# Patent

## Title
Ungulates with genetically modified immune systems

## 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.

## Background
This application is a continuation of pending prior application U.S. Non-provisional application Ser. No. 11/789,961, filed on Apr. 26, 2007, which is a continuation-in-part of U.S. Non-provisional application Ser. No. 11/257,817, filed on Oct. 24, 2005, which claims the benefit of U.S. Provisional Application No. 60/621,433, filed on Oct. 22, 2004. This application also claims the benefit of U.S. Provisional Patent Application No. 60/794,963, filed on Apr. 26, 2006. The contents of the foregoing U.S. patent applications are hereby incorporated by reference in their entireties.


FIELD OF THE INVENTION
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.
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 Vκ to Jκ joins; the transcriptional orientation of half of the human Vκ gene segments is opposite to that of the Jκ 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 VH and JH to distinguish them from the light-chain VL and JL), there is a third gene segment called the diversity or DH gene segment, which lies between the VH and JH gene segments. The process of recombination that generates a complete heavy-chain V region occurs in two separate stages. In the first, a DH gene segment is joined to a JH gene segment; then a VH gene segment rearranges to DJH to make a complete VH-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 Vλ gene segments is followed by four sets of Jλ gene segments each linked to a single Cλ gene. The κ light-chain locus is on chromosome 2 and the cluster of Vκ, gene segments is followed by a cluster of Jκ gene segments, and then by a single Cκ gene. The organization of the heavy-chain locus, on chromosome 14, resembles that of the κ locus, with separate clusters of VH, DH, and JH gene segments and of CH genes. 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 107 possibilities. 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 VH genes 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 VH genes of each member of the latter group belong to a single VH gene 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 JH segments, only a single JH is 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 JH gene. 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 VH, 20-30 DH and four to six JH gene segments is a major mechanism of generating the antibody repertoire in humans, species with fewer VH, DH or JH segments 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 Cγ2 constant 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 XenoMouseTM 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 VH genes, one or more DH genes, one or more JH genes, 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
tctagaagacgctggagagaggccagacttcctcgga


 



acagctcaaagagctctgtcaaagccagatcccatca


 



cacgtgggcaccaataggccatgccagcctccaaggg


 



ccgaactgggttctccacggcgcacatgaagcctgca


 



gcctggcttatcctcttccgtggtgaagaggcaggcc


 



cgggactggacgaggggctagcagggtgtggtaggca


 



ccttgcgccccccaccccggcaggaaccagagaccct


 



ggggctgagagtgagcctccaaacaggatgccccacc


 



cttcaggccacctttcaatccagctacactccacctg


 



ccattctcctctgggcacagggcccagcccctggatc


 



ttggccttggctcgacttgcacccacgcgcacacaca


 



cacttcctaacgtgctgtccgctcacccctccccagc


 



gtggtccatgggcagcacggcagtgcgcgtccggcgg


 



tagtgagtgcagaggtcccttcccctcccccaggagc


 



cccaggggtgtgtgcagatctgggggctcctgtccct


 



tacaccttcatgcccctcccctcatacccaccctcca


 



ggcgggaggcagcgagacctttgcccagggactcagc


 



caacgggcacacgggaggccagccctcagcagctggc


 



tcccaaagaggaggtgggaggtaggtccacagctgcc


 



acagagagaaaccctgacggaccccacaggggccacg


 



ccagccggaaccagctccctcgtgggtgagcaatggc


 



cagggccccgccggccaccacggctggccttgcgcca


 



gctgagaactcacgtccagtgcagggagactcaagac


 



agcctgtgcacacagcctcggatctgctcccatttca


 



agcagaaaaaggaaaccgtgcaggcagccctcagcat


 



ttcaaggattgtagcagcggccaactattcgtcggca


 



gtggccgattagaatgaccgtggagaagggcggaagg


 



gtctctcgtgggctctgcggccaacaggccctggctc


 



cacctgcccgctgccagcccgaggggcttgggccgag


 



ccaggaaccacagtgctcaccgggaccacagtgactg


 



accaaactcccggccagagcagccccaggccagccgg


 



gctctcgccctggaggactcaccatcagatgcacaag


 



ggggcgagtgtggaagagacgtgtcgcccgggccatt


 



tgggaaggcgaagggaccttccaggtggacaggaggt


 



gggacgcactccaggcaagggactgggtccccaaggc


 



ctggggaaggggtactggcttgggggttagcctggcc


 



agggaacggggagcggggcggggggctgagcagggag


 



gacctgacctcgtgggagcgaggcaagtcaggcttca


 



ggcagcagccgcacatcccagaccaggaggctgaggc


 



aggaggggcttgcagcggggcgggggcctgcctggct


 



ccgggggctcