc

	75181	tctgtcggtc accttgcccc tcttgctgat
		gcaatttccc cgtaattgca gattcagcag

	75241	gaggaatgct tcgggccttt gcacctgacc
		gcatgagcag aggtcacggc cagccccctt

	75301	ggatctcagt ccagctcggc cgcttggccg
		tgacgttcca ggtcacaggg cctgccggca

	75361	cagaggagca ggcccttcag tgccgtcgag
		cactcggagc tgctgcctcc gctgagttca

	75421	ctcagtgtct acgcacagag cgcccactgt
		gtaccaggcc ctattccacg ttccccagtc

	75481	accgagcccc cagggctggt ggggacctgc
		cctcgggtac actgtgtccc gtcacgtggc

	75541	tttacgtgtg tctctgaggg aggctggcat
		tgcggtccac ctctcagcac aaacatctgt

	75601	cccctgggaa gggggtccca tttctgggtg
		cgagcagccc cctggggtcc gtgtctcctc

	75661	cttacctggc tcaaggcccc ggctcctggg
		tcctggacag cagggagccc acccctcggg

	75721	gctgtggagg gggaccttgc ttctggaggc
		cacgccgagg gcccaggcgc cgcctccggc

	75781	cgtcgccctg agggagcagg cccgacgcca
		gcgcggctcc tctgtgaggc ccgggaaacc

	75841	ctgcctgagg gtgcgggtgg gcaggtgccc
		ctgcccccag gctctcctgt gtgagtgaca

	75901	ctcaccagcc agctctggat gccacccatc
		cgggttctcc aggaggcact catagcgggt

	75961	ggggtcccct ccctcccccc tctgtggagg
		gagggagtct gatcactggg aggctggtgg

	76021	tccgtacccg cccccccgac tctggacgtg
		tttactaccc ccgcctgggc tcaggacagg

	76081	gcattggatg ggaaggacag ggctgggtcc
		tggccaggct gggggctctg cagggcatgg

	76141	gtgcccctgt ctcttcttat attccaacgt
		cactgcaggg gggcgcaaat cttggacccc

	76201	acttactgat gatctgcatc aggacatagg
		tcccccctcc tgcagcgggg ggctggccac

	76261	ggagggcgct ggggaaggcc cctcctccag
		cccctcggcg aggctcacca ggtgcccatc

	76321	ctcagccagc agggcgacgc tcgctgggag
		ggcggagagg gaggcagggc agggctggta

	76381	cgacccccgc tggggcgggg gggccctcag
		ccggtcctcc agcacccttg ctgccccccc

	76441	tcaccgtcag ggggcacctg gccgctctgc
		ctcaggtggg cggtgagggt cccaaggcca

	76501	caccaggtgt tcaccagctc ccagcagctg
		gctgtgggag aggggcagag gtgggcgcat

	76561	ggcacccgcc ttccccccag accaggatgc
		tctgccttcc tcccgcccat ctccccagac

	76621	atctgaagga ctcttgcctc caccatgcag
		ccccgcctcc accagaagct caggttcccc

	76681	gccccccctc cccgaagctg caggacccct
		gaccagcgaa gagatgggac agttggaaca

	76741	cacgctcccc cagcagcggc acagcagctg
		tgtggcccag aagagcccgc ctgtttccct

	76801	caagcaactc cccatggatg tcatcccatg
		gacaccccct tccccacacc gcctcctcgt

	76861	tctccccctc caaggcagag ggaacgcacc
		cccacctgtc tgctaggaca ggggacccca

	76921	cttacctccg aacatcacct tgataaacat
		ggccgtggtg gggacagatc cctccgaccc

	76981	ccaacttccg acctggggaa ggagctgggg
		tggagctcga ctgcagggtg gggccctgtg

	77041	ggaggtgtac gggtggagag ggtgatgggt
		gggtgggctc aagcggagct ccttgctcag

	77101	tccaggcggt ccctgcagct agtccaggat
		cctcagcctt ctccccctca ctggatcagg

	77161	gaagactgag gttccctccc ctgccccccc
		acccagcttc caagctggtc tctgtggcag

	77221	tgggagctgc caagaggtct gagcggccag
		tatccgggta acggggtttg tggagggtcc

	77281	gggcattccc ggtgcagggc tctagtgggg
		gctggagcct cgggcccaga gctgtccaga

	77341	gaccagtgcc ctcccaccgc cgccgcccgc
		aaggagagac agagctccca ggcggggagt

	77401	cggaggttcc tggaggggga gcatcctcaa
		ctctgcaggc ccccttccca ggcgcactcc

	77461	cggcctcccc gtcttctgtc ccctgctctt
		gttgaagtat gattggcata cagttcacag

	77521	ccactcttcg gagtgttctc cacactaagg
		atacagaaca tgtccctcgt ccccccaaac

	77581	tcccagccag gctgtcacga agagggaggc
		ggccgacggg gcagggcctt gcactcctgc

	77641	gtgtggggtc cacaggggtc gtccccgtgt
		cggtggcccc ttcctctcac gccaggaggg

	77701	tccccttgcc tggaggtgcc gtggatccgc
		tcgctgcctg ctctttgggt tgtttcccgc

	77761	atggggtgat gatgaagagg ccagtacaga
		cactcgccag caggtctctg ggtgaacagg

	77821	catttatttc tctttcctga gggcagatcc
		tgggagtggg gtgccggacc gtccggggag

	77881	agtatgcttc tgtttctaag aagctgccgt
		gttctccagt gtgctgcacc atgtcacggc

	77941	ccctctgtgc gtctggactc aggagacctc
		cttctcagcg gccctccccc ccaggtggtc

	78001	aggccatctg tgcccttctg ggggcagagc
		tcagcgccgg aggcgggagg aggcccagat

	78061	cccagcgcag cccaccagcg ttgctctgct
		tccctcggca ttcatagctg gagaaagggc

	78121	aaggagcacc ggctgaagcc ccacctggag
		gacgcacttc gatggcagca ggtgctcaga

	78181	ggtggccccg ggcagcattc cccagacgca
		caggccagtg ctttcttccc aggacaccac

	78241	tgtgtctggg gacccgagtc ctgcagcacg
		gtcgggagcg gctgtgccca gattccggcc

	78301	tgcacccttg gctccagcca ccacccctgt
		ttgtcaaggg gtttttgtct ttcgagccgc

	78361	cgaggaggga gtcttttgtc tgcagtgtca
		cagaagtgcc ataaagaggg gcccacagtg

	78421	ggagctttat aacattggtg cggagggctg
		taacaggtca gggaggcact tgagggagcc

	78481	ttctagggcg atggagatgt tctaaaattt
		ggtctgggta caggctacag agatgtgtgg

	78541	gtgtgtgtgt gtgtgtgtgt aaaaccctcg
		agccacacgt gtgaggtctg tgcatgtgac

	78601	cgtacacagg agacctcggt ggaaagcagc
		cacctgctct gactgcacct gtggatttcc

	78661	agctcctgcc ctcaggcggc cctgcggggc
		ccactggctg acggggagac ggcaccgccc

	78721	tcccccgctg tcagggtggg ggggctgacg
		atttgcatgt cgtgtcaggg tccagcggcc

	78781	tcccttgcgt ggaggtcccg aagcacctgg
		agcgccgccc gcagaacagc ggactcctgc

	78841	ctgcctccct gcctctggcc atggcctgcc
		cgcctctggc cctctttctg ctcggggccc

	78901	tcctggcagg tgagccctcc caaggcctgg
		ctcacctagg ggtgtgtaag acagcacggg

	78961	gctctagaag taaatcgcgg ggaagtaaat
		cgtagtgggc aggggggatg gtttccgaag

	79021	gggccctgag ggggacagga gacctggcct
		cagtttcccc actggtgagt gaccagatag

	79081	ccagggtacc tttggactct gactctgggg
		ggctctcaga gactggtctc ctactcagtt

	79141	tttcagaggg gaagctggtg tggccttgtc
		actgccctgc agggcctcag ggacaagcta

	79201	tccctgagga ggtctccagc agtcagtggc
		cggaggctga gccgatggat atagtaacag

	79261	cccaggcggc ctcttggggg tggtcagcct
		gtagccaggt tttggacgag ccgaagtgac

	79321	ctaagtgatg ggggtctgca gagcaaggga
		tgagggtggg cagcaggagg acccagagcc

	79381	caccagccca ccctctgaat tctggaccct
		tagctgcatg tggctccttg ggaagacggg

	79441	gcttaagggt tgcccgctct gtggcccaca
		cagtgctgat tccacagcac tggctgtgag

	79501	cttttgggag cagattctcc cggggagtct
		gacccaggct ttgtggggca ggggctggag

	79561	ggaaggggcc caggccagac ctgagtgtgt
		gtctctcagc ctcccagcca gccctgacca

	79621	agccagaagc actgctggtc ttcccaggac
		aagtggccca actgtcctgc acgatcagcc

	79681	cccattacgc catcgtcggg gacctcggcg
		tgtcctggta tcagcagcga gcaggcagcg

	79741	ccccccgcct gctcctctac taccgctcag
		aggagcacca acaccgggcc cccggcattc

	79801	cggaccgctt ctctgcagct gcggatgcag
		cccacaacac ctgcatcctg accatcagcc

	79861	ccgtgcagcc cgaagatgac gccgattatt
		actgctttgt gggtgactta ttctaggggt

	79921	gtgggatgag tgtcttccgt ctgcctgcca
		cttctactcc tgaccttggg accctctctc

	79981	tgagcctcag ttttcctcct ctgtgaaatg
		ggttaataac actcaccatg tcaacaataa

	80041	ctgctctgag ggttatgaga tccctgtggc
		tcggggtgtg ggggtaggga tggtcctggg

	80101	gattactgca gaagaggaag cacctgagac
		ccttggcgtg gggcccagcc tccccaccag

	80161	cccccagggg cccagactgg tggctcttgc
		cttcctgtga cgggaggagc tggagtgaga

	80221	gaaaaaggaa ccagcctttg ctggtcccgg
		ctctgcatgg ctggttgggt tccaacactc

	80281	aacgagggga ctggaccggg tcttcgggag
		cccctgccta ctcctgggtg gggcaagggg

	80341	gcaggtgtga gtgtgtgtgt ggggtgcaga
		cactcagagg cacctgaagg caggtgggca

	80401	gagggcaggg gaggcatggg cagcagccct
		cctggggtag agaggcaggc ttgccaccag

	80461	aagcagaact tagccctggg aggggggtgg
		gggggttgaa gaacacagct ctcttctctc

	80521	ccggttcctc taagaggcgc cacatgaaca
		gggggactac ccatcagatg nnnnnnnnnn

	80581	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	80641	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		agagggtggg tgggtggaat ttaatatagt

	80701	ggtgcgcgtg gagcgtgggc ggcgcattta
		aggcggtcat ctaaaatagt ggataggggg

	80761	tggtgtgaca ataacgggtg gtggatgtgg
		tttacggggg gtgcaatagt tctgagtftg

	80821	ttagtgtctt cttgatgggg ttgcggcgtg
		tggacctacg ccttgagtat gtgggggggg

	80881	aaaagcagtg agggtagtag ggatgggaaa
		tattggtgga ggttctttgt tggtgtattt

	80941	tttggtatta tgttgggtgg tggagtggtg
		ggttgggtgt aatttcgctt gcgttatgtg

	81001	ttttttttct ttttcgtgtc gtgggttggg
		ttggttggtg ctttgtggtg gtggtgggtt

	81061	gtggtataaa aaaaaatgtg tggttgtgct
		cagcttagcc ctataacggt cggctttgtt

	81121	tcttgtttgt tctgtgggcg tgagcggatg
		gctcgggcct ccgtgctccg cggcgcggcc

	81181	tcgcgcgccc tcctgctccc gctgctgctg
		ctgctgctgc tcccgccgcc gccgctgctg

	81241	ctggcccggg ccccgcggcc gccggtgagt
		gcccgccgtc ctccagcccc cccgccccgc

	81301	cccgccctcc acgccgaggg gcgccggctc
		gcagagctgg atccaagggg gtgcccggga

	81361	gtggcccggc gcggcccgtt accccgaaac
		gctgtctggg tgccccgggg gtgtggtgga

	81421	tagtgagctt cccgtccctg gaagtatgca
		agtgaagccg gcgccgggat cgctcgggct

	81481	ggctggtgag cgggcgggac tcggtcgggc
		gctagacgca cgccgccagc cccccagctc

	81541	ccagacctgc ccactccgcg cccgcccggc
		cgcgatcccg ggtgtgtgtg tgtgttgcag

	81601	gggagggaca gcgggagtgg ctacagggct
		cccgactcac cgcagggaca aagacccgcg

	81661	ggtccccagc tggcgtcagc cgccaggtgt
		gtggcctcgg tgagcacacc tccaggcggg

	81721	agggttgagg gaagcgctgt ggggagggca
		tgcggggtct gagcctggaa gagacggatg

	81781	ctaccgcctg ggacctgtga gtggcgggat
		tgggaggcta tggaatcagg aggcagccta

	81841	agcgtgagag ctccggtgtg gcctggcggg
		ggtggtaggg gggggacgcc cctgtgtgtg

	81901	ccagcctgcg tgtgccctaa aggctgcgcc
		ctcccccact gctggggctt cgggggacca

	81961	gtcacagcct aggctactgc aggcgcacag
		ctccccggga gcccggccca cgcgggtgtg

	82021	ccgctgagcc tccagcctgt cggggcaggg
		gtggggggca gggatggggt cgttagcggg

	82081	gttgggggca gacgcccagg cagactctct
		gggcacagct ccggtgacaa gggaggtctg

	82141	gcaagcctgg gccccttctg tccagccacg
		ccagctctgc cctggccagt cttgccccct

	82201	ggcagtgctg gggatggaag ggggagcggg
		tacctcagtc tgggggccct gcctcctccc

	82261	cagccccgcc cggcccccta ggcctagggg
		cagagtctag gggtcaccct ggggagctgc

	82321	tgaatccgcg ggtttaggaa ccggagggac
		ctgggctttt gaaccacgtg gccctaggtg

	82381	agccctccgg cgcctcggta gccctcaccc
		ccagccttgt ccaggtgggc gggtgggagg

	82441	cgacagtgcc cactgctggg ctgaacagcg
		tctgcaggga ggccaggaga gctgggcaca

	82501	cggacacgtt ccatcacctg gagctgccac
		tgtgccactt gtgcggggtc aggcggggtc

	82561	tgagccgggc tgtcatctgt cacgccacag
		atatgcaggg ggcactcggg gtcgcctcgg

	82621	acatgcttat ccctggacgg ctgttggcag
		ggccgggaag gctctgtaaa tatttatcca

	82681	tcccagctca cagctttcag ggttgatgaa
		agccccgccg cccgcccact gtgggggacc

	82741	ccgccttccc ttctggagcc agcggggtga
		gggggtgggg gagatggacc tgcctgccca

	82801	ggagcaggcg gtgtgactct ggcaggtcac
		ttgacctctc tgagcctcag ggagggcccg

	82861	ggatggtgtg cggatgctct ctgccttcct
		cccagcctga ccagtgtcct cccctcgggg

	82921	tcgcctcctg cccaccgcag agggggtggc
		tatggggacc tgggccgatg gcaggcaggc

	82981	cggagagggc atgcccggct cagccgtgcc
		cagcacttcc cagtccaggg gcccccgcca

	83041	ctcccagccg ctggctgcct cccattttcc
		cgattgcagg ttggccccga ggctgaccgg

	83101	agcctctggc tcagctggga gactgaattc
		cccaagcaat tcctcaagga tgtgtgaggc

	83161	tgtggtgtgg tgcctatccg ggagaggtgg
		ggtgagcgga ctgggcacct ccgcccaggg

	83221	caggcccagg gagacgctgg ctgacgagca
		ggcaggcctg caaggaggac gagcagccat

	83281	ctcaggaatg tgggttttgg agacaagcca
		cagctggggg ggtggggggg ccatgggtgg

	83341	ggaggcctga tccccaggtc taggtccagc
		tctgggctcc ctcgccgtgt gaccctgggc

	83401	caagacctgg acctctctgg gccccgtctc
		ttcccctggg aggtggggcg atgcctgctc

	83461	cccaatcccc cagggctgtg gatgaggcag
		acgaggtgtg tgctcatccc cacctcactg

	83521	ccttccagca gccccgggcg gggggggtgg
		tggggactgg cgcacccagg tgaggatcag

	83581	gccttggagc tagggagggc cccccagccc
		caggccagaa aggacacggg gagacagaat

	83641	gcaggagggc ggcagagcag gggccagcgg
		tggggaaact gaggccaaga gcctgtggac

	83701	gatgtgctcc aggaaaggac ctcgctgcct
		ggggcctgga tcctagagcc tccaggagcg

	83761	gtgaccatga cgtgggcagg gaaccggagg
		ccccggcttg caggtggacc cggcgcgagt

	83821	cactcttcct ctctggccct gagagcttcc
		ttccagctgc cgctcctgtg ttctaatgtc

	83881	aagtctggag gcctgggggg caggtggggg
		ctgactgcca ggtgggggag ggcaggaatt

	83941	tggcagagca gcgtcccaga gtgggagaag
		ccagcccatg gaggggactc tctccatgcc

	84001	tgctgcccca aagggcgtta tagagagagg
		tcggttaccc cttcgccatg gccccgttcc

	84061	cattgaacag atgggaaagt ggaggctgag
		agaaggctgt gacttgccca gggtctccgt

	84121	ggcatggaac tgggcctgct gagtctcagg
		ccggggatct cgctgctgca ctgagcacgc

	84181	caggatgcag gggtctgggc ctggacctag
		cgcctcgtgg gggcaagaga ggaaggcacg

	84241	ctgggcctgc ctgtcaccct ccaccccacc
		gtggcttgtt gctcaggcct tcctgggggc

	84301	agaggagagg ggagatttca ctcgctggca
		ggctaggccc tgggctctct ggggctccgg

	84361	gggaacaatg cagccctggt ctttctgagg
		agggtccttg gacctccacc agggttgagg

	84421	aaaggatttc tgttcctcct ggaggtcacg
		gagccgacat ggggaggagc aggggcaggc

	84481	ccggggccca catcctcagt gtgagacctg
		gacgtgtgtc ctcccacctg acgctggggg

	84541	tggggggtgg gggccggggg ggatccagtg
		aaccctgccc ccaaattgtc tggaagacag

	84601	cgggtacttg gtcatttccc cttcctcctc
		ttcgtttgcc ctggtgggga cagtccctcc

	84661	cctggggaag ggggacccca gcctgaagaa
		cagagcagag ctggggtcag gggtgtgctg

	84721	ggagcgcaga gagcctcctg ctctgcctgc
		tggtcattcc tggtggctct ggagtcggca

	84781	gctggtgggg agcggctggg gtgctcgtct
		gagctctggg gtgcccaggg cctgggagag

	84841	ttgccagagg ctgaggccga gggtggggcc
		ctggcggccc ggctcctgcc ccaaatatgg

	84901	ctcgggaagg ccacagcggc actgagcaga
		caggccgggc cagacgggcg ctgaggctcc

	84961	cggcctctcc cccagctccg ctgtgaccct
		cacctgcggc ccggggtgcc agggcccccg

	85021	cttggttctg ccgtgtcttt gcaggctgat
		cccacgggct ctccctgcct ctctgagctt

	85081	ccgccttttc caggcagggg aaccgcgacc
		tccaggctgg gacgcgggga gggtgtatgc

	85141	gccaggtcag aatcacccct ccaccgggag
		agcgtggtcc aggggccctg gcagggtggg

	85201	gaccgagcat ctgggaactg ccagccaccc
		ccacccatgc agaggggaca tacagaccac

	85261	acggaggctg tgcctccgct gcagcaactg
		gagaacaccc agccgcggcc aaacataaat

	85321	aactaaataa taaaagtttt aaagatcgtt
		acttaaaaaa acaagtgtgc cccagtgatc

	85381	ggaccccagt tcccggtgcc ctgagtggtg
		ccggccctgt gctgagcatg gcctggttgg

	85441	ttcaccccca gatccacact aaagggtggg
		atcaccccta ctagtcaggt gagcagatgc

	85501	agggggggag ggcggcagcc cctccatgct
		ggtgggtggc cgtggtgggt gtcctgggca

	85561	ggagccagct cacggagctg gagaggacag
		acctgggggg ttgggggcgc ccaggaagaa

	85621	acgcaggggg agaggtgtct gccgggggtg
		ggggtccctt cgaggctgtg cgtgaagagg

	85681	gcaggcgggc ctgcagcccc acctacccgt
		ccccggccca aacggcggga gtaagtgacc

	85741	ctgggcacct ggggccctcc aggagggggc
		gggaggcctt gggatcagca tctggacgcc

	85801	agtcagcccg cgccagagcg ccatgctccc
		cgacggcctc cgctggagtg aggctgcgct

	85861	gacacccaca ccgctgaccc gggcctctct
		cccgctcagg atgccccccg ccgccacccc

	85921	gtgagcagag ggccacagcc ctggcccgac
		gcccctcccg acagtgacgc ccccgccctg

	85981	gccacccagg aggccctccc gcttgctggc
		cgccccagac ctccccgctg cggcgtgcct

	86041	gacctgcccg atgggccgag tgcccgcaac
		cgacagaagc ggttcgtgct gtcgggcggg

	86101	cgctgggaga agacggacct cacctacagg
		tagggccagt ggccacgagc tggcctttga

	86161	tctccacctg ctgtctgaga cacgctggag
		ctggggggag ggcagatccc tatggccaac

	86221	aggctggagt gtcccccaac tcccgtgccc
		actgctcaac accccaaacc cacacttaga

	86281	tgcactccca tgccctccct tgggagcacg
		gtctccacac ccacctggcc accccacaca

	86341	cccgtggggc acggccgtta gtcacccacg
		caacctctgc gggcaccgtg ctgcgggcca

	86401	ggccctggga ctctcagtga gggaggcaga
		cacggcccct cctccggggg agcgaggtgc

	86461	tccccacgcc cggttcagct ctagcaccgc
		actcgggacc ctcacaggga gggacccact

	86521	ggggcaggcc aggtgacggc tcgggtgacc
		tcggcccctg gcgctgagac tacacttcct

	86581	gcagtgggcg gcgaagatgg gtgtggtgtc
		ccacgtcgtt gcagcgggga ctcctggggc

	86641	ctcggaagtg tcctgggcgg ggagcctggg
		gagcaggaag ggcaggtctt ggggtccaag

	86701	gcctccccac ggtcaggtct gggagggggc
		ctcggggctc ttgggtcctt tccgcccagt

	86761	gcagaccctc gcggccacct aagggcacac
		agaccacaca aagctgtgcc catgcagtgt

	86821	ggggagtggt gcgcaccctc agagcacact
		gggcccacat cacgcacgcc tgccccctca

	86881	ctgtgcatcc ggggaaactc ctggccccga
		cagccagcgg ggctgacgct accccgtgag

	86941	ccagacccag gcccccctca ccgcccctgt
		cctccccagg atcctccggt tcccatggca

	87001	gctgctgcgg gaacaggtgc ggcagacggt
		ggcggaggcc ctccaggtgt ggagcgatgt

	87061	cacaccgctc accttcaccg aggtgcacga
		gggccgcgcc gacatcgtga tcgacttcac

	87121	caggtgagcg ggggcctgag ggcaccccca
		ccctgggaag gaaacccatc tgccggcagc

	87181	cactgactct gcccctaccc accccccgac
		aggtactggc acggggacaa tctgcccttt

	87241	gatggacctg ggggcatcct ggcccacgcc
		ttcttcccca agacccaccg agaaggggat

	87301	gtccacttcg actatgatga gacctggacc
		atcggggaca accagggtag gggctggggc

	87361	cccactttcc ggaggggccc tgtcgaggcc
		ccggagccgg gcccgggctc tgcgtccgct

	87421	ggggagctcg cgcattgccg ggctgtctcc
		ctcttccagg cacggatctc ctgcaggtgg

	87481	cggcacacga gtttggccac gtgctcgggc
		tgcagcacac gacagctgcg aaggccctga

	87541	tgtccccctt ctacaccttc cgctacccac
		tgagcctcag cccagacgac cgcaggggca

	87601	tccagcagct gtacggccgg cctcagctag
		ctcccacgtc caggcctccg gacctgggcc

	87661	ctggcaccgg ggcggacacc aacgagatcg
		cgccgctgga ggtgaggccc tgctccccct

	87721	gcccacggct gcctctgcag ctccaacatg
		ggctcctcct aacccttcgc tctcacccca

	87781	gccggacgcc ccaccggatg cctgccaggt
		ctcctttgac gcagccgcca ccatccgtgg

	87841	cgagctcttc ttcttcaagg caggctttgt
		gtggcggctg cgcgggggcc ggctgcagcc

	87901	tggctaccct gcgctggcct ctcgccactg
		gcaggggctg cccagccctg tggatgcagc

	87961	cttcgaggac gcccagggcc acatctggtt
		cttccaaggt gagtgggagc cgggtcacac

	88021	tcaggagact gcagggagcc aggaacgtca
		tggccaaggg tagggacaga cagacgtgat

	88081	gagcagatgg acagacggag ggggtcccgg
		agttttgggg cccaggaaga gcgtgactca

	88141	ctcctctggg cacagctggg aggcttcctg
		gaggaggcgg ttctcgaagc gggagtagga

	88201	taaaaggtat tgcaccccat gaagcacgtg
		tgatccttgc ccctagagac aaggctctgg

	88261	ggctcagagg tggtgaagtg acccacatga
		gggcacagct tggagaatgt cgggagggat

	88321	gtgagctcag tgtgccagag atgggagcct
		ggagcatgcc aaggggcagg gcctgctgcc

	88381	tgagagctgg cactggggtg ggcagccaag
		tgcagggatg gagcgggcgc ccaggtggcc

	88441	tctttgctgc tcagaacgac ctttcccatg
		tatacctccc agcgccgctg gcattgccca

	88501	gtgtccttct tgggggcagg agtaccaagc
		aggcattatt actggccttt tgtgttttat

	88561	ggacaacgaa actgaggctg ggaaggtccg
		aggtggtgtt ggtggcggaa ggtggccgct

	88621	gggcagccct gttgcagcac acacccccca
		cccaccgttt ctccaacagg agctcagtac

	88681	tgggtgtatg acggtgagaa gccggtcctg
		ggccccgcgc ccctctccga gctgggcctg

	88741	caggggtccc cgatccatgc cgccctggtg
		tggggctccg agaagaacaa gatctacttc

	88801	ttccgaagtg gggactactg gcgcttccag
		cccagcgccc gccgcgtgga cagccctgtg

	88861	ccgcgccggg tcaccgactg gcgaggggtg
		ccctcggaga tcgacgcggc cttccaggat

	88921	gctgaaggtg tgcagggggc aggccctctg
		cccagccccc tcccattccg cccctcctcc

	88981	tgccaaggac tgtgctaact ccctgtgctc
		catctttgtg gctgtgggca ccaggcacgg

	89041	catggagact gaggcccgtg cccaggtccc
		ttggatgtgg ctagtgaaat cagtccgagg

	89101	ctccagcctc tgtcaggctg ggtggcagct
		cagaccagac cctgagggca ggcagaaggg

	89161	ctcgcccaag ggtagaaaga ccctggggct
		tccttggtgg ctcagacagt aaagcgtctg

	89221	cctgcaatgc gggagacctg gattcgatcc
		ctgggtcagg gagatcccct ggagaaggaa

	89281	atggcaatgc cctccggtac tgttgcctgg
		aaaattccat ggacagagca gcctggaagc

	89341	tccatggggt cgcgaagagt cagacacaat
		ggagcgactt cactgtctta agggccacct

	89401	gaggtcctca ggtttcaagg aacccagcag
		tggccaaggc ctgtgcccat ccctctgtcc

	89461	acttaccagg ccctgaccct cctgtctcct
		caggcttcgc ctacttcctg cgtggccgcc

	89521	tctactggaa gtttgacccc gtgaaggtga
		aagccctgga gggcttcccc cggctcgtgg

	89581	gccccgactt cttcagctgt actgaggctg
		ccaacacttt ccgctgatca ccgcctggct

	89641	gtcctcaggc cctgacacct ccacacagga
		gaccgtggcc gtgcctgtgg ctgtaggtac

	89701	caggcagggc acggagtcgc ggctgctatg
		ggggcaaggc agggcgctgc caccaggact

	89761	gcagggaggg ccacgcgggt cgtggccact
		gccagcgact gtctgagact gggcaggggg

	89821	gctctggcat ggaggctgag ggtggtcttg
		ggctggctcc acgcagcctg tgcaggtcac

	89881	atggaaccca gctgcccatg gtctccatcc
		acacccctca gggtcgggcc tcagcagggc

	89941	tgggggagct ggagccctca ccgtcctcgc
		tgtggggtcc catagggggc tggcacgtgg

	90001	gtgtcagggt cctgcgcctc ctgcctccca
		caggggttgg ctctgcgtag gtgctgcctt

	90061	ccagtttggt ggttctggag acctattccc
		caagatcctg gccaaaaggc caggtcagct

	90121	ggtgggggtg cttcctgcca gagaccctgc
		accctggggg ccccagcata cctcagtcct

	90181	atcacgggtc agatcctcca aagccatgta
		aatgtgtaca gtgtgtataa agctgttttg

	90241	tttttcattt tttaaccgac tgtcattaaa
		cacggtcgtt ttctacctgc ctgctggggt

	90301	gtctctgtga gtgcaaggcc agtatagggt
		ggaactggac cagggagttg ggaggcttgg

	90361	ctggggaccc gctcagtccc ctggtcctca
		gggctgggtg ttggttcagg gctccccctg

	90421	ctccatctca tcctgcttga atgcctacag
		tggcttcaca gtctgctccc catctcccca

	90481	gcggcctctc agaccgtcgt ccaccaagtg
		ctgctcacgt tttccgatcc agccactgtc

	90541	aggacacaga accgaactca aggttactgt
		ggctgactcc tcactctctg gggtctactt

	90601	gcctgccacc ctcagagagc caaggatccg
		cctgtgatgc aggagtgagt gaagtcgctc

	90661	agccgagtcc gactctttgc aaccccatag
		gactgtagcc taccaggctc ctctgtctat

	90721	gggatttttc aggcaagagt gctggagtgg
		gttgccattt ccttctccag gggatcttcc

	90781	caaccctggt ctcccgcata gcaggcagac
		tctttactgt ctgagccacc aggcaatgca

	90841	ggagacctag gttcagtctc tgggtgggga
		agatcccctg gagaagggaa tgacaacctg

	90901	cttcagtatt cttgattggg gaatcccatg
		gacaaaggag cctggaggcc tacagcccat

	90961	agggtgcaaa gagacacgac tgagcaagtc
		acacacacag agccctacgt ggatgctcat

	91021	agcggcacct catagctgcc atgtatcagg
		tgttggcatg ggcagccatc agcagggggc

	91081	catttctgac ccactgcctt gttccaccgg
		atacacgggt gccttcctgt gtgtcgggcc

	91141	cactcggctg tcagcgccca agggcagggc
		tgtcgggagg cacagggcac agagttaagg

	91201	aggggatggg gacgttagct cctccccagc
		tctcagcgga tgcagcaggc aaaacaaacg

	91261	ctaggaatcc tgccaaaccc ggtagtctct
		gcccatgctc gccccatccc cagagccaca

	91321	agaacgggag ctggggggtg gcccggagct
		gggatactgg tccctgggcc cgcccatgtg

	91381	ctcggccgca cagcgtcctc cgggcgggga
		aactgaggca cgggcgcctc cggcttcctc

	91441	cccgccttcc gggcctcgcc tcgttcctcc
		tcaccagggc agtattccag ccccggctgt

	91501	gagacggaga agggcgccgt tcgagtcagg
		gccgcggctg ttatttctgc cggtgagcgg

	91561	ccttccctgg tacctccact tgagaggcgg
		ccgggaaggc cgagaaacgg gccgaggctc

	91621	ctttaagggg cccgtggggg cgcgcccggc
		ccttttgtcc gggtggcggc ggcggcgacg

	91681	cgcgcgtcag cgtcaacgcc cgcgcctgcg
		cactgagggc ggcctgcttg tcgtctgcgg

	91741	cggcggcggc ggcggcggcg gaggaggcga
		accccatctg gcttggcaag agactgagnn

	91801	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	91861	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnct gcaggtgccg gcggtgacgc

	91921	ggacgtacac cgcggcctgc gtcctcacca
		ccgccgccgt ggtaaccgcc cccgggggtt

	91981	gccaaggtta cgattggacc ctccccgccc
		cgaccctgct cccctagggt gggtgggtcg

	92041	gggggcagtt tctaagatct cctggttccg
		cagcagctgg aactcctcag tcccttccag

	92101	ctctacttca acccgcacct cgtgttccgg
		aagttccagg tgaggccgcc ccgccccttg

	92161	cacttgctgg cccaacccct cccgcccagc
		gctggcctga ccgcccccca ccccgcccac

	92221	cccacgcagg tttggaggct catcaccaac
		ttcctcttct tcgggcccct gggattcagc

	92281	ttcttcttca acatgctctt cgtgtatcct
		gcgccgtggt ggaagcggga ggagggcggg

	92341	gcgggggacc gggcgggagg cagcgggccc
		cgggaagctg agaccctcca aggggcacgc

	92401	ttcctatacc aaagccgcag gttccgctac
		tgccgcatgc tggaggaggg ctccttccgc

	92461	ggccgcacgg ccgacttcgt cttcatgttt
		ctcttcgggg gcgtcctgat gactgtatcc

	92521	ttcccgggct cggggaccta tgggtccggg
		cctctgctgg ccctgaggcc ctgcttgagc

	92581	gcatgccaca gagggagagt tgcgaccccg
		agctgagggt gtttttgagc gtacatcacg

	92641	tgctcagctg caggtgcccc tgtcgaactc
		cagggctaca cccaaaatac cacagggcag

	92701	ggtgcccagg ggctgagtcc tgaatgcagg
		tagccaggag gatctagggc tgggcccggg

	92761	ggctggggtg aagtggagag gcagggccga
		tcagggggcc cctggaggcc accgtttggt

	92821	cttagagtgg gaagcgaaac caacctgctt
		gagggtttca ggggtttagg aagtcagagg

	92881	ggccctgggc agggcacaag accttgactc
		tggcccagct actggggctc ctgggtagcc

	92941	tcttcttcct gggccaggcc ctcacggcca
		tgctggtgta cgtgtggagc cgccgcagcc

	93001	ctggggtgag ggtcaacttc tttggcctcc
		tcaccttcca ggcgccgttc ctgccctggg

	93061	cgctcatggg cttttcaatg ctgctgggca
		actccatcct ggtggacctg ctgggtgagc

	93121	ctgctgtcca gggagcctgc cccaagctgg
		gtgtgctggg ccagagccct ggtcctctcc

	93181	ccgcccccac ccctcttccc cactcctggc
		gcccccatcc ttccagcccc tccaacaagt

	93241	cagcctatag gttttactta ttcgagcctg
		acccatttgc tgacgcttgt gtggggcccg

	93301	acccggtagg gatgggtggc tcagggtgcc
		tgctcacagc tccacttctt ctgacgtcct

	93361	caggcctgac ctcctcccag gttctgccta
		ctctgggcca agcctggccc cacgctgggc

	93421	tggctggccg tgcagggcat cagaccccca
		tgctttgggg gcttcagggc tgtggagggt

	93481	ggcctcggca ttggcgcctc tcccacaggg
		attgcggtgg gccacgtcta ctacttcctg

	93541	gaggacgtct tccccaacca gcctggaggc
		aagaggctgc tgctgacccc cagcttcctg

	93601	tgagtgctga cagccttccc cacccccttc
		cccagatggc tctctacccc atgagggggg

	93661	gggaccctgc cagctgccgc tcagcgtggg
		ctcctcccca caggaaactg ctactggatg

	93721	ccccagagga ggaccccaat tacctgcccc
		tccccgagga gcagccagga cccctgcagc

	93781	agtgaggacg acctcaccca gagccgggtc
		ccccaccccc acccctggcc tgcaacgcag

	93841	ctccctgtcc tggaggccgg gcctgggccc
		agggcccccg ccctgaataa acaagtgacc

	93901	tgcagcctgt tcgccacagc actggctctc
		ctgccgcggc cagcctctcc acgcggggca

	93961	ggtgctgctg gccgagagcc agggccacca
		agcctgacgt gctctccgac ccagaacatt

	94021	ggcacagctg gaggcccaga gagggtccag
		aacctgccca ctcgccagca gaactctgag

	94081	cacagagggc agccctgctg gggttctcat
		ccctgccctg cctgtgccgt aattcagctt

	94141	ccactgatgg ggctcacatc tcaggggcgg
		ggctgggact gggatgctgg gttgtgctga

	94201	gctttggccg tgggggccct cctgtcccga
		actagcaacc cccaagggga cctctgcttc

	94261	atttcccagc caggccactg aaggacgggc
		caggtgcaga agagggccag gccctttctg

	94321	tgactccgaa gcctcaagtg tcagtgtttg
		cagagtccag tggctgaggc agaggcctct

	94381	gggaagctct gcccctgccg tttgcagctg
		aggccggcag gagcctcacc tggtccccag

	94441	ctcacgggca ttggaggacc agtccgcacg
		gtggtttact cctgggtcgg caccagccgc

	94501	cgccggctgt ccctttcaca gaggataaaa
		gtactcgctc tggagttgga ctttaatgtt

	94561	gtcatgaaac ctctggccca gcagcgggct
		ccgcagtggg tggcaggtga aggcccctcc

	94621	ccgggcctct ccaggcaggt gccgcctggc
		cagcagggaa ggcaggcagt gtcatccccc

	94681	actggctctg gggctcaggc tacctcctgc
		tgtggccgga acatctcccc cagtggtgga

	94741	gcccagtgtc cgtgaggcca gctgggcctg
		aaaccttcct ctctgaagcc ccgctgtccc

	94801	cttgccctgt atggagggca gaggctggag
		cgcaagttcc taggatgtgc ttgcgagacc

	94861	cccgagccca ggggcgaggc ccatctcagc
		ccacccccga actggaaacc cttggagctc

	94921	tgcccctcgt ggtgtgaggc ccctgctatg
		cgaccctcag ccctgccagc aacggaaggt

	94981	gcagggcccg ggcccacggg cttaacgcaa
		ctgggcctgg gtcacctgcg gggcctggtc

	95041	ccaggaggaa gacccaggtg ccaccctcct
		gggtgccacg tccaggtcac gtggggaccc

	95101	gtccatgtca cagaagatgc agggtcaccc
		ggtgagctgg cgccgggccc tgccagagca

	95161	ccagccgcgg gtggaggtgg gccccagctc
		tcctgtcagg cacgtggtgc tgggaggtgc

	95221	ggccggagca gtgcccacca gctgcagcag
		gacaggtggg cacaggccca ccagcagtgc

	95281	ccgcacggga tgggcccctg caagggccag
		agaagccacg ctcctggctg ggggctgggc

	95341	tgggactgac aggtggccct gccctctgcg
		ccccactact tcccagccac ccgggactcc

	95401	aaggacttgc tgagctgggc aggtgggacg
		ccgaggggag tcaaactgct cgtgggggca

	95461	ggaggggcgg tccacagggc tgagccctga
		gctgaaccct ggccctgctc gtggttgtgg

	95521	gggtgggggg gtccagtggc gccctagccc
		tgctgaggcc cagctgggac gtgcgcgccg

	95581	gagggcgagg ggccagccca tgccatgctg
		tcccccgttc tcagctccat gctaccactt

	95641	tgaagaaaca gaacctgttg cctttttatt
		tagaaagtgt tgcttgccct gcctggggct

	95701	tctatacaaa aaacaaacac agctcaacgt
		ggcctctcct gaccagagac gggcggtggg

	95761	gactggggct cagcagacgg aatgtgtccc
		cggcggcggg agaccaggag gcccctggcc

	95821	cgctcctcag gacggctggg ctgtccccac
		ctggtcccct ccgagccaga agatggagga

	95881	gaggtgggct gatctccaga tgctccctgg
		gagccaagcg ccacggggtg gtcaccaggc

	95941	cggggccgtg ttggccagac gcctcatccg
		cctgtgggag ggggagggca gcaacccccg

	96001	gatctctcag gcaaccgagt gaggaggcag
		gagcccccag cccctccctc ggccgctctg

	96061	ctgcgtgggg ccctgaagtc gtcctctgtc
		tcgcccccct ccccagggag agtgagcctg

	96121	ttctgggctg tggtcagacc tgcccgaggg
		ccagcctcgc ccggggccct gtcctgcctg

	96181	gaaggggctg gggcagcacc ttgtgttccg
		gtcctggtcc cggatcttct tctccatctc

	96241	tgcatccgtc agggtctcca gcagcgggca
		ccactggtca gcgtcgcctg tgttccggat

	96301	ggcaatctcc accgtgggca gggggttctc
		actgtggagg acgagagagg tagacggctc

	96361	acagagcagc tgcaggagag gcccctagaa
		agcagtgtcc accccgctgc gggcagacag

	96421	gacatggagc ctggtttctg cacccggctc
		ccgacacagg gcggccgggc acgctgccaa

	96481	catggcatct ccgggtctgc atgtggggag
		gggtccacag gacagtgctg caggtccagc

	96541	cattcccagt ggacttgctg ggaggaggag
		ggccgtccgc cccgctcagt gtccaggaga

	96601	aaggagagca aaggagtcca tccacccagg
		agtggagtcc cagggcccct gccctgacca

	96661	gcctgcaggg ggcccctcgg cccacatcac
		aggggcccag aatccataag ccctgactgc

	96721	tccaccccgg ggcccctcaa agacgcgcct
		agactccgtc cgagggccac ctgcacaccc

	96781	tctggcgaag tggactcagg gctgggggtc
		agcctcggtg aggccgcaaa ggctggggac

	96841	tcctggccga gctgctgcct ctgccaggag
		ccaggcccag cctgccggcg agcctcagcc

	96901	acgccctcac ccaccctgcc cgcggcgcca
		cgctggcctc cgggtcctct cctctggcct

	96961	cctgctgggc cactggtgct cagccccagc
		agtcggcctg ccaggagccc tgcagagtca

	97021	gcccccagag ggaggagggg gcccggggga
		acagcacagg aacaaacaga cccctggcct

	97081	tagttttagc tcctcatctg gaaaatgggg
		acagtgtcct tgctgcgagg ggtttcagag

	97141	gaccactgcc atgcaacacc cagcacacac
		ccactgcgtg ggggctcggg cccgagccgg

	97201	tgcccccgag tcccaggctg gtggctgggc
		cgccccagcc accctgccga cagctgcttc

	97261	ccagccgggc ggtgctgcgg cagtccagaa
		gccagcactg cagacccaaa tgtcactcct

	97321	cacgttgcgg gctcccagct gccttccttg
		ggggcagcag acacgaaagt caccaagccc

	97381	acgccgacgg gagcaaacac gtcttcctct
		taaacaagtg cgggtcccgg aggccctgtg

	97441	tttacctccc tgtggctccg ggaagattgc
		atcccagggg gttgttctaa accaagggct

	97501	gctcgggcca ggcctggaag gaggggcctg
		gagccaggag cccaccctta cgggcattcg

	97561	gcttcctggg tctcaaggcc ggctgggacc
		ctgcattccc accacccgcc aggtgcaagc

	97621	agggaggccg tgtcggagga ggcagagggc
		ctggagggtc gtcttcgacg tgacctcact

	97681	tttacaacct cacaggtgcg gcaggccagc
		tgggaggcat ggctgtgccc tcctggtaga

	97741	tgagaacaag actgcaggga gtgatccccc
		tgaacttccc caaccaggag gagacaaaac

	97801	tcggtgtcgc cctcctgctt aagatcaact
		gactctggac aaggggccca gcccacccga

	97861	tggggaaagg gcagtccttc caacaagcgg
		tgctgggacg ggacccggca ggccatggtt

	97921	tctcagctat gacaccagca gcacaagcac
		cccgagaaaa acagctaagc tgggcactgt

	97981	cacacaagtg aactccaaac ccaagaaaac
		cacaaaaagc ctgcggatct tcagatatgt

	98041	gggaagggac ctgtatctgg aatgtataac
		gaactcctga aaagtgaaag tgttagtcac

	98101	tcagtctgtt cagctctttg caaccccatg
		gacggtagcc tgccaggctc ctctgcccat

	98161	gggattctct aggcaagaat actggagtgg
		gttgccatgc cttcctccag gggatcttcc

	98221	caacccaggg attgaacctg tgtctctctt
		gcactggcag gcgggttctt taccagtagc

	98281	gccacctgag tagaaacact ccaggtgccc
		tgagtgtcag agcaggaggg actcggccca

	98341	ggcctgtgag gggaccctct ccgagtcccc
		tgctgcacag cagtgagagg tgcgttctga

	98401	gtcagcctcc agggatgagg gacttggtgt
		cgacatcact cccaggacct caggatctgc

	98461	tctgggaagc gaggctcccc aggctggccc
		caggcccgct ggcctcagct cgtgagccgt

	98521	gcgtggacag gtgccatgag caggcctccc
		acgggactcg gggcgcggcc tggaccccgg

	98581	ggctgccagt ggtcgcgggg ggccccgtgt
		ggcggctgtt ccctctcttg ctccgagtcc

	98641	taggaacatg gtgggcgctg cctcctgggg
		tttctggaga agcagctgag atgcaaacag

	98701	ccccacgcgc tccctcagct gttccctgtc
		acgggtggcc ccttggtgac ggcctccatg

	98761	cagggacggt gacagctcga gcagccgcgt
		aaaaccacac ggggacggtg gcagctcgag

	98821	cagccgcgta aagcctgaca tccaatttgg
		aagcctcccg cagtggaaga ggggcccggg

	98881	gacggggctg cccggggcga gctccaccgg
		gtcgggggtc acgaggagcc cacccgcgtc

	98941	cccgccacca gcacctggga ccagataccc
		tccccgctct gagggcggcc tgaacgccgc

	99001	cccctcccac gggggcgccc accgcctgct
		cgtggactga acaagaggcg gcagtggcct

	99061	ccagaccccc tcgggggagg gcagacctgt
		ccgagactga gcacaagtcc agggaatgag

	99121	caagggtctc agtaatgtcc ccaccgggac
		gggacgggag gaggcgacag aggccgctga

	99181	ggtgcggggc agccctcagt agctggcatc
		aaggccccag gcagtcccgg ggcatccccg

	99241	cagggggcgg gggcgaccac cggcccgagc
		ccaggcagtc ccggggcatc cctgcagcgg

	99301	gcgggggcga ccaccggccc gagccctacc
		tgaaggcgta ggtcttctga tgccagctca

	99361	gctgtccccg gatgctgtag gcgatggtgg
		tgacgaactc cccgcccagc cccagctcgg

	99421	agcacagctt cagagcgaac ttctcgggcg
		agttctcctt ctccgacatg tcccactcga

	99481	actggtccac caaggagatg ttccccacgt
		ggatgttcag ctggcccggg agcacagaca

	99541	tgagccagag cggccccctc tggggccagg
		ccgcaccctc accacccctt ctccccggaa

	99601	catccccgcc tcgttcttgg ccgcgcccct
		gtgctgctac ttggggtaag gaaaacaacc

	99661	cccatctctc tgaaaagggt taactagcga
		ggaagatgcg ctggtaactg gaaaactccc

	99721	tacaaagaaa gcttggatct gatggcttca
		ctggtgaatt ccaccaaaca tttcaagcac

	99781	taacaccaat ccttatcaaa tcctgccaaa
		aaactgaaaa ggaaggaaca catcataact

	99841	ccctgccttg ataccaaagc cagacaaaga
		tactacgaga aaggaaaggt gcagaccggc

	99901	acttactgtg gacattgatg tgaaacctca
		gcagacacga gcaaaactac attcaccagc

	99961	acgtcagaag aatcacacac cgttataaat
		gatgggatga tgacacaacc acattataaa

	100021	cggtggggct tactctggtg atgtaaggac
		ggctcagtaa gaaaaccggt caatgccatg

	100081	aaccacttga acagagtgaa ggacaaaaac
		cacacagtca tcttgataat tggaggaaaa

	100141	tcattagaca aacttcaacg tgctttcacg
		ataaaagcac tcagtaaact aagatcagat

	100201	ggaaaccaca tcaacaagat taattcagtc
		aaaaaattca ctgcaagtat cacccacaat

	100261	ggcagaagac tggtaacttt tcctctaaga
		tcaggaacga gccaaagata cccagtcttg

	100321	ccacttttgt tcaatatagc gttggaattt
		ctactcagtg cagtgcagtc gctcagtcgt

	100381	gtccgactct tttcgacccc atggatcaca
		gcacgccagg cctccctgtc catcaccaac

	100441	tcccggagtt cacccaaact catgtgcact
		gagtcagtga tgccatccag ccatctcatc

	100501	ctctgtcgtc cccttctcct cctgcctcca
		atcccttcca gcagttaggc aagaaaaata

	100561	aatcaaaggt atccacctgg aatggaagaa
		gtaaaactat ctctggtccg agatgttaca

	100621	atcttatatg cagagtttaa gatgctaaca
		aaatactatt agaactaatg aatgaattca

	100681	gcaaggtacc aggatacaaa gtcaacgtgc
		aaaaatcagc cgcatttcta catgctaaca

	100741	ctgcacaatc tgaagaagaa aggatgaaca
		aattacaata acataaaaaa gaataaaatc

	100801	cttagaaatt aacttgatca aagagatgta
		caatgaacaa tataaaacat actgaaagaa

	100861	attgaagata taaataaatg gaaaaacatc
		ctatgtccat ggattggaag acttaaaatt

	100921	attaagctgt caaggctatg gtttttccag
		tggtcatgta tggatgtgag agttggacta

	100981	taaagaaagc tgagcaccga agaagtgatg
		cttttgaact gtggtgttgg agaagactct

	101041	tgagaggtcc ttggactgca aggagatcca
		accagtccat cctaaaggag atcagtcctg

	101101	ggtgttcatt ggaaggactg atgttaaagc
		tgaaactcca atactttggc cacctgatgc

	101161	gaagagctga ctcatttgaa aagaccctga
		tgctgggtaa gattgagggc gggaggggaa

	101221	ggggacaaca gaggatgaga tggttggatg
		gcatcaccga ctcaatggac atgggtttgg

	101281	gtggactctg gaagttggtg atggacaggg
		aggcctggcg tgctgcggtt catggggttg

	101341	tgaggagtcg gacacgactg agcgactgaa
		ctgaactgaa catgaatacc caaagcaatc

	101401	tacaaagcca aatgtaatcc ctatcaaaat
		cccaatagca tttctgcaga aacaggaaaa

	101461	aaaatcttaa aattcatatg gaatctaagg
		aaaagcaaag gatgtctggt caaaacaatg

	101521	acgaaaagaa caacaaagct ggaagactca
		cacttcctga tttcagaact tactgcaaag

	101581	atacaataat gaaaacactg tgggactaac
		gtaaaagcag acacgtgggc caacgggaca

	101641	gcccagaaat aaactctcaa ataagcagtc
		aaatgatttt caacagagat gccaagacca

	101701	ctcagtgaag gaaagtgttt gcaaccaacg
		gttttgggaa aaaagaaccc acatgcgaaa

	101761	gaatgaagtg ggacccttac ccagccccat
		ctacagaaat caactcaaaa cagacagaac

	101821	atatggctca agccataaaa cgctcagaaa
		aacagagcaa agctttatga tgttggattt

	101881	ggcggtgatt tctcagatat gacgtcaaag
		gcataggtga taagcgaaaa aataaactgg

	101941	acttcaccaa aatacaacac ttctatgcat
		ccaaggacac taccgacagc ataacaaggc

	102001	agcccaggga aaggaggaaa catccgcaaa
		tcacagcatc tgggaacaga ccgctgcctg

	102061	tgagatacag ggaaccgata aaaacaagaa
		aacagcaaaa cccggactca aaaatgggaa

	102121	ggactccagc agacacagga gacagacaag
		ccgccagcag gtcactaatc agcaagcaag

	102181	gcccgcaaag gcccgtatcc aaggctgtgg
		tttttccagt ggtcatgtag gaaagagagc

	102241	tggatcgtaa gaaagctgag cgctgaagaa
		ttgattgaac tgtggtgttg gagaagactc

	102301	ttgagagtcc cttggactgc aagatcaaac
		cagtccattc tgaaggagat cagtcccgaa

	102361	tagtcactga aggactgatg ctgtagctcc
		aatactttgg ccacctgatt cgaagaactg

	102421	actcattggc aaagaccctg atgctgggaa
		agattgaagg caggaggaga aggggacgac

	102481	agaggatgag atggttggat ggcatcactg
		actccatgga catgagcttg ggcaagctcc

	102541	gggagagagt gaaggacagg gaagcctggc
		gtgctgcagc ccgtgggtcc caaatctttg

	102601	gaccaagcga ctgaacaata acaaatcaac
		agggaaatgc aaatcaaaac cacagtgaga

	102661	tactgtccac caccaggcag gcgttcttca
		gcggggttcg gggcaggtgg tgccctcttc

	102721	tctcgtaacg cccccaggac cgcgggggct
		gctgagacag catggggtgt gcttggccta

	102781	gcctgcccat gacaagagtg gcagtgtgct
		cgcctcactg cgcccttccc tgctctgccc

	102841	accagctggg ccacccctgg gaccacccag
		cttccgctcc gtggacggca aggccgcagc

	102901	agcgcccgga cacgcccaga acgtggtgcc
		ctcctcagaa gtcggcctgt gcccttcctg

	102961	ggacaagccg cccaagagac agtcttccag
		agccctgccc cacaacacgg accccagaca

	103021	ggctcctgtg gaggcctcca cgcacctccg
		cacctcgcaa gccccgagga caaggcaggc

	103081	ccgctgcggg tgaggagccg cctaccttga
		taatgacgcg ctggtctgac tggtcttcca

	103141	ggatgctgtc cgtggggtag gactcgatct
		gctgtctgat ggcagaggca atggctggca

	103201	cgaatgtcag tgggttcaga tccaggtcgt
		cacagagaat ctctgagaac atctccgggg

	103261	tcatcagctt ctctgaaacg atgacggagc
		gggggaaccc ccagtggacc acagggccta

	103321	cggtcagcgt gctcagcccc ggcctccccc
		agccttgcct cctctgccac cgcccccccg

	103381	ggtgacgaca ggaccccctg gcagcacgca
		gacagagctg agtgcacgcc agccagggcg

	103441	gcggacggac cattcatgtt ccaggtaaag
		gcatcccgca gcttctgccc gtcaatctcc

	103501	atgtccagtc ggatggggac cagcacctcg
		ggctgggacg cgttctcgtg gatcacggct

	103561	gggtcgtggt cgtcgaagct ggaaggggag
		cggccgcgtg ctcagcaaag cgggctgggc

	103621	ccctgtgccc agggcctccc tctctgcacc
		actggtcgct gagacctgcc cagagaggac

	103681	ctgtccacta cgggccgggc cggcagaaac
		agggctggcg ggggtccacg cggggcggga

	103741	ggggagctgc cgactcggca gcgggacaag
		ctcagaggtt ccctgcagga agagaggttt

	103801	aagccccaga gcaggcagga ttctcccagc
		agctgtgggg aagaaagggt atgtccagaa

	103861	gaagaaaccc tggaacaaag gccgaggggc
		aggagggttg aggagctgct tggagagcag

	103921	tgaagggggg ctgggcggct ggggggtgct
		ggggagcctc ggtggccaag cacccagggc

	103981	tccccacctg cagcctggac cccgagggag
		ccccagagga cggagagcaa ggcagctccg

	104041	cactcacacc tgccctttag gatggggaag
		agggaagaga cgggggctgc ggggggcaag

	104101	gaaaccaggc acgccccgct tagacccggg
		ggcgagaacc actttccaag aacgcagggg

	104161	cgccaatgat gaacaatggg tagcagcccg
		caggcgggag gcccggtggc cgaggcccct

	104221	caccagagcg ggaaggtccg cttcttgtcg
		cggcccatgc ggttcctgtt gatggtggtg

	104281	gagcagggca cggcgtccag gtggtgcgag
		ctgttgggca gggtgggcac ccactggctg

	104341	ttcctcttgg ccttctgttc cctgggagac
		acagacgccc gtccgctcag cctatgggcc

	104401	aaaagccgcc ccccagccgc caggttgtgg
		ccagtggacg cccgccatgc ccctctgggc

	104461	ccaggccccc atggggacct ctgtgcgccc
		agctccgcgg tggttattcc ccaggctcca

	104521	agcggcacct gctcggggtc accagtttta
		ggggaggagg agagggcagg ggccccagcc

	104581	cagtctgtga gctgtcaccc ccaggctcca
		agcggcacct gctcggggtc accagtttta

	104641	ggggaggagg agagggcagg ggccccagcc
		cagtctgtga gctgtcaccc ccaggctcca

	104701	agcggcacct gctcggggtc accagtttta
		ggggaggagg agagggcagg ggccccagcc

	104761	cagtctgtga gctgtcaccc ccaggctcca
		agcggcacct gctcggggtc accagtttta

	104821	ggggaggagg agagggcagg ggccccagcc
		cagtctgtga gctgtcaccc gtgctatgtg

	104881	ctgggctggg cactcaggaa agagggtcag
		ggttcacggg ggggtggcgc gcagatttcc

	104941	aggagagccc cgagggcagc agagaggagg
		ctcaggtcaa tggttgggca gggggccagg

	105001	gctggagaca cagagagggt cccgattcgg
		gggggtgccc tcagcaggtg gctgggagtc

	105061	cctgggggtt tgcacacttt cgatcaggct
		gttatttcag acgcttggtc cagcctgaga

	105121	caggtaatgc ctctggcctc cgggccttca
		gggatggaaa gatactctag aaagcgggac

	105181	tcaaagtaac tcaaggaact cgcgtcccac
		agtggggagc ccttctctcc aatttacatg

	105241	gggcgtttac tacgaggaaa ataccgaagg
		ccgttttgag ctgaggctcc cgggccgggc

	105301	tgtccgtttg tgagactgct cgtcacccct
		gggccacatc cctggtggcc aagggggcaa

	105361	tcagtgcggt gactgcacga cacacctctg
		cagccctgcc ccacagctgt caccatcggt

	105421	gacgtccacc ccctggagaa cctgaccact
		gcccggtttc ccgctaaaac agcgcccttc

	105481	caggatgggg ggcagaggga gaggccttgg
		ccttttcact cctcttctgc agcgggggcc

	105541	cctcgcaccc cagtgcccgg gcccaggagc
		gccccttggg gtggggcagg gagggatcca

	105601	cacaccaagg ggagccagga cccccccaaa
		tctgctgccc tgccctgata cccgagacct

	105661	ggggaaacgg gggactgggg ctgatgcggg
		caggaccaag aactgaggcg gtgagacggg

	105721	gtccccacca caggccatct ggctggcagt
		ttctactccg ggcctgcagg ccaagaggga

	105781	aaaggtgccc cactcagatc aggcgcctcc
		cgtccccagg gagggcctac aaggtcagat

	105841	cctttgtaac ttccacgggc aaaactggct
		tgctgggcct gtgcgggccg catgggcgtg

	105901	gaccaccaca cctttcccca ctgagtctcc
		agccggagct gtcacccagg tccccccagg

	105961	ccagccccac cccgccacct tgcagtagcc
		tctcgtatcc aggccgaggc tgcccggtcg

	106021	acccctcctg cctgatggcc tcaagtggac
		aatgcgagtc acgttgcagc acgtgagtgg

	106081	gacgggcagc gccacgcggg gtccgggcat
		ccgagtccca ccactcagcc tcccttccgc

	106141	tgcagagagg tctgtccaag agccctgggg
		gccatccagc ccctgtccga cctggccggt

	106201	gtggaagagg gggtgtgcca cccctcctgg
		ggggctggct gggcgctggg caggcccctc

	106261	ctaagagtgg agcccactgg tggttttcct
		gcagccccac ctccacacag cagttctcac

	106321	tgcccagtaa caggaggcta ctggcctagc
		tctctccctc gtgtgatgga ctcaaccagg

	106381	agcgttcacg gccccacaca gggttctcgg
		ctgctgcatg aggatctcaa agccccatcc

	106441	acgtgcatgt aatctcctcc ggtaacttct
		ctagggaagc ccggctatcc tgccatcctc

	106501	accgcaccac cagggcgaga aaagccatct
		ccagcgctca catccacaat gggccaggcc

	106561	gtgagcacac caccttcttc gggaggttgt
		gggggcgggn nnnnnnnnnn nnnnnnnnnn

	106621	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
		nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn

	106681	nnnnnnnnnn nnnnnnnnng cgcgcccccc
		ccccccgcgg cgccggcacc ccgggcggcg

	106741	gcccccggcg ctgggagcag gtgcggggcc
		gcggccgctc gtgagcctcc agcccggagg

	106801	acgggccccg ggggccggcc cggtgcccag
		gccctgggag ccccggaggc cagagtgcca

	106861	gagggccgga ggacccggga aggcccgaga
		gaggtgggaa gcacggggtt ccagccctag

	106921	gccatttcag ccccaaagcc atcggtgaaa
		ccattgctgg ccccagataa aagcgtcgcc

	106981	aactttttca ccccggcgga gactttagcg
		ggtagctgcc ccctaggggg aatggaaaaa

	107041	ccaggattta ccaggtgggt ggaggtcaca
		actgcccaga tcctgagaaa gaggggtcag

	107101	tggggcggga agattagtgg ggagaggagc
		tttcagaacc caagggaatg aaacgaggct

	107161	tgaggttggt tatccagcag ccgccccctg
		ccccgtgagt gagcgaaggc tgggcccctt

	107221	attgtcacat cttccagctc ttcgctagaa
		aacctagagt tttaaatact gtggcagctg

	107281	agtcaaacaa taaggaaaag cccgactctt
		tgagagccag gcacaaggcg tctgtgacag

	107341	ggtctccagg ctgcccattt gcagtctctg
		aaacggaggg tttttcgaga aggaggtctt

	107401	ggggtgcctg ccagaattgg aggggggggc
		gcgggaagtg aggacccaga agagagggct

	107461	tggcccgctg caaggaggtc actggacact
		ggagctgaag cgccagccga aactggaaac

	107521	tcgaaatctg tctccgtgcc agccacaagg
		cctatgattt tccttggcga cgttcagcat

	107581	cttaggagga gctggcgggg gaggcgggta
		gttcgtgggc ggttgcagca gggcaggaag

	107641	gtgaggaacc tgaggctggt cagagagctg
		gttggagtga tgcccatcgg tggacccgct

	107701	ggagaaggcc tgagtagaga aggtctaagc
		ttaacgggga aggggtgggc cagggtggaa

	107761	atggggtggg aagtttgagg agggggagca
		gtggagatgg gggttgtgag gaatgggagt

	107821	gagcttagac gtcttgagga tactgcagtt
		ctgtgctttt tttcacacct ggctgaaaat

	107881	tcactgaaaa caaaacaacc cttgctctgt
		gacagcctag aggggtggga gggaggctta

	107941	agagggaggg gacgtgcgtg tgcctatggg
		cgattcatgt gggtgtacgg cagaaagcaa

	108001	cacagtatgt aattaccctc caattaaaga
		tcaagtacaa cttaaaaacc ccaaacacaa

	108061	cattgtaagt cagctagact ccagtaaaca
		tttcagtaag aagattcaac tgggaatgag

	108121	ttccgccgtg actatcctga tgaatttccc
		gtgtcttctt gaggccattc ctctttgaac

	108181	ttccgtgttt ggggaagcgt gcctttgtat
		ggagtcctga ggagtaaatg agacgggctt

	108241	gtagaaggcc tagtagtgcc ttgcacgcgg
		cagatgctca ataacctcga gttgtcacca

	108301	ttatggtacc tcaagagtct ccttggagct
		tgcacggttt ctgaatgggg tcctgcgggg

	108361	ctcccttggg gctcccacat ggggttgggg
		ggctgagtgg ggtgtccccg ctccttgctt

	108421	gtcccctgtg gaacaccccc ttccacccga
		gcagctctgc ttttgtctct tgtgtttgtt

	108481	tatatctcct agattgttgt tcagtcgctc
		agtcgtgtcc aactctccga ccccatggac

	108541	tgcagcacac caggccttct gccttcacca
		tctcccggag cttgctcaaa ctcctgtcca

	108601	ttgagttgct gatgccgtcc aaccatctcg
		tcctctgtcg tccccttctc cttttgacct

	108661	cagtctttcc cagcatcagg gtcttttcca
		atgagtcagc tctttgactc aggtggccaa

	108721	gtattggagc ttcagcttca ttatcagtcc
		ttccaatgaa tattcagggt tgatttcttt

	108781	taggattgag tgacttgatc tccttgcagt
		ccaagggact ctcaagagtc ttcaacacca

	108841	cagttcaaaa gcatcagttc ttcggcactc
		agccttcttt atgatccaac gcccacatcg

	108901	gtacatgact actggaaaaa ctttggctca
		gagataattg acttgattga atacaaagtt

	108961	ctttggcaaa aaataaaagt gtggcaagca
		gtactgacac aaaagcaagt ggcttttcct

	109021	ccgttgagtc atttatttat tcagtgggtg
		tgtgcgtgta gagacggagc ggctgtgctg

	109081	ggagctgggg cttccacttc agaggagccc
		cggacctgcc ctcggggagt tcacaggcag

	109141	tgctgcgggg ggtcctgcca ggacgcctgc
		cctgcgagtg cccagtgctg tgatggatgc

	109201	gtgtcccgca tctgcggcca ctggggccac
		gtgcccgaga ttgtccgggt ctgagggtgc

	109261	agagaagagg aggcatttgg actgagtctg
		gaaaaatgag catgtggcca cgtgagaagc

	109321	cagtggtgag gggaccagtc aggcggagga
		aagagcggct catacgagtt gtggagctgg

	109381	aagcatgagg gtgtgtggaa gcagaggccg
		gggacagggc cgcagggccg gccatggagg

	109441	gcgtgggctg ctgcaggctc ctgagaaggg
		ggacgctgcc atcatgaccg ggtttaggtg

	109501	tttgaccctg gtgtccacgt agaggacaga
		tgtgtggggg gggagctgga gatgggcatc

	109561	catcgggagt cagcctggag agaggcagag
		accccgtcag tgggccctca ggacgtggat

	109621	ggggcggatg ttgggaagat ctgactcctg
		ggttccggct ggggctccgg gctggagggg

	109681	tgccgcccac cgagcacagg aggcaaacag
		atgccctctc ccagcaagac cccagcccca

	109741	gcaccctccg gggccggact ccgcccctct
		tccagaatgg ctcccttgct gtcctcgccc

	109801	atctttccgg tgccctgagc ctctagagtc
		tggacaccag cgtccgcctt gcgcttgttt

	109861	ctgggaagtc tctggcttgt ctctgactca
		cccaggaccg tcttcgaggg caaggttgtg

	109921	tccttggttc catctgcttt ggggtccggc
		tcctcgctgc ttgacctgct gatgtgacag

	109981	tgtctcttgt tttcttttca gaatccgaga
		gcagctgtgt gtgtcccaga cagacccagc

	110041	cgctgggatg acgggcccct ctgtggagat
		ccccccggcc gccaagctgg gtgaggcttt

	110101	cgtgtttgcc ggcgggctgg acatgcaggc
		agacctgttc gcggaggagg acctgggggc

	110161	cccctttctt caggggaggg ctctggagca
		gatggccgtc atctacaagg agatccctct

	110221	cggggagcaa ggcagggagc aggacgatta
		ccggggggac ttcgatctgt gctccagccc

	110281	tgttccgcct cagagcgtcc ccccgggaga
		cagggcccag gacgatgagc tgttcggccc

	110341	gaccttcctc cagaaaccag acccgactgc
		gtaccggatc acgggcagcg gggaagccgc

	110401	cgatccgcct gccagggagg cggtgggcag
		gggtgacttg gggctgcagg ggccgcccag

	110461	gaccgcgcag cccgccaagc cctacgcgtg
		tcgggagtgc ggcaaggcct tcagccagag

	110521	ctcgcacctg ctccggcacc tggtgattca
		caccggggag aagccgtatg agtgcggcga

	110581	gtgcggcaag gccttcagcc agagctcgca
		cctgctccgg caccaggcca tccacaccgg

	110641	ggagaagccg tacgagtgcg gcgagtgcgg
		caaggccttc cggcagagct cggccctggc

	110701	gcagcacgcg aagacgcaca gcgggaggcg
		gccgtacgtc tgccgcgagt gcggcaagga

	110761	cttcagccgc agctccagcc tgcgcaagca
		cgagcgcatc cacaccgggg agaagcccta

	110821	cgcgtgccag gagtgcggca aggccttcaa
		ccagagctcg ggcctgagcc agcaccgcaa

	110881	gatccactcg ctgcagaggc cgcacgcctg
		cgagctgtgc gggaaggcct tctgccaccg

	110941	ctcgcacctg ctgcggcacc agcgcgtcca
		cacgggcaag aagccgtacg cctgcgcgga

	111001	ctgcggcaag gccttcagcc agagctccaa
		cctcatcgag caccgcaaga cgcacacggg

	111061	cgagaggccc taccggtgcc acaagtgcgg
		caaggccttc agccagagct cggcgctcat

	111121	cgagcaccag cgcacccaca cgggcgagag
		gccttacgag tgcggccagt gcggcaaggc

	111181	cttccgccac agctcggcgc tcatccagca
		ccagcgcacg cacacgggcc gcaagcccta

	111241	cgtgtgcaac gagtgcggca aggccttccg
		ccaccgctcg gcgctcatcg agcactacaa

	111301	gacgcacacg cgcgagcggc cctacgagtg
		caaccgctgc ggcaaggcct tccggggcag

	111361	ctcgcacctc ctccgccacc agaaggtcca
		cgcggcggac aagctctagg gtccgcccgg

	111421	ggcgagggca cgccggccct ggcgcccccg
		gcccagcggg tggacctggg gggccagccg

	111481	gacggcggaa tcccggccgg ctcttctctg
		ccgtgacccc ggggggttgg ttttgccctc

	111541	cattcgcttt ttctaaagtg cagacgaata
		cacgtcagag ggacgaagtg gggttaagcc

	111601	cccgggagac gtccggcgag ctctaacgtc
		agacacttga agaagtgaag cggactcgca

	111661	gcccgtacag cccggggaag atgagtccaa
		agtcgagggt caccttggcc actgcagggt

	111721	cgctcggcgg tggggcggag cgggtgcagg
		agggctcctc ctgggcttgg ggtggcaggc

	111781	gaggaccccg cgcctctcag ccctcggcct
		gggttggctg agggcgggcc tggctgtagg

	111841	ccctccagcg gaggtggagg cgctgcccgg
		ctcagccagg cacaggaccc tgccacgagg

	111901	agtagccctc cgccagaccc ggcgtccagg
		ctggggcgcc tgcggggcct ccgttctgtg

	111961	gctgggcagc ctgcgccctg tccagggatg
		aaggggttcc ggtctgaagg gctgggttca

	112021	gggtccagct ctggcccctc ctgccttggt
		gtcctggagg aagccccaag gctccgtttc

	112081	cctctccagg aggtggggac gttgggaatg
		ccacattccc ctggggggtg tgtgtgtgtg

	112141	ttcaaggctc ccattcagac tgggactggg
		cactcacgag ctttggcaac tggcaactga

	112201	ggacggagac ccagggtgac accccacctc
		ctgctgcggc ccccccggca ggggagacac

	112261	aggcccgtct ggttcccaag atggcagggc
		ccctccccct ccagcttgtg ccctgggtgt

	112321	ggtgcctggg gctacagcga ccctttccgg
		ttccccgggc cagttcagct gggcatcctc

	112381	agggcggggc tctgagggtg ccatgtttcc
		agagctcctc ctcctcccac cagtagcagg

	112441	cgggcggcca gctcccaggc agccccctgg
		catcgcctag gtgcacacct gcccgctgtg

	112501	acccagcaag gcttgaaggt ggccatccca
		gttaagtccc ctgcccctgg cccaggaatg

	112561	ggctcgggca gggccgcatc tggctgcccc
		agaagcgtct gtccctggcc tctgggagtt

	112621	ggcggtggtc tctggtactg tccctcgcag
		ggccccttag cactgctcgg ggaggaggtg

	112681	ggctgaactg attttgaagt tttacatgtc
		tgcggccgca gtcctacgag cccgtcaggg

	112741	tcatgctggt tatttcagca gatggggctt
		ggctcggcag ctaggatggt cctgaataaa

	112801	aatgggaagg ccagagctgt tcctccatca
		gcaggcttgg cagctgggga cgttgaaagg

	112861	acaggtctgc tggtctgggg agaccagctc
		tgtgcagccc ctgctgtccg tgggggtact

	112921	aaaccagccc ctgtgtgcgc ccatctgagt
		ggcagcccgc ctggaggatc gcccatcact

	112981	tgtgagaatt gagagaatgc tgacaccccc
		gcttggtgca gggggacagg gccccctaag

	113041	atctacctcc ttgccccacc cccgggaccc
		cctcagcctt ggccaggact gtccttactg

	113101	ggcagggcag tcatccactt ccaacctttg
		ccgtctcctc cgcgcgctgt gctcccagcc

	113161	aaattgtttt atttttttcc aagcatcact
		ttgcacacgt caccactctc cttaaaacca

	113221	cccttccgga gtctcctgct cgtaaatcgc
		cggtttcagc caacctgggt cgccccccaa

	113281	gcccagcaag cctgctgagc cccgcgcctc
		ccagctactt cacgctcgcc tcaagcttct

	113341	aaacgcggac cttctccccc ccacccccat
		ccctttcttt tctgatttat gtaacacggc

	113401	aggtaagact cctctcctga agggttgaca
		gactcacaca aaaccgtggt cagaccaggc

	113461	aagtgctttt tttcagaagt gtgagcggaa
		cctagtcttc agctcatgct ctttccttgt

	113521	tttcttatgt gttctaagtc ctttgacttg
		ggctcccaga cagcgacgtt gtaagaggcc

	113581	gtcctggtag catttgaatt gtcctcgagt
		ttcgttgtcg gattttgttt tattgtctta

	113641	gttttccctt cttttagcag acgttgttga
		ctgtcgtaaa gctccagttc ttggttctgt

	113701	ttactaatca aattgttttg tcaaagtaca
		tgtattctgc tcttttcttt atcttttttg

	113761	ttgcttaata ttaacacttt acatttctaa
		gattaattat ttaggtaatt aataattttt

	113821	aacatttcta gtaaacgtgg gtacttgggt
		ctgtgtttgt tttcttgtag ttacagcttt

	113881	ttctgctcta tactgttgac gtctgggttt
		ttttttgctc ttaggaattt ccctttgacc

	113941	ccattattat tattttaatt agtatttttt
		aataattaaa aattagtgtt tttaaattaa

	114001	ccctaatcct aaccccagtg atgactgctt
		cagtcattgc tgttacttat tatgtgctgg

	114061	tgtcaggatt tttaagtgtc catagacatt
		ctctgagcct gaatatatta tcagttttat

	114121	acagcatttg tgtactctca agaaacgtgt
		tttcactctg tcagttcggt ttgttacctc

	114181	agtctttatg ttattttgct ccagtccgca
		cttgctctaa cttgtcttcc cttcgaggtg

	114241	tgaggacgcc tggcagccgg tgagcatgcc
		ggggtccggg gtcgtgggcc caggcgccca

	114301	gcaaagccct gtgggtgtgt gcacggctgg
		gctgctccgg gaggaagcct gtggccccac

	114361	ggtagttagg agcgctggtt tacctggtca
		caccacggtc tggttttgtg tgcttttccc

	114421	tgacgtgttt ctgttttgcc ttggtttcta
		ttctgtttta tgagtgccgt ttacgctttg

	114481	ttagtcatgc cgttatctcg atagacaggg
		tgtacgtgat caagtgatta ccgtatttgg

	114541	agcagatgtc tatttaacag agatgaactg
		agaacctgtg cctttgcatg ccctctttgc

	114601	ctcttttaat gcttctagct tcaacttctc
		ttttccaaac attataatgg aaaccccttg

	114661	cttttttttt tttaatttgc atttgcatga
		gagtttattt agctcggcat tttattttta

	114721	aaatttgtgt atatattttt gctatatatc
		tgtaacttat aaacagcaaa ttattggatt

	114781	ttgctttctg attctttctg taattcttct
		tacataagaa gttctcctat gagtaacatt

	114841	gctgtttaga gtgaggcatg atttatttcc
		agcttagtat gtattgggtc ggttaacccc

	114901	caaaggtcat gctcatcccc gccccatctc
		tgtgagttat tgtccgagtg tggagcgccc

	114961	tgtctaggcc gacgagagac ccaccatcgg
		gcacacctgc ccctcctggt ctggtcagtg

	115021	ccgggctctg tcctgagtcc actcctgatg
		tcacaggctg gtgcttcagc gacctcggct

	115081	gtgacacgga gggtgtgatg gcactgccca
		gccccatggg gcttggagga ctaaaggatg

	115141	cacacctgcc tggcagactg agggcacagg
		tgtttctcac actgtcagcg ttttgaaata

	115201	ttcctttgat tttctaccct aactcccaaa
		ggccgttcaa cataagctag aatgctacgt

	115261	ggtgcttgat tacattttag aaaagtttca
		gcaaatacca cgagatgcag caaagaacta

	115321	gacctcacag atcaggccgc ctgcataagg
		gagcccacac agtcgtggga gacggggacc

	115381	ctctcccacg tcctgtctgt cccaggatgg
		tcccctcacc cgccccctct ctcccctcgc

	115441	cctcctgtgg tgggggccgg ccaccatcac
		agctgcagag cctcaagaag ggggtcgccc

	115501	tggccactcc cgtggcagga gggacacgag
		ggcaggagct taccgcgggt gcagtggtct

	115561	cggatcagct cagctggccg ctgcggggtc
		ggggggacag ttcagtggga ggcaggagcc

	115621	cccactacag ctgccaggac ttctcagagg
		tgacaagggg gttcagtcac ctcagcccag

	115681	gtggaaacca aatggcctct tgcgcggctc
		ctggggccac gcggaggttc gctgggatca

	115741	caggtatctg gatgtgtgcg ccatggacat
		gcaccacctt cggggggtaa ggggtgggga

	115801	aaggcagccc ctttcttttg ggggaccccc
		tcttcagtgt ctgataacca ggaaaccaaa

	115861	tcagaaggtg gtctgggggt gctgagcagg
		gtgtctccta caccacaggc cacacactca

	115921	cacagcctcc aggactccag tggggctgag
		cgctggagac tcacccacgt ttgctacccc

	115981	cccacccaag gccatcccag aacagctgcc
		tgcgtcctca cggctggccc ctcccctctg

	116041	gtctaaccca gtgtgggtgg gccggcctgg
		ggtctccacc tgcctcctgc tgttccctgg

	116101	gctgctggct gtctgcagat gcggggccct
		ggcccggaga agccccatca gagcccagag

	116161	gacgggagtg gagcggggag gtgagccccg
		gagtctcgag gggccagagg caaaatactg

	116221	ggctgtgtcc ctggaaggca gtttcccatg
		aaaccttcaa tataggccgc cccagacgat

	116281	cagcctcatc tgctacgtgg attcctcccc
		gtagcgaatg gtgattgggt tctacatgga

	116341	cccgggactt ctgtttgaat tataatcttt
		cccccactgc ccctccaggg atctggaaaa

	116401	tggaggcctg ggctagacgg aagcttcctc
		caagattctt tattgaaggg attcgaagag

	116461	aaacaggtgg tcagtaatct gtgggggatg
		gaggggtgag cgctacgtgt aacggtttta

	116521	ctgttgctac gggaccagtt ttgatgtctt
		tccccttcaa gaagcagacc caaacaccga

	116581	gatgctgagg ttagcagcac agagcgggtt
		catccacaag gcaaccaggc agggagacca

	116641	gagacgctct ggaatctgcc tccctatggg
		cacgggctgg gtgctcacgg atgaagacca

	116701	agcagcaggt ggcgtggggc gtggggagcc
		tgcggaaagc gatggacaag gtgcgggacc

	116761	gcggtccgcg cggtggaccc aagctccgcc
		tctgcgctgc agcgcgagct gggggcggag

	116821	cttccaggga cccgcgaccg cgcccagtgg
		gagggtccgc ggtccaccca gtcctaacag

	116881	ctcagctcca gctagacgcc gctgagtccg
		gctttctaga gagcaacccc ggcgggtatt

	116941	ttatggttct ggcttcctga ttggaggaca
		cgcgagtctt agaacaccct tgattagtgc

	117001	gggcaggcgg aatggatttg actgatcacg
		atctgcagtt tcaccatctc aggggccgcc

	117061	ctcaccccca cctatcctgc caaagggggg
		gcctcggtgc tgagatcggg gccacacgtg

	117121	cactagacgg tcggtcagcg ctgctgctga
		gcggacccgg ggccatcctc acaccgccac

	117181	tggcccctgt gctcaataaa aggaaggaaa
		gcgggaaaag cgctttctgg ccgcggtggc

	117241	ctcgcgcgtt cctccatcgc catctgctgg
		cagagcccgg catggcaccc gctgcacaga

	117301	aacctcggtg tccgtttggg tgccccatcc
		ttgaccccga gagagcaccc tccgtccaaa

	117361	atgaaaaaca gctgctccca agagtcatta
		taatcacagc caattgtgtt aattcgtcct

	117421	cggatccact cacagttcca cggaacattc
		tgctaacctc tgacaactcc tacataaagc

	117481	aatactgaga agaaaagaac gtggttgata
		aatacaaagg catacaacaa taaggagcaa

	117541	agaaaaaaga cagtcctcgc agttctgttt
		tgttcatctc tcatgagtag gatggcagat

	117601	aaaacacaga atgcccagtg aataatttta
		gtctaagtat gtccccaata ctgcctaatc

	117661	ttcaaatcta accttatttt taaaatatat
		attttttgct ggtcactcat cagttcatgc

	117721	accaaagcct ttgtttcttg actcctaact
		ttttgacccc tctggggtga ggagcacccc

	117781	taacctcgag agcccatcac acagtcccct
		tgggactaga cccttctttg cccatcacag

	117841	ctgaccggaa gggccagccc atggccagcg
		ctcgcgcccc ctggcggaca gactctgcgc

	117901	ggcagccccg ggagcccagg tgcgaccccg
		cggtctctgg cgccctctag tgtggaaaga

	117961	tctcctcctg gtgttcccag tcattgggct
		gtattttatt agagaagatg ctcgcgtgac

	118021	gatgatgatg gtcctttacc gggaggcacg
		tttggggcgc gtcggctcag gggccgagct

	118081	attagcctgc atcgcgccca caggcatcgc
		gtccccctga gccgggtcag ctgtgggctg

	118141	tcctgacacg ggtttccccc agtctctggc
		ccgctgtccc tcccaggtca gtgtccagcg

	118201	ttgcccttct ggttgtggac ttgtgcagcg
		gtctcagcag atggaggggc gaccctaaag

	118261	gatgtattga ggcatctcag cactgtcctc
		cgcccaggtt tgctggtcag cagtgaagtg

	118321	accgggaaaa ggggctgtct tggggtcctt
		tcagaggcct gggttagacc aaagttttct

	118381	agaagattca ccattgcagg gagtcaaaga
		caaaactagg gtggtcagca atctgtgggg

	118441	gattcggcgg tgagggaatt ctgaatgcta
		catgtaatgg ttttactatt gttagggaac

	118501	atttttcccc cctacaaaca gcaggccaaa
		atactgagat gtcaggtttg catcaaagag

	118561	cgggttcatc cacaaggcaa ccagagaacg
		ctctggaatc tgcctccctg cgggcacagg

	118621	ctgggtgctc acggatgaag accaagcagc
		aggtggcgtg gggagtgggg agcctgggga

	118681	aagcgatgga caaggtgcga ggacctccgg
		cgcgagctgg aggcggagct tccagggaca

	118741	cgcggccacg cccagtggga gggtcagcgg
		tccatccagt cctaacagct cagctccaac

	118801	tagacgctgc tgagtctggc tttctagaga
		acactccggg cgggtatttt attgttttgg

	118861	cttcgtgact ggaggacgtt caagtcttaa
		aacacccttg attagtgcgg ggaggcggaa

	118921	tggatttgac tgatcacgac ccgcagtttc
		accatctcag gggccgccct caccccctcc

	118981	taccctacca aaggtggggg catcggtgct
		gagatctggg gtgacacata aaatcaggtg

	119041	aagtcttagg acagggggcc gattccaggt
		cctagggtgc agaaaaaacc tacctggccc

	119101	cgggctagac agcgtggagg gcgtggcccg
		ggctggtgca cagaagtggc ccccaactgg

	119161	tcagaaggtg tgggagccca gggctggtct
		actgcagaag gggtcgcctg gtggacagag

	119221	tggggcctga gtgcctgctg aactggtccg
		tcagggctgc tgagcagaca cgggccatca

	119281	tcactggctc ctgtgctcga tagaagggag
		ggaaaccagg aaagcaaagg cgctttatgg

	119341	ccgcttttgt gtttcgcgtt cctctagcac
		cgtctgccgg cagaacgcgg cattacatcc

	119401	gctggccaaa cctcggggtc cggcttggat
		gtccccatcc ttgtctcgga gatctcacct

	119461	ctcagcagtt cccctgggga caatgtcgag
		aagatgcgac cttgacccgg agctcggtgg

	119521	agagggtgcc ctgggttctt tccgcagttg
		cttggagtgg aggtgcctca tgttgggctg

	119581	ggaacgggag gaaggaaaca ggtcatgatt
		gagatgctct agacagactg tccctgctct

	119641	tgccaaattt cagaagattg tctttaataa
		atattccatt ttttgtatgc ccttaggtct

	119701	atttccagac actttaaata tattgaaaga
		ctttaaatat ttatataaaa atattattta

	119761	tagactgtat aaaaggaaca gttagaactg
		gacttggaac aacagactgg ttccaaatag

	119821	gaaaaggagt acgtcaaggc tgtatattgt
		caccctgctt atttaactta tatgcagagt

	119881	acatcatgag aaacgctggg ctggaagaaa
		cacaagctgg aatcaagatt gccgggagaa

	119941	atatcaataa cctcagatat gcagatgaca
		ccacccttat ggcagaaagt gaagaggaac

	120001	tcaaaagcct cttgatgaag gtgaaagagg
		agagcgaaaa agttggctta aagctcaaca

	120061	tttagaaaac gaagatcatg gcatctggtc
		ccatcacttc atggaaatag atggggaaac

	120121	agttgagaca gtgtcagact ttatttttgg
		gggctccaat gaaattaaaa gacgcttact

	120181	tcttggaagg aaagttatga ccaacctaga
		cagcatatta aaaagcagag acactacttt

	120241	gccagcaaag gtccgtctag tcaaggctat
		ggtttttcca gtggtcatgt atggatgtga

	120301	gagttggact gtgaagaagg ctgagcaccg
		aagaagtgat gcttttgaac tgtggtgttg

	120361	gagaagactc ttgagaggcc cttggactgc
		aaggagatcc aaccagtcca tcgtaaagga

	120421	gatcaccccc tgggtggtca ttggaaggac
		tgatgttgaa gctgaaactc cagtactttg

	120481	gctacctaat gcgaagagct gactcattgg
		aaaagaccct gatgctggga aagattgaag

	120541	gtgggaggag aaggggacaa cagaggatga
		gatggttgga ttgcatcact gactcgatgg

	120601	acgtgagtct gagtgaagtc tgggagttgg
		tgatggccag ggaggccctg gcgtgctggc

	120661	ggttcatggg gtcgcaaaga gtcggccatg
		actgagtgac tgaactgaac tgatccagaa

	120721	atttaaaatt aatatataaa ccaaatccat
		gcagacaatt ataagcatat attataaatg

	120781	cataattata agcaagtata tgttatattt
		ataatagttt ataatgtatt tataagcaag

	120841	tatatattat tataagcata attgtaagta
		gaagtaactt tgggctttcc tggtggctca

	120901	gacagtaaag aatctgcctg cagtacagga
		gaccgggttc gatccctggt ttggggaaat

	120961	tccctggaga agggaatggc aaccaactcc
		aacatgtttg cctggagaat tccatggaca

	121021	gaggagcccg gaaggttgca gtccatgggg
		ttgcaaagag ctggatacaa cagagtgact

	121081	aacacatgta tataaataaa tttacctata
		tattgtatat atatttataa acatattcag

	121141	atattataaa taattagaaa catattatac
		atgtatttaa atactgttat aaacataaat

	121201	ttaaaaaata attttcagcc ctttggcttg
		ggggtgtgtt tgtggacgtc tttgtgctac

	121261	tgttcctgaa gtggagctct cccctcccaa
		accagctttt gaaatgactg ggaaagcaat

	121321	ggaatacata agcatcagga agatagcaac
		agagctgtca ttcttcacag agggtgtgct

	121381	tgagtgtgta gcaagtcccg cagaatgtag
		acagattaat atagtctatt aaaaatagtg

	121441	tagcaaattt acgaggtgcg atttcaagta
		taaagactta ctgggtctct cagttcagtt

	121501	cagtcgcttg gttgtgtccg actctttttg
		accccatgga ccgcagcacg ccaggcctcc

	121561	ctgtccatca ccaactcctg gagttcactc
		aaactcatgt ccatcgagtc ggtgatgcca

	121621	tccaaccatc tcatcctctg gcgtcccctt
		ctcctcccac cttcaatctt tcccagcatc

	121681	agggtctttc ccagtgagtc agttctttgc
		atcaggtggc cagagtagtg gagtttcagc

	121741	ttcagcatcg gtccttccaa tgaatattct
		ggactgattt cctttaggat tgactggttg

	121801	gatctccttg cagttcaagg gactctcaag
		agtcttctcc aacagcacag tctatgaata

	121861	gaatagcaaa tgaatagaga ataacattta
		cgaggatata ttttaccatt gcataaaata

	121921	tatcagcttg tagagaacag acttgttccc
		aggggagagg gtgggtaggg atggagtggg

	121981	agtttgngat cancagaagc gagctgttat
		atagaagatg gataaaaagg atacacaaca

	122041	atgtcctact gtgtggcacc gggacctata
		ttcagtagct tgtgagaaac cataatcgac

	122101	aagactgagg aaaagtatat atatatgtat
		gtacttgagt tgctttgctg tacagaagaa

	122161	attaacacaa cattgtaaat cgatatttca
		atagaatcca cccccccaaa tatataagtt

	122221	tcctggagat ggagacggca acccactcca
		tttcttgcac ccaatattct tgcctggagg

	122281	atcccatgga tagaggatcg caaagactcg
		gacataaccc agcgactaac actttccctt

	122341	tcaaatgtgt aggtttacta gcgtgaatct
		acagagatgc ccaagacatt cgtttatgag

	122401	gaaaactcca cacgcagctt cactgagaat
		tattaaacct attaaaggga gagagcgcca

	122461	ggatattcat ggattgaaag attcgatgtg
		gtcaagttgc cagttttccc caaactgatt

	122521	ggtaaattcc ccaggagctg gctcaaggcg
		caaaattccc tttacctttt tttaagagac

	122581	gaagccaagg agccgattct ggttgagaga
		cgctcaggtc ctcctgcggg agagcagccc

	122641	tcttcctccc ggtcgcctgg gcagtttcga
		ggccacgacc agaaggactt ggctccctgt

	122701	gtcgcgcact cagaagtctc cctctccgtc
		ccaaggactc agaagctggg cgtcctgccc

	122761	gcagcagagg aggcagcctg gaggggcccc
		gcgggcacag cggtccgggt ttcagccgag

	122821	ttgcccgccc cgcccctcta cctgggcgct
		gccgcccggc tccggggccg gccgtgccct

	122881	ccgtggccgc aaggcgtcgc tgtccccccg
		ctggaagtgc tgacccggag gaaggggccc

	122941	agacggaggg actcggagcc tccgagtgac
		accctgggac tccgagcgct ggagcctggc

	123001	gtcaccccag gcaggggcag tgggggcccg
		gggcggggtc aggggcctcc cccggttctc

	123061	atttgacacc gcgggggtgc gctgggcaca
		gtgtccaggg gccacgttcc gagcaggggc

	123121	gcgatgcagg cccgggcgcg gcctgtcccg
		ggcgcgagtc cagctgcttt gcagaggtgg

	123181	cggcaggtcg cagtgaccct cacagagacg
		ccccactctg cggctccagg tgggcctgtg

	123241	ccccccagaa gtgctgacct gtgcaccggg
		aaggcacagg gccccccagc catgtctgcg

	123301	atggaagagc cggaaccgcg ccatgcccgt
		cctcgctgac cggcaggcac ccgccgtgtg

	123361	tccacacgct gagccatctg gctccccttg
		cttgacatac acccaggacc tgagtgtgca

	123421	ggaagttaga aggggcaggt gtggtgacac
		gatgccatcc agcatcacct gagaacctgg

	123481	acaaacctca ggggcccagc ctgctctgtg
		aggccccgag ggccggcccc tccccggacc

	123541	cctgccttga atccggccac actgcccgcc
		ttcctgctcc tgcggcttgt cagacacgcc

	123601	tgagcccagg gcctgtgcac tcgctgtccc
		ttctgccagg actgctcctc cccaggctct

	123661	tgctggggct ccccttcttc attcgggggt
		ggcctctctt gttcagtggc tcagctgtgc

	123721	ccagtctttg caaccccatg gactgcagca
		cgccaggctt ccctgtcctt cactagctcc

	123781	tggagtttgc tcaaactcat gtccattgag
		tcagtgatgc tatccaacca tctcatcctt

	123841	tgctgcccac ttcttctcct gctctcaatc
		tttcccagca tcagggtctt ttccaatgag

	123901	ttagctctct gcatcaggag gccaaagtat
		tggagcttca gcatcagtcc ttccagtgaa

	123961	tatgcgaggt tgatttccct tagaattgac
		tggttggatc tccttcctgt ccagagaact

	124021	ctcaagagtc ttctccagca ccacagtcgg
		agagcatcag ttcttcagtg atcaggtttc

	124081	tttatagccc agctctcaca tcggtacatg
		actattggaa aacccatagc tttgattaga

	124141	tggaccttca ttggcaaagt gatgggcctt
		cattggccct gctttttaat acaccatcta

	124201	ggtttgtcgt agctttcctt ccaaagagca
		aacatctttt aatttcctgg ctgcagtaac

	124261	catccatagt gattttggag cccaagaaaa
		taaaatctgc cactgtttcc actttttccc

	124321	cttctatttg ctatgaagtg aggggactgg
		atgccatgat cttagtttaa accagcagtt

	124381	gtcaccccga ccgcttcctt tcctaaagag
		ctcatcacac ctcccactgg aatgcaatgt

	124441	gttgcctgtc cgcctgcttc acctcctggg
		actttgctgc aggtcttggt ctctgaggcc

	124501	cctgccgtat ccccagggcc cagagcagtg
		ctgggcttcg agtccgatca gggactatgt

	124561	gtgtggactg gatggtgctt gcttcttctg
		gggaacgaga gacctgggcc tggggaacga

	124621	ggggacctgg tgtgaccgga tctcctccct
		cgggagagga gccaagcgag tggacacagg

	124681	tcagtgtgtc ttgctcctgt gtggcaggtg
		tcccgtctgt gtctgtcatc ttggcatttc

	124741	ggtgtttctg tgaacccagc ccctcccctc
		ctgatacccc atcccatcag cacagaggag

	124801	actgggcttg gggactctct ggtcctgaga
		ttcctctccg catgtgactc ccccctcctg

	124861	gggggagcag gcaccgtgtg tgaggagggt
		ggaagctttt caagaccccc agcttttctg

	124921	tcccaggggg ctctggcagg gccttgggag
		ctggaatgag ctggaatctg ggccagtggg

	124981	ggtttccctg gtggtaaaga acccgcctgc
		ccatgcacga ggcataagag acgcgggttc

	125041	gatcactggg tcgggaagat cccctacagg
		agggcatggc aacccactcc agtattcttt

	125101	cctgaagaat cccttggaca gaggagcctg
		gtgggctaca gtctctgggg tggcaaggag

	125161	tcggacacga ctgaagcgac ttaccatgca
		cgcacgcggg gtcaggggtc agggccgcgc

	125221	tgcttacctg ctgtgtgacc ttagccaggt
		cacacccccc aggctgtgaa agagaacagt

	125281	cttcccagac tcgggcatcc aggtctttac
		agacgtgcct gtgagctttg tgactctggc

	125341	tctgtggccg ctagagggcg ctgtccgccg
		ggccctatgt gcgtgcacgc atgtgagcat

	125401	gttcgcatac gtgtgtgcat ctgtcggggg
		cgcacggtgc ggggacacgg gcacgcggtc

	125461	aggaacgcag cccggacacc tccacgtggc
		ccgcgagtac cgtcaggtgg gggctgtggc

	125521	tccgctgtgt gggtgacccg ccctcccccc
		gcgaacgtgg tgcatagtga ccgcctggct

	125581	gggctcctga gctcagccat cctgcccccc
		gggtcagctc ccgacaggcc cagctctagg

	125641	ccccaggcgt ggaccgaggc ccccaggccc
		cggcctgtga gatgggacct ccgtctgggg

	125701	ggctcattct gctcccggag gcctggcagg
		cccctcctct ttggcattgc ataccctcgc

	125761	attggggtgg gtaagcacag taccccatgc
		ctgtggcccc gtgggagcgg cctgctcagg

	125821	gaggccggag cctcagctac agggctgtca
		caccgggctg cagaggaaga agacgggagc

	125881	gaggcctaca ggaacctagc caggccctgg
		cccactgagc cgacaggagc ctggccagag

	125941	gcctgcacag gacggggtgg cggggggggt
		ggggtggggt gctgggcccc gtggccttga

	126001	ctgcagaccc cgagggctcc tcagcttaga
		acggccaagc ctgagtcttg ggggtgcagg

	126061	tcaggggg

Primers
In another embodiment, primers are provided to generate 3′ and 5′ sequences of a targeting vector. The oligonucleotide primers can be capable of hybridizing to porcine immunoglobulin genomic sequence, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. In a particular embodiment, the primers hybridize under stringent conditions to Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. Another embodiment provides oligonucleotide probes capable of hybridizing to porcine heavy chain, kappa light chain or lambda light chain nucleic acid sequences, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The polynucleotide primers or probes can have at least 14 bases, 20 bases, 30 bases, or 50 bases which hybridize to a polynucleotide of the present invention. The probe or primer can be at least 14 nucleotides in length, and in a particular embodiment, are at least 15, 20, 25, 28, or 30 nucleotides in length.
In one embodiment, primers are provided to amplify a fragment of porcine Ig heavy-chain that includes the functional joining region (the J6 region). In one non-limiting embodiment, the amplified fragment of heavy chain can be represented by Seq ID No 4 and the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 2, to produce the 5′ recombination arm and complementary to a portion of Ig heavy-chain mu constant region, such as, but not limited to Seq ID No 3, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 4) can be subcloned and assembled into a targeting vector.
In other embodiments, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the constant region. In another embodiment, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the J region. In one non-limiting embodiment, the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 21 or 10, to produce the 5′ recombination arm and complementary to genomic sequence 3′ of the constant region, such as, but not limited to Seq ID No 14, 24 or 18, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 20) can be subcloned and assembled into a targeting vector.
II. Genetic Targeting of the Immunoglobulin Genes
The present invention provides cells that have been genetically modified to inactivate immunoglobulin genes, for example, immunoglobulin genes described above. Animal cells that can be genetically modified can be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disaggregated preparation of a whole or part of an embryo, fetus, or adult animal. In one embodiment of the invention, cells can be selected from the group consisting of, but not limited to, epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, Islets of Langerhans cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, leydig cells, peritubular cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, and osteoclasts. In one alternative embodiment, embryonic stem cells can be used. An embryonic stem cell line can be employed or embryonic stem cells can be obtained freshly from a host, such as a porcine animal. The cells can be grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF).
In a particular embodiment, the cells can be fibroblasts; in one specific embodiment, the cells can be fetal fibroblasts. Fibroblast cells are a suitable somatic cell type because they can be obtained from developing fetuses and adult animals in large quantities. These cells can be easily propagated in vitro with a rapid doubling time and can be clonally propagated for use in gene targeting procedures.
Targeting Constructs
Homologous Recombination
In one embodiment, immunoglobulin genes can be genetically targeted in cells through homologous recombination. Homologous recombination permits site-specific modifications in endogenous genes and thus novel alterations can be engineered into the genome. In homologous recombination, the incoming DNA interacts with and integrates into a site in the genome that contains a substantially homologous DNA sequence. In non-homologous (“random” or “illicit”) integration, the incoming DNA is not found at a homologous sequence in the genome but integrates elsewhere, at one of a large number of potential locations. In general, studies with higher eukaryotic cells have revealed that the frequency of homologous recombination is far less than the frequency of random integration. The ratio of these frequencies has direct implications for “gene targeting” which depends on integration via homologous recombination (i.e. recombination between the exogenous “targeting DNA” and the corresponding “target DNA” in the genome).
A number of papers describe the use of homologous recombination in mammalian cells. Illustrative of these papers are Kucherlapati et al., Proc. Natl. Acad. Sci. USA 81:3153-3157, 1984; Kucherlapati et al., Mol. Cell. Bio. 5:714-720, 1985; Smithies et al, Nature 317:230-234, 1985; Wake et al., Mol. Cell. Bio. 8:2080-2089, 1985; Ayares et al., Genetics 111:375-388, 1985; Ayares et al., Mol. Cell. Bio. 7:1656-1662, 1986; Song et al., Proc. Natl. Acad. Sci. USA 84:6820-6824, 1987; Thomas et al. Cell 44:419-428, 1986; Thomas and Capecchi, Cell 51:503-512, 1987; Nandi et al., Proc. Natl. Acad. Sci. USA 85:3845-3849, 1988; and Mansour et al., Nature 336:348-352, 1988. Evans and Kaufman, Nature 294:146-154, 1981; Doetschman et al., Nature 330:576-578, 1987; Thoma and Capecchi, Cell 51:503-512, 4987; Thompson et al., Cell 56:316-321, 1989.
The present invention can use homologous recombination to inactivate an immunoglobulin gene in cells, such as the cells described above. The DNA can comprise at least a portion of the gene(s) at the particular locus with introduction of an alteration into at least one, optionally both copies, of the native gene(s), so as to prevent expression of functional immunoglobulin. The alteration can be an insertion, deletion, replacement or combination thereof. When the alteration is introduce into only one copy of the gene being inactivated, the cells having a single unmutated copy of the target gene are amplified and can be subjected to a second targeting step, where the alteration can be the same or different from the first alteration, usually different, and where a deletion, or replacement is involved, can be overlapping at least a portion of the alteration originally introduced. In this second targeting step, a targeting vector with the same arms of homology, but containing a different mammalian selectable markers can be used. The resulting transformants are screened for the absence of a functional target antigen and the DNA of the cell can be further screened to ensure the absence of a wild-type target gene. Alternatively, homozygosity as to a phenotype can be achieved by breeding hosts heterozygous for the mutation.
Targeting Vectors
In another embodiment, nucleic acid targeting vector constructs are also provided. The targeting vectors can be designed to accomplish homologous recombination in cells. These targeting vectors can be transformed into mammalian cells to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. In one embodiment, the targeting vectors can contain a 3′ recombination arm and a 5′ recombination arm (i.e. flanking sequence) that is homologous to the genomic sequence of ungulate heavy chain, kappa light chain or lambda light chain genomic sequence, for example, sequence represented by Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The homologous DNA sequence can include at least 15 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence, particularly contiguous sequence, homologous to the genomic sequence. The 3′ and 5′ recombination arms can be designed such that they flank the 3′ and 5′ ends of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. The targeting of a functional region can render it inactive, which results in the inability of the cell to produce functional immunoglobulin molecules. In another embodiment, the homologous DNA sequence can include one or more intron and/or exon sequences. In addition to the nucleic acid sequences, the expression vector can contain selectable marker sequences, such as, for example, enhanced Green Fluorescent Protein (eGFP) gene sequences, initiation and/or enhancer sequences, poly A-tail sequences, and/or nucleic acid sequences that provide for the expression of the construct in prokaryotic and/or eukaryotic host cells. The selectable marker can be located between the 5′ and 3′ recombination arm sequence.
Modification of a targeted locus of a cell can be produced by introducing DNA into the cells, where the DNA has homology to the target locus and includes a marker gene, allowing for selection of cells comprising the integrated construct. The homologous DNA in the target vector will recombine with the chromosomal DNA at the target locus. The marker gene can be flanked on both sides by homologous DNA sequences, a 3′ recombination arm and a 5′ recombination arm. Methods for the construction of targeting vectors have been described in the art, see, for example, Dai et al., Nature Biotechnology 20: 251-255, 2002; WO 00/51424.
Various constructs can be prepared for homologous recombination at a target locus. The construct can include at least 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous with the target locus. The sequence can include any contiguous sequence of an immunoglobulin gene.
Various considerations can be involved in determining the extent of homology of target DNA sequences, such as, for example, the size of the target locus, availability of sequences, relative efficiency of double cross-over events at the target locus and the similarity of the target sequence with other sequences.
The targeting DNA can include a sequence in which DNA substantially isogenic flanks the desired sequence modifications with a corresponding target sequence in the genome to be modified. The substantially isogenic sequence can be at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the corresponding target sequence (except for the desired sequence modifications). In a particular embodiment, the targeting DNA and the target DNA can share stretches of DNA at least about 75, 150 or 500 base pairs that are 100% identical. Accordingly, targeting DNA can be derived from cells closely related to the cell line being targeted; or the targeting DNA can be derived from cells of the same cell line or animal as the cells being targeted.
Porcine Heavy Chain Targeting
In particular embodiments of the present invention, targeting vectors are provided to target the porcine heavy chain locus. In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J6 region of the porcine immunoglobulin heavy chain locus. Since the J6 region is the only functional joining region of the porcine immunoglobulin heavy chain locus, this will prevent the expression of a functional porcine heavy chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the J6 region, optionally including J1-4 and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the J6 region, including the mu constant region (a “J6 targeting construct”), see for example, FIG. 1. Further, this J6 targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No S and FIG. 1. In other particular embodiments, the 5′ targeting arm can contain sequence 5′ of J1, such as depicted in Seq ID No. 1 and/or Seq ID No 4. In another embodiments, the 5′ targeting arm can contain sequence 5′ of J1, J2 and/or J3, for example, as depicted in approximately residues 1-300, 1-500, 1-750, 1-1000 and/or 1-1500 Seq ID No 4. In a further embodiment, the 5′ targeting arm can contain sequence 5′ of the constant region, for example, as depicted in approximately residues 1-300, 1-500, 1-750, 1-1000, 1-1500 and/or 1-2000 or any fragment thereof of Seq ID No 4 and/or any contiguous sequence of Seq ID No. 4 or fragment thereof. In another embodiment, the 3′ targeting arm can contain sequence 3′ of the constant region and/or including the constant region, for example, such as resides 7000-8000 and/or 8000-9000 or fragment thereof of Seq ID No 4. In other embodiments, targeting vector can contain any contiguous sequence or fragment thereof of Seq ID No 4. sequence In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the diversity region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the diversity region of the porcine heavy chain locus. In a further embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the mu constant region and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the mu constant region of the porcine heavy chain locus.
In further embodiments, the targeting vector can include, but is not limited to any of the following sequences: the Diversity region of heavy chain is represented, for example, by residues 1089-1099 of Seq ID No 29 (D(pseudo)), the Joining region of heavy chain is represented, for example, by residues 1887-3352 of Seq ID No 29 (for example: J(psuedo): 1887-1931 of Seq ID No 29, J(pseudo): 2364-2411 of Seq ID No 29, J(pseudo): 2756-2804 of Seq ID No 29, J (functional J): 3296-3352 of Seq ID No 29), the recombination signals are represented, for example, by residues 3001-3261 of Seq ID No 29 (Nonamer), 3292-3298 of Seq ID No 29 (Heptamer), the Constant Region is represented by the following residues: 3353-9070 of Seq ID No 29 (J to C mu intron), 5522-8700 of Seq ID No 29 (Switch region), 9071-9388 of Seq ID No 29 (Mu Exon 1), 9389-9469 of Seq ID No 29 (Mu Intron A), 9470-9802 of Seq ID No 29 (Mu Exon 2), 9830-10069 of Seq ID No 29 (Mu Intron B), 10070-10387 of Seq ID No 29 (Mu Exon 3), 10388-10517 of Seq ID No 29 (Mu Intron C), 10815-11052 of Seq ID No 29 (Mu Exon 4), 11034-11039 of Seq ID No 29 (Poly(A) signal) or any fragment or combination thereof. Still further, any contiguous sequence at least about 17, 20, 30, 40, 50, 100, 150, 200 or 300 nucleotides of Seq ID No 29 or fragment and/or combination thereof can be used as targeting sequence for the heavy chain targeting vector. It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.

In other embodiments, targeting vectors designed to disrupt the expression of porcine heavy chain genes can contain recombination arms, for example, the 3′ or 5′ recombination arm, that target the constant region of heavy chain. In one embodiment, the recombination arm can target the mu constant region, for example, the C mu sequences described above or as disclosed in Sun & Butler Immunogenetics (1997) 46: 452-460. In another embodiment, the recombination arm can target the delta constant region, such as the sequence disclosed in Zhao et al. (2003) J immunol 171: 1312-1318, or the alpha constant region, such as the sequence disclosed in Brown & Butler (1994) Molec Immunol 31: 633-642.


Seq ID No. 5

GGCCAGACTTCCTCGGAACAGCTCAAAGAGCTCTGTCAAAGCCAGATCCC

ATCACACGTGGGCACCAATAGGCCATGCCAGCCTGCAAGGGCCGAACTGG

GTTCTCCACGGCGCACATGAAGCCTGCAGCCTGGCTTATCCTCTTCCGTG

GTGAAGAGGCAGGCCCGGGACTGGACGAGGGGCTAGCAGGGTGTGGTAGG

CACCTTGCGCCCCCCACCCCGGCAGGAACCAGAGACCCTGGGGCTGAGAG

TGAGCCTCCAAACAGGATGCCCCACCCTTCAGGCCACCTTTCAATCCAGC

TACACTCCACCTGCCATTCTCCTCTGGGCACAGGGCCCAGCCCCTGGATC

TTGGCCTTGGCTCGACTTGCACCCACGCGCACACACACACTTCCTAACGT

GCTGTCCGCTCACCCCTCCCCAGCGTGGTCCATGGGCAGCACGGCAGTGC

GCGTCCGGCGGTAGTGAGTGCAGAGGTCCCTTCCCCTCCCCCAGGAGCCC

CAGGGGTGTGTGCAGATCTGGGGGCTCCTGTCCCTTACACCTTCATGCCC

CTCCCCTCATACCCACCCTCCAGGCGGGAGGCAGCGAGACCTTTGCCCAG

GGACTCAGCCAACGGGCACACGGGAGGCCAGCCCTCAGCAGCTGGCTCCC

AAAGAGGAGGTGGGAGGTAGGTCCACAGCTGCCACAGAGAGAAACCCTGA

CGGACCCCACAGGGGCCACGCCAGCCGGAACCAGCTCCCTCGTGGGTGAG

CAATGGCCAGGGCCCCGCCGGCCACCACGGCTGGCCTTGCGCCAGCTGAG

AACTCACGTCCAGTGCAGGGAGACTCAAGACAGCCTGTGCACACAGCCTC

GGATCTGCTCCCATTTCAAGCAGAAAAAGGAAACCGTGCAGGCAGCCCTC

AGCATTTCAAGGATTGTAGCAGCGGCCAACTATTCGTCGGCAGTGGCCGA

TTAGAATGACCGTGGAGAAGGGCGGAAGGGTCTCTCGTGGGCTCTGCGGC

CAACAGGCCCTGGCTCCACCTGCCCGCTGCCAGCCCGAGGGGCTTGGGCC

GAGCCAGGAACCACAGTGCTCACCGGGACCACAGTGACTGACCAAACTCC

CGGCCAGAGCAGCCCCAGGCCAGCCGGGCTCTCGCCCTGGAGGACTCACC

ATCAGATGCACAAGGGGGCGAGTGTGGAAGAGACGTGTCGCCCGGGCCAT

TTGGGAAGGCGAAGGGACCTTCCAGGTGGACAGGAGGTGGGACGCACTCC

AGGCAAGGGACTGGGTCCCCAAGGCCTGGGGAAGGGGTACTGGCTTGGGG

GTTAGCCTGGCCAGGGAACGGGGAGCGGGGCGGGGGGCTGAGCAGGGAGG

ACCTGACCTCGTGGGAGCGAGGCAAGTCAGGCTTCAGGCAGCAGCCGCAC

ATCCCAGACCAGGAGGCTGAGGCAGGAGGGGCTTGCAGCGGGGCGGGGGC

CTGCCTGGCTCCGGGGGCTCCTGGGGGACGCTGGCTCTTGTTTCCGTGTC

CCGCAGCACAGGGCCAGCTCGCTGGGCCTATGCTTACCTTGATGTCTGGG

GCCGGGGCGTCAGGGTCGTCGTCTCCTCAGGGGAGAGTCCCCTGAGGCTA

CGCTGGGG*GGGGACTATGGCAGCTCCACCAGGGGCCTGGGGACCAGGGG

CCTGGACCAGGCTGCAGCCCGGAGGACGGGCAGGGCTCTGGCTCTCCAGC

ATCTGGCCCTCGGAAATGGCAGAACCCCTGGCGGGTGAGCGAGCTGAGAG

CGGGTCAGACAGACAGGGGCCGGCCGGAAAGGAGAAGTTGGGGGCAGAGC

CCGCCAGGGGCCAGGCCCAAGGTTCTGTGTGCCAGGGCCTGGGTGGGCAC

ATTGGTGTGGCCATGGCTACTTAGACGCGTGATCAAGGGCGAATTCCAGC

ACACTGGCGGCCGTTACTAGTggatcccggcgcgccctaccgggtagggg

aggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctg

ggcacttggcgctacacaagtggcctctggcctcgcacacattccacatc

caccggtaggcgccaaccggctccgttctttggtggccccttcgcgccac

cttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcg

tcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgca

gatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcg

gccaatagcagctttggctccttcgctttctgggctcagaggctgggaag

gggtgggtccgggggcgggctcaggggcgggctcaggggcggggcgggcg

cccgaaggtcctccggaagcccggcattctgcacgcttcaaaagcgcacg

tctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgcag

ccaatatgggatcggccattgaacaagatggattgcacgcaggttctccg

gccgcttgggtggagaggctattcggctatgactgggcacaacagacaat

cggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccgg

ttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggac

gaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagc

tgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcg

aagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaa

gtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggc

tacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgta

ctcggatggaagccggtcttgtcaatcaggatgatctggacgaagagcat

caggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcc

cgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaata

tcatggtggaaaatggccgcttttctggattcatcgactgtggccggctg

ggtgtggcggatcgctatcaggacatagcgttggctacccgtgatattgc

tgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggta

tcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgag

ttcttctgaggggatcaattcTCTAGATGCATGCTCGAGCGGCCGCCAGT

GTGATGGATATCTGCAGAATTCGCCCTtCCAGGCGTTGAAGTCGTCGTGT

CCTCAGGTAAGAACGGCCCTCCAGGGCCTTTAATTTCTGCTCTCGTCTGT

GGGCTTTTCTGACTCTGATCCTCGGGAGGCGTCTGTGCCCCGCCCGGGGA

TGAGGCCGGCTTGCCAGGAGGGGTCAGGGACCAGGAGCCTGTGGGAAGTT

CTGACGGGGGCTGCAGGCGGGAAGGGCCCCACCGGGGGGCGAGCCCCAGG

CCGCTGGGCGGCAGGAGACCCGTGAGAGTGCGCCTTGAGGAGGGTGTCTG

CGGAAGCACGAACGCCGGCCGGGAAGGGCTTGCTGCAATGCGGTCTTCAG

ACGGGAGGCGTCTTCTGCCCTCACCGTCTTTCAAGCCCTTGTGGGTCTGA

AAGAGCCATGTCGGAGAGAGAAGGGACAGGCCTGTCCCGACCTGGCCGAG

AGCGGGCAGCCCCGGGGGAGAGCGGGGCGATCGGCCTGGGCTCTGTGAGG

CCAGGTCCAAGGGAGGACGTGTGGTCCTCGTGACAGGTGCACTTGCGAAA

CCTTAGAAGACGGGGTATGTTGGAAGCGGCTCCTGATGTTTAAGAAAAGG

GAGACTGTAAAGTGAGCAGAGTCCTCAAGTGTGTTAAGGTTTTAAAGGTC

AAAGTGTTTTAAACCTTTGTGACTGCAGTTAGCAAGCGTGCGGGGAGTGA

ATGGGGTGCCAGGGTGGCCGAGAGGCAGTACGAGGGCCGTGCCGTCCTCT

AATTCAGGGCTTAGTTTTGCAGAATAAAGTCGGCCTGTTTTCTAAAAGCA

TTGGTGGTGCTGAGCTGGTGGAGGAGGCCGCGGGCAGCCCTGGCCACCTG

CAGCAGGTGGCAGGAAGCAGGTCGGCCAAGAGGCTATTTTAGGAAGCCAG

AAAACACGGTCGATGAATTTATAGCTTCTGGTTTCCAGGAGGTGGTTGGG

CATGGCTTTGCGCAGCGCCACAGAACCGAAAGTGCCCACTGAGAAAAAAC

AACTCCTGCTTAATTTGCATTTTTCTAAAAGAAGAAACAGAGGCTGACGG

AAACTGGAAAGTTCCTGTTTTAACTACTCGAATTGAGTTTTCGGTCTTAG

CTTATCAACTGCTCACTTAGATTCATTTTCAAAGTAAACGTTTAAGAGCC

GAGGCATTCCTATCCTCTTCTAAGGCGTTATTCCTGGAGGCTCATTCACC

GCCAGCACCTCCGCTGCCTGCAGGCATTGCTGTCACCGTCACCGTGACGG

CGCGCACGATTTTCAGTTGGCCCGCTTCCCCTCGTGATTAGGACAGACGC

GGGCACTCTGGCCCAGCCGTCTTGGCTCAGTATCTGCAGGCGTCCGTCTC

GGGACGGAGCTCAGGGGAAGAGCGTGACTCCAGTTGAACGTGATAGTCGG

TGCGTTGAGAGGAGACCCAGTCGGGTGTCGAGTCAGAAGGGGCCCGGGGC

CCGAGGCCCTGGGCAGGACGGCCCGTGCCCTGCATCACGGGCCCAGCGTC

CTAGAGGCAGGACTCTGGTGGAGAGTGTGAGGGTGCCTGGGGCCCCTCCG

GAGCTGGGGCCGTGCGGTGCAGGTTGGGCTCTCGGCGCGGTGTTGGCTGT

TTCTGCGGGATTTGGAGGAATTCTTCCAGTGATGGGAGTCGCCAGTGACC

GGGCACCAGGCTGGTAAGAGGGAGGCCGCCGTCGTGGCCAGAGCAGCTGG

GAGGGTTCGGTAAAAGGCTCGCCCGTTTCCTTTAATGAGGACTTTTCCTG

GAGGGCATTTAGTCTAGTCGGGACCGTTTTCGACTCGGGAAGAGGGATGC

GGAGGAGGGCATGTGCCCAGGAGCCGAAGGCGCCGCGGGGAGAAGCCCAG

GGCTCTCCTGTCCCCACAGAGGCGACGCCACTGCCGCAGACAGACAGGGC

CTTTCCCTCTGATGACGGCAAAGGCGCCTCGGCTCTTGCGGGGTGCTGGG

GGGGAGTCGCCCCGAAGCCGCTCACCCAGAGGCCTGAGGGGTGAGACTGA

CCGATGCCTCTTGGCCGGGCCTGGGGCCGGACCGAGGGGGACTCCGTGGA

GGCAGGGCGATGGTGGCTGCGGGAGGGAACCGACCCTGGGCCGAGCCCGG

CTTGGCGATTCCCGGGCGAGGGCCCTCAGCCGAGGCGAGTGGGTCCGGCG

GAACCACCCTTTCTGGCCAGCGCCACAGGGCTCTCGGGACTGTCCGGGGC

GACGCTGGGCTGCCCGTGGCAGGCCTGGGCTGACCTGGACTTCACCAGAC

AGAACAGGGCTTTCAGGGCTGAGCTGAGCCAGGTTTAGCGAGGCCAAGTG

GGGCTGAACCAGGCTCAACTGGCCTGAGCTGGGTTGAGCTGGGCTGACCT

GGGCTGAGCTGAGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCT

GGACTGGCTGAGCTGAGCTGGGTTGAGCTGAGCTGAGCTGGCCTGGGTTG

AGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGTTGAGCTGGGTTG

ATCTGAGCTGAGCTGGGCTGAGCTGAGCTAGGCTGGGGTGAGCTGGGCTG

AGCTGGTTTGAGTTGGGTTGAGCTGAGCTGAGCTGGGCTGTGCTGGCTGA

GCTAGGCTGAGCTAGGCTAGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAG

GCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCGTTGAGCTGG

CTGGGCTGGATTGAGCTGGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCT

GGGTTGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGTTGAGCT

GTCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCT

CAGCAGAGCTGGGTTGGGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCT

GGCCTGGGTTGAGCTGGGCTGAGCTGAGCTGGGCTGAGCTGGCCTGTGTT

GAGCTGGGCTGGGTTGAGCTGGGCTGAGCTGGATTGAGCTGGGTTGAGCT

GAGCTGGGCTGGGCTGTGCTGACTGAGCTGGGCTGAGCTAGGCTGGGGTG

AGCTGGGCTGAGCTGATCCGAGCTAGGCTGGGCTGGTTTGGGCTGAGCTG

AGCTGAGCTAGGCTGGATTGATCTGGCTGAGCTGGGTTGAGCTGAGCTGG

GCTGAGCTGGTCTGAGCTGGCCTGGGTCGAGCTGAGCTGGACTGGTTTGA

GCTGGGTCGATCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGA

GCTGAGCTGGGTTGAGCTGGGCTGAGCTGAGGGCTGGGGTGAGCTGGGCT

GAACTAGCCTAGCTAGGTTGGGCTGAGCTGGGCTGGTTTGGGCTGAGCTG

AGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCAGGCCTGG

GGTGAGCTGGGCTAGGTGGAGCTGAGCTGGGTCGAGCTGAGTTGGGCTGA

GCTGGCCTGGGTTGAGGTAGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGA

GCTGGCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGG

TTGAGCTGGGCTCGGTTGAGCTGGGCTGAGCTGAGCCGACCTAGGCTGGG

ATGAGCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAG

CAGGCTGAGCTGGGCCTGGAGCCTGGCCTGGGGTGAGCTGGGCTGAGCTG

CGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAA

GCTGGGCCGAGCTGGCCTGGGATGAGCTGGGCCGGTTTGGGCTGAGCTGA

GCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGG

GTGAGCTGGGCTGAGCTAAGCTGAGCTGGGCTGGTTTGGGCTGAGCTGGC

TGAGCTGGGTCCTGCTGAGCTGGGCTGAGCTGACCAGGGGTGAGCTGGGC

TGAGTTAGGCTGGGCTCAGCTAGGCTGGGTTGATCTGGCAGGGCTGGTTT

GCGCTGGGTCAAGCTCCCGGGAGATGGCCTGGGATGAGCTGGGCTGGTTT

GGGCTGAGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTG

GGCTGAGCTGGCCTGGGGTGAGCTGGGCTGGGTGGAGCTGAGCTGGGCTG

AACTGGGCTAAGCTGGCTGAGCTGGATCGAGCTGAGCTGGGCTGAGCTGG

CCTGGGGTTAGCTGGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTGG

CTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGAG

CTGGGCTGGGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGGCTGGGCTGAG

CTGAGCTAGGCTGCATTGAGCTGGCTGGGATGGATTGAGCTGGCTGAGCT

GGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGG

GTTGAGCTGAGCTGGGCTGAGCTGGGCTCAGCAGAGCTGGGTTGAGCTGA

GCTGGGTTGAGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGTTGAGCTGA

GCTGGGTTGAGCTGGGCTCGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTG

AGCTGGGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTG

AGCTAGCTGGGCTCAGCTAGGCTGGGTTGAGCTGAGCTGGGCTGAACTGG

GCTGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCTGGGCTGAGCAGA

GCTGGGCTGAGCAGAGCTGGGTTGGTCTGAGCTGGGTTGAGCTGGGCTGA

GCTGGGCTGAGCAGAGTTGGGTTGAGCTGAGCTGGGTTCAGCTGGGCTGA

GCTAGGCTGGGTTGAGCTGGGTTGAGTTGGGCTGAGCTGGGCTGGGTTGA

GCGGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCGGAACTGGGTTGA

TCTGAATTGAGCTGGGCTGAGCCGGGCTGAGCCGGGCTGAGCTGGGCTAG

GTTGAGCTTGGGTGAGCTTGCCTCAGCTGGTCTGAGCTAGGTTGGGTGGA

GCTAGGCTGGATTGAGCTGGGCTGAGGTGAGCTGATCTGGCCTCAGCTGG

GCTGAGGTAGGCTGAACTGGGCTGTGCTGGGCTGAGCTGAGCTGAGCCAG

TTTGAGCTGGGTTGAGCTGGGCTGAGCTGGGCTGTGTTGATCTTTCCTGA

ACTGGGCTGAGCTGGGCTGAGCTGGCCTAGCTGGATTGAACGGGGGTAAG

CTGGGCCAGGCTGGACTGGGCTGAGCTGAGCTAGGCTGAGCTGAGTTGAA

TTGGGTTAAGCTGGGCTGAGATGGGCTGAGCTGGGCTGAGCTGGGTTGAG

CCAGGTCGGACTGGGTTACCCTGGGCCACACTGGGCTGAGCTGGGCGGAG

CTCGATTAACCTGGTCAGGCTGAGTCGGGTCCAGCAGACATGCGCTGGCC

AGGCTGGCTTGACCTGGACACGTTCGATGAGCTGCCTTGGGATGGTTCAC

CTCAGCTGAGCCAGGTGGCTCCAGCTGGGCTGAGCTGGTGACCCTGGGTG

ACCTCGGTGACCAGGTTGTCCTGAGTCCGGGCCAAGCCGAGGCTGCATCA

GACTCGCCAGACCCAAGGCCTGGGCCCCGGCTGGCAAGCCAGGGGCGGTG

AAGGCTGGGCTGGCAGGACTGTCCCGGAAGGAGGTGCACGTGGAGCCGCC

CGGACCCCGACCGGCAGGACCTGGAAAGACGCCTCTCACTCCCCTTTCTC

TTCTGTCCCCTCTCGGGTCCTCAGAGAGCCAGTCTGCCCCGAATCTCTAC

CCCCTCGTCTCCTGCGTCAGCCCCCCGTCCGATGAGAGCCTGGTGGCCCT

GGGCTGCCTGGCCCGGGACTTCCTGCCCAGCTCCGTCACCTTCTCCTGGAA

Porcine Kappa Chain Targeting
In particular embodiments of the present invention, targeting vectors are provided to target the porcine kappa chain locus. In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the constant region of the porcine immunoglobulin kappa chain locus. Since the present invention discovered that there is only one constant region of the porcine immunoglobulin kappa light chain locus, this will prevent the expression of a functional porcine kappa light chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the constant region, optionally including the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the constant region, optionally including at least part of the enhancer region (a “Kappa constant targeting construct”), see for example, FIG. 2. Further, this kappa constant targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 20 and FIG. 2. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the joining region of the porcine kappa light chain locus. In other embodiments, the 5′ arm of the targeting vector can include Seq ID No 12 and/or Seq ID No 25 or any contiguous sequence or fragment thereof. In another embodiment, the 3′ arm of the targeting vector can include Seq ID No 15, 16 and/or 19 or any contiguous sequence or fragment thereof.

In further embodiments, the targeting vector can include, but is not limited to any of the following sequences: the coding region of kappa light chain is represented, for example by residues 1-549 of Seq ID No 30 and 10026-10549 of Seq ID No 30, whereas the intronic sequence is represented, for example, by residues 550-10025 of Seq ID No 30, the Joining region of kappa light chain is represented, for example, by residues 5822-7207 of Seq ID No 30 (for example, J1:5822-5859 of Seq ID No 30, J2:6180-6218 of Seq ID No 30, J3:6486-6523 of Seq ID No 30, J4:6826-6863 of Seq ID No 30, J5:7170-7207 of Seq ID No 30), the Constant Region is represented by the following residues: 10026-10549 of Seq ID No 30 (C exon) and 10026-10354 of Seq ID No 30 (C coding), 10524-10529 of Seq ID No 30 (Poly(A) signal) and 11160-11264 of Seq ID No 30 (SINE element) or any fragment or combination thereof. Still further, any contiguous sequence at least about 17, 20, 30, 40, 50, 100, 150, 200 or 300 nucleotides of Seq ID No 30 or fragment and/or combination thereof can be used as targeting sequence for the heavy chain targeting vector. It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.


Seq ID No. 20

ctcaaacgtaagtggctttttccgactgattctttgctgtttctaattgt

tggttggctttttgtccatttttcagtgttttcatcgaattagttgtcag

ggaccaaacaaattgccttcccagattaggtaccagggaggggacattgc

tgcatgggagaccagagggtggctaatttttaacgtttccaagccaaaat

aactggggaagggggcttgctgtcctgtgagggtaggtttttatagaagt

ggaagttaaggggaaatcgctatggttcacttttggctcggggaccaaag

tggagcccaaaattgagtacattttccatcaattatttgtgagatttttg

tcctgttgtgtcatttgtgcaagtttttgacattttggttgaatgagcca

ttcccagggacccaaaaggatgagaccgaaaagtagaaaagagccaactt

ttaagctgagcagacagaccgaattgttgagtttgtgaggagagtagggt

ttgtagggagaaaggggaacagatcgctggctttttctctgaattagcct

ttctcatgggactggcttcagagggggtttttgatgagggaagtgttcta

gagccttaactgtgggttgtgttcggtagcgggaccaagctggaaatcaa

acgtaagtgcacttttctactcctttttctttcttatacgggtgtgaaat

tggggacttttcatgtttggagtatgagttgaggtcagttctgaagagag

tgggactcatccaaaaatctgaggagtaagggtcagaacagagttgtctc

atggaagaacaaagacctagttagttgatgaggcagctaaatgagtcagt

tgacttgggatccaaatggccagacttcgtctgtaaccaacaatctaatg

agatgtagcagcaaaaagagatttccattgaggggaaagtaaaattgtta

atattgtggatcacctttggtgaagggacatccgtggagattgaacgtaa

gtattttttctctactaccttctgaaatttgtctaaatgccagtgttgac

ttttagaggcttaagtgtcagttttgtgaaaaatgggtaaacaagagcat

ttcatatttattatcagtttcaaaagttaaactcagctccaaaaatgaat

ttgtagacaaaaagattaatttaagccaaattgaatgattcaaaggaaaa

aaaaattagtgtagatgaaaaaggaattcttacagctccaaagagcaaaa

gcgaattaattttctttgaactttgccaaatcttgtaaatgatttttgtt

ctttacaatttaaaaaggttagagaaatgtatttcttagtctgttttctc

tcttctgtctgataaattattatatgagataaaaatgaaaattaatagga

tgtgctaaaaaatcagtaagaagttagaaaaatatatgtttatgttaaag

ttgccacttaattgagaatcagaagcaatgttatttttaaagtctaaaat

gagagataaactgtcaatacttaaattctgcagagattctatatcttgac

agatatctcctttttcaaaaatccaatttctatggtagactaaatttgaa

atgatcttcctcataatggagggaaaagatggactgaccccaaaagctca

gatttaagaaaacctgtttaaggaaagaaaataaaagaactgcatttt

ttaaaggcccatgaatttgtagaaaaataggaaatattttaataagtgta

ttcttttattttcctgttattacttgatggtgtttttataccgccaagga

ggccgtggcaccgtcagtgtgatctgtagaccccatggcggccttttttc

gcgattgaatgaccttggcggtgggtccccagggctctggtggcagcgca

ccagccgctaaaagccgctaaaaactgccgctaaaggccacagcaacccc

gcgaccgcccgttcaactgtgctgacacagtgatacagataatgtcgcta

acagaggagaatagaaatatgacgggcacacgctaatgtggggaaaagag

ggagaagcctgatttttattttttagagattctagagataaaattcccag

tattatatccttttaataaaaaatttctattaggagattataaagaattt

aaagctatttttttaagtggggtgtaattctttcagtagtctcttgtcaa

atggatttaagtaatagaggcttaatccaaatgagagaaatagacgcata

accctttcaaggcaaaagctacaagagcaaaaattgaacacagcagccag

ccatctagccactcagattttgatcagttttactgagtttgaagtaaata

tcatgaaggtataattgctgataaaaaaataagatacaggtgtgacacat

ctttaagtttcagaaatttaatggcttcagtaggattatatttcacgtat

acaaagtatctaagcagataaaaatgccattaatggaaacttaatagaaa

tatatttttaaattccttcattctgtgacagaaattttctaatctgggtc

ttttaatcacctaccctttgaaagagtttagtaatttgctatttgccatc

gctgtttactccagctaatttcaaaagtgatacttgagaaagattatttt

tggtttgcaaccacctggcaggactattttagggccattttaaaactctt

ttcaaactaagtattttaaactgttctaaaccatttagggccttttaaaa

atcttttcatgaatttcaaacttcgttaaaagttattaaggtgtctggca

agaacttccttatcaaatatgctaatagtttaatctgttaatgcaggata

taaaattaaagtgatcaaggcttgacccaaacaggagtatcttcatagca

tatttcccctcctttttttctagaattcatatgattttgctgccaaggct

attttatataatctctggaaaaaaaatagtaatgaaggttaaaagagaag

aaaatatcagaacattaagaattcggtattttactaactgcttggttaac

atgaaggtttttattttattaaggtttctatctttataaaaatctgttcc

cttttctgctgatttctccaagcaaaagattcttgatttgttttttaact

cttactctcccacccaagggcctgaatgcccacaaaggggacttccagga

ggccatctggcagctgctcaccgtcagaagtgaagccagccagttcctcc

tgggcaggtggccaaaattacagttgacccctcctggtctggctgaacct

tgccccatatggtgacagccatctggccagggcccaggtctccctctgaa

gcctttgggaggagagggagagtggctggcccgatcacagatgcggaagg

ggctgactcctcaaccggggtgcagactctgcagggtgggtctgggccca

acacacccaaagcacgcccaggaaggaaaggcagcttggtatcactgccc

agagctaggagaggcaccgggaaaatgatctgtccaagacccgttcttgc

ttctaaactccgagggggtcagatgaagtggttttgtttcttggcctgaa

gcatcgtgttccctgcaagaagcggggaacacagaggaaggagagaaaag

atgaactgaacaaagcatgcaaggcaaaaaaggGGGTCTAGCCGCGGTCT

AGGAAGCTTTCTAGGGTACCTCTAGGGATCCCGGCGCGCCCTACCGGGTA

GGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCC

GCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCA

CATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCG

CCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCT

CGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCG

TGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGC

AGCGGCCAATAGCAGCTTTGGCTCCTTCGCTTTCTGGGCTCAGAGGCTGG

GAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCG

GGCGCCCGAAGGTCCTCCGGAAGCCCGGCATTCTGCACGCTTCAAAAGCG

CACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCT

GCAGCCAATATGGGATCGGCCATTGAACAAGATGGATTGCACGCAGGTTC

TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGA

CAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGC

CCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCA

GGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCG

CAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTG

GGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGA

GAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATC

CGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCA

CGTACTCGGATGGAAGCCGGTCTTGTCAATCAGGATGATCTGGACGAAGA

GCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCA

TGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCG

AATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG

GCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTGGCTACCCGTGATA

TTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGGTTCCTCGTGCTTTAC

GGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGA

CGAGTTCTTCTGAGGGGATCAATTCTCTAGAGCTCGCTGATCAGCCTCGA

CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT

TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA

GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG

GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT

GCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGGGGAAAGAACCAGCTG

GGGGCGCGCCCctcgagcggccgccagtgtgatggatatctgcagaattc

gcccttggatcaaacacgcatcctcatggacaatatgttgggttcttagc

ctgctgagacacaacaggaactcccctggcaccactttagaggccagaga

aacagcacagataaaattccctgccctcatgaagcttatagtctagctgg

ggagatatcataggcaagataaacacatacaaatacatcatcttaggtaa

taatatatactaaggagaaaattacaggggagaaagaggacaggaattgc

tagggtaggattataagttcagatagttcatcaggaacactgttgctgag

aagataacatttaggtaaagaccgaagtagtaaggaaatggaccgtgtgc

ctaagtgggtaagaccattctaggcagcaggaacagcgatgaaagcactg

aggtgggtgttcactgcacagagttgttcactgcacagagttgtgtgggg

aggggtaggtcttgcaggctcttatggtcacaggaagaattgttttactc

ccaccgagatgaaggttggtggattttgagcagaagaataattctgcctg

gtttatatataacaggatttccctgggtgctctgatgagaataatctgtc

aggggtgggatagggagagatatggcaataggagccttggctaggagccc

acgacaataattccaagtgagaggtggtgctgcattgaaagcaggactaa

caagacctgctgacagtgtggatgtagaaaaagatagaggagacgaaggt

gcatctagggttttctgcctgaggaattagaaagataaagctaaagctta

tagaagatgcagcgctctggggagaaagaccagcagctcagttttgatcc

atctggaattaattttggcataaagtatgaggtatgtgggttaacattat

ttgttttttttttttccatgtagctatccaactgtcccagcatcatttat

tttaaaagactttcctttcccctattggattgttttggcaccttcactga

agatcaactgagcataaaattgggtctatttctaagctcttgattccatt

ccatgacctatttgttcatctttaccccagtagacactgccttgatgatt

aaagcccctgttaccatgtctgttttggacatggtaaatctgagatgcct

attagccaaccaagcaagcacggcccttagagagctagatatgagagcct

ggaattcagacgagaaaggtcagtcctagagacatacatgtagtgccatc

accatgcggatggtgttaaaagccatcagactgcaacagactgtgagagg

gtaccaagctagagagcatggatagagaaacccaagcactgagctgggag

gtgctcctacattaagagattagtgagatgaaggactgagaagattgatc

agagaagaaggaaaatcaggaaaatggtgctgtcctgaaaatccaaggga

agagatgttccaaagaggagaaaactgatcagttgtcagctagcgtcaat

tgggatgaaaatggaccattggacagagggatgtagtgggtcatgggtga

atagataagagcagcttctatagaatggcaggggcaaaattctcatctga

tcggcatgggttctaaagaaaacgggaagaaaaaattgagtgcatgacca

gtcccttcaagtagagaggtggaaaagggaaggaggaaaatgaggccacg

acaacatgagagaaatgacagcatttttaaaaattttttattttatttta

tttatttatttttgctttttagggctgcccctgcaacatatggaggttcc

caggttaggggtctaatcagagctatagctgccagcctacaccacagcca

tagcaatgccagatctacatgacctacaccacagctcacagcaacgccgg

atccttaacccactgagtgaggccagagatcaaacccatatccttatgga

tactagtcaggttcattaccactgagccaaaatgggaaatcctgagtaat

gacagcattttttaatgtgccaggaagcaaaacttgccaccccgaaatgt

ctctcaggcatgtggattattttgagctgaaaacgattaaggcccaaaaa

acacaagaagaaatgtggaccttcccccaacagcctaaaaaatttagatt

gagggcctgttcccagaatagagctattgccagacttgtctacagaggct

aagggctaggtgtggtggggaaaccctcagagatcagagggacgtttatg

taccaagcattgacatttccatctccatgcgaatggccttcttcccctct

gtagccccaaaccaccacccccaaaatcttcttctgtctttagctgaaga

tggtgttgaaggtgatagtttcagccactttggcgagttcctcagttgtt

ctgggtctttcctccTgatccacattattcgactgtgtttgattttctcc

tgtttatctgtctcattggcacccatttcattcttagaccagcccaaaga

acctagaagagtgaaggaaaatttcttccaccctgacaaatgctaaatga

gaatcaccgcagtagaggaaaatgatctggtgctgcgggagatagaagag

aaaatcgctggagagatgtcactgagtaggtgagatgggaaaggggtgac

acaggtggaggtgttgccctcagctaggaagacagacagttcacagaaga

gaagcgggtgtccgtggacatcttgcctcatggatgaggaaaccgaggct

aagaaagactgcaaaagaaaggtaaggattgcagagaggtcgatccatga

ctaaaatcacagtaaccaaccccaaaccaccatgttttctcctagtctgg

cacgtggcaggtactgtgtaggttttcaatattattggtttgtaacagta

cctattaggcctccatcccctcctctaatactaacaaaagtgtgagactg

gtcagtgaaaaatggtcttctttctctatgaatctttctcaagaagatac

ataactttttattttatcataggcttgaagagcaaatgagaaacagcctc

caacctatgacaccgtaacaaaatgtttatgatcagtgaagggcaagaaa

caaaacatacacagtaaagaccctccataatattgtgggtggcccaacac

aggccaggttgtaaaagctttttattctttgatagaggaatggatagtaa

tgtttcaacctggacagagatcatgttcactgaatccttccaaaaattca

tgggtagtttgaattataaggaaaataagacttaggataaatactttgtc

caagatcccagagttaatgccaaaatcagttttcagactccaggcagcct

gatcaagagcctaaactttaaagacacagtcccttaataactactattca

cagttgcactttcagggcgcaaagactcattgaatcctacaatagaatga

gtttagatatcaaatctctcagtaatagatgaggagactaaatagcgggc

atgacctggtcacttaaagacagaattgagattcaaggctagtgttcttt

ctacctgttttgtttctacaagatgtagcaatgcgctaattacagacctc

tcagggaaggaa

Porcine Lambda Chain Targeting
In particular embodiments of the present invention, targeting vectors are provided to target the porcine lambda chain locus. In one embodiment, lambda can be targeted by designing a targeting construct that contains a 5′ arm containing sequence located 5′ to the first JC unit and a 3′ arm containing sequence 3′ to the last JC unit of the J/C cluster region, thus preventing functional expression of the lambda locus (see, FIGS. 3-4). In one embodiment, the targeting vector can contain any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof. Seq ID No 28. In one embodiment, the 5′ targeting arm can contain Seq ID No. 32, which includes 5′ flanking sequence to the first lambda J/C region of the porcine lambda light chain genomic sequence or any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof (see also, for example FIG. 5). In another embodiment, the 3′ targeting arm can contain, but is not limited to one or more of the following: Seq ID No. 33, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, from approximately 200 base pairs downstream of lambda J/C; Seq ID No. 34, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, approximately 11.8 Kb downstream of the J/C cluster, near the enhancer; Seq ID No. 35, which includes approximately 12 Kb downstream of lambda, including the enhancer region; Seq ID No. 36, which includes approximately 17.6 Kb downstream of lambda; Seq ID No. 37, which includes approximately 19.1 Kb downstream of lambda; Seq ID No. 38, which includes approximately 21.3 Kb downstream of lambda; and Seq ID No. 39, which includes approximately 27 Kb downstream of lambda, or any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof of Seq ID Nos 32-39 (see also, for example FIG. 6). It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.
Seq ID No. 48 (as shown in Example 4) provides a representative, non-limiting example of a targeting construct that contains a 5′ arm containing sequence located 5′ to the first JC unit and a 3′ arm containing sequence 3′ to the last JC unit of the J/C cluster region. Representative 5′ and 3′ arms are shown in Seq ID No. 49 and 50 (also in Example 4).
In another embodiment, lambda is targeted using two targeting vectors. The two lambda targeting vectors, i.e., a vector pair, are utilized in a two step strategy to delete the entire J/C region of porcine lambda. In the first step, a first targeting vector is inserted upstream of the J/C region (or alternatively downstream of the J/C region). If the first targeting vector is inserted upstream of the J/C region, the 5′ and 3′ recombination arms of the first targeted vector contain homologous sequence to the 5′ flanking sequence of the first J/C unit of the J/C cluster region. See FIG. 5, which shows 7 JC units in the J/C cluster region. If the first targeting vector is inserted downstream of the J/C cluster region, the 5′ and 3′ recombination arms of the first targeting vector contain homologous sequence to the 3′ region of the last J/C unit in the JC region.
The first-step vectors are designed with lox sites that flank a fusion gene which can provide both positive and negative selection. Selection of the targeting event utilizes the Tn5 APHII gene commonly described as Neo resistance. Once targeting events are isolated, Cre is provided transiently to facilitate deletion of the selectable marker located between two lox sites. Negative selection is then provided by the Herpes simplex thymidine kinase coding region. This step selects for targeted cells that have deleted the selectable marker and retains a single lox site upstream (alternatively downstream) of the J/C region.
The second step is performed in the same lineage as the first step. The second targeting step also inserts a marker that provides both positive and negative selection. However, the second step inserts the marker on the opposite site of the J/C region in comparison to the first step. That is, if the first vector was inserted upstream of the J/C region, the second targeting vector is inserted downstream, and vice versa. FIG. 6 shows a second targeting vector inserted downstream of the J/C region. In addition, the second targeting vector has a single lox site that is located distally compared to the first vector. In other words, for the first strategy, the second vector has a single lox site located downstream of the marker gene (the alternative vector has the lox site upstream of the marker). After Cre mediated deletion, the region between the first targeting event (which left a lox remnant) and the second targeting event (which has a lox site outside of the marker) is deleted. Cells that have deleted the entire J/C cluster region are thus obtained.
In a representative, non-limiting example, the vector pair is Seq. ID No. 44 (step 1) and Seq. ID No. 45 (step 2).

In a further, non-limiting example, the vector pair is Seq. ID No. 46 (step 1) and Seq. ID No. 47 (step 2).


SEQ. ID	taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgctgagcaggccctggcctccctggcc

44	gagggcggtttgcgtattagaggcctaaatggccgaattcagcggataacaatttcacacaggaaacagctatgaccatg

	attatctagtaactataacggtcctaaggtagcgagcgatcgcttaattaacctgcagggatatcccatgggggccgccag

	tgtgatggatatctgcagaattcgcccttgatattaagagaagggcaagtcagcttaagtttgggggtagaggggaacag

	ggagtgaggagatctggcctgagagataggagccctggtggccacaggaggactctttgggtcctgtcggatggacac

	agggcggcccgggggcatgttggagcccggctggttcttaccagaggcagggggcaccctctgacacgggagcagg

	gcatgttccatacatgacacacccctctgctccagggcaggtgggtggcggcacagaggagccagggactctgagcaa

	ggggtccaccagtggggcagttggatccagacttctctgggccagcgagagtctagccctcagccgttctctgtccagg

	aggggggtggggcaggcctgggcggccagagctcatccctcaagggttcccagggtcctgccagacccagatttccg

	accgcagccaccacaagaggatgtggtctgctgtggcagctgccaagaccttgcagcaggtgcagggtgggggggtg

	ggggcacctgggggcagctggggtcactgagttcagggaaaaccccttttttcccctaaacctggggccatccctaggg

	gaaaccacaacttctgagccctgggcagtggctgctgggagggaagagcttcatcctggaccctgggggggaaccca

	gctccaaaggtgcaaggggcccaggtccaaggctagagtgggccaagcaccgcaatggccagggagtgggggagg

	tggagctggactggatcagggcctccttgggactccctacaccctgtgtgacatgttagggtacccacaccccatcacca

	gtcagggcctggcccatctccagggccagggatgtgcatgtaagtgtgtgtgagtgtgtgtgtgtggtgtagtacacccct

	tggcatccggttccgaggccttgggttcctccaaagttgctctctgaattaggtcaaactgtgaggtcctgatcgccatcatc

	aacttcgttctccccacctcccatcattatcaagagctggggagggtctgggatttcttcccacccacaagccaaaagata

	agcctgctggtgatggcagaagacacaggatcctgggtcagagacaaaggccagtgtgtcacagcgagagaggcag

	ccggactatcagctgtcacagagaggccttagtccgctgaactcaggccccagtgactcctgttccactgggcactggcc

	cccctccacagcgcccccaggccccagggagaggcgtcacagcttagagatggccctgctgaacagggaacaagaa

	caggtgtgccccatccagcgccccaggggtgggacaggtgggctggatttggtgtgaagcccttgagccctggaaccc

	aaccacagcagggcagttggtagatgccatttggggagaggccccaggagtaagggccatgggcccttgagggggc

	caggagctgaggacagggacagagacggcccaggcagaggacagggccatgaggggtgcactgagatggccact

	gccagcaggggcagctgccaacccgtccagggaacttattcagcagtcagctggaggtgccattgaccctgagggca

	gatgaagcccaggccaggctaggtgggctgtgaagaccccaggggacagagctctgtccctgggcagcactggcctc

	tcattctgcagggcttgacgggatcccaaggcctgctgcccctgatggtagtggcagtaccgcccagagcaggacccc

	agcatggaaaccccaacgggacgcagcctgcggagcccacaaaaccagtaaggagccgaagcagtcatggcacgg

	ggagtgtggacttccctttgatggggcccaggcatgaaggacagaatgggacagcggccatgagcagaaaatcagcc

	ggaggggatgggcctaggcagacgctggctttatttgaagtgttggcattttgtctggtgtgtattgttggtattgattttatttt

	agtatgtcagtgacatactgacatattatgtaacgacatattattatgtgttttaagaagcactccaagggaacaggctgtctg

	taatgtgtccagagaagagagcaagagcttggctcagtctcccccaaggaggtcagttcctcaacaggggtcctaaatgt

	ttcctggagccaggcctgaatcaagggggtcatatctacacgtggggcagacccatggaccattttcggagcaataagat

	ggcagggaggataccaagctggtcttacagatccagggctttgacctgtgacgcgggcgctcctccaggcaaagggag

	aagccagcaggaagctttcagaactggggagaacagggtgcagacctccagggtcttgtacaacgcaccctttatcctg

	gggtccaggaggggtcactgagggatttaagtgggggaccatcagaaccaggtttgtgttttggaaaaatggctccaaa

	gcagagaccagtgtgaggccagattagatgatgaagaagaggcagtggaaagtcgatgggtggccaggtagcaaga

	gggcctatggagttggcaagtgaatttaaagtggtggcaccagagggcagatggggaggagcaggcactgtcatgga

	ctgtctatagaaatctaaaatgtataccctttttagcaatatgcagtgagtcataaaagaacacatatatatttcctttggccgg

	ccggcgcgccacgcgtataacttcgtatagcatacattatacgaagttatcttaagggctatggcagggcctgccgcccc

	gacgttggctgcgagccctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtatt

	ggccccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaacgacc

	caacaccgtgcgttttattctgtctttttattgccgtcatagcgcgggttccttccggtatgtctccttccgtgtttcactcgagt

	tagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgagga

	agcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccac

	acccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgg

	gtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgc

	tcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtc

	gaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagca

	aggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcga

	gcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagggcac

	cggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagcc

	gattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttc

	aatggccgatcccattccagatctgttagcctcccccatctcccgtgcaaacgtgcgcgccaggtcgcagatcgtcggtat

	ggagcctggggtggtgacgtgggtctggatcatcccggaggtaagttgcagcagggcgtcccggcagccggcgggc

	gattggtcgtaatccaggataaagacgtgcatgggacggaggcgtttggtcaagacgtccaaggcccaggcaaacacg

	ttgtacaggtcgccgttgggggccagcaactcgggggcccgaaacagggtaaataacgtgtccccgatatggggtcgt

	gggcccgcgttgctctggggctcggcaccctggggcggcacggccgtccccgaaagctgtccccaatcctcccgcca

	cgacccgccgccctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcgaatcgcggccagcatagcca

	ggtcaagccgctcgccggggcgctggcgtttggccaggcggtcgatgtgtctgtcctccggaagggcccccaacacg

	atgtttgtgccgggcaaggtcggcgggatgagggccacgaacgccagcacggcctggggggtcatgctgcccataag

	gtatcgcgcggccgggtagcacaggagggcggcgatgggatggcggtcgaagatgagggtgagggccgggggcg

	gggcatgtgagctcccagcctcccccccgatatgaggagccagaacggcgtcggtcacggcataaggcatgcccattg

	ttatctgggcgcttgtcattaccaccgccgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtggtgtagat

	gttcgcgattgtctcggaagcccccagcacccgccagtaagtcatcggctcgggtacgtagacgatatcgtcgcgcgaa

	cccagggccaccagcagttgcgtggtggtggttttccccatcccgtggggaccgtctatataaacccgcagtagcgtgg

	gcattttctgctccgggcggacttccgtggcttcttgctgccggcgagggcgcaacgccgtacgtcggttgctatggccg

	cgagaacgcgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaagccatggtggctctagaggtcga

	aaggcccggagatgaggaagaggagaacagcgcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttccg

	gaggaccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccggacccaccccttcccagcctctg

	agcccagaaagcgaaggagccaaagctgctattggccgctgccccaaaggcctacccgcttccattgctcagcggtgc

	tgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcgagctgcggggcgggggg

	gaacttcctgactaggggaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcctaccg

	gtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccagcggggctgctaaagcgcatgct

	ccagactgccttgggaaaagcgcctcccctacccggtagggatccgcgttacataacttacggtaaatggcccgcctgg

	ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattg

	acgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt

	gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct

	acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgggg

	atttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacggttaacaagcttataacttcgtat

	agcatacattatacgaagttattacgtagcggccgcgtcgacgataaattgtgtaattccacttctaaggattcatcccaagg

	ggggaaaataatcaaagatgtaaccaaaggtttacaaacaagaactcatcattaatcttccttgttgttatttcaacgatattat

	tattattactattattattattattattttgtctttttgcattttctagggccactcccacggcatagagaggttcccaggctagggg

	tcaaatcggagctacagctgccggcctacgccagagccacagcaacgcaggatctgagccacagcaatgcaggatct

	acaccacagctcatggtaacgctggatccttaacccaatgagtgaggccagggatcgaacctgtaacttcatggttcctag

	tcggattcattaaccactgagccacgacaggaactccaacattattaatgatgggagaaaactggaagtaacctaaatatc

	cagcagaaagggtgtggccaaatacagcatggagtagccatcataaggaatcttacacaagcctccaaaattgtgtttctg

	aaattgggtttaaagtacgtttgcattttaaaaagcctgccagaaaatacagaaaaatgtctgtgatatgtctctggctgatag

	gattttgcttagttttaattttggctttataattttctatagttatgaaaatgttcacaagaagatatatttcattttagcttctaaaata

	attataacacagaagtaatttgtgctttaaaaaaatattcaacacagaagtatataaagtaaaaattgaggagttcccatcgtg

	gctcagtgattaacaaacccaactagtatccatgaggatatggatttgatccctggccttgctcagtgggttgaggatccag

	tgttgctgtgagctgtggtgtaggttgcagacacagcactctggcgttgctgtgactctggcgtaggccggcagctacag

	ctccatttggacccttagcctgggaacctccatatgcctgagatacggccctaaaaagtcaaaagccaaaaaaatagtaa

	aaattgagtgtttctacttaccacccctgcccacatcttatgctaaaacccgttctccagagacaaacatcgtcaggtgggtc

	tatatatttccagccctcctcctgtgtgtgtatgtccgtaaaacacacacacacacacacacacgcacacacacacacacg

	tatctaattagcattggtattagtttttcaaaagggaggtcatgctctaccttttaggcggcaaatagattatttaaacaaatctg

	ttgacattttctatatcaacccataagatctcccatgttcttggaaaggctttgtaagacatcaacatctgggtaaaccagcat

	ggtttttagggggtttgtggatttttttcatattttttagggcacacctgcagcatatggaggttcccaggctaggggttgaat

	cagagctgtagctgccggcctacaccacagccacagcaacgccagatccttaacccactgagaaaggccagggattga

	acctgcatcctcatggatgctggtcagatttatttctgctgagccacaacaggaactccctgaaccagaatgcttttaaccat

	tccactttgcatggacatttagattgtttccatttaaaaatacaaattacaaggagttcccgtcgtggctcagtggtaacgaatt

	ggactaggaaccatgaggtttcgggttcgatccctggccttgctcggtgggttaaggatccagcattgatgtgagatatgg

	tgtaggtcgcagacgtggctcggatcccacgttgctgtggctctggcgtaggccggcaacaacagctccgattcgaccc

	ctagcctgggaacctccatgtgccacaggagcagccctagaaaaggcaaaaagacaaaaaaataaaaaattaaaatga

	aaaaataaaataaaaatacaaattacaagagacggctacaaggaaatccccaagtgtgtgcaaatgccatatatgtataaa

	atgtactagtgtctcctcgcgggaaagttgcctaaaagtgggttggctggacagagaggacaggctttgacattctcatag

	gtagtagcaatgggcttctcaaaatgctgttccagtttacactcaccatagcaaatgacagtgcctcttcctctccacccttg

	ccaataatgtgacaggtggatctttttctattttgtgtatctgacaagcaaaaaatgagaacaggagttcctgtcgtggtgca

	gtggagacaaatctgactaggaaccatgaaatttcgggttcaatccctggcctcactcagtaggtaaaggatccagggttg

	cagtgagctgtggggtaggtcgcagacacagtgcaaatttggccctgttgtggctgtggtgtaggccggcagctatagct

	ccaattggacccctagcctgggaacctccttatgccgtgggtgaggccctaaaaaaaagagtgcaaaaaaaaaaaataa

	gaacaaaaatgatcatcgtttaattctttatttgatcattggtgaaacttattttccttttatatttttattgactgattttatttctcctat

	gaatttaccggtcatagttttgcctgggtgtttttactccggttttagttttggttggttgtattttcttagagagctatagaaactct

	tcatctatttggaatagtaattcctcattaagtatttgtgctgcaaaaaattttccctgatctgttttatgcttttgtttgtggggtctt

	tcacgagaaagcctttttagtttttacacctcagcttggttgtttttcttgattgtgtctgtaatctgcggccaacataggaaaca

	catttttactttagtgtttttttcctattttcttcaagtacgtccattgttttggtgtctgattttactttgcctggggtttgtttttgtgtg

	gcaggaatataaacttatgtattttccaaatggagagccaatggttgtatatttgttgaattcaaatgcaactttatcaaacacc

	aaatcatcgatttatcacaactcttctctggtttattgatctaatgatcaattcctgttccacgctgttttaattattttagctttgtgg

	attttggtgcctggtagagaacaaagcctccattattttcattcaaaatagtcccgtctattatctgccattgttgtagtattaga

	ctttaaaatcaatttactgattttcaaaagttattcctttggtgatgtggaatactttatacttcataaggtacatggattcatttgtg

	gggaattgatgtctttgctattgtggccatttgtcaagttgtgtaatattttacccatgccaactttgcatattgtatgtgagtttat

	tcccagggtttttaataggatgtttattgaagttgtcagtgtttccacaatttcatcgcctcagtgcttactgtttgcataaaagg

	aaacctactcacttttgcctattgctcttgtattcaatcattttagttaactcttgtgttaattttgagagtttttcagctgactgtctg

	gggttttctttaatagactagccctttgtctgtaaagaataattttatcgaatttttcttaacactcacactctccccacccccacc

	cccgctcatctcctttcattgggtcaaatctgtagaatacaataaaagtaagagtgggaaccttagcctttaagtcgattttgc

	ctttaaatgtgaatgttgctatgtttcgggacattctctttatcaagttgcggatgtttccttagataattaacttaataaaagact

	ggatgtttgctttcttcaaatcagaattgtgttgaatttatattgctattctgtttaattttgtttcaaaaaatttacatgcacacctta

	aagataaccatgaccaaatagtcctcctgctgagagaaaatgttggccccaatgccacaggttacctcccgactcagata

	aactacaatgggagataaaatcagatttggcaaagcctgtggattcttgccataactctcagagcatgacttgggtgttttttc

	cttttctaagtattttaatggtatttttgtgttacaataggaaatctaggacacagagagtgattcaatgaggggaacgcattct

	gggatgactctaggcctctggtttggggagagctctattgaagtaaagacaatgagaggaagcaagtttgcagggaact

	gtgaggaatttagatggggaatgttgggtttgaggtttctatagggcacgcaagcagagatgcactcaggaggaagaag

	gagcataaatctagtggcgctgccggcaagcttgctggaggaggccaattgggagctgctggaatgcatggaggcggc

	gctctcgaggctggaggaggccagctgatttaaatcggtccgcgtacgatgcatattaccctgttatccctaccgcggtta

	ctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgatgctcttctcccggtgaaaacctctgacacatggctct

	tctaaatccggagtttaaacgcttccttcatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgtt

	gctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccg

	acaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgga

	tacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt

	cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtc

	caacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcgg

	tgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcca

	gttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagc

	agcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaa

	aactcacgttaagggattttggtcatgcctaggtggcaaacagctattatgggtattatgggtctaccggtgcatgagattat

	caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctga

	cagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgt

	gtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggct

	ccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcc

	agtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggc

	atcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccat

	gttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggtta

	tggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctg

	agaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaa

	aagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacc

	cactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgcc

	gcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcaggg

	ttattgtctcgggagcggatacatatttgaatgtatttagaaaaa

SEQ ID 45	taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgctgagcaggccctggcctccctggcc

	gagggcggtttgcgtattagaggcctaaatggccgaattcagcggataacaatttcacacaggaaacagctatgaccatg

	attatctagtaactataacggtcctaaggtagcgagcgatcgcttaattaacctgcagggataaccactgacccatgacgg

	gaactcccagggctcagctcttgactccaggttcgcagctgccctcaaagcaatgcaaccctggctggccccgcctcat

	gcatccggcctcctccccaaagagctctgagcccacctgggcctaggtcctcctccctgggactcatggcctaagggta

	cagagttactggggctgatgaagggaccaatggggacaggggcctcaaatcaaagtggctgtctctctcatgtcccttcc

	tctcctcagggtccaaaatcagggtcagggccccagggcaggggctgagagggcctctttctgaaggccctgtctcagt

	gcaggttatgggggtctgggggagggtcaatgcagggctcacccttcagtgccccaaagcctagagagtgagtgcctg

	ccagtggcttcccaggcccaatcccttgactgcctgggaatgctcaaatgcaggaactgtcacaacaccttcagtcaggg

	gctgctctgggaggaaaaacactcagaattgggggttcagggaaggcccagtgccaagcatagcaggagctcaggtg

	gctgcagatggtgtgaaccccaggagcaggatggccggcactccccccagaccctccagagccccaggttggctgcc

	ctcttcactgccgacacccctgggtccacttctgccctttcccacctaaaacctttagggctcccactttctcccaaatgtga

	gacatcaccacggctcccagggagtgtccagaagggcatctggctgagaggtcctgacatctgggagcctcaggcccc

	acaatggacagacgccctgccaggatgctgctgcagggctgttagctaggcggggtggagatggggtactttgcctctc

	agaggccccggccccaccatgaaacctcagtgacaccccatttccctgagttcacatacctgtatcctactccagtcacct

	tccccacgaacccctgggagcccaggatgatgctggggctggagccacgaccagcccacgagtgatccagctctgcc

	aatcagcagtcatttcccaagtgttccagccctgccaggtcccactacagcagtaatggaggccccagacaccagtcca

	gcagttagagggctggactagcaccagctttcaagcctcagcatctcaaggtgaatggccagtgcccctccccgtggcc

	atcacaggatcgcagatatgaccctaggggaagaaatatcctgggagtaaggaagtgcccatactcaaggatggcccct

	ctgtgacctaacctgtccctgaggattgtacttccaggcgttaaaacagtagaacgcctgcctgtgaacccccgccaagg

	gactgcttggggaggccccctaaaccagaacacaggcactccagcaggacctctgaactctgaccaccctcagcaagt

	gggcaccccccgcagcttccaaggcaccccagggctcaccacagcggcccctcctggcagcccctcacccaggccc

	agaccctctaagatggcacatctaagccaatccacctccttgtcattcctcctgtccccacccaggacccttctcagatgaa

	accttcgctccagccgctgggccctctctcctgcccctctggcagttctccagggactccgcctcccactctctgtctctcc

	ctgcactcctaggaacaagcgacctccaggaagcccagtccaattatcccctctgtgtcctccccaatctctgcctctggg

	tggatttgagcaccacatcctgttctcttcgacctgaaactccttggccccggtgtccgctctcctgggccctcttttctctcct

	cccctcttccgtgccccgtttgtttggtgttacaggcaggccccggggagccgtccctccagctgctcttccttgtctgtctc

	aggagccagaaactggcagcatctaaaaagggctcctgtttcttcatctgcccagcctcctagcccaaccagggctctgg

	cctcactccagagggtgggctccagagggcaggggttgcaccctcttagtgcctcagaggctcagctgggtgcaggat

	gggggggccctcagggagcccctcagtgactgctgatcacttactgcaggactgttcccagctcttcccaatcattggaat

	gacaatacctagttctgctccatcatagtgatgcaggaaaaatgttactgaaatcctggttcttgtttagcaatcgaagaatg

	aattccgcgaacacacaggcagcaagcaagcgaagcctttattaaaggaaagcagatagctcccagggctgcaggga

	gcggggagaagagctccccactctctattgtcctatagggctttttaccccttaaagttggggggatacaaaaaaaataga

	agaaaaagggagttcccgtcagggcacagcagaaacaaatccaactaggaaccatgaggttgggggttcgattcctgg

	cctctctcagtgggttaaggatgcagcgttgccgtgagctatgatacaggtcacagatgcagctcagatctactagtcaatt

	gacaggcgccggagcaggagctaggcctttggccggccggcgcgccagatctcttaagggctatggcagggcctgcc

	gccccgacgttggctgcgagccctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcggg

	cgtattggccccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaa

	cgacccaacaccgtgcgttttattctgtctttttattgccgtcatagcgcgggttccttccggtattgtctccttccgtgtttcact

	cgagttagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacg

	aggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccg

	ccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgcc

	atgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctg

	atgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggt

	ggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcagg

	agcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacg

	tcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagg

	gcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagc

	agccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatctt

	gttcaatggccgatcccattccagatctgttagcctcccccatctcccgtgcaaacgtgcgcgccaggtcgcagatcgtcg

	gtatggagcctggggtggtgacgtgggtctggatcatcccggaggtaagttgcagcagggcgtcccggcagccggcg

	ggcgattggtcgtaatccaggataaagacgtgcatgggacggaggcgtttggtcaagacgtccaaggcccaggcaaac

	acgttgtacaggtcgccgttgggggccagcaactcgggggcccgaaacagggtaaataacgtgtccccgatatggggt

	cgtgggcccgcgttgctctggggctcggcaccctggggcggcacggccgtccccgaaagctgtccccaatcctcccg

	ccacgacccgccgccctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcgaatcgcggccagcatag

	ccaggtcaagccgctcgccggggcgctggcgtttggccaggcggtcgatgtgtctgtcctccggaagggcccccaaca

	cgatgtttgtgccgggcaaggtcggcgggatgagggccacgaacgccagcacggcctggggggtcatgctgcccata

	aggtatcgcgcggccgggtagcacaggagggcggcgatgggatggcggtcgaagatgagggtgagggccggggg

	cggggcatgtgagctcccagcctcccccccgatatgaggagccagaacggcgtcggtcacggcataaggcatgccca

	ttgttatctgggcgcttgtcattaccaccgccgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtggtgta

	gatgttcgcgattgtctcggaagcccccagcacccgccagtaagtcatcggctcgggtacgtagacgatatcgtcgcgc

	gaacccagggccaccagcagttgcgtggtggtggttttccccatcccgtggggaccgtctatataaacccgcagtagcgt

	gggcattttctgctccgggcggacttccgtggcttcttgctgccggcgagggcgcaacgccgtacgtcggttgctatggc

	cgcgagaacgcgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaagccatggtggctctagaggtc

	gaaaggcccggagatgaggaagaggagaacagcgcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttc

	cggaggaccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccggacccaccccttcccagcct

	ctgagcccagaaagcgaaggagccaaagctgctattggccgctgccccaaaggcctacccgcttccattgctcagcgg

	tgctgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcgagctgcggggcgggg

	gggaacttcctgactaggggaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcctac

	cggtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccagcggggctgctaaagcgcatg

	ctccagactgccttgggaaaagcgcctcccctacccggtagggatccgcgttacataacttacggtaaatggcccgcctg

	gctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccatt

	gacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctat

	tgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc

	tacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggg

	gatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacggttaacaagcttataacttcgta

	tagcatacattatacgaagttattacgtagcggccgcgtcgacgatatcgctgccggagcccccggggccgctgccgga

	agatctggcattgctgtgactgtggtgtaggccggcagctggagctctgattagacccctcacctgggaatctccatatgc

	tgcacgtgcggccctaaaaagacaaaagacaaaaaaaaaaaaaaaaaaaaaaaatcaaaaaaaaacatagggggtta

	ccaacgtggggtccagaaagatgtggttttctcccattggccttgcccagttacctatatcagtccttgtccaacaggggttt

	taggggggaaatgccccataaattttacggtttctttgcccttctcttcctttagactgagtcaccattgctctcattccttttcta

	tcagttgaggagtgggttagagattaaggtccatgtggtggaggtacacttcttatagtaaacaaggcctatggggaattac

	tctctggagcccttaaaccacaaatgataatccatgccacatcaaagatgcatcgaagcccatgctcctacactgactacct

	gagttagcattctgcctcaacaggactgaccatccccagctctggggcagatatcctctctctgccacaagggcagtgac

	ccccatgctgtctgagggtcacgctttaccccccccccacccctgccgtgaccccccagaccaccccaggaggtgggc

	actaatatccctcattaccccatagatgaggaaacagaggttcccccggggtcccacaggtgctcagggtcacatgcacc

	gtgggcacccaggccccatcccaaggccaccctccctcctcaggaagctgtgctgcgctgggccagaaggtactgcac

	acgactcctcagcctccggtggtgggaggcagcctcaagcctctgagtgggggggcacccgggctcctcaatctatact

	gactcctgggggtgggagaaggggagggggagctgtggcctctgagtccactaagcaaatcagggtgggcaatgcg

	ggcccatttcaaggaggagagaaccgaggctctgacagcaggccgggggtccagggacctgcccagggtcataggc

	tgaactgctggctgacctgccttgggttctttccttggctcctcagccctgtgtgatgtgacaggtcattcattcactcactcg

	ctcattcattcagcaaaccctcagtgagccctgctgggagcaggtgctaggggcaaggagacaggacctcttgccctgg

	aacagctgaagcactgggggacaggcagtggcagggaggtgcgtgatcaccgctgaccccattccatcctccagccc

	ccaggtcagtttccacccaccattgaccccaccatgtcctccatccccaaggtcagtttcccgcccaaggagcatctcctt

	acacactagggacaaaatttcacggctgtcactgggcatctctccacgctcatcacagccctctagcagccttgaagtcct

	gtagagcccttcccatttcacagaagggacaagactatgagggccacaccgtgagccatgagccttaggctgtgagccg

	ggacagcccctgcaggactggtggcctcagggcactgggtggggagggtgcacagtgggtgggccccttgtggaata

	gagaggagtgtcaggtcaggggagggggcttggcctggccctggcctgcctggtgtgcaaccctaggcagcccctcct

	tcccaggcctcctacttcctggaggccaagcctcagggaggtaattgagtcaggtgggggagggggggttgtggctttc

	ttcacagcagaaaaacagagcccacaatagtgtccactgagacagaggggtcctgggggaggggaggggtgggagg

	tgactgctgagccctgtgggagggagggagcaactactgagctgagctgggtgactctcccatctgccccgccccctgt

	ggggccagcagagtcaccgagagaacatgacccagccaggcctggacagggggacacccatgtcctttaccccaca

	gggttcactgagcctatctgccccaagcctgtgtctccctgggacggagaccctcactcccaaccacaaaggtctaaact

	caagttcccaacagccttgaaaatacagcttccgggggcctccaaggagcagtcagccgtccactgccaggctcgctg

	gctcagtgacacaggacacatcctgatgacggtccacctgtctccaagcaggttctcctctgccgatggggcaacgagct

	cctcctgtggctccctggctggatgcgtgggaggcggggtgggggggcaggcggtgttcctggccgcacacaaggag

	cacccccaccagcatccgaagacgggggcccggtctttccccaaaacactgcttgcgggagactttgtgacgtttccag

	gggccatgctcccttcgggcagcttgggggacttctgctcctatgtggtcacctgcagggactccccccaggccttgggg

	acaaacaaagtgatgagagggagggttagtgggtcggggcagggccagtctttggaccggtttatctgaaaagccagtt

	ggtcaccgggaaccacagcaaacctaaacccatttggccaggcatctcccagggacagtctcccccaggatgcgggg

	cccaggggggctccaggggtgacctgcgtcctggatttccctgatgctcccagttcgtgcctctgtccaagcatgattttta

	atagtgccccttccactcccagaaatgtccaagtgtgggcaataaattctggtcacctgagctcagtgtaactgtttgctgaa

	tgacacttactgtaacaggttaaaatgggaggcccaaggccacgcagagccatcgaaggctctgtgtgtcccagccctg

	atagaagcatcaggatggggactgtggcctcaccaggggccacatccaggcggtcaccatggggttcctggtctccgt

	gggccttgactggagcccctggtgtgagctcaccccatcccagcctgtgagaggcctggatgtgggcctgacatcatttc

	ccacccagtgacagcactgcatgtgatggggcctctgggcagcctttttcccgggggaaactggcaggaatcaggacc

	accaggacaggggtcaggggagaggcgatgctgggcaccagagcctggaccaccctcgggttctcagcgatgggca

	acccctgccacccagggccccgccttcctggggagacatcggggtttccaggccatcctgggaggagggtgggagcc

	tcagctagaccccagctggcttgcccccccatgccccggccaagagagggtcttggagggaagggggaccccagac

	cagcctggcgagcccatcctcagggtctctggtcagacaggggctcagctgagctccagggtagaccaaggccctgc

	gtggatgaggccagtgtggtcactgcccagagcaaagccacctctcagcagccctttcctgagcaccttctgtgtgcggg

	gacatcagcagtggcaacacagccatgctggggactcagggctagagacaggggaccagcctatggagagtgggta

	gtgtcctgcagggcaggcttgtgccctggagaaaacaaaccagggtgaggccagggacgctggccgggttcacagg

	gtgatggctgagcacagagtgccaggggctggactgtcctgactctgggttggtggctgagggcctgtgtccctctatgc

	ctctgggttggtgataatggaaacttgctccctggagagacaggacgaatggttgatgggaaatgaatgtttgcttgtcact

	tggttgactgttgttgccgttagcattgggcttcttgggccaggcagcctcaggccagcactgctgggctccccacaggc

	ccgacaccctcagccctgtgcagctggcctggcgaaaccaagaggccctgatgcccaaaatagccgggaaaccccaa

	ccagcccagccctggcagcaggtgcctcccatttgcctgggctgggggaggggtggctctggttctggaagtttctgcc

	agtccagctggagaagggacctgtatcccagcacccaggccgcccaagcccctgcaccagggcctgggccaggcag

	agttgacatcaatcaattgggagctgctggaatgcatggaggcggcgctctcgaggctggaggaggccagctgatttaa

	atcggtccgcgtacgatgcatattaccctgttatccctaccgcggttactggccgtcgttttacaacgtcgtgactgggaaa

	accctggcgatgctcttctcccggtgaaaacctctgacacatggctcttctaaatccggagtttaaacgcttccttcatgtga

	gcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgac

	gagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct

	ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtg

	gcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccc

	ccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg

	cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta

	cggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga

	tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctca

	agaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcctaggt

	ggcaaacagctattatgggtattatgggtctaccggtgcatgagattatcaaaaaggatcttcacctagatccttttaaattaa

	aaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct

	cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatct

	ggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccgga

	agggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagt

	agttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttca

	ttcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctc

	cgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccat

	ccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgc

	ccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcg

	aaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttac

	tttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaat

	gttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcgggagcggatacatatttgaatgtat

	ttagaaaaa

SEQ ID 46	taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgctgagcaggccctggcctccctggcc

	gagggcggtttgcgtattagaggcctaaatggccgaattcagcggataacaatttcacacaggaaacagctatgaccatg

	attatctagtaactataacggtcctaaggtagcgagcgatcgcttaattaacctgcagggatatcccatgggggccgccag

	tgtgatggatatctgcagaattcgcccttgatattaagagaagggcaagtcagcttaagtttgggggtagaggggaacag

	ggagtgaggagatctggcctgagagataggagccctggtggccacaggaggactctttgggtcctgtcggatggacac

	agggcggcccgggggcatgttggagcccggctggttcttaccagaggcagggggcaccctctgacacgggagcagg

	gcatgttccatacatgacacacccctctgctccagggcaggtgggtggcggcacagaggagccagggactctgagcaa

	ggggtccaccagtggggcagttggatccagacttctctgggccagcgagagtctagccctcagccgttctctgtccagg

	aggggggtggggcaggcctgggcggccagagctcatccctcaagggttcccagggtcctgccagacccagatttccg

	accgcagccaccacaagaggatgtggctgctgtggcagctgccaagaccttgcagcaggtgcagggtgggggggtg

	ggggcacctgggggcagctggggtcactgagttcagggaaaaccccttttttcccctaaacctggggccatccctaggg

	gaaaccacaacttctgagccctgggcagtggctgctgggagggaagagcttcatcctggaccctgggggggaaccca

	gctccaaaggtgcaaggggcccaggtccaaggctagagtgggccaagcaccgcaatggccagggagtgggggagg

	tggagctggactggatcagggcctccttgggactccctacaccctgtgtgacatgttagggtacccacaccccatcacca

	gtcagggcctggcccatctccagggccagggatgtgcatgtaagtgtgtgtgagtgtgtgtgtgtggtgtagtacacccct

	tggcatccggttccgaggccttgggttcctccaaagttgctctctgaattaggtcaaactgtgaggtcctgatcgccatcatc

	aacttcgttctccccacctcccatcattatcaagagctggggagggtctgggatttcttcccacccacaagccaaaagata

	agcctgctggtgatggcagaagacacaggatcctgggtcagagacaaaggccagtgtgtcacagcgagagaggcag

	ccggactatcagctgtcacagagaggccttagtccgctgaactcaggccccagtgactcctgttccactgggcactggcc

	cccctccacagcgcccccaggccccagggagaggcgtcacagcttagagatggccctgctgaacagggaacaagaa

	caggtgtgccccatccagcgccccaggggtgggacaggtgggctggatttggtgtgaagcccttgagccctggaaccc

	aaccacagcagggcagttggtagatgccatttggggagaggccccaggagtaagggccatgggcccttgagggggc

	caggagctgaggacagggacagagacggcccaggcagaggacagggccatgaggggtgcactgagatggccact

	gccagcaggggcagctgccaacccgtccagggaacttattcagcagtcagctggaggtgccattgaccctgagggca

	gatgaagcccaggccaggctaggtgggctgtgaagaccccaggggacagagctctgtccctgggcagcactggcctc

	tcattctgcagggcttgacgggatcccaaggcctgctgcccctgatggtagtggcagtaccgcccagagcaggacccc

	agcatggaaaccccaacgggacgcagcctgcggagcccacaaaaccagtaaggagccgaagcagtcatggcacgg

	ggagtgtggacttccctttgatggggcccaggcatgaaggacagaatgggacagcggccatgagcagaaaatcagcc

	ggaggggatgggcctaggcagacgctggctttatttgaagtgttggcattttgtctggtgtgtattgttggtattgattttatttt

	agtatgtcagtgacatactgacatattatgtaacgacatattattatgtgttttaagaagcactccaagggaacaggctgtctg

	taatgtgtccagagaagagagcaagagcttggctcagtctcccccaaggaggtcagttcctcaacaggggtcctaaatgt

	ttcctggagccaggcctgaatcaagggggtcatatctacacgtggggcagacccatggaccattttcggagcaataagat

	ggcagggaggataccaagctggtcttacagatccagggctttgacctgtgacgcgggcgctcctccaggcaaagggag

	aagccagcaggaagctttcagaactggggagaacagggtgcagacctccagggtcttgtacaacgcaccctttatcctg

	gggtccaggaggggtcactgagggatttaagtgggggaccatcagaaccaggtttgtgttttggaaaaatggctccaaa

	gcagagaccagtgtgaggccagattagatgatgaagaagaggcagtggaaagtcgatgggtggccaggtagcaaga

	gggcctatggagttggcaagtgaatttaaagtggtggcaccagagggcagatggggaggagcaggcactgtcatgga

	ctgtctatagaaatctaaaatgtataccctttttagcaatatgcagtgagtcataaaagaacacatatatatttcctttggccgg

	ccggcgcgccacgcgtataacttcgtatagcatacattatacgaagttatcttaagggctatggcagggcctgccgcccc

	gacgttggctgcgagccctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtatt

	ggccccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaacgacc

	caacaccgtgcgttttattctgtctttttattgccgtcatagcgcgggttccttccggtattgtctccttccgtgtttcactcgagt

	tagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgagga

	agcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccac

	acccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgg

	gtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgc

	tcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtc

	gaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagca

	aggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcga

	gcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagggcac

	cggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagcc

	gattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttc

	aatggccgatcccattccagatctgttagcctcccccatctcccgtgcaaacgtgcgcgccaggtcgcagatcgtcggtat

	ggagcctggggtggtgacgtgggtctggatcatcccggaggtaagttgcagcagggcgtcccggcagccggcgggc

	gattggtcgtaatccaggataaagacgtgcatgggacggaggcgtttggtcaagacgtccaaggcccaggcaaacacg

	ttgtacaggtcgccgttgggggccagcaactcgggggcccgaaacagggtaaataacgtgtccccgatatggggtcgt

	gggcccgcgttgctctggggctcggcaccctggggcggcacggccgtccccgaaagctgtccccaatcctcccgcca

	cgacccgccgccctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcgaatcgcggccagcatagcca

	ggtcaagccgctcgccggggcgctggcgtttggccaggcggtcgatgtgtctgtcctccggaagggcccccaacacg

	atgtttgtgccgggcaaggtcggcgggatgagggccacgaacgccagcacggcctggggggtcatgctgcccataag

	gtatcgcgcggccgggtagcacaggagggcggcgatgggatggcggtcgaagatgagggtgagggccgggggcg

	gggcatgtgagctcccagcctcccccccgatatgaggagccagaacggcgtcggtcacggcataaggcatgcccattg

	ttatctgggcgcttgtcattaccaccgccgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtggtgtagat

	gttcgcgattgtctcggaagcccccagcacccgccagtaagtcatcggctcgggtacgtagacgatatcgtcgcgcgaa

	cccagggccaccagcagttgcgtggtggtggttttccccatcccgtggggaccgtctatataaacccgcagtagcgtgg

	gcattttctgctccgggcggacttccgtggcttcttgctgccggcgagggcgcaacgccgtacgtcggttgctatggccg

	cgagaacgcgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaagccatggtggctctagaggtcga

	aaggcccggagatgaggaagaggagaacagcgcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttccg

	gaggaccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccggacccaccccttcccagcctctg

	agcccagaaagcgaaggagccaaagctgctattggccgctgccccaaaggcctacccgcttccattgctcagcggtgc

	tgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcgagctgcggggcgggggg

	gaacttcctgactaggggaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcctaccg

	gtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccagcggggctgctaaagcgcatgct

	ccagactgccttgggaaaagcgcctcccctacccggtagggatccgcgttacataacttacggtaaatggcccgcctgg

	ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattg

	acgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt

	gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct

	acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgggg

	atttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacggttaacaagcttagatctgcggc

	cgcgtcgacgataaattgtgtaattccacttctaaggattcatcccaaggggggaaaataatcaaagatgtaaccaaaggt

	ttacaaacaagaactcatcattaatcttccttgttgttatttcaacgatattattattattactattattattattattattttgtctttttg

	cattttctagggccactcccacggcatagagaggttcccaggctaggggtcaaatcggagctacagctgccggcctacg

	ccagagccacagcaacgcaggatctgagccacagcaatgcaggatctacaccacagctcatggtaacgctggatcctt

	aacccaatgagtgaggccagggatcgaacctgtaacttcatggttcctagtcggattcattaaccactgagccacgacag

	gaactccaacattattaatgatgggagaaaactggaagtaacctaaatatccagcagaaagggtgtggccaaatacagca

	tggagtagccatcataaggaatcttacacaagcctccaaaattgtgtttctgaaattgggtttaaagtacgtttgcattttaaaa

	agcctgccagaaaatacagaaaaatgtctgtgatatgtctctggctgataggattttgcttagttttaattttggctttataattn

	ctatagttatgaaaatgttcacaagaagatatatttcattttagcttctaaaataattataacacagaagtaatttgtgctttaaaa

	aatattcaacacagaagtatataaaaaaattgaggagttcccatcgtggctcagtgattaacaaacccaactagtatc

	catgaggatatggatttgatccctggccttgctcagtgggttgaggatccagtgttgctgtgagctgtggtgtaggttgcag

	acacagcactctggcgttgctgtgactctggcgtaggccggcagctacagctccatttggacccttagcctgggaacctc

	catatgcctgagatacggccctaaaaagtcaaaagccaaaaaaatagtaaaaattgagtgtttctacttaccacccctgcc

	cacatcttatgctaaaacccgttctccagagacaaacatcgtcaggtgggtctatatatttccagccctcctcctgtgtgtgta

	tgtccgtaaaacacacacacacacacacacacgcacacacacacacacgtatctaattagcattggtattagtttttcaaaa

	gggaggtcatgctctaccttttaggcggcaaatagattatttaaacaaatctgttgacattttctatatcaacccataagatctc

	ccatgttcttggaaaggctttgtaagacatcaacatctgggtaaaccagcatggtttttagggggttgtgtggatttttttcata

	ttttttagggcacacctgcagcatatggaggttcccaggctaggggttgaatcagagctgtagctgccggcctacaccac

	agccacagcaacgccagatccttaacccactgagaaaggccagggattgaacctgcatcctcatggatgctggtcagat

	ttatttctgctgagccacaacaggaactccctgaaccagaatgcttttaaccattccactttgcatggacatttagattgtttcc

	atttaaaaatacaaattacaaggagttcccgtcgtggctcagtggtaacgaattggactaggaaccatgaggtttcgggttc

	gatccctggccttgctcggtgggttaaggatccagcattgatgtgagatatggtgtaggtcgcagacgtggctcggatccc

	acgttgctgtggctctggcgtaggccggcaacaacagctccgattcgacccctagcctgggaacctccatgtgccacag

	gagcagccctagaaaaggcaaaaagacaaaaaaataaaaaattaaaatgaaaaaataaaataaaaatacaaauacaag

	agacggctacaaggaaatccccaagtgtgtgcaaatgccatatatgtataaaatgtactagtgtctcctcgcgggaaagtt

	gcctaaaagtgggttggctggacagagaggacaggctttgacattctcataggtagtagcaatgggcttctcaaaatgctg

	ttccagtttacactcaccatagcaaatgacagtgcctcttcctctccacccttgccaataatgtgacaggtggatctttttctatt

	ttgtgtatctgacaagcaaaaaatgagaacaggagttcctgtcgtggtgcagtggagacaaatctgactaggaaccatga

	aatttcgggttcaatccctggcctcactcagtaggtaaaggatccagggttgcagtgagctgtggggtaggtcgcagaca

	cagtgcaaatttggccctgttgtggctgtggtgtaggccggcagctatagctccaattggacccctagcctgggaacctcc

	ttatgccgtgggtgaggccctaaaaaaaagagtgcaaaaaaaaaaaataagaacaaaaatgatcatcgtttaattctttattt

	gatcattggtgaaacttattttccttttatatttttattgactgattttatttctcctatgaatttaccggtcatagttttgcctgggtgtt

	tttactccggttttagttttggttggttgtattttcttagagagctatagaaactcttcatctatttggaatagtaattcctcattaagt

	atttgtgctgcaaaaaattttccctgatctgttttatgcttttgtttgtggggtctttcacgagaaagcctttttagtttttacacctc

	agcttggttgtttttcttgattgtgtctgtaatctgcggccaacataggaaacacatttttactttagtgtttttttcctattttcttca

	agtacgtccattgttttggtgtctgattttactttgcctggggtttgtttttgtgtggcaggaatataaacttatgtattttccaaatg

	gagagccaatggttgtatatttgttgaattcaaatgcaactttatcaaacaccaaatcatcgatttatcacaactcttctctggtt

	tattgatctaatgatcaattcctgttccacgctgttttaattattttagctttgtggattttggtgcctggtagagaacaaagcctc

	cattattttcattcaaaatagtcccgtctattatctgccattgttgtagtattagactttaaaatcaatttactgattttcaaaagttat

	tcctttggtgatgtggaatactttatacttcataaggtacatggattcatttgtggggaattgatgtctttgctattgtggccattt

	gtcaagttgtgtaatattttacccatgccaactttgcatattgtatgtgagtttattcccagggtttttaataggatgtttattgaag

	ttgtcagtgtttccacaatttcatcgcctcagtgcttactgtttgcataaaaggaaacctactcacttttgcctattgctcttgtatt

	caatcattttagttaactcttgtgttaattttgagagtttttcagctgactgtctggggttttctttaatagactagccctttgtctgt

	aaagaataattttatcgaatttttcttaacactcacactctccccacccccacccccgctcatctcctttcattgggtcaaatct

	gtagaatacaataaaagtaagagtgggaaccttagcctttaagtcgattttgcctttaaatgtgaatgttgctatgtttcggga

	cattctctttatcaagttgcggatgtttccttagataattaacttaataaaagactggatgtttgctttcttcaaatcagaattgtgt

	tgaatttatattgctattctgtttaattttgtttcaaaaaatttacatgcacaccttaaagataaccatgaccaaatagtcctcctg

	ctgagagaaaatgttggccccaatgccacaggttacctcccgactcagataaactacaatgggagataaaatcagatttg

	gcaaagcctgtggattcttgccataactctcagagcatgacttgggtgttttttccttttctaagtattttaatggtatttttgtgtta

	caataggaaatctaggacacagagagtgattcaatgaggggaacgcattctgggatgactctaggcctctggtttgggga

	gagctctattgaagtaaagacaatgagaggaagcaagtttgcagggaactgtgaggaatttagatggggaatgttgggttt

	gaggtttctatagggcacgcaagcagagatgcactcaggaggaagaaggagcataaatctagtggcgctgccggcaa

	gcttgctggaggaggccaattgggagctgctggaatgcatggaggcggcgctctcgaggctggaggaggccagctga

	tttaaatcggtccgcgtacgatgcatattaccctgttatccctaccgcggttactggccgtcgttttacaacgtcgtgactgg

	gaaaaccctggcgatgctcttctcccggtgaaaacctctgacacatggctcttctaaatccggagtttaaacgcttccttcat

	gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccc

	tgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc

	ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagc

	gtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac

	cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccac

	tggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaa

	ctacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctct

	tgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatct

	caagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcctag

	gtggcaaacagctattatgggtattatgggtctaccggtgcatgagattatcaaaaaggatcttcacctagatccttttaaatt

	aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcaccta

	tctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccat

	ctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccg

	gaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa

	gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggctt

	cattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtc

	ctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgc

	catccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctct

	tgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggg

	gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctt

	ttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacgga

	aatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcgggagcggatacatatttgaat

	gtatttagaaaaa

SEQ ID 47	taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgctgagcaggccctggcctccctggcc

	gagggcggtttgcgtattagaggcctaaatggccgaattcagcggataacaatttcacacaggaaacagctatgaccatg

	attatctagtaactataacggtcctaaggtagcgagcgatcgcttaattaacctgcagggataaccactgacccatgacgg

	gaactcccagggctcagctcttgactccaggttcgcagctgccctcaaagcaatgcaaccctggctggccccgcctcat

	gcatccggcctcctccccaaagagctctgagcccacctgggcctaggtcctcctccctgggactcatggcctaagggta

	cagagttactggggctgatgaagggaccaatggggacaggggcctcaaatcaaagtggctgtctctctcatgtcccttcc

	tctcctcagggtccaaaatcagggtcagggccccagggcaggggctgagagggcctctttctgaaggccctgtctcagt

	gcaggttatgggggtctgggggagggtcaatgcagggctcacccttcagtgccccaaagcctagagagtgagtgcctg

	ccagtggcttcccaggcccaatcccttgactgcctgggaatgctcaaatgcaggaactgtcacaacaccttcagtcaggg

	gctgctctgggaggaaaaacactcagaattgggggttcagggaaggcccagtgccaagcatagcaggagctcaggtg

	gctgcagatggtgtgaaccccaggagcaggatggccggcactccccccagaccctccagagccccaggttggctgcc

	ctcttcactgccgacacccctgggtccacttctgccctttcccacctaaaacctttagggctcccactttctcccaaatgtga

	gacatcaccacggctcccagggagtgtccagaagggcatctggctgagaggtcctgacatctgggagcctcaggcccc

	acaatggacagacgccctgccaggatgctgctgcagggctgttagctaggcggggtggagatggggtactttgcctctc

	agaggccccggccccaccatgaaacctcagtgacaccccatttccctgagttcacatacctgtatcctactccagtcacct

	tccccacgaacccctgggagcccaggatgatgctggggctggagccacgaccagcccacgagtgatccagctctgcc

	aatcagcagtcatttcccaagtgttccagccctgccaggtcccactacagcagtaatggaggccccagacaccagtcca

	gcagttagagggctggactagcaccagctttcaagcctcagcatctcaaggtgaatggccagtgcccctccccgtggcc

	atcacaggatcgcagatatgaccctaggggaagaaatatcctgggagtaaggaagtgcccatactcaaggatggcccct

	ctgtgacctaacctgtccctgaggattgtacttccaggcgttaaaacagtagaacgcctgcctgtgaacccccgccaagg

	gactgcttggggaggccccctaaaccagaacacaggcactccagcaggacctctgaactctgaccaccctcagcaagt

	gggcaccccccgcagcttccaaggcaccccagggctcaccacagcggcccctcctggcagcccctcacccaggccc

	agaccctctaagatggcacatctaagccaatccacctccttgtcattcctcctgtccccacccaggacccttctcagatgaa

	accttcgctccagccgctgggccctctctcctgcccctctggcagttctccagggactccgcctcccactctctgtctctcc

	ctgcactcctaggaacaagcgacctccaggaagcccagtccaattatcccctctgtgtcctccccaatctctgcctctggg

	tggatttgagcaccacatcctgttctcttcgacctgaaactccttggccccggtgtccgctctcctgggccctcttttctctcct

	cccctcttccgtgccccgtttgtttggtgttacaggcaggccccggggagccgtccctccagctgctcttccttgtctgtctc

	aggagccagaaactggcagcatctaaaaagggctcctgtttcttcatctgcccagcctcctagcccaaccagggctctgg

	cctcactccagagggtgggctccagagggcaggggttgcaccctcttagtgcctcagaggctcagctgggtgcaggat

	gggggggccctcagggagcccctcagtgactgctgatcacttactgcaggactgttcccagctcttcccaatcattggaat

	gacaatacctagttctgctccatcatagtgatgcaggaaaaatgttactgaaatcctggttcttgtttagcaatcgaagaatg

	aattccgcgaacacacaggcagcaagcaagcgaagcctttattaaaggaaagcagatagctcccagggctgcaggga

	gcggggagaagagctccccactctctattgtcctatagggctttttaccccttaaagttggggggatacaaaaaaaataga

	agaaaaagggagttcccgtcagggcacagcagaaacaaatccaactaggaaccatgaggttgggggttcgattcctgg

	cctctctcagtgggttaaggatgcagcgttgccgtgagctatgatacaggtcacagatgcagctcagatctactagtcaatt

	gacaggcgccggagcaggagctaggcctttggccggccggcgcgccacgcgtataacttcgtatagcatacattatac

	gaagttatcttaagggctatggcagggcctgccgccccgacgttggctgcgagccctgggccttcacccgaacttgggg

	ggtggggtggggaaaaggaagaaacgcgggcgtattggccccaatggggtctcggtggggtatcgacagagtgcca

	gccctgggaccgaaccccgcgtttatgaacaaacgacccaacaccgtgcgttttattctgtctttttattgccgtcatagcgc

	gggttccttccggtattgtctccttccgtgtttcactcgagttagaagaactcgtcaagaaggcgatagaaggcgatgcgct

	gcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcac

	gggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccat

	tttccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgag

	cctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatcc

	gagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccg

	cattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgccc

	aatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccac

	gatagccgcgctgcctcgtcctgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccct

	gcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccac

	ccaagcggccggagaacctgcgtgcaatccatcttgttcaatggccgatcccattccagatctgttagcctcccccatctc

	ccgtgcaaacgtgcgcgccaggtcgcagatcgtcggtatggagcctggggtggtgacgtgggtctggatcatcccgga

	ggtaagttgcagcagggcgtcccggcagccggcgggcgattggtcgtaatccaggataaagacgtgcatgggacgga

	ggcgtttggtcaagacgtccaaggcccaggcaaacacgttgtacaggtcgccgttgggggccagcaactcgggggcc

	cgaaacagggtaaataacgtgtccccgatatggggtcgtgggcccgcgttgctctggggctcggcaccctggggcggc

	acggccgtccccgaaagctgtccccaatcctcccgccacgacccgccgccctgcagataccgcaccgtattggcaagc

	agcccgtaaacgcggcgaatcgcggccagcatagccaggtcaagccgctcgccggggcgctggcgtttggccaggc

	ggtcgatgtgtctgtcctccggaagggcccccaacacgatgtttgtgccgggcaaggtcggcgggatgagggccacga

	acgccagcacggcctggggggtcatgctgcccataaggtatcgcgcggccgggtagcacaggagggcggcgatgg

	gatggcggtcgaagatgagggtgagggccgggggcggggcatgtgagctcccagcctcccccccgatatgaggagc

	cagaacggcgtcggtcacggcataaggcatgcccattgttatctgggcgcttgtcattaccaccgccgcgtccccggcc

	gatatctcaccctggtcaaggcggtgttgtgtggtgtagatgttcgcgattgtctcggaagcccccagcacccgccagtaa

	gtcatcggctcgggtacgtagacgatatcgtcgcgcgaacccagggccaccagcagttgcgtggtggtggttttccccat

	cccgtggggaccgtctatataaacccgcagtagcgtgggcattttctgctccgggcggacttccgtggcttcttgctgccg

	gcgagggcgcaacgccgtacgtcggttgctatggccgcgagaacgcgcagcctggtcgaacgcagacgcgtgctgat

	ggccggggtacgaagccatggtggctctagaggtcgaaaggcccggagatgaggaagaggagaacagcgcggcag

	acgtgcgcttttgaagcgtgcagaatgccgggcttccggaggaccttcgggcgcccgccccgcccctgagcccgcccc

	tgagcccgcccccggacccaccccttcccagcctctgagcccagaaagcgaaggagccaaagctgctattggccgct

	gccccaaaggcctacccgcttccattgctcagcggtgctgtccatctgcacgagactagtgagacgtgctacttccatttgt

	cacgtcctgcacgacgcgagctgcggggcgggggggaacttcctgactaggggaggagtagaaggtggcgcgaag

	gggccaccaaagaacggagccggttggcgcctaccggtggatgtggaatgtgtgcgaggccagaggccacttgtgta

	gcgccaagtgcccagcggggctgctaaagcgcatgctccagactgccttgggaaaagcgcctcccctacccggtagg

	gatccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatga

	cgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggc

	agtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgccca

	gtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag

	tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttg

	gcaccaaaatcaacggttaacaagcttataacttcgtatagcatacattatacgaagttattacgtagcggccgcgtcgacg

	atatcgctgccggagcccccggggccgctgccggaagatctggcattgctgtgactgtggtgtaggccggcagctgga

	gctctgattagacccctcacctgggaatctccatatgctgcacgtgcggccctaaaaagacaaaagacaaaaaaaaaaa

	aaaaaaaaaaaaatcaaaaaaaaacatagggggttaccaacgtggggtccagaaagatgtggttttctcccattggcctt

	gcccagttacctatatcagtccttgtccaacaggggttttaggggtggaaatgccccataaattttacggtttctttgcccttct

	cttcctttagactgagtcaccattgctctcattccttttctatcagttgaggagtgggttagagattaaggtccatgtggtggag

	gtacacttcttatagtaaacaaggcctatggggaattactctctggagcccttaaaccacaaatgataatccatgccacatc

	aaagatgcatcgaagcccatgctcctacactgactacctgagttagcattctgcctcaacaggactgaccatccccagctc

	tggggcagatatcctctctctgccacaagggcagtgacccccatgctgtctgagggtcacgctttaccccccccccaccc

	ctgccgtgaccccccagaccaccccaggaggtgggcactaatatccctcattaccccatagatgaggaaacagaggttc

	ccccggggtcccacaggtgctcagggtcacatgcaccgtgggcacccaggccccatcccaaggccaccctccctcctc

	aggaagctgtgctgcgctgggccagaaggtactgcacacgactcctcagcctccggtggtgggaggcagcctcaagc

	ctctgagtgggggggcacccgggctcctcaatctatactgactcctgggggtgggagaaggggagggggagctgtgg

	cctctgagtccactaagcaaatcagggtgggcaatgcgggcccatttcaaggaggagagaaccgaggctctgacagca

	ggccgggggtccagggacctgcccagggtcataggctgaactgctggctgacctgccttgggttctttccttggctcctc

	agccctgtgtgatgtgacaggtcattcattcactcactcgctcattcattcagcaaaccctcagtgagccctgctgggagca

	ggtgctaggggcaaggagacaggacctcttgccctggaacagctgaagcactgggggacaggcagtggcagggag

	gtgcgtgatcaccgctgaccccattccatcctccagcccccaggtcagtttccacccaccattgaccccaccatgtcctcc

	atccccaaggtcagtttcccgcccaaggagcatctccttacacactagggacaaaatttcacggctgtcactgggcatctc

	tccacgctcatcacagccctctagcagccttgaagtcctgtagagcccttcccatttcacagaagggacaagactatgag

	ggccacaccgtgagccatgagccttaggctgtgagccgggacagcccctgcaggactggtggcctcagggcactggg

	tggggagggtgcacagtgggtgggccccttgtggaatagagaggagtgtcaggtcaggggagggggcttggcctggc

	cctggcctgcctggtgtgcaaccctaggcagcccctccttcccaggcctcctacttcctggaggccaagcctcagggag

	gtaattgagtcaggtgggggagggggggttgtggctttcttcacagcagaaaaacagagcccacaatagtgtccactga

	gacagaggggtcctgggggaggggaggggtgggaggtgactgctgagccctgtgggagggagggagcaactactg

	agctgagctgggtgactctcccatctgccccgccccctgtggggccagcagagtcaccgagagaacatgacccagcca

	ggcctggacagggggacacccatgtcctttaccccacagggttcactgagcctatctgccccaagcctgtgtctccctgg

	gacggagaccctcactcccaaccacaaaggtctaaactcaagttcccaacagccttgaaaatacagcttccgggggcct

	ccaaggagcagtcagccgtccactgccaggctcgctggctcagtgacacaggacacatcctgatgacggtccacctgt

	ctccaagcaggttctcctctgccgatggggcaacgagctcctcctgtggctccctggctggatgcgtgggaggcggggt

	gggggggcaggcggtgttcctggccgcacacaaggagcacccccaccagcatccgaagacgggggcccggtctttc

	cccaaaacactgcttgcgggagactttgtgacgtttccaggggccatgctcccttcgggcagcttgggggacttctgctcc

	tatgtggtcacctgcagggactccccccaggccttggggacaaacaaagtgatgagagggagggttagtgggtcgggg

	cagggccagtctttggaccggtttatctgaaaagccagttggtcaccgggaaccacagcaaacctaaacccatttggcca

	ggcatctcccagggacagtctcccccaggatgcggggcccaggggggctccaggggtgacctgcgtcctggatttccc

	tgatgctcccagttcgtgcctctgtccaagcatgatttttaatagtgccccttccactcccagaaatgtccaagtgtgggcaa

	taaattctggtcacctgagctcagtgtaactgtttgctgaatgacacttactgtaacaggttaaaatgggaggcccaaggcc

	acgcagagccatcgaaggctctgtgtgtcccagccctgatagaagcatcaggatggggactgtggcctcaccaggggc

	cacatccaggcggtcaccatggggttcctggtctccgtgggccttgactggagcccctggtgtgagctcaccccatccca

	gcctgtgagaggcctggatgtgggcctgacatcatttcccacccagtgacagcactgcatgtgatggggcctctgggca

	gcctttttcccgggggaaactggcaggaatcaggaccaccaggacaggggtcaggggagaggcgatgctgggcacc

	agagcctggaccaccctcgggttctcagcgatgggcaacccctgccacccagggccccgccttcctggggagacatc

	ggggtttccaggccatcctgggaggagggtgggagcctcagctagaccccagctggcttgcccccccatgccccggc

	caagagagggtcttggagggaagggggaccccagaccagcctggcgagcccatcctcagggtctctggtcagacag

	gggctcagctgagctccagggtagaccaaggccctgcgtggatgaggccagtgtggtcactgcccagagcaaagcca

	cctctcagcagccctttcctgagcaccttctgtgtgcggggacatcagcagtggcaacacagccatgctggggactcag

	ggctagagacaggggaccagcctatggagagtgggtagtgtcctgcagggcaggcttgtgccctggagaaaacaaac

	cagggtgaggccagggacgctggccgggttcacagggtgatggctgagcacagagtgccaggggctggactgtcct

	gactctgggttggtggctgagggcctgtgtccctctatgcctctgggttggtgataatggaaacttgctccctggagagac

	aggacgaatggttgatgggaaatgaatgtttgcttgtcacttggttgactgttgttgccgttagcattgggcttcttgggccag

	gcagcctcaggccagcactgctgggctccccacaggcccgacaccctcagccctgtgcagctggcctggcgaaacca

	agaggccctgatgcccaaaatagccgggaaaccccaaccagcccagccctggcagcaggtgcctcccatttgcctgg

	gctgggggaggggtggctctggttctggaagtttctgccagtccagctggagaagggacctgtatcccagcacccagg

	ccgcccaagcccctgcaccagggcctgggccaggcagagttgacatcaatcaattgggagctgctggaatgcatggag

	gcggcgctctcgaggctggaggaggccagctgatttaaatcggtccgcgtacgatgcatattaccctgttatccctaccg

	cggttactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgatgctcttctcccggtgaaaacctctgacacat

	ggctcttctaaatccggagtttaaacgcttccttcatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggc

	cgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaa

	acccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgctta

	ccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtag

	gtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtctt

	gagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta

	ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctg

	aagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt

	gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtgg

	aacgaaaactcacgttaagggattttggtcatgcctaggtggcaaacagctattatgggtattatgggtctaccggtgcatg

	agattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttg

	gtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccc

	cgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctca

	ccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct

	ccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgcta

	caggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatc

	ccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactc

	atggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaag

	tcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcag

	aactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcga

	tgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca

	aaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcattt

	atcagggttattgtctcgggagcggatacatatttgaatgtatttagaaaaa

The two-step strategy outline above, utilizing a vector pair, can be used to delete the entire J/C cluster region (i.e., all J/C units), multiple J/C units or an individual J/C unit.
Selectable Marker Genes
The DNA constructs can be designed to modify the endogenous, target immunoglobulin gene. The homologous sequence for targeting the construct can have one or more deletions, insertions, substitutions or combinations thereof. The alteration can be the insertion of a selectable marker gene fused in reading frame with the upstream sequence of the target gene.
Suitable selectable marker genes include, but are not limited to: genes conferring the ability to grow on certain media substrates, such as the tk gene (thymidine kinase) or the hprt gene (hypoxanthine phosphoribosyltransferase) which confer the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine); the bacterial gpt gene (guanine/xanthine phosphoribosyltransferase) which allows growth on MAX medium (mycophenolic acid, adenine, and xanthine). See, for example, Song, K-Y., et al. Proc. Nat'l Acad. Sci. U.S.A. 84:6820-6824 (1987); Sambrook, J., et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), Chapter 16. Other examples of selectable markers include: genes conferring resistance to compounds such as antibiotics, genes conferring the ability to grow on selected substrates, genes encoding proteins that produce detectable signals such as luminescence, such as green fluorescent protein, enhanced green fluorescent protein (eGFP). A wide variety of such markers are known and available, including, for example, antibiotic resistance genes such as the neomycin resistance gene (neo) (Southern, P., and P. Berg, J. Mol. Appl. Genet. 1:327-341 (1982)); and the hygromycin resistance gene (hyg) (Nucleic Acids Research 11:6895-6911 (1983), and Te Riele, H., et al., Nature 348:649-651 (1990)). Other selectable marker genes include: acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline.
Methods for the incorporation of antibiotic resistance genes and negative selection factors will be familiar to those of ordinary skill in the art (see, e.g., WO 99/15650; U.S. Pat. No. 6,080,576; U.S. Pat. No. 6,136,566; Niwa et al., J. Biochem. 113:343-349 (1993); and Yoshida et al., Transgenic Research 4:277-287 (1995)).
Combinations of selectable markers can also be used. For example, to target an immunoglobulin gene, a neo gene (with or without its own promoter, as discussed above) can be cloned into a DNA sequence which is homologous to the immunoglobulin gene. To use a combination of markers, the HSV-tk gene can be cloned such that it is outside of the targeting DNA (another selectable marker could be placed on the opposite flank, if desired). After introducing the DNA construct into the cells to be targeted, the cells can be selected on the appropriate antibiotics. In this particular example, those cells which are resistant to G418 and gancyclovir are most likely to have arisen by homologous recombination in which the neo gene has been recombined into the immunoglobulin gene but the tk gene has been lost because it was located outside the region of the double crossover.
Deletions can be at least about 50 bp, more usually at least about 100 bp, and generally not more than about 20 kbp, where the deletion can normally include at least a portion of the coding region including a portion of or one or more exons, a portion of or one or more introns, and can or can not include a portion of the flanking non-coding regions, particularly the 5′-non-coding region (transcriptional regulatory region). Thus, the homologous region can extend beyond the coding region into the 5′-non-coding region or alternatively into the 3′-non-coding region. Insertions can generally not exceed 10 kbp, usually not exceed 5 kbp, generally being at least 50 bp, more usually at least 200 bp.
The region(s) of homology can include mutations, where mutations can further inactivate the target gene, in providing for a frame shift, or changing a key amino acid, or the mutation can correct a dysfunctional allele, etc. The mutation can be a subtle change, not exceeding about 5% of the homologous flanking sequences. Where mutation of a gene is desired, the marker gene can be inserted into an intron or an exon.
The construct can be prepared in accordance with methods known in the art, various fragments can be brought together, introduced into appropriate vectors, cloned, analyzed and then manipulated further until the desired construct has been achieved. Various modifications can be made to the sequence, to allow for restriction analysis, excision, identification of probes, etc. Silent mutations can be introduced, as desired. At various stages, restriction analysis, sequencing, amplification with the polymerase chain reaction, primer repair, in vitro mutagenesis, etc. can be employed.
The construct can be prepared using a bacterial vector, including a prokaryotic replication system, e.g. an origin recognizable by E. coli, at each stage the construct can be cloned and analyzed. A marker, the same as or different from the marker to be used for insertion, can be employed, which can be removed prior to introduction into the target cell. Once the vector containing the construct has been completed, it can be further manipulated, such as by deletion of the bacterial sequences, linearization, introducing a short deletion in the homologous sequence. After final manipulation, the construct can be introduced into the cell.
The present invention further includes recombinant constructs containing sequences of immunoglobulin genes. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. The construct can also include regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmiacia), viral origin vectors (M13 vectors, bacterial phage 1 vectors, adenovirus vectors, and retrovirus vectors), high, low and adjustable copy number vectors, vectors which have compatible replicons for use in combination in a single host (pACYC184 and pBR322) and eukaryotic episomal replication vectors (pCDM8). Other vectors include prokaryotic expression vectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Corp.), pGEMEX-1, and pGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A, pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and pProEx-HT (Invitrogen, Corp.) and variants and derivatives thereof. Other vectors include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3′SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.) and variants or derivatives thereof. Additional vectors that can be used include: pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificial chromosomes), BAC's (bacterial artificial chromosomes), P1 (Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH116A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1 (Invitrogen), pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(−)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZ□, pGAPZ, pGAPZ□, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen; □ ExCell, □ gt11, pTrc99A, pKK223-3, pGEX-1□T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia; pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg, pET-32LIC, pET-30LIC, pBAC-2 cp LIC, pBACgus-2 cp LIC, pT7Blue-2 LIC, pT7Blue-2, □SCREEN-1, □BlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3 cp, pBACgus-2 cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg, and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, p□gal-Basic, p□gal-Control, p□gal-Promoter, p□gal-Enhancer, pCMV□, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3, pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTrip1Ex, □gt10, □gt11, pWE15, and □Trip1Ex from Clontech; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS +/−, pBluescript II SK +/−, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS +/−, pBC KS +/−, pBC SK +/−, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, pOG45, pFRT□GAL, pNEO□GAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 from Stratagene and variants or derivatives thereof. Two-hybrid and reverse two-hybrid vectors can also be used, for example, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof. Any other plasmids and vectors may be used as long as they are replicable and viable in the host.
Techniques which can be used to allow the DNA construct entry into the host cell include, for example, calcium phosphate/DNA co precipitation, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, or any other technique known by one skilled in the art. The DNA can be single or double stranded, linear or circular, relaxed or supercoiled DNA. For various techniques for transfecting mammalian cells, see, for example, Keown et al., Methods in Enzymology Vol. 185, pp. 527-537 (1990).
In one specific embodiment, heterozygous or homozygous knockout cells can be produced by transfection of primary fetal fibroblasts with a knockout vector containing immunoglobulin gene sequence isolated from isogenic DNA. In another embodiment, the vector can incorporate a promoter trap strategy, using, for example, IRES (internal ribosome entry site) to initiate translation of the Neor gene.
Site Specific Recombinases
In additional embodiments, the targeting constructs can contain site specific recombinase sites, such as, for example, lox. In one embodiment, the targeting arms can insert the site specific recombinase target sites into the targeted region such that one site specific recombinase target site is located 5′ to the second site specific recombinase target site. Then, the site specific recombinase can be activated and/or applied to the cell such that the intervening nucleotide sequence between the two site specific recombinase sites is excised.
Site-specific recombinases include enzymes or recombinases that recognize and bind to a short nucleic acid site or sequence-specific recombinase target site, i.e., a recombinase recognition site, and catalyze the recombination of nucleic acid in relation to these sites. These enzymes include recombinases, transposases and integrases. Examples of sequence-specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites and frt sites. Non-limiting examples of site-specific recombinases include, but are not limited to, bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage λ, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, and the Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and the β-lactamase transposons, and the immunoglobulin recombinases.
In one embodiment, the recombination site can be a lox site that is recognized by the Cre recombinase of bacteriophage P1. Lox sites refer to a nucleotide sequence at which the product of the cre gene of bacteriophage P1, the Cre recombinase, can catalyze a site-specific recombination event. A variety of lox sites are known in the art, including the naturally occurring loxP, loxB, loxL and loxR, as well as a number of mutant, or variant, lox sites, such as loxP511, loxP514, lox.DELTA.86, lox.DELTA.117, loxC2, loxP2, loxP3 and lox P23. Additional example of lox sites include, but are not limited to, loxB, loxL, loxR, loxP, loxP3, loxP23, loxΔ86, loxΔ117, loxP511, and loxC2.
In another embodiment, the recombination site is a recombination site that is recognized by a recombinases other than Cre. In one embodiment, the recombinase site can be the FRT sites recognized by FLP recombinase of the 2 pi plasmid of Saccharomyces cerevisiae. FRT sites refer to a nucleotide sequence at which the product of the FLP gene of the yeast 2 micron plasmid, FLP recombinase, can catalyze site-specific recombination. Additional examples of the non-Cre recombinases include, but are not limited to, site-specific recombinases include: att sites recognized by the Int recombinase of bacteriophage λ (e.g. att1, att2, att3, attP, attB, attL, and attR), the recombination sites recognized by the resolvase family, and the recombination site recognized by transposase of Bacillus thruingiensis.
In particular embodiments of the present invention, the targeting constructs can contain: sequence homologous to a porcine immunoglobulin gene as described herein, a selectable marker gene and/or a site specific recombinase target site.
Selection of Homologously Recombined Cells
The cells can then be grown in appropriately-selected medium to identify cells providing the appropriate integration. The presence of the selectable marker gene inserted into the immunoglobulin gene establishes the integration of the target construct into the host genome. Those cells which show the desired phenotype can then be further analyzed by restriction analysis, electrophoresis, Southern analysis, polymerase chain reaction, etc to analyze the DNA in order to establish whether homologous or non-homologous recombination occurred. This can be determined by employing probes for the insert and then sequencing the 5′ and 3′ regions flanking the insert for the presence of the immunoglobulin gene extending beyond the flanking regions of the construct or identifying the presence of a deletion, when such deletion is introduced. Primers can also be used which are complementary to a sequence within the construct and complementary to a sequence outside the construct and at the target locus. In this way, one can only obtain DNA duplexes having both of the primers present in the complementary chains if homologous recombination has occurred. By demonstrating the presence of the primer sequences or the expected size sequence, the occurrence of homologous recombination is supported.
The polymerase chain reaction used for screening homologous recombination events is known in the art, see, for example, Kim and Smithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al., Nature 338:153-156, 1989. The specific combination of a mutant polyoma enhancer and a thymidine kinase promoter to drive the neomycin gene has been shown to be active in both embryonic stem cells and EC cells by Thomas and Capecchi, supra, 1987; Nicholas and Berg (1983) in Teratocarcinoma Stem Cell, eds. Siver, Martin and Strikland (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (pp. 469-497); and Linney and Donerly, Cell 35:693-699, 1983.
The cell lines obtained from the first round of targeting are likely to be heterozygous for the targeted allele. Homozygosity, in which both alleles are modified, can be achieved in a number of ways. One approach is to grow up a number of cells in which one copy has been modified and then to subject these cells to another round of targeting using a different selectable marker. Alternatively, homozygotes can be obtained by breeding animals heterozygous for the modified allele, according to traditional Mendelian genetics. In some situations, it can be desirable to have two different modified alleles. This can be achieved by successive rounds of gene targeting or by breeding heterozygotes, each of which carries one of the desired modified alleles.
Identification of Cells that have Undergone Homologous Recombination
In one embodiment, the selection method can detect the depletion of the immunoglobulin gene directly, whether due to targeted knockout of the immunoglobulin gene by homologous recombination, or a mutation in the gene that results in a nonfunctioning or nonexpressed immunoglobulin. Selection via antibiotic resistance has been used most commonly for screening (see above). This method can detect the presence of the resistance gene on the targeting vector, but does not directly indicate whether integration was a targeted recombination event or a random integration. Certain technology, such as Poly A and promoter trap technology, increase the probability of targeted events, but again, do not give direct evidence that the desired phenotype, a cell deficient in immunoglobulin gene expression, has been achieved. In addition, negative forms of selection can be used to select for targeted integration; in these cases, the gene for a factor lethal to the cells is inserted in such a way that only targeted events allow the cell to avoid death. Cells selected by these methods can then be assayed for gene disruption, vector integration and, finally, immunoglobulin gene depletion. In these cases, since the selection is based on detection of targeting vector integration and not at the altered phenotype, only targeted knockouts, not point mutations, gene rearrangements or truncations or other such modifications can be detected.
Animal cells believed to lacking expression of functional immunoglobulin genes can be further characterized. Such characterization can be accomplished by the following techniques, including, but not limited to: PCR analysis, Southern blot analysis, Northern blot analysis, specific lectin binding assays, and/or sequencing analysis.
PCR analysis as described in the art can be used to determine the integration of targeting vectors. In one embodiment, amplimers can originate in the antibiotic resistance gene and extend into a region outside the vector sequence. Southern analysis can also be used to characterize gross modifications in the locus, such as the integration of a targeting vector into the immunoglobulin locus. Whereas, Northern analysis can be used to characterize the transcript produced from each of the alleles.
Further, sequencing analysis of the cDNA produced from the RNA transcript can also be used to determine the precise location of any mutations in the immunoglobulin allele.
In another aspect of the present invention, ungulate cells lacking at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the process, sequences and/or constructs described herein are provided. These cells can be obtained as a result of homologous recombination. Particularly, by inactivating at least one allele of an ungulate heavy chain, kappa light chain or lambda light chain gene, cells can be produced which have reduced capability for expression of porcine antibodies. In other embodiments, mammalian cells lacking both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be produced according to the process, sequences and/or constructs described herein. In a further embodiment, porcine animals are provided in which at least one allele of an ungulate heavy chain, kappa light chain and/or lambda light chain gene is inactivated via a genetic targeting event produced according to the process, sequences and/or constructs described herein. In another aspect of the present invention, porcine animals are provided in which both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene are inactivated via a genetic targeting event. The gene can be targeted via homologous recombination. In other embodiments, the gene can be disrupted, i.e. a portion of the genetic code can be altered, thereby affecting transcription and/or translation of that segment of the gene. For example, disruption of a gene can occur through substitution, deletion (“knock-out”) or insertion (“knock-in”) techniques. Additional genes for a desired protein or regulatory sequence that modulate transcription of an existing sequence can be inserted.
In embodiments of the present invention, alleles of ungulate heavy chain, kappa light chain or lambda light chain gene are rendered inactive according to the process, sequences and/or constructs described herein, such that functional ungulate immunoglobulins can no longer be produced. In one embodiment, the targeted immunoglobulin gene can be transcribed into RNA, but not translated into protein. In another embodiment, the targeted immunoglobulin gene can be transcribed in an inactive truncated form. Such a truncated RNA may either not be translated or can be translated into a nonfunctional protein. In an alternative embodiment, the targeted immunoglobulin gene can be inactivated in such a way that no transcription of the gene occurs. In a further embodiment, the targeted immunoglobulin gene can be transcribed and then translated into a nonfunctional protein.
III. Insertion of Artificial Chromosomes Containing Human Immunoglobulin Genes
Artificial Chromosomes
One aspect of the present invention provides ungulates and ungulate cells that lack at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the processes, sequences and/or constructs described herein, which are further modified to express at least part of a human antibody (i.e. immunoglobulin (Ig)) locus. This human locus can undergo rearrangement and express a diverse population of human antibody molecules in the ungulate. These cloned, transgenic ungulates provide a replenishable, theoretically infinite supply of human antibodies (such as polyclonal antibodies), which can be used for therapeutic, diagnostic, purification, and other clinically relevant purposes.
In one particular embodiment, artificial chromosome (ACs) can be used to accomplish the transfer of human immunoglobulin genes into ungulate cells and animals. ACs permit targeted integration of megabase size DNA fragments that contain single or multiple genes. The ACs, therefore, can introduce heterologous DNA into selected cells for production of the gene product encoded by the heterologous DNA. In a one embodiment, one or more ACs with integrated human immunoglobulin DNA can be used as a vector for introduction of human Ig genes into ungulates (such as pigs).
First constructed in yeast in 1983, ACs are man-made linear DNA molecules constructed from essential cis-acting DNA sequence elements that are responsible for the proper replication and partitioning of natural chromosomes (Murray et al. (1983), Nature 301:189-193). A chromosome requires at least three elements to function. Specifically, the elements of an artificial chromosome include at least: (1) autonomous replication sequences (ARS) (having properties of replication origins—which are the sites for initiation of DNA replication), (2) centromeres (site of kinetochore assembly that is responsible for proper distribution of replicated chromosomes at mitosis and meiosis), and (3) telomeres (specialized structures at the ends of linear chromosomes that function to both stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule).
In one embodiment, the human Ig can be maintained as an independent unit (an episome) apart from the ungulate chromosomal DNA. For example, episomal vectors contain the necessary DNA sequence elements required for DNA replication and maintenance of the vector within the cell. Episomal vectors are available commercially (see, for example, Maniatis, T. et al., Molecular Cloning, A Laboratory Manual (1982) pp. 368-369). The AC can stably replicate and segregate along side endogenous chromosomes. In an alternative embodiment, the human IgG DNA sequences can be integrated into the ungulate cell's chromosomes thereby permitting the new information to be replicated and partitioned to the cell's progeny as a part of the natural chromosomes (see, for example, Wigler et al. (1977), Cell 11:223). The AC can be translocated to, or inserted into, the endogenous chromosome of the ungulate cell. Two or more ACs can be introduced to the host cell simultaneously or sequentially.
ACs, furthermore, can provide an extra-genomic locus for targeted integration of megabase size DNA fragments that contain single or multiple genes, including multiple copies of a single gene operatively linked to one promoter or each copy or several copies linked to separate promoters. ACs can permit the targeted integration of megabase size DNA fragments that contain single or multiple human immunoglobulin genes. The ACs can be generated by culturing the cells with dicentric chromosomes (i.e., chromosomes with two centromeres) under such conditions known to one skilled in the art whereby the chromosome breaks to form a minichromosome and formerly dicentric chromosome.
ACs can be constructed from humans (human artificial chromosomes: “HACs”), yeast (yeast artificial chromosomes: “YACs”), bacteria (bacterial artificial chromosomes: “BACs”), bacteriophage P1-derived artificial chromosomes: “PACs”) and other mammals (mammalian artificial chromosomes: “MACs”). The ACs derive their name (e.g., YAC, BAC, PAC, MAC, HAC) based on the origin of the centromere. A YAC, for example, can derive its centromere from S. cerevisiae. MACs, on the other hand, include an active mammalian centromere while HACs refer to chromosomes that include human centromeres. Furthermore, plant artificial chromosomes (“PLACs”) and insect artificial chromosomes can also be constructed. The ACs can include elements derived from chromosomes that are responsible for both replication and maintenance. ACs, therefore, are capable of stably maintaining large genomic DNA fragments such as human Ig DNA.
In one embodiment, ungulates containing YACs are provided. YACs are genetically engineered circular chromosomes that contain elements from yeast chromosomes, such as S. cerevisiae, and segments of foreign DNAs that can be much larger than those accepted by conventional cloning vectors (e.g., plasmids, cosmids). YACs allow the propagation of very large segments of exogenous DNA (Schlessinger, D. (1990), Trends in Genetics 6:248-253) into mammalian cells and animals (Choi et al. (1993), Nature Gen 4:117-123). YAC transgenic approaches are very powerful and are greatly enhanced by the ability to efficiently manipulate the cloned DNA. A major technical advantage of yeast is the ease with which specific genome modifications can be made via DNA-mediated transformation and homologous recombination (Ramsay, M. (1994), Mol Biotech 1:181-201). In one embodiment, one or more YACs with integrated human Ig DNA can be used as a vector for introduction of human Ig genes into ungulates (such as pigs).
The YAC vectors contain specific structural components for replication in yeast, including: a centromere, telomeres, autonomous replication sequence (ARS), yeast selectable markers (e.g., TRP1, URA3, and SUP4), and a cloning site for insertion of large segments of greater than 50 kb of exogenous DNA. The marker genes can allow selection of the cells carrying the YAC and serve as sites for the synthesis of specific restriction endonucleases. For example, the TRP1 and URA3 genes can be used as dual selectable markers to ensure that only complete artificial chromosomes are maintained. Yeast selectable markers can be carried on both sides of the centromere, and two sequences that seed telomere formation in vivo are separated. Only a fraction of one percent of a yeast cell's total DNA is necessary for replication, however, including the center of the chromosome (the centromere, which serves as the site of attachment between sister chromatids and the sites of spindle fiber attachment during mitosis), the ends of the chromosome (telomeres, which serve as necessary sequences to maintain the ends of eukaryotic chromosomes), and another short stretch of DNA called the ARS which serves as DNA segments where the double helix can unwind and begin to copy itself.
In one embodiment, YACs can be used to clone up to about 1, 2, or 3 Mb of immunoglobulin DNA. In another embodiment, at least 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, or 95 kilobases.
Yeast integrating plasmids, replicating vectors (which are fragments of YACs), can also be used to express human Ig. The yeast integrating plasmid can contain bacterial plasmid sequences that provide a replication origin and a drug-resistance gene for growth in bacteria (e.g., E. coli), a yeast marker gene for selection of transformants in yeast, and restriction sites for inserting Ig sequences. Host cells can stably acquire this plasmid by integrating it directly into a chromosome. Yeast replicating vectors can also be used to express human Ig as free plasmid circles in yeast. Yeast or ARS-containing vectors can be stabilized by the addition of a centromere sequence. YACs have both centromeric and telomeric regions, and can be used for cloning very large pieces of DNA because the recombinant is maintained essentially as a yeast chromosome.
YACs are provided, for example, as disclosed in U.S. Pat. Nos. 6,692,954, 6,495,318, 6,391,642, 6,287,853, 6,221,588, 6,166,288, 6,096,878, 6,015,708, 5,981,175, 5,939,255, 5,843,671, 5,783,385, 5,776,745, 5,578,461, and 4,889,806; European Patent Nos. 1 356 062 and 0 648 265; PCT Publication Nos. WO 03/025222, WO 02/057437, WO 02/101044, WO 02/057437, WO 98/36082, WO 98/12335, WO 98/01573, WO 96/01276, WO 95/14769, WO 95/05847, WO 94/23049, and WO 94/00569.
In another embodiment, ungulates containing BACs are provided. BACs are F-based plasmids found in bacteria, such as E. Coli, that can transfer approximately 300 kb of foreign DNA into a host cell. Once the Ig DNA has been cloned into the host cell, the newly inserted segment can be replicated along with the rest of the plasmid. As a result, billions of copies of the foreign DNA can be made in a very short time. In a particular embodiment, one or more BACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs).
The BAC cloning system is based on the E. coli F-factor, whose replication is strictly controlled and thus ensures stable maintenance of large constructs (Willets, N., and R. Skurray (1987), Structure and function of the F-factor and mechanism of conjugation. In Escherichia coli and Salmonella Typhimurium: Cellular and Molecular Biology (F. C. Neidhardt, Ed) Vol. 2 pp 1110-1133, Am. Soc. Microbiol., Washington, D.C.). BACs have been widely used for cloning of DNA from various eukaryotic species (Cai et al. (1995), Genomics 29:413-425; Kim et al. (1996), Genomics 34:213-218; Misumi et al. (1997), Genomics 40:147-150; Woo et al. (1994), Nucleic Acids Res 22:4922-4931; Zimmer, R. and Gibbins, A.M.V. (1997), Genomics 42:217-226). The low occurrence of the F-plasmid can reduce the potential for recombination between DNA fragments and can avoid the lethal overexpression of cloned bacterial genes. BACs can stably maintain the human immunoglobulin genes in a single copy vector in the host cells, even after 100 or more generations of serial growth.
BAC (or pBAC) vectors can accommodate inserts in the range of approximately 30 to 300 kb pairs. One specific type of BAC vector, pBeloBac11, uses a complementation of the lacZ gene to distinguish insert-containing recombinant molecules from colonies carrying the BAC vector, by color. When a DNA fragment is cloned into the lacZ gene of pBeloBac11, insertional activation results in a white colony on X-Gal/IPTG plates after transformation (Kim et al. (1996), Genomics 34:213-218) to easily identify positive clones.
For example, BACs can be provided such as disclosed in U.S. Pat. Nos. 6,713,281, 6,703,198, 6,649,347, 6,638,722, 6,586,184, 6,573,090, 6,548,256, 6,534,262, 6,492,577, 6,492,506, 6,485,912, 6,472,177, 6,455,254, 6,383,756, 6,277,621, 6,183,957, 6,156,574, 6,127,171, 5,874,259, 5,707,811, and 5,597,694; European Patent Nos. 0 805 851; PCT Publication Nos. WO 03/087330, WO 02/00916, WO 01/39797, WO 01/04302, WO 00/79001, WO 99/54487, WO 99/27118, and WO 96/21725.
In another embodiment, ungulates containing bacteriophage PACs are provided. In a particular embodiment, one or more bacteriophage PACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs). For example, PACs can be provided such as disclosed in U.S. Pat. Nos. 6,743,906, 6,730,500, 6,689,606, 6,673,909, 6,642,207, 6,632,934, 6,573,090, 6,544,768, 6,489,458, 6,485,912, 6,469,144, 6,462,176, 6,413,776, 6,399,312, 6,340,595, 6,287,854, 6,284,882, 6,277,621, 6,271,008, 6,187,533, 6,156,574, 6,153,740, 6,143,949, 6,017,755, and 5,973,133; European Patent Nos. 0 814 156; PCT Publication Nos. WO 03/091426, WO 03/076573, WO 03/020898, WO 02/101022, WO 02/070696, WO 02/061073, WO 02/31202, WO 01/44486, WO 01/07478, WO 01/05962, and WO 99/63103.
In a further embodiment, ungulates containing MACs are provided. MACs possess high mitotic stability, consistent and regulated gene expression, high cloning capacity, and non-immunogenicity. Mammalian chromosomes can be comprised of a continuous linear strand of DNA ranging in size from approximately 50 to 250 Mb. The DNA construct can further contain one or more sequences necessary for the DNA construct to multiply in yeast cells. The DNA construct can also contain a sequence encoding a selectable marker gene. The DNA construct can be capable of being maintained as a chromosome in a transformed cell with the DNA construct. MACs provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway, a very large gene [e.g., such as the cystic fibrosis (CF) gene (−600 kb)], or several genes [e.g., a series of antigens for preparation of a multivalent vaccine] can be stably introduced into a cell.
Mammalian artificial chromosomes [MACs] are provided. Also provided are artificial chromosomes for other higher eukaryotic species, such as insects and fish, produced using the MACS are provided herein. Methods for generating and isolating such chromosomes. Methods using the MACs to construct artificial chromosomes from other species, such as insect and fish species are also provided. The artificial chromosomes are fully functional stable chromosomes. Two types of artificial chromosomes are provided. One type, herein referred to as SATACs [satellite artificial chromosomes] are stable heterochromatic chromosomes, and the another type are minichromosomes based on amplification of euchromatin. As used herein, a formerly dicentric chromosome is a chromosome that is produced when a dicentric chromosome fragments and acquires new telomeres so that two chromosomes, each having one of the centromeres, are produced. Each of the fragments can be replicable chromosomes.
Also provided are artificial chromosomes for other higher eukaryotic species, such as insects and fish, produced using the MACS are provided herein. In one embodiment, SATACs [satellite artificial chromosomes] are provided. SATACs are stable heterochromatic chromosomes. In another embodiment, minichromosomes are provided wherein the minichromosomes are based on amplification of euchromatin.
In one embodiment, artificial chromosomes can be generated by culturing the cells with the dicentric chromosomes under conditions whereby the chromosome breaks to form a minichromosome and formerly dicentric chromosome. In one embodiment, the SATACs can be generated from the minichromosome fragment, see, for example, in U.S. Pat. No. 5,288,625. In another embodiment, the SATACs can be generated from the fragment of the formerly dicentric chromosome. The SATACs can be made up of repeating units of short satellite DNA and can be fully heterochromatic. In one embodiment, absent insertion of heterologous or foreign DNA, the SATACs do not contain genetic information. In other embodiments, SATACs of various sizes are provided that are formed by repeated culturing under selective conditions and subcloning of cells that contain chromosomes produced from the formerly dicentric chromosomes. These chromosomes can be based on repeating units 7.5 to 10 Mb in size, or megareplicons. These megareplicaonscan be tandem blocks of satellite DNA flanked by heterologous non-satellite DNA. Amplification can produce a tandem array of identical chromosome segments [each called an amplicon] that contain two inverted megareplicons bordered by heterologous [“foreign”] DNA. Repeated cell fusion, growth on selective medium and/or BrdU [5-bromodeoxyuridine] treatment or other genome destabilizing reagent or agent, such as ionizing radiation, including X-rays, and subcloning can result in cell lines that carry stable heterochromatic or partially heterochromatic chromosomes, including a 150-200 Mb “sausage” chromosome, a 500-1000 Mb gigachromosome, a stable 250-400 Mb megachromosome and various smaller stable chromosomes derived therefrom. These chromosomes are based on these repeating units and can include human immunoglobulin DNA that is expressed. (See also U.S. Pat. No. 6,743,967
In other embodiments, MACs can be provided, for example, as disclosed in U.S. Pat. Nos. 6,743,967, 6,682,729, 6,569,643, 6,558,902, 6,548,287, 6,410,722, 6,348,353, 6,297,029, 6,265,211, 6,207,648, 6,150,170, 6,150,160, 6,133,503, 6,077,697, 6,025,155, 5,997,881, 5,985,846, 5,981,225, 5,877,159, 5,851,760, and 5,721,118; PCT Publication Nos. WO 04/066945, WO 04/044129, WO 04/035729, WO 04/033668, WO 04/027075, WO 04/016791, WO 04/009788, WO 04/007750, WO 03/083054, WO 03/068910, WO 03/068909, WO 03/064613, WO 03/052050, WO 03/027315, WO 03/023029, WO 03/012126, WO 03/006610, WO 03/000921, WO 02/103032, WO 02/097059, WO 02/096923, WO 02/095003, WO 02/092615, WO 02/081710, WO 02/059330, WO 02/059296, WO 00/18941, WO 97/16533, and WO 96/40965.
In another aspect of the present invention, ungulates and ungulate cells containing HACs are provided. In a particular embodiment, one or more HACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs). In a particular embodiment, one or more HACs with integrated human Ig DNA are used to generate ungulates (for example, pigs) by nuclear transfer which express human Igs in response to immunization and which undergo affinity maturation.
Various approaches may be used to produce ungulates that express human antibodies (“human Ig”). These approaches include, for example, the insertion of a HAC containing both heavy and light chain Ig genes into an ungulate or the insertion of human B-cells or B-cell precursors into an ungulate during its fetal stage or after it is born (e.g., an immune deficient or immune suppressed ungulate) (see, for example, WO 01/35735, filed Nov. 17, 2000, US 02/08645, filed Mar. 20, 2002). In either case, both human antibody producing cells and ungulate antibody-producing B-cells may be present in the ungulate. In an ungulate containing a HAC, a single B-cell may produce an antibody that contains a combination of ungulate and human heavy and light chain proteins. In still other embodiments, the total size of the HAC is at least to approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Mb.
For example, HACs can be provided such as disclosed in U.S. Pat. Nos. 6,642,207, 6,590,089, 6,566,066, 6,524,799, 6,500,642, 6,485,910, 6,475,752, 6,458,561, 6,455,026, 6,448,041, 6,410,722, 6,358,523, 6,277,621, 6,265,211, 6,146,827, 6,143,566, 6,077,697, 6,025,155, 6,020,142, and 5,972,649; U.S. Pat. Application No. 2003/0037347; PCT Publication Nos. WO 04/050704, WO 04/044156, WO 04/031385, WO 04/016791, WO 03/101396, WO 03/097812, WO 03/093469, WO 03/091426, WO 03/057923, WO 03/057849, WO 03/027638, WO 03/020898, WO 02/092812, and WO 98/27200.
Additional examples of ACs into which human immunoglobulin sequences can be inserted for use in the invention include, for example, BACs (e.g., pBeloBAC11 or pBAC108L; see, e.g., Shizuya et al. (1992), Proc Natl Acad Sci USA 89(18):8794-8797; Wang et al. (1997), Biotechniques 23(6):992-994), bacteriophage PACs, YACs (see, e.g., Burke (1990), Genet Anal Tech Appl 7(5):94-99), and MACs (see, e.g., Vos (1997), Nat. Biotechnol. 15(12):1257-1259; Ascenzioni et al. (1997), Cancer Lett 118(2):135-142), such as HACs, see also, U.S. Pat. Nos. 6,743,967, 6,716,608, 6,692,954, 6,670,154, 6,642,207, 6,638,722, 6,573,090, 6,492,506, 6,348,353, 6,287,853, 6,277,621, 6,183,957, 6,156,953, 6,133,503, 6,090,584, 6,077,697, 6,025,155, 6,015,708, 5,981,175, 5,874,259, 5,721,118, and 5,270,201; European Patent Nos. 1 437 400, 1 234 024, 1 356 062, 0 959 134, 1 056 878, 0 986 648, 0 648 265, and 0 338 266; PCT Publication Nos. WO 04/013299, WO 01/07478, WO 00/06715, WO 99/43842, WO 99/27118, WO 98/55637, WO 94/00569, and WO 89/09219. Additional examples includes those AC provided in, for example, PCT Publication No. WO 02/076508, WO 03/093469, WO 02/097059; WO 02/096923; US Publication Nos US 2003/0113917 and US 2003/003435; and U.S. Pat. No. 6,025,155.
In other embodiments of the present invention, ACs transmitted through male gametogenesis in each generation. The AC can be integrating or non-integrating. In one embodiment, the AC can be transmitted through mitosis in substantially all dividing cells. In another embodiment, the AC can provide for position independent expression of a human immunogloulin nucleic acid sequence. In a particular embodiment, the AC can have a transmittal efficiency of at least 10% through each male and female gametogenesis. In one particular embodiment, the AC can be circular. In another particular embodiment, the non-integrating AC can be that deposited with the Belgian Coordinated Collections of Microorganisms—BCCM on Mar. 27, 2000 under accession number LMBP 5473 CB. In additional embodiments, methods for producing an AC are provided wherein a mitotically stable unit containing an exogenous nucleic acid transmitted through male gametogenesis is identified; and an entry site in the mitotically stable unit allows for the integration of human immunoglobulin genes into the unit.
In other embodiments, ACs are provided that include: a functional centromere, a selectable marker and/or a unique cloning site. Tin other embodiments, the AC can exhibit one or more of the following properties: it can segregate stably as an independent chromosome, immunoglobulin sequences can be inserted in a controlled way and can expressed from the AC, it can be efficiently transmitted through the male and female germline and/or the transgenic animals can bear the chromosome in greater than about 30, 40, 50, 60, 70, 80 or 90% of its cells.
In particular embodiments, the AC can be isolated from fibroblasts (such as any mammalian or human fibroblast) in which it was mitotically stable. After transfer of the AC into hamster cells, a lox (such as loxP) site and a selectable marker site can be inserted. In other embodiments, the AC can maintain mitotic stability, for example, showing a loss of less than about 5, 2, 1, 0.5 or 0.25 percent per mitosis in the absence of selection. See also, US 2003/0064509 and WO 01/77357.
Xenogenous Immunoglobulin Genes
In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In another embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
Human immunoglobulin genes, such as the Ig heavy chain gene (human chromosome 414), Ig kappa chain gene (human chromosome #2) and/or the Ig lambda chain gene (chromosome #22) can be inserted into Acs, as described above. In a particular embodiment, any portion of the human heavy, kappa and/or lambda Ig genes can be inserted into ACs. In one embodiment, the nucleic acid can be at least 70, 80, 90, 95, or 99% identical to the corresponding region of a naturally-occurring nucleic acid from a human. In other embodiments, more than one class of human antibody is produced by the ungulate. In various embodiments, more than one different human Ig or antibody is produced by the ungulate. In one embodiment, an AC containing both a human Ig heavy chain gene and Ig light chain gene, such as an automatic human artificial chromosome (“AHAC,” a circular recombinant nucleic acid molecule that is converted to a linear human chromosome in vivo by an endogenously expressed restriction endonuclease) can be introduced. In one embodiment, the human heavy chain loci and the light chain loci are on different chromosome arms (i.e., on different side of the centromere). In one embodiments, the heavy chain can include the mu heavy chain, and the light chain can be a lambda or kappa light chain. The Ig genes can be introduced simultaneously or sequentially in one or more than one ACs.
In particular embodiments, the ungulate or ungulate cell expresses one or more nucleic acids encoding all or part of a human Ig gene which undergoes rearrangement and expresses more than one human Ig molecule, such as a human antibody protein. Thus, the nucleic acid encoding the human Ig chain or antibody is in its unrearranged form (that is, the nucleic acid has not undergone V(D)J recombination). In particular embodiments, all of the nucleic acid segments encoding a V gene segment of an antibody light chain can be separated from all of the nucleic acid segments encoding a J gene segment by one or more nucleotides. In a particular embodiment, all of the nucleic acid segments encoding a V gene segment of an antibody heavy chain can be separated from all of the nucleic acid segments encoding a D gene segment by one or more nucleotides, and/or all of the nucleic acid segments encoding a D gene segment of an antibody heavy chain are separated from all of the nucleic acid segments encoding a J gene segment by one or more nucleotides. Administration of an antigen to a transgenic ungulate containing an unrearranged human Ig gene is followed by the rearrangement of the nucleic acid segments in the human Ig gene locus and the production of human antibodies reactive with the antigen.
In one embodiment, the AC can express a portion or fragment of a human chromosome that contains an immunoglobulin gene. In one embodiment, the AC can express at least 300 or 1300 kb of the human light chain locus, such as described in Davies et al. 1993 Biotechnology 11:911-914.
In another embodiment, the AC can express a portion of human chromosome 22 that contains at least the λ light-chain locus, including V_λ gene segments, J_λ gene segments, and the single C_λ gene. In another embodiment, the AC can express at least one V_λ gene segment, at least one J_λ gene segment, and the C_λ gene. In other embodiment, ACs can contain portions of the lambda locus, such as described in Popov et al. J Exp Med. 1999 May 17; 189(10):1611-20.
In another embodiment, the AC can express a portion of human chromosome 2 that contains at least the κ light-chain locus, including V_κ gene segments, J_κ gene segments and the single C_κ gene. In another embodiment, the AC can express at least one V_κ gene segment, at least one J_κ gene segment and the C_κ gene. In other embodiments, AC containing portions of the kappa light chain locus can be those describe, for example, in Li et al. 2000 J Immunol 164: 812-824 and Li S Proc Natl Acad Sci USA. 1987 June; 84(12):4229-33. In another embodiment, AC containing approximately 1.3 Mb of human kappa locus are provided, such as described in Zou et al FASEB J. 1996 August; 10(10):1227-32.
In further embodiments, the AC can express a portion of human chromosome 14 that contains at least the human heavy-chain locus, including V_H, D_H, J_Hand C_Hgene segments. In another embodiment, the AC can express at least one V_Hgene segment, at least one D_Hgene segment, at least one J_Hgene segment and at least one at least one C_Hgene segment. In other embodiments, the AC can express at least 85 kb of the human heavy chain locus, such as described in Choi et al. 1993 Nat Gen 4:117-123 and/or Zou et al. 1996 PNAS 96: 14100-14105.
In other embodiments, the AC can express portions of both heavy and light chain loci, such as, at least 220, 170, 800 or 1020 kb, for example, as disclosed in Green et al. 1994 Nat Gen 7:13-22; Mendez et al 1995 Genomics 26: 294-307; Mendez et al. 1997 Nat Gen 15: 146-156; Green et al. 1998 J Exp Med 188: 483-495 and/or Fishwild et al. 1996 Nat Biotech 14: 845-851. In another embodiment, the AC can express megabase amounts of human immunoglobulin, such as described in Nicholson J Immunol. 1999 Dec. 15; 163(12):6898-906 and Popov Gene. 1996 Oct. 24; 177(1-2):195-201. In addition, in one particular embodiment, MACs derived from human chromosome #14 (comprising the Ig heavy chain gene), human chromosome #2 comprising the Ig kappa chain gene) and human chromosome #22 (comprising the Ig lambda chain gene) can be introduced simultaneously or successively, such as described in US Patent Publication No. 2004/0068760 to Robl et al. In another embodiments, the total size of the MAC is less than or equal to approximately 10, 9, 8, or 7 megabases.
In a particular embodiment, human Vh, human Dh, human Jh segments and human mu segments of human immunoglobulins in germline configuration can be inserted into an AC, such as a YAC, such that the Vh, Dh, Jh and mu DNA segments form a repertoire of immunoglobulins containing portions which correspond to the human DNA segments, for example, as described in U.S. Pat. No. 5,545,807 to the Babraham Instititute. Such ACs, after insertion into ungulate cells and generation of ungulates can produce heavy chain immunoglobulins. In one embodiment, these immunoglobulins can form functional heavy chain-light chain immunoglobulins. In another embodiment, these immunoglobulins can be expressed in an amount allowing for recovery from suitable cells or body fluids of the ungulate. Such immunoglobulins can be inserted into yeast artificial chromosome vectors, such as described by Burke, D T, Carle, G F and Olson, M V (1987) “Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors” Science, 236, 806-812, or by introduction of chromosome fragments (such as described by Richer, J and Lo, C W (1989) “Introduction of human DNA into mouse eggs by injection of dissected human chromosome fragments” Science 245, 175-177).
Additional information on specific ACs containing human immunoglobulin genes can be found in, for example, recent reviews by Giraldo & Montoliu (2001) Transgenic Research 10: 83-103 and Peterson (2003) Expert Reviews in Molecular Medicine 5: 1-25.
AC Transfer Methods
The human immunoglobulin genes can be first inserted into ACs and then the human-immunoglobulin-containing ACs can be inserted into the ungulate cells. Alternatively, the ACs can be transferred to an intermediary mammalian cell, such as a CHO cell, prior to insertion into the ungulate call. In one embodiment, the intermediary mammalian cell can also contain and AC and the first AC can be inserted into the AC of the mammalian cell. In particular, a YAC containing human immunoglobulin genes or fragments thereof in a yeast cell can be transferred to a mammalian cell that harbors an MAC. The YAC can be inserted into the MAC. The MAC can then be transferred to an ungulate cell. The human Ig genes can be inserted into ACs by homologous recombination. The resulting AC containing human Ig genes, can then be introduced into ungulate cells. One or more ungulate cells can be selected by techniques described herein or those known in the art, which contain an AC containing a human Ig.
Suitable hosts for introduction of the ACs are provided herein, which include but are not limited to any animal or plant, cell or tissue thereof, including, but not limited to: mammals, birds, reptiles, amphibians, insects, fish, arachnids, tobacco, tomato, wheat, monocots, dicots and algae. In one embodiment, the ACs can be condensed (Marschall et al Gene Ther. 1999 Sep.; 6(9):1634-7) by any reagent known in the art, including, but not limited to, spermine, spermidine, polyethylenimine, and/or polylysine prior to introduction into cells. The ACs can be introduced by cell fusion or microcell fusion or subsequent to isolation by any method known to those of skill in this art, including but not limited to: direct DNA transfer, electroporation, nuclear transfer, microcell fusion, cell fusion, spheroplast fusion, lipid-mediated transfer, lipofection, liposomes, microprojectile bombardment, microinjection, calcium phosphate precipitation and/or any other suitable method. Other methods for introducing DNA into cells, include nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells. Polycations, such as polybrene and polyornithine, may also be used. For various techniques for transforming mammalian cells, see e.g., Keown et al. Methods in Enzymology (1990) Vol. 185, pp. 527-537; and Mansour et al. (1988) Nature 336:348-352.
The ACs can be introduced by direct DNA transformation; microinjection in cells or embryos, protoplast regeneration for plants, electroporation, microprojectile gun and other such methods known to one skilled in the art (see, e.g., Weissbach et al. (1988) Methods for Plant Molecular Biology, Academic Press, N.Y., Section VIII, pp. 421-463; Grierson et al. (1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9; see, also U.S. Pat. Nos. 5,491,075; 5,482,928; and 5,424,409; see, also, e.g., U.S. Pat. No. 5,470,708,).
In particular embodiments, one or more isolated YACs can be used that harbor human Ig genes. The isolated YACs can be condensed (Marschall et al Gene Ther. 1999 September; 6(9):1634-7) by any reagent known in the art, including, but not limited to spermine, spermidine, polyethylenimine, and/or polylysine. The condensed YACs can then be transferred to porcine cells by any method known in the art (for example, microinjection, electroporation, lipid mediated transfection, etc). Alternatively, the condensed YAC can be transferred to oocytes via sperm-mediated gene transfer or intracytoplasmic sperm injection (ICSI) mediated gene transfer. In one embodiment, spheroplast fusion can be used to transfer YACs that harbor human Ig genes to porcine cells.
In other embodiments of the invention, the AC containing the human Ig can be inserted into an adult, fetal, or embryonic ungulate cell. Additional examples of ungulate cells include undifferentiated cells, such as embryonic cells (e.g., embryonic stem cells), differentiated or somatic cells, such as epithelial cells, neural cells epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, muscle cells, cells from the female reproductive system, such as a mammary gland, ovarian cumulus, granulosa, or oviductal cell, germ cells, placental cell, or cells derived from any organ, such as the bladder, brain, esophagus, fallopian tube, heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, and uterus or any other cell type described herein.
Site Specific Recombinase Mediated Transfer
In particular embodiments of the present invention, the transfer of ACs containing human immunoglobulin genes to porcine cells, such as those described herein or known in the art, can be accomplished via site specific recombinase mediated transfer. In one particular embodiment, the ACs can be transferred into porcine fibroblast cells. In another particular embodiment, the ACs can be YACs.
In other embodiments of the present invention, the circularized DNA, such as an AC, that contain the site specific recombinase target site can be transferred into a cell line that has a site specific recombinase target site within its genome. In one embodiment, the cell's site specific recombinase target site can be located within an exogenous chromosome. The exogenous chromosome can be an artificial chromosome that does not integrate into the host's endogenous genome. In one embodiment, the AC can be transferred via germ line transmission to offspring. In one particular embodiment, a YAC containing a human immunoglobulin gene or fragment thereof can be circularized via a site specific recombinase and then transferred into a host cell that contains a MAC, wherein the MAC contains a site specific recombinase site. This MAC that now contains human immunoglobulin loci or fragments thereof can then be fused with a porcine cell, such as, but not limited to, a fibroblast. The porcine cell can then be used for nuclear transfer.
In certain embodiments of the present invention, the ACs that contain human immunoglobulin genes or fragments thereof can be transferred to a mammalian cell, such as a CHO cell, prior to insertion into the ungulate call. In one embodiment, the intermediary mammalian cell can also contain and AC and the first AC can be inserted into the AC of the mammalian cell. In particular, a YAC containing human immunoglobulin genes or fragments thereof in a yeast cell can be transferred to a mammalian cell that harbors a MAC. The YAC can be inserted in the MAC. The MAC can then be transferred to an ungulate cell. In particular embodiments, the YAC harboring the human Ig genes or fragments thereof can contain site specific recombinase target sites. The YAC can first be circularized via application of the appropriate site specific recombinase and then inserted into a mammalian cell that contains its own site specific recombinase target site. Then, the site specific recombinase can be applied to integrate the YAC into the MAC in the intermediary mammalian cell. The site specific recombinase can be applied in cis or trans. In particular, the site specific recombinase can be applied in trans. In one embodiment, the site specific recombinase can be expressed via transfection of a site specific recombinase expression plasmid, such as a Cre expression plasmid. In addition, one telomere region of the YAC can also be retrofitted with a selectable marker, such as a selectable marker described herein or known in the art. The human Ig genes or fragments thereof within the MAC of the intermediary mammalian cell can then be transferred to an ungulate cell, such as a fibroblast.
Alternatively, the AC, such as a YAC, harboring the human Ig genes or fragments thereof can contain site specific recombinase target sites optionally located near each telomere. The YAC can first be circularized via application of the appropriate site specific recombinase and then inserted into an ungulate cell directly that contains its own site specific recombinase target site within it genome. Alternatively, the ungulate cell can harbor its own MAC, which contains a site specific recombinase target site. In this embodiment, the YAC can be inserted directly into the endogenous genome of the ungulate cell. In particular embodiments, the ungulate cell can be a fibroblast cell or any other suitable cell that can be used for nuclear transfer. See, for example, FIG. 7; Call et al., Hum Mol Genet. 2000 Jul. 22; 9(12):1745-51.
In other embodiments, methods to circularize at least 100 kb of DNA are provided wherein the DNA can then be integrated into a host genome via a site specific recombinase. In one embodiment, at least 100, 200, 300, 400, 500, 1000, 2000, 5000, 10,000 kb of DNA can be circularized. In another embodiment, at least 1000, 2000, 5000, 10,000, or 20,000 megabases of DNA can be circularized. In one embodiment, the circularization of the DNA can be accomplished by attaching site specific recombinase target sites at each end of the DNA sequence and then applying the site specific recombinase to result in circularization of the DNA. In one embodiment, the site specific recombinase target site can be lox. In another embodiment, the site specific recombinase target site can be Flt. In certain embodiments, the DNA can be an artificial chromosome, such as a YAC or any AC described herein or known in the art. In another embodiment, the AC can contain human immunoglobulin loci or fragments thereof.
In another preferred embodiment, the YAC can be converted to, or integrated within, an artificial mammalian chromosome. The mammalian artificial chromosome is either transferred to or harbored within a porcine cell. The artificial chromosome can be introduced within the porcine genome through any method known in the art including but not limited to direct injection of metaphase chromosomes, lipid mediated gene transfer, or microcell fusion.
Site-specific recombinases include enzymes or recombinases that recognize and bind to a short nucleic acid site or sequence-specific recombinase target site, i.e., a recombinase recognition site, and catalyze the recombination of nucleic acid in relation to these sites. These enzymes include recombinases, transposases and integrases. Examples of sequence-specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites and frt sites. Non-limiting examples of site-specific recombinases include, but are not limited to, bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage λ, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, and the Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and the β-lactamase transposons, and the immunoglobulin recombinases.
In one embodiment, the recombination site can be a lox site that is recognized by the Cre recombinase of bacteriophage P1. Lox sites refer to a nucleotide sequence at which the product of the cre gene of bacteriophage P1, the Cre recombinase, can catalyze a site-specific recombination event. A variety of lox sites are known in the art, including the naturally occurring loxP, loxB, loxL and loxR, as well as a number of mutant, or variant, lox sites, such as loxP511, loxP514, lox.DELTA.86, lox.DELTA.117, loxC2, loxP2, loxP3 and lox P23. Additional example of lox sites include, but are not limited to, loxB, loxL, loxR, loxP, loxP3, loxP23, loxΔ86, loxΔ117, loxP511, and loxC2.
In another embodiment, the recombination site is a recombination site that is recognized by a recombinases other than Cre. In one embodiment, the recombinase site can be the FRT sites recognized by FLP recombinase of the 2 pi plasmid of Saccharomyces cerevisiae. FRT sites refer to a nucleotide sequence at which the product of the FLP gene of the yeast 2 micron plasmid, FLP recombinase, can catalyze site-specific recombination. Additional examples of the non-Cre recombinases include, but are not limited to, site-specific recombinases include: att sites recognized by the Int recombinase of bacteriophage λ (e.g. att1, att2, att3, attP, attB, attL, and attR), the recombination sites recognized by the resolvase family, and the recombination site recognized by transposase of Bacillus thruingiensis.
IV. Production of Genetically Modified Animals
In additional aspects of the present invention, ungulates that contain the genetic modifications described herein can be produced by any method known to one skilled in the art. Such methods include, but are not limited to: nuclear transfer, intracytoplasmic sperm injection, modification of zygotes directly and sperm mediated gene transfer.
In another embodiment, a method to clone such animals, for example, pigs, includes: enucleating an oocyte, fusing the oocyte with a donor nucleus from a cell in which at least one allele of at least one immunoglobulin gene has been inactivated, and implanting the nuclear transfer-derived embryo into a surrogate mother.
Alternatively, a method is provided for producing viable animals that lack any expression of functional immunoglobulin by inactivating both alleles of the immunoglobulin gene in embryonic stem cells, which can then be used to produce offspring.
In another aspect, the present invention provides a method for producing viable animals, such as pigs, in which both alleles of the immunoglobulin gene have been rendered inactive. In one embodiment, the animals are produced by cloning using a donor nucleus from a cell in which both alleles of the immunoglobulin gene have been inactivated. In one embodiment, both alleles of the immunoglobulin gene are inactivated via a genetic targeting event.
Genetically altered animals that can be created by modifying zygotes directly. For mammals, the modified zygotes can be then introduced into the uterus of a pseudopregnant female capable of carrying the animal to term. For example, if whole animals lacking an immunoglobulin gene are desired, then embryonic stem cells derived from that animal can be targeted and later introduced into blastocysts for growing the modified cells into chimeric animals. For embryonic stem cells, either an embryonic stem cell line or freshly obtained stem cells can be used.
In a suitable embodiment of the invention, the totipotent cells are embryonic stem (ES) cells. The isolation of ES cells from blastocysts, the establishing of ES cell lines and their subsequent cultivation are carried out by conventional methods as described, for example, by Doetchmann et al., J. Embryol. Exp. Morph. 87:27-45 (1985); Li et al., Cell 69:915-926 (1992); Robertson, E. J. “Tetracarcinomas and Embryonic Stem Cells: A Practical Approach,” ed. E. J. Robertson, IRL Press, Oxford, England (1987); Wurst and Joyner, “Gene Targeting: A Practical Approach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); Hogen et al., “Manipulating the Mouse Embryo: A Laboratory Manual,” eds. Hogan, Beddington, Costantini and Lacy, Cold Spring Harbor Laboratory Press, New York (1994); and Wang et al., Nature 336:741-744 (1992). In another suitable embodiment of the invention, the totipotent cells are embryonic germ (EG) cells. Embryonic Germ cells are undifferentiated cells functionally equivalent to ES cells, that is they can be cultured and transfected in vitro, then contribute to somatic and germ cell lineages of a chimera (Stewart et al., Dev. Biol. 161:626-628 (1994)). EG cells are derived by culture of primordial germ cells, the progenitors of the gametes, with a combination of growth factors: leukemia inhibitory factor, steel factor and basic fibroblast growth factor (Matsui et al., Cell 70:841-847 (1992); Resnick et al., Nature 359:550-551 (1992)). The cultivation of EG cells can be carried out using methods described in the article by Donovan et al., “Transgenic Animals, Generation and Use,” Ed. L. M. Houdebine, Harwood Academic Publishers (1997), and in the original literature cited therein.
Tetraploid blastocysts for use in the invention may be obtained by natural zygote production and development, or by known methods by electrofusion of two-cell embryos and subsequently cultured as described, for example, by James et al., Genet. Res. Camb. 60:185-194 (1992); Nagy and Rossant, “Gene Targeting: A Practical Approach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); or by Kubiak and Tarkowski, Exp. Cell Res. 157:561-566 (1985).
The introduction of the ES cells or EG cells into the blastocysts can be carried out by any method known in the art. A suitable method for the purposes of the present invention is the microinjection method as described by Wang et al., EMBO J. 10:2437-2450 (1991).
Alternatively, by modified embryonic stem cells transgenic animals can be produced. The genetically modified embryonic stem cells can be injected into a blastocyst and then brought to term in a female host mammal in accordance with conventional techniques. Heterozygous progeny can then be screened for the presence of the alteration at the site of the target locus, using techniques such as PCR or Southern blotting. After mating with a wild-type host of the same species, the resulting chimeric progeny can then be cross-mated to achieve homozygous hosts.
After transforming embryonic stem cells with the targeting vector to alter the immunoglobulin gene, the cells can be plated onto a feeder layer in an appropriate medium, e.g., fetal bovine serum enhanced DMEM. Cells containing the construct can be detected by employing a selective medium, and after sufficient time for colonies to grow, colonies can be picked and analyzed for the occurrence of homologous recombination. Polymerase chain reaction can be used, with primers within and without the construct sequence but at the target locus. Those colonies which show homologous recombination can then be used for embryo manipulating and blastocyst injection. Blastocysts can be obtained from superovulated females. The embryonic stem cells can then be trypsinized and the modified cells added to a droplet containing the blastocysts. At least one of the modified embryonic stem cells can be injected into the blastocoel of the blastocyst. After injection, at least one of the blastocysts can be returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct. The blastocysts are selected for different parentage from the transformed ES cells. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected, and then genotyping can be conducted to probe for the presence of the modified immunoglobulin gene.
In other embodiments, sperm mediated gene transfer can be used to produce the genetically modified ungulates described herein. The methods and compositions described herein to either eliminate expression of endogenous immunoglobulin genes or insert xenogenous immunoglobulin genes can be used to genetically modify the sperm cells via any technique described herein or known in the art. The genetically modified sperm can then be used to impregnate a female recipient via artificial insemination, intracytoplasmic sperm injection or any other known technique. In one embodiment, the sperm and/or sperm head can be incubated with the exogenous nucleic acid for a sufficient time period. Sufficient time periods include, for example, about 30 seconds to about 5 minutes, typically about 45 seconds to about 3 minutes, more typically about 1 minute to about 2 minutes. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via intracytoplasmic sperm injection.
The potential use of sperm cells as vectors for gene transfer was first suggested by Brackett et al., Proc., Natl. Acad. Sci. USA 68:353-357 (1971). This was followed by reports of the production of transgenic mice and pigs after in vitro fertilization of oocytes with sperm that had been incubated by naked DNA (see, for example, Lavitrano et al., Cell 57:717-723 (1989) and Gandolfi et al. Journal of Reproduction and Fertility Abstract Series 4, 10 (1989)), although other laboratories were not able to repeat these experiments (see, for example, Brinster et al. Cell 59:239-241 (1989) and Gavora et al., Canadian Journal of Animal Science 71:287-291 (1991)). Since then, there have been several reports of successful sperm mediated gene transfer in chicken (see, for example, Nakanishi and Iritani, Mol. Reprod. Dev. 36:258-261 (1993)); mice (see, for example, Maione, Mol. Reprod. Dev. 59:406 (1998)); and pigs (see, for example, Lavitrano et al. Transplant. Proc. 29:3508-3509 (1997); Lavitrano et al., Proc. Natl. Acad. Sci. USA 99:14230-5 (2002); Lavitrano et al., Mol. Reprod. Dev. 64-284-91 (2003)). Similar techniques are also described in U.S. Pat. No. 6,376,743; issued Apr. 23, 2002; U.S. Patent Publication Nos. 20010044937, published Nov. 22, 2001, and 20020108132, published Aug. 8, 2002.
In other embodiments, intracytoplasmic sperm injection can be used to produce the genetically modified ungulates described herein. This can be accomplished by co-inserting an exogenous nucleic acid and a sperm into the cytoplasm of an unfertilized oocyte to form a transgenic fertilized oocyte, and allowing the transgenic fertilized oocyte to develop into a transgenic embryo and, if desired, into a live offspring. The sperm can be a membrane-disrupted sperm head or a demembranated sperm head. The co-insertion step can include the substep of preincubating the sperm with the exogenous nucleic acid for a sufficient time period, for example, about 30 seconds to about 5 minutes, typically about 45 seconds to about 3 minutes, more typically about 1 minute to about 2 minutes. The co-insertion of the sperm and exogenous nucleic acid into the oocyte can be via microinjection. The exogenous nucleic acid mixed with the sperm can contain more than one transgene, to produce an embryo that is transgenic for more than one transgene as described herein. The intracytoplasmic sperm injection can be accomplished by any technique known in the art, see, for example, U.S. Pat. No. 6,376,743. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via intracytoplasmic sperm injection.
Any additional technique known in the art may be used to introduce the transgene into animals. Such techniques include, but are not limited to pronuclear microinjection (see, for example, Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (see, for example, Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (see, for example, Thompson et al., 1989, Cell 56:313-321; Wheeler, M. B., 1994, WO 94/26884); electroporation of embryos (see, for example, Lo, 1983, Mol Cell. Biol. 3:1803-1814); cell gun; transfection; transduction; retroviral infection; adenoviral infection; adenoviral-associated infection; liposome-mediated gene transfer; naked DNA transfer; and sperm-mediated gene transfer (see, for example, Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see, for example, Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via these techniques.
Somatic Cell Nuclear Transfer to Produce Cloned, Transgenic Offspring
In a further aspect of the present invention, ungulate, such as porcine or bovine, cells lacking one allele, optionally both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be used as donor cells for nuclear transfer into recipient cells to produce cloned, transgenic animals. Alternatively, ungulate heavy chain, kappa light chain and/or lambda light chain gene knockouts can be created in embryonic stem cells, which are then used to produce offspring. Offspring lacking a single allele of a functional ungulate heavy chain, kappa light chain and/or lambda light chain gene produced according to the process, sequences and/or constructs described herein can be breed to further produce offspring lacking functionality in both alleles through mendelian type inheritance.
In another embodiment, the present invention provides a method for producing viable pigs that lack any expression of functional alpha-1,3-GT by breeding a male pig heterozygous for the alpha-1,3-GT gene with a female pig heterozygous for the alpha-1,3-GT gene. In one embodiment, the pigs are heterozygous due to the genetic modification of one allele of the alpha-1,3-GT gene to prevent expression of that allele. In another embodiment, the pigs are heterozygous due to the presence of a point mutation in one allele of the alpha-1,3-GT gene. In another embodiment, the point mutation can be a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene. In one specific embodiment, a method to produce a porcine animal that lacks any expression of functional alpha-1,3-GT is provided wherein a male pig that contains a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene is bred with a female pig that contains a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene, or vise versa.
The present invention provides a method for cloning an animal, such as a pig, lacking a functional immunoglobulin gene via somatic cell nuclear transfer. In general, the animal can be produced by a nuclear transfer process comprising the following steps: obtaining desired differentiated cells to be used as a source of donor nuclei; obtaining oocytes from the animal; enucleating said oocytes; transferring the desired differentiated cell or cell nucleus into the enucleated oocyte, e.g., by fusion or injection, to form NT units; activating the resultant NT unit; and transferring said cultured NT unit to a host animal such that the NT unit develops into a fetus.
Nuclear transfer techniques or nuclear transplantation techniques are known in the art(Dai et al. Nature Biotechnology 20:251-255; Polejaeva et al Nature 407:86-90 (2000); Campbell et al, Theriogenology, 43:181 (1995); Collas et al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, U.S. Pat. Nos. 4,944,384 and 5,057,420).
A donor cell nucleus, which has been modified to alter the immunoglobulin gene, is transferred to a recipient oocyte. The use of this method is not restricted to a particular donor cell type. The donor cell can be as described herein, see also, for example, Wilmut et al Nature 385 810 (1997); Campbell et al Nature 380 64-66 (1996); Dai et al., Nature Biotechnology 20:251-255, 2002 or Cibelli et al Science 280 1256-1258 (1998). All cells of normal karyotype, including embryonic, fetal and adult somatic cells which can be used successfully in nuclear transfer can be employed. Fetal fibroblasts are a particularly useful class of donor cells. Generally suitable methods of nuclear transfer are described in Campbell et al Theriogenology 43 181 (1995), Dai et al. Nature Biotechnology 20:251-255, Polejaeva et al Nature 407:86-90 (2000), Collas et al Mol. Reprod. Dev. 38 264-267 (1994), Keefer et al Biol. Reprod. 50 935-939 (1994), Sims et al Proc. Nat'l. Acad. Sci. USA 90 6143-6147 (1993), WO-A-9426884, WO-A-9424274, WO-A-9807841, WO-A-9003432, U.S. Pat. No. 4,994,384 and U.S. Pat. No. 5,057,420. Differentiated or at least partially differentiated donor cells can also be used. Donor cells can also be, but do not have to be, in culture and can be quiescent. Nuclear donor cells which are quiescent are cells which can be induced to enter quiescence or exist in a quiescent state in vivo. Prior art methods have also used embryonic cell types in cloning procedures (Campbell et al (Nature, 380:64-68, 1996) and Stice et al (Biol. Reprod., 20 54:100-110, 1996).
Somatic nuclear donor cells may be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disaggregated preparation of a whole or part of an embryo, fetus, or adult animal. In a suitable embodiment of the invention, nuclear donor cells are selected from the group consisting of epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, extended cells, cumulus cells, epidermal cells or endothelial cells. In another embodiment, the nuclear donor cell is an embryonic stem cell. In a particular embodiment, fibroblast cells can be used as donor cells.
In another embodiment of the invention, the nuclear donor cells of the invention are germ cells of an animal. Any germ cell of an animal species in the embryonic, fetal, or adult stage may be used as a nuclear donor cell. In a suitable embodiment, the nuclear donor cell is an embryonic germ cell.
Nuclear donor cells may be arrested in any phase of the cell cycle (G0, G1, G2, S, M) so as to ensure coordination with the acceptor cell. Any method known in the art may be used to manipulate the cell cycle phase. Methods to control the cell cycle phase include, but are not limited to, G0 quiescence induced by contact inhibition of cultured cells, G0 quiescence induced by removal of serum or other essential nutrient, G0 quiescence induced by senescence, G0 quiescence induced by addition of a specific growth factor; G0 or G1 quiescence induced by physical or chemical means such as heat shock, hyperbaric pressure or other treatment with a chemical, hormone, growth factor or other substance; S-phase control via treatment with a chemical agent which interferes with any point of the replication procedure; M-phase control via selection using fluorescence activated cell sorting, mitotic shake off, treatment with microtubule disrupting agents or any chemical which disrupts progression in mitosis (see also Freshney, R. I., “Culture of Animal Cells: A Manual of Basic Technique,” Alan R. Liss, Inc, New York (1983).
Methods for isolation of oocytes are well known in the art. Essentially, this can comprise isolating oocytes from the ovaries or reproductive tract of an animal. A readily available source of oocytes is slaughterhouse materials. For the combination of techniques such as genetic engineering, nuclear transfer and cloning, oocytes must generally be matured in vitro before these cells can be used as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell to develop into an embryo. This process generally requires collecting immature (prophase I) oocytes from mammalian ovaries, e.g., bovine ovaries obtained at a slaughterhouse, and maturing the oocytes in a maturation medium prior to fertilization or enucleation until the oocyte attains the metaphase II stage, which in the case of bovine oocytes generally occurs about 18-24 hours post-aspiration. This period of time is known as the “maturation period”. In certain embodiments, the oocyte is obtained from a gilt. A “gilt” is a female pig that has never had offspring. In other embodiments, the oocyte is obtained from a sow. A “sow” is a female pig that has previously produced offspring.
A metaphase II stage oocyte can be the recipient oocyte, at this stage it is believed that the oocyte can be or is sufficiently “activated” to treat the introduced nucleus as it does a fertilizing sperm. Metaphase II stage oocytes, which have been matured in vivo have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes can be collected surgically from either non-superovulated or superovulated animal 35 to 48, or 39-41, hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone. The oocyte can be placed in an appropriate medium, such as a hyaluronidase solution.
After a fixed time maturation period, which ranges from about 10 to 40 hours, about 16-18 hours, about 40-42 hours or about 39-41 hours, the oocytes can be enucleated. Prior to enucleation the oocytes can be removed and placed in appropriate medium, such as HECM containing 1 milligram per milliliter of hyaluronidase prior to removal of cumulus cells. The stripped oocytes can then be screened for polar bodies, and the selected metaphase II oocytes, as determined by the presence of polar bodies, are then used for nuclear transfer. Enucleation follows.
Enucleation can be performed by known methods, such as described in U.S. Pat. No. 4,994,384. For example, metaphase II oocytes can be placed in either HECM, optionally containing 7.5 micrograms per milliliter cytochalasin B, for immediate enucleation, or can be placed in a suitable medium, for example an embryo culture medium such as CR1aa, plus 10% estrus cow serum, and then enucleated later, such as not more than 24 hours later, or not more than 16-18 hours later.
Enucleation can be accomplished microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes can then be screened to identify those of which have been successfully enucleated. One way to screen the oocytes is to stain the oocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, and then view the oocytes under ultraviolet irradiation for less than 10 seconds. The oocytes that have been successfully enucleated can then be placed in a suitable culture medium, for example, CR1aa plus 10% serum.
A single mammalian cell of the same species as the enucleated oocyte can then be transferred into the perivitelline space of the enucleated oocyte used to produce the NT unit. The mammalian cell and the enucleated oocyte can be used to produce NT units according to methods known in the art. For example, the cells can be fused by electrofusion. Electrofusion is accomplished by providing a pulse of electricity that is sufficient to cause a transient breakdown of the plasma membrane. This breakdown of the plasma membrane is very short because the membrane reforms rapidly. Thus, if two adjacent membranes are induced to breakdown and upon reformation the lipid bilayers intermingle, small channels can open between the two cells. Due to the thermodynamic instability of such a small opening, it enlarges until the two cells become one. See, for example, U.S. Pat. No. 4,997,384 by Prather et al. A variety of electrofusion media can be used including, for example, sucrose, mannitol, sorbitol and phosphate buffered solution. Fusion can also be accomplished using Sendai virus as a fusogenic agent (Graham, Wister Inot. Symp. Monogr., 9, 19, 1969). Also, the nucleus can be injected directly into the oocyte rather than using electroporation fusion. See, for example, Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994). After fusion, the resultant fused NT units are then placed in a suitable medium until activation, for example, CR1aa medium. Typically activation can be effected shortly thereafter, for example less than 24 hours later, or about 4-9 hours later, or optimally 1-2 hours after fusion. In a particular embodiment, activation occurs at least one hour post fusion and at 40-41 hours post maturation.
The NT unit can be activated by known methods. Such methods include, for example, culturing the NT unit at sub-physiological temperature, in essence by applying a cold, or actually cool temperature shock to the NT unit. This can be most conveniently done by culturing the NT unit at room temperature, which is cold relative to the physiological temperature conditions to which embryos are normally exposed. Alternatively, activation can be achieved by application of known activation agents. For example, penetration of oocytes by sperm during fertilization has been shown to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer. Also, treatments such as electrical and chemical shock can be used to activate NT embryos after fusion. See, for example, U.S. Pat. No. 5,496,720, to Susko-Parrish et al. Fusion and activation can be induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Additionally, activation can be effected by simultaneously or sequentially by increasing levels of divalent cations in the oocyte, and reducing phosphorylation of cellular proteins in the oocyte. This can generally be effected by introducing divalent cations into the oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in the form of an ionophore. Other methods of increasing divalent cation levels include the use of electric shock, treatment with ethanol and treatment with caged chelators. Phosphorylation can be reduced by known methods, for example, by the addition of kinase inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethyl-aminopurine, staurosporine, 2-aminopurine, and sphingosine. Alternatively, phosphorylation of cellular proteins can be inhibited by introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.
The activated NT units, or “fused embryos”, can then be cultured in a suitable in vitro culture medium until the generation of cell colonies. Culture media suitable for culturing and maturation of embryos are well known in the art. Examples of known media, which can be used for embryo culture and maintenance, include Ham's F-10+10% fetal calf serum (FCS), Tissue Culture Medium-199 (TCM-199)+10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media, and, in one specific example, the activated NT units can be cultured in NCSU-23 medium for about 1-4 h at approximately 38.6° C. in a humidified atmosphere of 5% CO2.
Afterward, the cultured NT unit or units can be washed and then placed in a suitable media contained in well plates which can contain a suitable confluent feeder layer. Suitable feeder layers include, by way of example, fibroblasts and epithelial cells. The NT units are cultured on the feeder layer until the NT units reach a size suitable for transferring to a recipient female, or for obtaining cells which can be used to produce cell colonies. These NT units can be cultured until at least about 2 to 400 cells, about 4 to 128 cells, or at least about 50 cells.
Activated NT units can then be transferred (embryo transfers), zero(0)-144 hours post activation, to the oviduct of an female pigs. In one embodiment, the female pigs can be an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/Landrace) (280-400 lbs) can be used. The gilts can be synchronized as recipient animals by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into the feed. Regu-Mate can be fed for 14 consecutive days. One thousand units of Human Chorionic Gonadotropin (hCG, Intervet America, Millsboro, Del.) can then be administered i.m. about 105 h after the last Regu-Mate treatment. Embryo transfers can then be performed about 22-26 h after the hCG injection. In one embodiment, the pregnancy can be brought to term and result in the birth of live offspring. In another embodiment, the pregnancy can be terminated early and embryonic cells can be harvested.
Breeding for Desired Homozygous Knockout Animals
In another aspect, the present invention provides a method for producing viable animals that lack any expression of a functional immunoglobulin gene is provided by breeding a male heterozygous for the immunoglobulin gene with a female heterozygous for the immunoglobulin gene. In one embodiment, the animals are heterozygous due to the genetic modification of one allele of the immunoglobulin gene to prevent expression of that allele. In another embodiment, the animals are heterozygous due to the presence of a point mutation in one allele of the alpha-immunoglobulin gene. In further embodiments, such heterozygous knockouts can be bred with an ungulate that expresses xenogenous immunoglobulin, such as human. In one embodiment, a animal can be obtained by breeding a transgenic ungulate that lacks expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof with an ungulate that expresses an xenogenous immunoglobulin. In another embodiment, a animal can be obtained by breeding a transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain with an ungulate that expresses an xenogenous, such as human, immunoglobulin. In a further embodiment, an animal can be obtained by breeding a transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain and expresses an xenogenous, such as human, immunoglobulin with another transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain with an ungulate and expresses an xenogenous, such as human, immunoglobulin to produce a homozygous transgenic ungulate that lacks expression of both alleles of heavy chain, kappa light chain and lambda light chain and expresses an xenogenous, such as human, immunoglobulin. Methods to produce such animals are also provided.
In one embodiment, sexually mature animals produced from nuclear transfer from donor cells that carrying a homozygous knockout in the immunoglobulin gene, can be bred and their offspring tested for the homozygous knockout. These homozygous knockout animals can then be bred to produce more animals.
In another embodiment, oocytes from a sexually mature homozygous knockout animal can be in vitro fertilized using wild type sperm from two genetically diverse pig lines and the embryos implanted into suitable surrogates. Offspring from these matings can be tested for the presence of the knockout, for example, they can be tested by cDNA sequencing, and/or PCR. Then, at sexual maturity, animals from each of these litters can be mated. In certain methods according to this aspect of the invention, pregnancies can be terminated early so that fetal fibroblasts can be isolated and further characterized phenotypically and/or genotypically. Fibroblasts that lack expression of the immunoglobulin gene can then be used for nuclear transfer according to the methods described herein to produce multiple pregnancies and offspring carrying the desired homozygous knockout.
Additional Genetic Modifications
In other embodiments, animals or cells lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can contain additional genetic modifications to eliminate the expression of xenoantigens. The additional genetic modifications can be made by further genetically modifying cells obtained from the transgenic cells and animals described herein or by breeding the animals described herein with animals that have been further genetically modified. Such animals can be modified to eliminate the expression of at least one allele of the alpha-1,3-galactosyltransferase gene, the CMP-Neu5Ac hydroxylase gene (see, for example, U.S. Ser. No. 10/863,116), the iGb3 synthase gene (see, for example, U.S. Patent Application 60/517,524), and/or the Forssman synthase gene (see, for example, U.S. Patent Application 60/568,922). In additional embodiments, the animals discloses herein can also contain genetic modifications to express fucosyltransferase, sialyltransferase and/or any member of the family of glucosyltransferases. To achieve these additional genetic modifications, in one embodiment, cells can be modified to contain multiple genetic modifications. In other embodiments, animals can be bred together to achieve multiple genetic modifications. In one specific embodiment, animals, such as pigs, lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can be bred with animals, such as pigs, lacking expression of alpha-1,3-galactosyl transferase (for example, as described in WO 04/028243).
In another embodiment, the expression of additional genes responsible for xenograft rejection can be eliminated or reduced. Such genes include, but are not limited to the CMP-NEUAc Hydroxylase Gene, the isoGloboside 3 Synthase gene, and the Forssman synthase gene. In addition, genes or cDNA encoding complement related proteins, which are responsible for the suppression of complement mediated lysis can also be expressed in the animals and tissues of the present invention. Such genes include, but are not limited to CD59, DAF, MCP and CD46 (see, for example, WO 99/53042; Chen et al. Xenotransplantation, Volume 6 Issue 3 Page 194-August 1999, which describes pigs that express CD59/DAF transgenes; Costa C et al, Xenotransplantation. 2002 January; 9(1):45-57, which describes transgenic pigs that express human CD59 and H-transferase; Zhao L et al.; Diamond L E et al. Transplantation. 2001 Jan. 15; 71(1):132-42, which describes a human CD46 transgenic pigs.
Additional modifications can include expression of tissue factor pathway inhibitor (TFPI), heparin, antithrombin, hirudin, TFPI, tick anticoagulant peptide, or a snake venom factor, such as described in WO 98/42850 and U.S. Pat. No. 6,423,316, entitled “Anticoagulant fusion protein anchored to cell membrane”; or compounds, such as antibodies, which down-regulate the expression of a cell adhesion molecule by the cells, such as described in WO 00/31126, entitled “Suppression of xenograft rejection by down regulation of a cell adhesion molecules” and compounds in which co-stimulation by signal 2 is prevented, such as by administration to the organ recipient of a soluble form of CTLA-4 from the xenogeneic donor organism, for example as described in WO 99/57266, entitled “Immunosuppression by blocking T cell co-stimulation signal 2 (B7/CD28 interaction)”.
In one embodiment, the animals or cells lacking expression of functional immunoglobulin, produced according to the present invention, can be further modified to transgenically express a cytoxic T-lymphocyte associated protein 4-immunoglobin (CTLA4). The animals or cells can be modified to express CTLA4 peptide or a biologically active fragment (e.g., extracellular domain, truncated form of the peptide in which at least the transmembrane domain has been removed) or derivative thereof. The peptide may be, e.g., human or porcine. The CTLA4 peptide can be mutated. Mutated peptides may have higher affinity than wildtype for porcine and/or human B7 molecules. In one specific embodiment, the mutated CTLA4 can be CTLA4 (Glu104, Tyr29). The CTLA4 peptide can be modified such that it is expressed intracellularly. Other modifications of the CTLA4 peptide include addition of a golgi retention signal to the N or C terminus. The golgi retention signal may be, e.g., the sequence KDEL. The CTLA4 peptide can be fused to a peptide dimerization domain or an immunoglobulin (Ig) molecule. The CTLA4 fusion peptides can include a linker sequence that can join the two peptides.
Certain aspects of the invention are described in greater detail in the non-limiting Examples that follow.

EXAMPLES

Example 1

Porcine Heavy Chain Targeting and Generation of Porcine Animals that Lack Expression of Heavy Chain

A portion of the porcine Ig heavy-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine heavy chain immunoglobulin can then be selected through hybridization of probes selective for porcine heavy chain immunoglobulin as described herein.
Sequence from a clone (Seq ID 1) was used to generate a primer complementary to a portion of the J-region (the primer is represented by Seq ID No. 2). Separately, a primer was designed that was complementary to a portion of Ig heavy-chain mu constant region (the primer is represented by Seq ID No. 3). These primers were used to amplify a fragment of porcine Ig heavy-chain (represented by Seq ID No. 4) that led the functional joining region (J-region) and sufficient flanking region to design and build a targeting vector. To maintain this fragment and subclones of this fragment in a native state, the E. coli (Stable 2, Invitrogen cat #1026-019) that harbored these fragments was maintained at 30° C. Regions of Seq. ID No. 4 were subcloned and used to assemble a targeting vector as shown in Seq. ID No. 5. This vector was transfected into porcine fetal fibroblasts that were subsequently subjected to selection with G418. Resulting colonies were screened by PCR to detect potential targeting events (Seq ID No. 6 and Seq ID No. 7, 5′ screen primers; and Seq ID No. 8 and Seq ID No. 9, 3′ screen primers). See FIG. 1 for a schematic illustrating the targeting. Targeting was confirmed by southern blotting. Piglets were generated by nuclear transfer using the targeted fetal fibroblasts as nuclear donors.
Nuclear Transfer.
The targeted fetal fibroblasts were used as nuclear donor cells. Nuclear transfer was performed by methods that are well known in the art (see, e.g., Dai et al., Nature Biotechnology 20: 251-255, 2002; and Polejaeva et al., Nature 407:86-90, 2000).
Enucleation of in vitro-matured oocytes (BoMed, Madison, Wis.; TransOva Genetics, Sioux City, Iowa) was begun between 40 and 42 hours post-maturation as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). For enucleation, we incubated the oocytes in calcium-free phosphate-buffered NCSU-23 medium containing 5 μg ml⁻¹cytochalasin B (Sigma) and 7.5 μg ml⁻¹Hoechst 33342 (Sigma) at 38° C. for 20 min. A small amount of cytoplasm from directly beneath the first polar body was then aspirated using an 18 μM glass pipette (Humagen, Charlottesville, Va.). We exposed the aspirated karyoplast to ultraviolet light to confirm the presence of a metaphase plate.
For nuclear transfer, a single fibroblast cell was placed under the zona pellucida in contact with each enucleated oocyte. Fusion and activation were induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Fused embryos were cultured in NCSU-23 medium for 1-4 h at 38.6° C. in a humidified atmosphere of 5% CO₂, and then transferred to the oviduct of an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/landrace) (280-400 lbs) were synchronized as recipients by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into their feed. Regu-Mate was fed for 14 consecutive days. Human chorionic gonadotropin (hCG, 1,000 units; Intervet America, Millsboro, Del.) was administered intra-muscularly 105 h after the last Regu-Mate treatment. Embryo transfers were done 22-26 h after the hCG injection.

Nuclear transfer produced 18 healthy piglets from four litters. These animals have one functional wild-type Ig heavy-chain locus and one disrupted Ig heavy chain locus.


Seq ID 2: primer from	ggccagacttcctcggaacagctca
Butler subclone to am-
plify J to C heavychain
(637Xba5′)

Seq ID 3: primer for C	ttccaggagaaggtgacggagct
to amplify J to C heavy-
chain (JM1L)

Seq ID 6: heavychain 5′	tctagaagacgctggagagaggccag
primer for 5′ screen
(HCKOXba5′2)

Seq ID 7: heavychain 3′	taaagcgcatgctccagactgcctt
primer for 5′ screen
(5′arm5′)

Seq ID 8: heavychain 5′	catcgccttctatcgccttctt
primer for 3′ screen
(NEO4425)

Seq ID 9: heavychain 3′	Aagtacttgccgcctctcagga
primer for 3′ screen
(650 + CA)

Southern blot analysis of cell and pig tissue samples. Cells or tissue samples were lysed overnight at 60° C. in lysis buffer (10mM Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% (w/v) Sarcosyl, 1 mg/ml proteinase K) and the DNA precipitated with ethanol. The DNA was then digested with NcoI or XbaI, depending on the probe to be used, and separated on a 1% agarose gel. After electrophoresis, the DNA was transferred to a nylon membrane and probed with digoxigenin-labeled probe (SEQ ID No 41 for NcoI digest, SEQ ID No 40 for XbaI digest). Bands were detected using a chemiluminescent substrate system (Roche Molecular Biochemicals).


Probes for Heavy Chain Southern:
HC J Probe (used with Xba I digest)

(Seq ID No 40)

CTCTGCACTCACTACCGCCGGACGCGCACTGCCGTGCTGCCCATGGACCA

CGCTGGGGAGGGGTGAGCGGACAGCACGTTAGGAAGTGTGTGTGTGCGCG

TGGGTGCAAGTCGAGCCAAGGCCAAGATCCAGGGGCTGGGCCCTGTGCCC

AGAGGAGAATGGCAGGTGGAGTGTAGCTGGATTGAAAGGTGGCCTGAAGG

GTGGGGCATCCTGTTTGGAGGCTCACTCTCAGCCCCAGGGTCTCTGGTTC

CTGCCGGGGTGGGGGGCGCAAGGTGCCTACCACACCCTGCTAGCCCCTCG

TCCAGTCCCGGGCCTGCCTCTTCACCACGGAAGAGGATAAGCCAGGCTGC

AGGCTTCATGTGCGCCGTGGAGAACCCAGTTCGGCCCTTGGAGG

HC Mu Probe (used with NcoI digest)

(Seq ID No 41)

GGCTGAAGTCTGAGGCCTGGCAGATGAGCTTGGACGTGCGCTGGGGAGTA

CTGGAGAAGGACTCCCGGGTGGGGACGAAGATGTTCAAGACGGGGGGCTG

CTCCTCTACGACTGCAGGCAGGAACGGGGCGTCACTGTGCCGGCGGCACC

CGGCCCCGCCCCCGCCACAGCCACAGGGGGAGCCCAGCTCACCTGGCCCA

GAGATGGACACGGACTTGGTGCCACTGGGGTGCTGGACCTCGCACACCAG

GAAGGCCTCTGGGTCCTGGGGGATGCTCACAGAGGGTAGGAGCACCCGGG

AGGAGGCCAAGTACTTGCCGCCTCTCAGGACGG

Example 2

Porcine Kappa Light Chain Targeting and Generation of Porcine Lacking Expression of Kappa Light Chain

A portion of the porcine Ig kappa-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine kappa chain immunoglobulin can then be selected through hybridization of probes selective for porcine kappa chain immunoglobulin as described herein.
A fragment of porcine Ig light-chain kappa was amplified using a primer complementary to a portion of the J-region (the primer is represented by Seq ID No. 10) and a primer complementary to a region of kappa C-region (represented by Seq ID No.11). The resulting amplimer was cloned into a plasmid vector and maintained in Stable2 cells at 30° C. (Seq ID No. 12). See FIG. 2 for a schematic illustration.
Separately, a fragment of porcine Ig light-chain kappa was amplified using a primer complementary to a portion of the C-region (Seq ID No. 13) and a primer complementary to a region of the kappa enhancer region (Seq ID No. 14). The resulting amplimer was fragmented by restriction enzymes and DNA fragments that were produced were cloned, maintained in Stable2 cells at 30 degrees C. and sequenced. As a result of this sequencing, two non-overlapping contigs were assembled (Seq ID No. 15, 5′ portion of amplimer; and Seq ID No. 16, 3′ portion of amplimer). Sequence from the downstream contig (Seq ID No. 16) was used to design a set of primers (Seq ID No. 17 and Seq ID No. 18) that were used to amplify a contiguous fragment near the enhancer (Seq ID No. 19). A subclone of each Seq ID No. 12 and Seq ID No. 19 were used to build a targeting vector (Seq ID No. 20). This vector was transfected into porcine fetal fibroblasts that were subsequently subjected to selection with G418. Resulting colonies were screened by PCR to detect potential targeting events (Seq ID No. 21 and Seq ID No. 22, 5′ screen primers; and Seq ID No. 23 and Seq Id No 43, 3′ screen primers, and Seq ID No. 24 and Seq Id No 24, endogenous screen primers). Targeting was confirmed by southern blotting. Southern blot strategy design was facilitated by cloning additional kappa sequence, it corresponds to the template for germline kappa transcript (Seq ID No. 25). Fetal pigs were generated by nuclear transfer.
Nuclear Transfer.
The targeted fetal fibroblasts were used as nuclear donor cells. Nuclear transfer was performed by methods that are well known in the art (see, e.g., Dai et al., Nature Biotechnology 20: 251-255, 2002; and Polejaeva et al., Nature 407:86-90, 2000).
Oocytes were collected 46-54 h after the hCG injection by reverse flush of the oviducts using pre-warmed Dulbecco's phosphate buffered saline (PBS) containing bovine serum albumin (BSA; 4 g⁻¹) (as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). Enucleation of in vitro-matured oocytes (BoMed, Madison, Wis.) was begun between 40 and 42 hours post-maturation as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). Recovered oocytes were washed in PBS containing 4 gl⁻¹BSA at 38° C., and transferred to calcium-free phosphate-buffered NCSU-23 medium at 38° C. for transport to the laboratory. For enucleation, we incubated the oocytes in calcium-free phosphate-buffered NCSU-23 medium containing 5 μg ml⁻¹cytochalasin B (Sigma) and 7.5 μg ml⁻¹Hoechst 33342 (Sigma) at 38° C. for 20 min. A small amount of cytoplasm from directly beneath the first polar body was then aspirated using an 18 μM glass pipette (Humagen, Charlottesville, Va.). We exposed the aspirated karyoplast to ultraviolet light to confirm the presence of a metaphase plate.
For nuclear transfer, a single fibroblast cell was placed under the zona pellucida in contact with each enucleated oocyte. Fusion and activation were induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Fused embryos were cultured in NCSU-23 medium for 1-4 h at 38.6° C. in a humidified atmosphere of 5% CO₂, and then transferred to the oviduct of an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/landrace) (280-400 lbs) were synchronized as recipients by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into their feed. Regu-Mate was fed for 14 consecutive days. Human chorionic gonadotropin (hCG, 1,000 units; Intervet America, Millsboro, Del.) was administered intra-muscularly 105 h after the last Regu-Mate treatment. Embryo transfers were done 22-26 h after the hCG injection.

Nuclear transfer using kappa targeted cells produced 33 healthy pigs from 5 litters. These pigs have one functional wild-type allele of porcine Ig light-chain kappa and one disrupted Ig light-chain kappa allele.


Seq ID 10: kappa J to C	caaggaqaccaagctggaactc
5′ primer (kjc5′1)

Seq ID 11: kappa J to C	tgatcaagcacaccacagagacag
3′ primer (kjc3′2)

Seq ID 13: 5′ primer for	gatgccaagccatccgtcttcatc
Kappa C to E (porKCS1)

Seq ID 14: 3′ primer for	tgaccaaagcagtgtgacggttgc
Kappa C to E (porKCA1)

Seq ID 17: kappa 5′	ggatcaaacacgcatcctcatggac
primer for amplification
of enhancer region
(K3′arm1S)

Seq ID 18: kappa 3′	ggtgattggggcatggttgagg
primer for amplification
of enhancer region
(K3′arm1A)

Seq ID 21: kappa screen,	cgaacccctgtgtatatagtt
5′ primer, 5′
(kappa5armS)

Seq ID 22: kappa screen,	gagatgaggaagaggagaaca
3′ primer, 5′
(kappaNeoA)

Seq ID 23: kappa screen,	gcattgtctgagtaggtgtcatt
5′ primer, 3′
(kappaNeoS)

Seq ID 24: kappa screen,	cgcttcttgcagggaacacgat
3′ primer, 5′
(kappa5armProbe3′)

Seq ID No 43, Kappa	GTCTTTGGTTTTTGCTGAGGGTT
screen, 3′ primer
(kappa3armA2)

Southern blot analysis of cell and pig tissue samples. Cells or tissue samples were lysed overnight at 60° C. in lysis buffer (10mM Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% (w/v) Sarcosyl, 1 mg/ml proteinase K) and the DNA precipitated with ethanol. The DNA was then digested with SacI and separated on a 1% agarose gel. After electrophoresis, the DNA was transferred to a nylon membrane and probed with digoxigenin-labeled probe (SEQ ID No 42). Bands were detected using a chemiluminescent substrate system (Roche Molecular Biochemicals).


Probe for Kappa Southern:
Kappa5ArmProbe 5′/3′

(SEQ ID No 42)

gaagtgaagccagccagttcctcctgggcaggtggccaaaattacagttg

acccctcctggtctggctgaaccttgccccatatggtgacagccatctgg

ccagggcccaggtctccctctgaagcctttgggaggagagggagagtggc

tggcccgatcacagatgcggaaggggctgactcctcaaccggggtgcaga

ctctgcagggtgggtctgggcccaacacacccaaagcacgcccaggaagg

aaaggcagcttggtatcactgcccagagctaggagaggcaccgggaaaat

gatctgtccaagacccgttcttgcttctaaactccgagggggtcagatga

agtggttttgtttcttggcctgaagcatcgtgttccctgcaagaagcgg

Example 3

Characterization of the Porcine Lambda Gene Locus

To disrupt or disable porcine lambda, a targeting strategy has been devised that allows for the removal or disruption of the region of the lambda locus that includes a concatamer of J to C expression cassettes. BAC clones that contain portions of the porcine genome can be generated. A portion of the porcine Ig lambda-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine lambda chain immunoglobulin can then be selected through hybridization of probes selective for porcine lambda chain immunoglobulin as described herein.
BAC clones containing a lambda J-C flanking region (see FIG. 3), can be independently fragmented and subcloned into a plasmid vector. Individual subdlones have been screened by PCR for the presence of a portion of the J to C intron. We have cloned several of these cassettes by amplifying from one C region to the next C region. This amplification was accomplished by using primers that are oriented to allow divergent extension within any one C region (Seq ID 26 and Seq ID 27). To obtain successful amplification, the extended products converge with extended products originated from adjacent C regions (as opposed to the same C region). This strategy produces primarily amplimers that extend from one C to the adjacent C. However, some amplimers are the result of amplification across the adjacent C and into the next C which lies beyond the adjacent C. These multi-gene amplimers contain a portion of a C, both the J and C region of the next J-C unit, the J region of the third J-C unit, and a portion of the C region of the third J-C unit. Seq ID 28 is one such amplimer and represents sequence that must be removed or disrupted.
Other porcine lambda sequences that have been cloned include: Seq ID No. 32, which includes 5′ flanking sequence to the first lambda J/C unit of the porcine lambda light chain genomic sequence; Seq ID No. 33, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, from approximately 200 base pairs downstream of lambda J/C; Seq ID No. 34, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, approximately 11.8 Kb downstream of the J/C cluster region, near the enhancer; Seq ID No. 35, which includes approximately 12 Kb downstream of lambda, including the enhancer region; Seq ID No. 36, which includes approximately 17.6 Kb downstream of lambda; Seq ID No. 37, which includes approximately 19.1 Kb downstream of lambda; Seq ID No. 38, which includes approximately 21.3 Kb downstream of lambda; and Seq ID No. 39, which includes approximately 27 Kb downstream of lambda.

Seq ID 26: 5′ primer for ccttcctcctgcacctgtcaac

lambda C to C amplimer

(lamC5′)

Seq ID 27: 3′ primer for tagacacaccagggtggccttg

lambda C to C amplimer

(lamC3′)

Example 4

Production of Targeting Vectors for the Lambda Gene

Following a first targeting strategy, shown in FIG. 4, a vector is designed and built with one targeting arm that is homologous to a region upstream of J1 (i.e., the first J/C unit or sequence) and the other arm homologous to a region that is downstream of the last C (i.e., the last J/C unit or sequence) This targeting vector utilizes a selectable marker (SM).

Seq ID No. 48 represents one example of a vector used in the first targeting strategy. Seq ID No. 48 is a lambda light chain knockout vector which includes both 5′ and 3′ homology arms and Neo resistance factor.


Seq ID	GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT

No. 48	TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC

	TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA

	GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA

	CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA

	AGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTC

	GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC

	CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT

	GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGC

	CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTA

	CAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG

	ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG

	AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG

	GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA

	AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTC

	TGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCA

	TGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA

	AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTG

	GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAG

	CGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTC

	GTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAG

	TGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATT

	TATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT

	GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG

	CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA

	ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG

	TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG

	AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT

	TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTA

	TCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCAT

	GCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCA

	AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGC

	CCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTT

	AAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCT

	CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT

	CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT

	TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGG

	GAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT

	TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAG

	CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGG

	TTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCAAACAG

	CTATGACCATGGCGGCCGCgtcgacAGGGTGTGGCCAAATACAG

	CATGGAGTAGCCATCATAAGGAATCTTACACAAGCCTCCAAAAT

	TGTGTTTCTGAAATTGGGTTTAAAGTACGTTTGCATTTTAAAAA

	GCCTGCCAGAAAATACAGAAAAATGTCTGTGATATGTCTCTGGC

	TGATAGGATTTTGCTTAGTTTTAATTTTGGCTTTATAATTTTCT

	ATAGTTATGAAAATGTTCACAAGAAGATATATTTCATTTTAGCT

	TCTAAAATAATTATAACACAGAAGTAATTTGTGCTTTAAAAAAA

	TATTCAACACAGAAGTATATAAAGTAAAAATTGAGGAGTTCCCA

	TCGTGGCTCAGTGATTAACAAACCCAACTAGTATCCATGAGGAT

	ATGGATTTGATCCCTGGCCTTGCTCAGTGGGTTGAGGATCCAGT

	GTTGCTGTGAGCTGTGGTGTAGGTTGCAGACACAGCACTCTGGC

	GTTGCTGTGACTCTGGCGTAGGCCGGCAGCTACAGCTCCATTTG

	GACCCTTAGCCTGGGAACCTCCATATGCCTGAGATACGGCCCTA

	AAAAGTCAAAAGCCAAAAAAATAGTAAAAATTGAGTGTTTCTAC

	TTACCACCCCTGCCCACATCTTATGCTAAAACCCGTTCTCCAGA

	GACAAACATCGTCAGGTGGGTCTATATATTTCCAGCCCTCCTCC

	TGTGTGTGTATGTCCGTAAAACACACACACACACACACACACGC

	ACACACACACACACGTATCTAATTAGCATTGGTATTAGTTTTTC

	AAAAGGGAGGTCATGCTCTACCTTTTAGGCGGCAAATAGATTAT

	TTAAACAAATCTGTTGACATTTTCTATATCAACCCATAAGATCT

	CCCATGTTCTTGGAAAGGCTTTGTAAGACATCAACATCTGGGTA

	AACCAGCATGGTTTTTAGGGGGTTGTGTGGATTTTTTTCATATT

	TTTTAGGGCACACCTGCAGCATATGGAGGTTCCCAGGCTAGGGG

	TTGAATCAGAGCTGTAGCTGCCGGCCTACACCACAGCCACAGCA

	ACGCCAGATCCTTAACCCACTGAGAAAGGCCAGGGATTGAACCT

	GCATCCTCATGGATGCTGGTCAGATTTATTTCTGCTGAGCCACA

	ACAGGAACTCCCTGAACCAGAATGCTTTTAACCATTCCACTTTG

	CATGGACATTTAGATTGTTTCCATTTAAAAATACAAATTACAAG

	GAGTTCCCGTCGTGGCTCAGTGGTAACGAATTGGACTAGGAACC

	ATGAGGTTTCGGGTTCGATCCCTGGCCTTGCTCGGTGGGTTAAG

	GATCCAGCATTGATGTGAGATATGGTGTAGGTCGCAGACGTGGC

	TCGGATCCCACGTTGCTGTGGCTCTGGCGTAGGCCGGCAACAAC

	AGCTCCGATTCGACCCCTAGCCTGGGAACCTCCATGTGCCACAG

	GAGCAGCCCTAGAAAAGGCAAAAAGACAAAAAAATAAAAAATTA

	AAATGAAAAAATAAAATAAAAATACAAATTACAAGAGACGGCTA

	CAAGGAAATCCCCAAGTGTGTGCAAATGCCATATATGTATAAAA

	TGTACTAGTGTCTCCTCGCGGGAAAGTTGCCTAAAAGTGGGTTG

	GCTGGACAGAGAGGACAGGCTTTGACATTCTCATAGGTAGTAGC

	AATGGGCTTCTCAAAATGCTGTTCCAGTTTACACTCACCATAGC

	AAATGACAGTGCCTCTTCCTCTCCACCCTTGCCAATAATGTGAC

	AGGTGGATCTTTTTCTATTTTGTGTATCTGACAAGCAAAAAATG

	AGAACAGGAGTTCCTGTCGTGGTGCAGTGGAGACAAATCTGACT

	AGGAACCATGAAATTTCGGGTTCAATCCCTGGCCTCACTCAGTA

	GGTAAAGGATCCAGGGTTGCAGTGAGCTGTGGGGTAGGTCGCAG

	ACACAGTGCAAATTTGGCCCTGTTGTGGCTGTGGTGTAGGCCGG

	CAGCTATAGCTCCAATTGGACCCCTAGCCTGGGAACCTCCTTAT

	GCCGTGGGTGAGGCCCTAAAAAAAAGAGTGCAAAAAAAAAAAAT

	AAGAACAAAAATGATCATCGTTTAATTCTTTATTTGATCATTGG

	TGAAACTTATTTTCCTTTTATATTTTTATTGACTGATTTTATTT

	CTCCTATGAATTTACCGGTCATAGTTTTGCCTGGGTGTTTTTAC

	TCCGGTTTTAGTTTTGGTTGGTTGTATTTTCTTAGAGAGCTATA

	GAAACTCTTCATCTATTTGGAATAGTAATTCCTCATTAAGTATT

	TGTGCTGCAAAAAATTTTCCCTGATCTGTTTTATGCTTTTGTTT

	GTGGGGTCTTTCACGAGAAAGCCTTTTTAGTTTTTACACCTCAG

	CTTGGTTGTTTTTCTTGATTGTGTCTGTAATCTGCGGCCAACAT

	AGGAAACACATTTTTACTTTAGTGTTTTTTTCCTATTTTCTTCA

	AGTACGTCCATTGTTTTGGTGTCTGATTTTACTTTGCCTGGGGT

	TTGTTTTTGTGTGGCAGGAATATAAACTTATGTATTTTCCAAAT

	GGAGAGCCAATGGTTGTATATTTGTTGAATTCAAATGCAACTTT

	ATCAAACACCAAATCATCGATTTATCACAACTCTTCTCTGGTTT

	ATTGATCTAATGATCAATTCCTGTTCCACGCTGTTTTAATTATT

	TTAGCTTTGTGGATTTTGGTGCCTGGTAGAGAACAAAGCCTCCA

	TTATTTTCATTCAAAATAGTCCCGTCTATTATCTGCCATTGTTG

	TAGTATTAGACTTTAAAATCAATTTACTGATTTTCAAAAGTTAT

	TCCTTTGGTGATGTGGAATACTTTATACTTCATAAGGTACATGG

	ATTCATTTGTGGGGAATTGATGTCTTTGCTATTGTGGCCATTTG

	TCAAGTTGTGTAATATTTTACCCATGCCAACTTTGCATATTGTA

	TGTGAGTTTATTCCCAGGGTTTTTAATAGGATGTTTATTGAAGT

	TGTCAGTGTTTCCACAATTTCATCGCCTCAGTGCTTACTGTTTG

	CATAAAAGGAAACCTACTCACTTTTGCCTATTGCTCTTGTATTC

	AATCATTTTAGTTAACTCTTGTGTTAATTTTGAGAGTTTTTCAG

	CTGACTGTCTGGGGTTTTCTTTAATAGACTAGCCCTTTGTCTGT

	AAAGAATAATTTTATCGAATTTTTCTTAACACTCACACTCTCCC

	CACCCCCACCCCCGCTCATCTCCTTTCATTGGGTCAAATCTGTA

	GAATACAATAAAAGTAAGAGTGGGAACCTTAGCCTTTAAGTCGA

	TTTTGCCTTTAAATGTGAATGTTGCTATGTTTCGGGACATTCTC

	TTTATCAAGTTGCGGATGTTTCCTTAGATAATTAACTTAATAAA

	AGACTGGATGTTTGCTTTCTTCAAATCAGAATTGTGTTGAATTT

	ATATTGCTATTCTGTTTAATTTTGTTTCAAAAAATTTACATGCA

	CACCTTAAAGATAACCATGACCAAATAGTCCTCCTGCTGAGAGA

	AAATGTTGGCCCCAATGCCACAGGTTACCTCCCGACTCAGATAA

	ACTACAATGGGAGATAAAATCAGATTTGGCAAAGCCTGTGGATT

	CTTGCCATAACTCTCAGAGCATGACTTGGGTGTTTTTTCCTTTT

	CTAAGTATTTTAATGGTATTTTTGTGTTACAATAGGAAATCTAG

	GACACAGAGAGTGATTCAATGAGGGGAACGCATTCTGGGATGAC

	TCTAGGCCTCTGGTTTGGGGAGAGCTCTATTGAAGTAAAGACAA

	TGAGAGGAAGCAAGTTTGCAGGGAACTGTGAGGAATTTAGATGG

	GGAATGTTGGGTTTGAGGTTTCTATAGGGCACGCAAGCAGAGAT

	GCACTCAGGAGGAAGAAGGAGCATAAATCTAGAGGCAAAAAGAG

	AGGTCAGGACTGGAAATAGAGATGCGAGACACCAGGGTGGCAGT

	CAGAGAGCACAGTGTGGGTCAGAAGACAGTGGAAGAACACAAGG

	GACAGAGAGGGATCTCCAACTTCACTGGGATGAGGGCCTTGTTG

	GCCTTGACCTGAGAGATTTCCAGGAGTTGAGGGTGGGAAGGAGc

	cgcggTCTAGGAAGCTTTCTAGGGTACCTCTAGGGATCCGAACA

	ATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATACAT

	TATACGAAGTTATATTCGATGCGGCCGCAAGGGGTTCGCGTCAG

	CGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCA

	GAGCAGagatccCGGCGCGCCCTACCGGGTAGGGGAGGCGCTTT

	TCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGC

	ACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCAC

	ATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCT

	TCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCC

	GCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGC

	ACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAAT

	GGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGG

	CTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCG

	GGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGA

	AGGTCCTCCGGAAGCCCGGCATTCTGCACGCTTCAAAAGCGCAC

	GTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGA

	CCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATTGCA

	CGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATG

	ACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTC

	CGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGA

	CCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGC

	TATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTC

	GACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGA

	AGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCG

	AGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACG

	CTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCG

	CATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCAATC

	AGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAA

	CTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCT

	CGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGG

	AAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGT

	GTGGCGGATCGCTATCAGGACATAGCGTTGGCTACCCGTGATAT

	TGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGC

	TTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTAT

	CGCCTTCTTGACGAGTTCTTCTGAGGGGATCAATTCtctagtGA

	ACAATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATA

	CATTATACGAAGTTATATTCGATGCGGCCGCAAGGGGTTCGCGT

	CAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCA

	TCAGAGCAGtctagaGCTCGCTGATCAGCCTCGACTGTGCCTTC

	TAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT

	TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAAT

	GAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCT

	GGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG

	ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCT

	GAGGCGGAAAGAACCAGCTGGGGGCGCGCCCctcgagGGGAAGG

	TATCTCCCAGGAAACTGGCCAGGACACATTGGTCCTCCGCCCTC

	CCCTTCCTCCCACTCCTCCTCCAGACAGGACTGTGCCCACCCCC

	TGCCACCTTTCTGGCCAGAACTGTCCATGGCAGGTGACCTTCAC

	ATGAGCCCTTCCTCCCTGCCTGCCCTAGTGGGACCCTCCATACC

	TCCCCCTGGACCCCGTTGTCCTTTCTTTCCAGTGTGGCCCTGAG

	CATAACTGATGCCATCATGGGCTGCTGACCCACCCGGGACTGTG

	TTGTGCAGTGAGTCACTTCTCTGTCATCAGGGCTTTGTAATTGA

	TAGATAGTGTTTCATCATCATTAGGACCGGGTGGCCTCTATGCT

	CTGTTAGTCTCCAAACACTGATGAAAACCTTCGTTGGCATAGTC

	CCAGCTTCCTGTTGCCCATCCATAAATCTTGACTTAGGGATGCA

	CATCCTGTCTCCAAGCAACCACCCCTCCCCTAGGCTAACTATAA

	AACTGTCCCAATGGCCCTTGTGTGGTGCAGAGTTCATGCTTCCA

	GATCATTTCTCTGCTAGATCCATATCTCACCTTGTAAGTCATCC

	TATAATAAACTGATCCATTGATTATTTGCTTCTGTTTTTTCCAT

	CTCAAAACAGCTTCTCAGTTCAGTTCGAATTTTTTATTCCCTCC

	ATCCACCCATACTTTCCTCAGCCTGGGGAACCCTTGCCCCCAGT

	CCCATGCCCTTCCTCCCTCTCTGCCCAGCTCAGCACCTGCCCAC

	CCTCACCCTTCCTGTCACTCCCTAGGACTGGACCATCCACTGGG

	GCCAGGACACTCCAGCAGCCTTGGCTTCATGGGCTCTGAAATCC

	ATGGCCCATCTCTATTCCTCACTGGATGGCAGGTTCAGAGATGT

	GAAAGGTCTAGGAGGAAGCCAGGAAGGAAACTGTTGCATGAAAG

	GCCGGCCTGATGGTTCAGTACTTAAATAATATGAGCTCTGAGCT

	CCCCAGGAACCAAAGCATGGAGGGAGTATGTGCCTCAGAATCTC

	TCTGAGATTCAGCAAAGCCTTTGCTAGAGGGAAAATAGTGGCTC

	AACCTTGAGGGCCAGCATCTTGCACCACAGTTAAAAGTGGGTAT

	TTGTTTTACCTGAGGCCTCAGCATTATGGGAACCGGGCTCTGAC

	ACAAACACAGGTGCAGCCCGGCAGCCTCAGAACACAGCAACGAC

	CACAAGCTGGGACAGCTGCCCCTGAACGGGGAGTCCACCATGCT

	TCTGTCTCGGGTACCACCAGGTCACCATCCCTGGGGGAGGTAGT

	TCCATAGCAGTAGTCCCCTGATTTCGCCCCTCGGGCGTGTAGCC

	AGGCAAGCTCCTGCCTCTGGACCCAGGGTGGACCCTTGCTCCCC

	ACTACCCTGCACATGCCAGACAGTCAAGACCACTCCCACCTCTG

	TCTGAGGCCCCCTTGGGTGTCCCAGGGCCCCCGAGCTGTCCTCT

	ACTCATGGTTCTTCCACCTGGGTACAAAAGAGGCGAGGGACACT

	TTTCTCAGGTTTGCGGCTCAGAAAGGTACCTTCCTAGGGTTTGT

	CCACTGGGAGTCACCTCCCTTGCATCTCAATGTCAGTGGGGAAA

	ACTGGGTCCCATGGGGGGATTAGTGCCACTGTGAGGCCCCTGAA

	GTCTGGGGCCTCTAGACACTATGATGATGAGGGATGTGGTGAAA

	AACCCCACCCCAGCCCTTCTTGCCGGGACCCTGGGCTGTGGCTC

	CCCCATTGCACTTGGGGTCAGAGGGGTGGATGGTGGCTATGGTC

	AGGCATGTTTCCCATGAGCTGGGGGCACCCTGGGTGACTTTCTC

	CTGTGAATCCTGAATTAGCAGCTATAACAAATTGCCCAAACTCT

	TAGGCTTAAAACAACACACATTTATTCCTCTGGGTCCCAGGGTC

	AGAAGTCCAAAATGAGTCCTATAGGCTAAATTTGAGGTGTCTCT

	GGGTTGAGCTCCTCCTGGAAGCCTTTTCCAGCCTCTAGAGTCCC

	AAGTCCTTGGCTCTGGGCCCCTCCCTCAAGCTTCAAAGCCACAG

	AAGCTTCTAATCTCTCTCCCTTCCCCTCTGACCTCTGCTCCCAT

	CCTCATACCCTGTCCCCTCACTCTGACCCTCCTGCCTCCCTCTT

	TCCCTTATAAAGACCCTGCATGGGGCCACGGAGATAATCCAGGG

	TAATCGCCCCTCTTCCAGCCCTTAACTCCATCCCATCTGCAAAA

	TCCCTGTCACCCCATAATGGACCTACagatctCCTAGAGTTAAC

	ACTGGCCGTCGTTTTACCGGTCCGTAGTCAGGTTTAGTTCGTCC

	GGCGGCGCCAGAAATCCGCGCGGTGGTTTTTGGGGGTCGGGGGT

	GTTTGGCAGCCACAGACGCCCGGTGTTCGTGTCGCGCCAGTACA

	TGCGGTCCATGCCCAGGCCATCCAAAAACCATGGGTCTGTCTGC

	TCAGTCCAGTCGTGGACTGACCCCACGCAACGCCCAAAATAATA

	ACCCCCACGAACCATAAACCATTCCCCATGGGGGACCCCGTCCC

	TAACCCACGGGGCCCGTGGCTATGGCAGGCCTGCCGCCCGACGT

	TGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGT

	GGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCT

	CGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGC

	GTTTATGAACAAACGACCCAACACCCGTGCGTTTTATTCTGTCT

	TTTTATTGCCGACATAGCGCGGGTTCCTTCCGGTATTGTCTCCT

	TCCGTGTTTCAGTTAGCCTCCCCCATCTCCCGTGCAAACGTGCG

	CGCCAGGTCGCAGATCGTCGGTATGGAGCCTGGGGTGGTGACGT

	GGGTCTGGATCATCCCGGAGGTAAGTTGCAGCAGGGCGTCCCGG

	CAGCCGGCGGGCGATTGGTCGTAATCCAGGATAAAGACGTGCAT

	GGGACGGAGGCGTTTGGCCAAGACGTCCAAGGCCCAGGCAAACA

	CGTTGTACAGGTCGCCGTTGGGGGCCAGCAACTCGGGGGCCCGA

	AACAGGGTAAATAACGTGTCCCCGATATGGGGTCGTGGGCCCGC

	GTTGCTCTGGGGCTCGGCACCCTGGGGCGGCACGGCCGTCCCCG

	AAAGCTGTCCCCAATCCTCCCGCCACGACCCGCCGCCCTGCAGA

	TACCGCACCGTATTGGCAAGCAGCCCGTAAACGCGGCGAATCGC

	GGTCAGCATAGCCAGGTCAAGCCGCTCGCCGGGGCGCTGGCGTT

	TGGCCAGGCGGTCGATGTGTCTGTCCTCCGGAAGGGCCCCCAAC

	ACGATGTTTGTGCCGGGCAAGGTCGGCGGGATGAGGGCCACGAA

	CGCCAGCACGGCCTGGGGGGTCATGCTGCCCATAAGGTATCGCG

	CGGCCGGGTAGCACAGGAGGGCGGCGATGGGATGGCGGTCGAAG

	ATGAGGGTGAGGGCCGGGGGCGGGGCATGTGAGCTCCCAGCCTC

	CCCCCCGATATGAGGAGCCAGAACGGCGTCGGTCACGGTATAAG

	GCATGCCCATTGTTATCTGGGCGCTTGTCATTACCACCGCCGCG

	TCCCCGGCCGATATCTCACCCTGGTCAAGGCGGTGTTGTGTGGT

	GTAGATGTTCGCGATTGTCTCGGAAGCCCCCAGCACCCGCCAGT

	AAGTCATCGGCTCGGGTACGTAGACGATATCGTCGCGCGAACCC

	AGGGCCACCAGCAGTTGCGTGGTGGTGGTTTTCCCCATCCCGTG

	GGGACCGTCTATATAAACCCGCAGTAGCGTGGGCATTTTCTGCT

	CCGGGCGGACTTCCGTGGCTTCTTGCTGCCGGCGAGGGCGCAAC

	GCCGTACGTCGGTTGCTATGGCCGCGAGAACGCGCAGCCTGGTC

	GAACGCAGACGCGTGCTGATGGCCGGGGTACGAAGCCATACGCG

	CTTCTACAAGGCGCTGGCCGAAGAGGTGCGGGAGTTTCACGCCA

	CCAAGATGTGCGGCACGCTGTTGACGCTGTTAAGCGGGTCGCTG

	CAGGGTCGCTCGGTGTTCGAGGCCACACGCGTCACCTTAATATG

	CGAAGTGGACCTGGGACCGCGCCGCCCCGACTGCATCTGCGTGT

	TCCAATTCGCCAATGACAAGACGCTGGGCGGGGTTTGCTCGACA

	TTGGGTGGAAACATTCCAGGCCTGGGTGGAGAGGCTTTTTGCTT

	CCTCTTGCAAAACCACACTGCTCGACATTGGGTGGAAACATTCC

	AGGCCTGGGTGGAGAGGCTTTTTGCTTCCTCTTGAAAACCACAC

	TGCTCGACTCTACGGTCCG

Seq ID No. 49 is a lambda light chain 5′ arm sequence


Seq ID	AGGGTGTGGCCAAATACAGCATGGAGTAGCCATCATAAGGAATC

No. 49	TTACACAAGCCTCCAAAATTGTGTTTCTGAAATTGGGTTTAAAG

	TACGTTTGCATTTTAAAAAGCCTGCCAGAAAATACAGAAAAATG

	TCTGTGATATGTCTCTGGCTGATAGGATTTTGCTTAGTTTTAAT

	TTTGGCTTTATAATTTTCTATAGTTATGAAAATGTTCACAAGAA

	GATATATTTCATTTTAGCTTCTAAAATAATTATAACACAGAAGT

	AATTTGTGCTTTAAAAAAATATTCAACACAGAAGTATATAAAGT

	AAAAATTGAGGAGTTCCCATCGTGGCTCAGTGATTAACAAACCC

	AACTAGTATCCATGAGGATATGGATTTGATCCCTGGCCTTGCTC

	AGTGGGTTGAGGATCCAGTGTTGCTGTGAGCTGTGGTGTAGGTT

	GCAGACACAGCACTCTGGCGTTGCTGTGACTCTGGCGTAGGCCG

	GCAGCTACAGCTCCATTTGGACCCTTAGCCTGGGAACCTCCATA

	TGCCTGAGATACGGCCCTAAAAAGTCAAAAGCCAAAAAAATAGT

	AAAAATTGAGTGTTTCTACTTACCACCCCTGCCCACATCTTATG

	CTAAAACCCGTTCTCCAGAGACAAACATCGTCAGGTGGGTCTAT

	ATATTTCCAGCCCTCCTCCTGTGTGTGTATGTCCGTAAAACACA

	CACACACACACACACACGCACACACACACACACGTATCTAATTA

	GCATTGGTATTAGTTTTTCAAAAGGGAGGTCATGCTCTACCTTT

	TAGGCGGCAAATAGATTATTTAAACAAATCTGTTGACATTTTCT

	ATATCAACCCATAAGATCTCCCATGTTCTTGGAAAGGCTTTGTA

	AGACATCAACATCTGGGTAAACCAGCATGGTTTTTAGGGGGTTG

	TGTGGATTTTTTTCATATTTTTTAGGGCACACCTGCAGCATATG

	GAGGTTCCCAGGCTAGGGGTTGAATCAGAGCTGTAGCTGCCGGC

	CTACACCACAGCCACAGCAACGCCAGATCCTTAACCCACTGAGA

	AAGGCCAGGGATTGAACCTGCATCCTCATGGATGCTGGTCAGAT

	TTATTTCTGCTGAGCCACAACAGGAACTCCCTGAACCAGAATGC

	TTTTAACCATTCCACTTTGCATGGACATTTAGATTGTTTCCATT

	TAAAAATACAAATTACAAGGAGTTCCCGTCGTGGCTCAGTGGTA

	ACGAATTGGACTAGGAACCATGAGGTTTCGGGTTCGATCCCTGG

	CCTTGCTCGGTGGGTTAAGGATCCAGCATTGATGTGAGATATGG

	TGTAGGTCGCAGACGTGGCTCGGATCCCACGTTGCTGTGGCTCT

	GGCGTAGGCCGGCAACAACAGCTCCGATTCGACCCCTAGCCTGG

	GAACCTCCATGTGCCACAGGAGCAGCCCTAGAAAAGGCAAAAAG

	ACAAAAAAATAAAAAATTAAAATGAAAAAATAAAATAAAAATAC

	AAATTACAAGAGACGGCTACAAGGAAATCCCCAAGTGTGTGCAA

	ATGCCATATATGTATAAAATGTACTAGTGTCTCCTCGCGGGAAA

	GTTGCCTAAAAGTGGGTTGGCTGGACAGAGAGGACAGGCTTTGA

	CATTCTCATAGGTAGTAGCAATGGGCTTCTCAAAATGCTGTTCC

	AGTTTACACTCACCATAGCAAATGACAGTGCCTCTTCCTCTCCA

	CCCTTGCCAATAATGTGACAGGTGGATCTTTTTCTATTTTGTGT

	ATCTGACAAGCAAAAAATGAGAACAGGAGTTCCTGTCGTGGTGC

	AGTGGAGACAAATCTGACTAGGAACCATGAAATTTCGGGTTCAA

	TCCCTGGCCTCACTCAGTAGGTAAAGGATCCAGGGTTGCAGTGA

	GCTGTGGGGTAGGTCGCAGACACAGTGCAAATTTGGCCCTGTTG

	TGGCTGTGGTGTAGGCCGGCAGCTATAGCTCCAATTGGACCCCT

	AGCCTGGGAACCTCCTTATGCCGTGGGTGAGGCCCTAAAAAAAA

	GAGTGCAAAAAAAAAAAATAAGAACAAAAATGATCATCGTTTAA

	TTCTTTATTTGATCATTGGTGAAACTTATTTTCCTTTTATATTT

	TTATTGACTGATTTTATTTCTCCTATGAATTTACCGGTCATAGT

	TTTGCCTGGGTGTTTTTACTCCGGTTTTAGTTTTGGTTGGTTGT

	ATTTTCTTAGAGAGCTATAGAAACTCTTCATCTATTTGGAATAG

	TAATTCCTCATTAAGTATTTGTGCTGCAAAAAATTTTCCCTGAT

	CTGTTTTATGCTTTTGTTTGTGGGGTCTTTCACGAGAAAGCCTT

	TTTAGTTTTTACACCTCAGCTTGGTTGTTTTTCTTGATTGTGTC

	TGTAATCTGCGGCCAACATAGGAAACACATTTTTACTTTAGTGT

	TTTTTTCCTATTTTCTTCAAGTACGTCCATTGTTTTGGTGTCTG

	ATTTTACTTTGCCTGGGGTTTGTTTTTGTGTGGCAGGAATATAA

	ACTTATGTATTTTCCAAATGGAGAGCCAATGGTTGTATATTTGT

	TGAATTCAAATGCAACTTTATCAAACACCAAATCATCGATTTAT

	CACAACTCTTCTCTGGTTTATTGATCTAATGATCAATTCCTGTT

	CCACGCTGTTTTAATTATTTTAGCTTTGTGGATTTTGGTGCCTG

	GTAGAGAACAAAGCCTCCATTATTTTCATTCAAAATAGTCCCGT

	CTATTATCTGCCATTGTTGTAGTATTAGACTTTAAAATCAATTT

	ACTGATTTTCAAAAGTTATTCCTTTGGTGATGTGGAATACTTTA

	TACTTCATAAGGTACATGGATTCATTTGTGGGGAATTGATGTCT

	TTGCTATTGTGGCCATTTGTCAAGTTGTGTAATATTTTACCCAT

	GCCAACTTTGCATATTGTATGTGAGTTTATTCCCAGGGTTTTTA

	ATAGGATGTTTATTGAAGTTGTCAGTGTTTCCACAATTTCATCG

	CCTCAGTGCTTACTGTTTGCATAAAAGGAAACCTACTCACTTTT

	GCCTATTGCTCTTGTATTCAATCATTTTAGTTAACTCTTGTGTT

	AATTTTGAGAGTTTTTCAGCTGACTGTCTGGGGTTTTCTTTAAT

	AGACTAGCCCTTTGTCTGTAAAGAATAATTTTATCGAATTTTTC

	TTAACACTCACACTCTCCCCACCCCCACCCCCGCTCATCTCCTT

	TCATTGGGTCAAATCTGTAGAATACAATAAAAGTAAGAGTGGGA

	ACCTTAGCCTTTAAGTCGATTTTGCCTTTAAATGTGAATGTTGC

	TATGTTTCGGGACATTCTCTTTATCAAGTTGCGGATGTTTCCTT

	AGATAATTAACTTAATAAAAGACTGGATGTTTGCTTTCTTCAAA

	TCAGAATTGTGTTGAATTTATATTGCTATTCTGTTTAATTTTGT

	TTCAAAAAATTTACATGCACACCTTAAAGATAACCATGACCAAA

	TAGTCCTCCTGCTGAGAGAAAATGTTGGCCCCAATGCCACAGGT

	TACCTCCCGACTCAGATAAACTACAATGGGAGATAAAATCAGAT

	TTGGCAAAGCCTGTGGATTCTTGCCATAACTCTCAGAGCATGAC

	TTGGGTGTTTTTTCCTTTTCTAAGTATTTTAATGGTATTTTTGT

	GTTACAATAGGAAATCTAGGACACAGAGAGTGATTCAATGAGGG

	GAACGCATTCTGGGATGACTCTAGGCCTCTGGTTTGGGGAGAGC

	TCTATTGAAGTAAAGACAATGAGAGGAAGCAAGTTTGCAGGGAA

	CTGTGAGGAATTTAGATGGGGAATGTTGGGTTTGAGGTTTCTAT

	AGGGCACGCAAGCAGAGATGCACTCAGGAGGAAGAAGGAGCATA

	AATCTAGAGGCAAAAAGAGAGGTCAGGACTGGAAATAGAGATGC

	GAGACACCAGGGTGGCAGTCAGAGAGCACAGTGTGGGTCAGAAG

	ACAGTGGAAGAACACAAGGGACAGAGAGGGATCTCCAACTTCAC

	TGGGATGAGGGCCTTGTTGGCCTTGACCTGAGAGATTTCCAGGA

	GTTGAGGGTGGGAAGGAG

Seq. ID No. 50 is a lambda 3′ arm sequence


Seq. ID	GGGAAGGTATCTCCCAGGAAACTGGCCAGGACACATTGGTCC

No. 50	TCCGCCCTCCCCTTCCTCCCACTCCTCCTCCAGACAGGACTG

	TGCCCACCCCCTGCCACCTTTCTGGCCAGAACTGTCCATGGC

	AGGTGACCTTCACATGAGCCCTTCCTCCCTGCCTGCCCTAGT

	GGGACCCTCCATACCTCCCCCTGGACCCCGTTGTCCTTTCTT

	TCCAGTGTGGCCCTGAGCATAACTGATGCCATCATGGGCTGC

	TGACCCACCCGGGACTGTGTTGTGCAGTGAGTCACTTCTCTG

	TCATCAGGGCTTTGTAATTGATAGATAGTGTTTCATCATCAT

	TAGGACCGGGTGGCCTCTATGCTCTGTTAGTCTCCAAACACT

	GATGAAAACCTTCGTTGGCATAGTCCCAGCTTCCTGTTGCCC

	ATCCATAAATCTTGACTTAGGGATGCACATCCTGTCTCCAAG

	CAACCACCCCTCCCCTAGGCTAACTATAAAACTGTCCCAATG

	GCCCTTGTGTGGTGCAGAGTTCATGCTTCCAGATCATTTCTC

	TGCTAGATCCATATCTCACCTTGTAAGTCATCCTATAATAAA

	CTGATCCATTGATTATTTGCTTCTGTTTTTTCCATCTCAAAA

	CAGCTTCTCAGTTCAGTTCGAATTTTTTATTCCCTCCATCCA

	CCCATACTTTCCTCAGCCTGGGGAACCCTTGCCCCCAGTCCC

	ATGCCCTTCCTCCCTCTCTGCCCAGCTCAGCACCTGCCCACC

	CTCACCCTTCCTGTCACTCCCTAGGACTGGACCATCCACTGG

	GGCCAGGACACTCCAGCAGCCTTGGCTTCATGGGCTCTGAAA

	TCCATGGCCCATCTCTATTCCTCACTGGATGGCAGGTTCAGA

	GATGTGAAAGGTCTAGGAGGAAGCCAGGAAGGAAACTGTTGC

	ATGAAAGGCCGGCCTGATGGTTCAGTACTTAAATAATATGAG

	CTCTGAGCTCCCCAGGAACCAAAGCATGGAGGGAGTATGTGC

	CTCAGAATCTCTCTGAGATTCAGCAAAGCCTTTGCTAGAGGG

	AAAATAGTGGCTCAACCTTGAGGGCCAGCATCTTGCACCACA

	GTTAAAAGTGGGTATTTGTTTTACCTGAGGCCTCAGCATTAT

	GGGAACCGGGCTCTGACACAAACACAGGTGCAGCCCGGCAGC

	CTCAGAACACAGCAACGACCACAAGCTGGGACAGCTGCCCCT

	GAACGGGGAGTCCACCATGCTTCTGTCTCGGGTACCACCAGG

	TCACCATCCCTGGGGGAGGTAGTTCCATAGCAGTAGTCCCCT

	GATTTCGCCCCTCGGGCGTGTAGCCAGGCAAGCTCCTGCCTC

	TGGACCCAGGGTGGACCCTTGCTCCCCACTACCCTGCACATG

	CCAGACAGTCAAGACCACTCCCACCTCTGTCTGAGGCCCCCT

	TGGGTGTCCCAGGGCCCCCGAGCTGTCCTCTACTCATGGTTC

	TTCCACCTGGGTACAAAAGAGGCGAGGGACACTTTTCTCAGG

	TTTGCGGCTCAGAAAGGTACCTTCCTAGGGTTTGTCCACTGG

	GAGTCACCTCCCTTGCATCTCAATGTCAGTGGGGAAAACTGG

	GTCCCATGGGGGGATTAGTGCCACTGTGAGGCCCCTGAAGTC

	TGGGGCCTCTAGACACTATGATGATGAGGGATGTGGTGAAAA

	ACCCCACCCCAGCCCTTCTTGCCGGGACCCTGGGCTGTGGCT

	CCCCCATTGCACTTGGGGTCAGAGGGGTGGATGGTGGCTATG

	GTCAGGCATGTTTCCCATGAGCTGGGGGCACCCTGGGTGACT

	TTCTCCTGTGAATCCTGAATTAGCAGCTATAACAAATTGCCC

	AAACTCTTAGGCTTAAAACAACACACATTTATTCCTCTGGGT

	CCCAGGGTCAGAAGTCCAAAATGAGTCCTATAGGCTAAATTT

	GAGGTGTCTCTGGGTTGAGCTCCTCCTGGAAGCCTTTTCCAG

	CCTCTAGAGTCCCAAGTCCTTGGCTCTGGGCCCCTCCCTCAA

	GCTTCAAAGCCACAGAAGCTTCTAATCTCTCTCCCTTCCCCT

	CTGACCTCTGCTCCCATCCTCATACCCTGTCCCCTCACTCTG

	ACCCTCCTGCCTCCCTCTTTCCCTTATAAAGACCCTGCATGG

	GGCCACGGAGATAATCCAGGGTAATCGCCCCTCTTCCAGCCC

	TTAACTCCATCCCATCTGCAAAATCCCTGTCACCCCATAATG

	GACCTAC

In a second strategy, the targeting strategy utilizes a vector pair. One targeting vector is designed to target upstream of J1. See FIG. 5. This targeting vector utilizes a selectable marker that can be selected for or against. Any combination of positive and negative selectable markers described herein or known in the art can be used. A fusion gene composed of the coding region of Herpes simplex thymidine kinase (TK) and the Tn5 aminoglycoside phosphotransferase (Neo resistance) genes is used. This fusion gene is flanked by recognition sites for any site specific recombinase (SSRRS) described herein or known in the art, such as lox sites. Upon isolation of targeted cells through the use of G418 selection, Cre is supplied in trans to delete the marker gene (See FIG. 5). Cells that have deleted the marker gene are selected by addition of any drug known in the art that can be metabolized by TK into a toxic product, such as ganciclovir. The resulting genotype is then targeted with a second vector. The second targeting vector (FIG. 6) is designed to target downstream of last C and uses a positive/negative selection system that is flanked on only one side by a specific recombination site (lox). The recombination site is placed distally in relation to the first targeting event. Upon isolation of the targeted genotype, Cre is again supplied in trans to mediate deletion from recombination site provided in the first targeting event to the recombination site delivered in the second targeting event. The entire J to C cluster region will be removed. The appropriate genotype is again selected by administration of ganciclovir.
Two vector pairs, i.e., lambda targeting constructs, were designed and built to target the first and last J/C regions and to include site-specific recombination sites. The first vector pair was composed of Seq ID No. 44 (step 1 vector) and Seq ID No. 45 (step 2 vector). The second vector pair was composed of Seq ID No. 46 (step 2 vector) and Seq ID No. 47 (step 1 vector).
Overview of Seq ID No. 44 (upstream vector, step 1, double lox):
Feature Map
CDS (3 total)

- NEO (+STOP) CDS
  - Start: 3311 End: 4114 (Complementary)
- TK CDS (from VEC1198)
  - Start: 4118 End: 5251 (Complementary)
- AP(R)
  - Start: 11732 End: 12589 (Complementary)
  - bla gene-Ap(r) determinant

Enhancer (1 total)

- CMV Enhancer
  - Start: 5779 End: 6199 (Complementary)

Misc. Binding Site (2 total)

- Left Homology Arm
  - Start: 238 End: 2978
- Right Homology Arm
  - Start: 6269 End: 10600

Misc. Feature (5 total)

- loxP-1
  - Start: 3006 End: 3039
- HSVTK-polyA
  - Start: 3046 End: 3304 (Complementary)
- loxP-2
  - Start: 6212 End: 6245

Promoter Eukaryotic (1 total)

- Mus-PGK Promoter (correct)
  - Start: 5264 End: 5772 (Complementary)

Replication Origin (2 total)

- Replication Origin
  - Start: 10921 End: 11509 (Complementary)
    Overview of Seq ID No. 45 (Downstream vector, step 2, single lox
    Feature Map

CDS (3 total)

- NEO (+STOP) CDS
  - Start: 3115 End: 3918 (Complementary)
- TK CDS (from VEC1198)
  - Start: 3922 End: 5055 (Complementary)
- AP(R)
  - Start: 11322 End: 12179 (Complementary)
  - bla gene-Ap(r) determinant

Enhancer (1 total)

- CMV Enhancer
  - Start: 5583 End: 6003 (Complementary)

Misc. Binding Site (2 total)

- Left Homology Arm
  - Start: 222 End: 2774
- Right Homology Arm
  - Start: 6112 End: 10226

Misc. Feature (4 total)

- HSVTK-polyA
  - Start: 2850 End: 3108 (Complementary)
- loxP-2
  - Start: 6016 End: 6049

Promoter Eukaryotic (1 total)

- Mus-PGK Promoter (correct)
  - Start: 5068 End: 5576 (Complementary)

Replication Origin (2 total)

- ORI
  - Start: 10511 End: 10511
  - RNaseH cleavage point
- Replication Origin
  - Start: 10511 End: 11099 (Complementary)
    Overview of Seq ID No. 46 (upstream vector alternative, step 2, single lox)
    Feature Map

CDS (3 total)

- NEO (+STOP) CDS
  - Start: 3311 End: 4114 (Complementary)
- TK CDS (from VEC1198)
  - Start: 4118 End: 5251 (Complementary)
- AP(R)
  - Start: 11698 End: 12555 (Complementary)
  - bla gene-Ap(r) determinant

Enhancer (1 total)

- CMV Enhancer
  - Start: 5779 End: 6199 (Complementary)

Misc. Binding Site (2 total)

- Left Homology Arm
  - Start: 238 End: 2978
- Right Homology Arm
  - Start: 6235 End: 10566

Misc. Feature (4 total)

- loxP-1
  - Start: 3006 End: 3039
- HSVTK-polyA
  - Start: 3046 End: 3304 (Complementary)

Promoter Eukaryotic (1 total)

- Mus-PGK Promoter (correct)
  - Start: 5264 End: 5772 (Complementary)

Replication Origin (2 total)

- ORI
  - Start: 10887 End: 10887
  - RNaseH cleavage point
- Replication Origin
  - Start: 10887 End: 11475 (Complementary)
    Overview of Seq ID No. 47 (Downstream vector alternative, step 1, double lox)
    Feature Map

CDS (3 total)

- NEO (+STOP) CDS
  - Start: 3149 End: 3952 (Complementary)
- TK CDS (from VEC1198)
  - Start: 3956 End: 5089 (Complementary)
- AP(R)
  - Start: 11356 End: 12213 (Complementary)
  - bla gene-Ap(r) determinant

Enhancer (1 total)

- CMV Enhancer
  - Start: 5617 End: 6037 (Complementary)

Misc. Binding Site (2 total)

- Left Homology Arm
  - Start: 222 End: 2774
- Right Homology Arm
  - Start: 6146 End: 10260

Misc. Feature (5 total)

- loxP-1
  - Start: 2844 End: 2877
- HSVTK-polyA
  - Start: 2884 End: 3142 (Complementary)
- loxP-2
  - Start: 6050 End: 6083

Promoter Eukaryotic (1 total)

- Mus-PGK Promoter (correct)
  - Start: 5102 End: 5610 (Complementary)

Replication Origin (2 total)

- Replication Origin
  - Start: 10545 End: 11133 (Complementary)

The first vector pair is used to produce cells in which the entire J/cluster region is deleted.
The second vector pair is used to produce cells in which the entire J/C cluster region is deleted.

Example 5

Crossbreeding of Heavy Chain Single Knockout with Kappa Single Knockout Pigs

To produce pigs that have both one disrupted Ig heavy chain locus and one disrupted Ig light-chain kappa allele, single knockout animals were crossbred. The first pregnancy yielded four fetuses, two of which screened positive by both PCR and Southern for both heavy-chain and kappa targeting events (see examples 1 and 2 for primers). Fetal fibroblasts were isolated, expanded and frozen. A second pregnancy resulting from the mating of a kappa single knockout with a heavy chain single knockout produced four healthy piglets.
Fetal fibroblast cells that contain a heavy chain single knockout and a kappa chain single knockout will be used for further targeting. Such cells will be used to target the lambda locus via the methods and compositions described herein. The resulting offspring will be heterozygous knockouts for heavy chain, kappa chain and lambda chain. These animals will be further crossed with animals containing the human Ig genes as described herein and then crossbred with other single Ig knockout animals to produce porcine Ig double knockout animals with human Ig replacement genes.
This invention has been described with reference to its preferred embodiments. Variations and modifications of the invention, will be obvious to those skilled in the art from the foregoing detailed description of the invention.

Claims

1. A targeting vector comprising: (a) a first nucleotide sequence comprising at least 17 contiguous nucleic acids homologous to SEQ ID No 28 or 31; (b) a selectable marker gene; and (c) a second nucleotide sequence comprising at least 17 contiguous nucleic acids homologous to SEQ ID No 28 or 31, which does not overlap with the first nucleotide sequence.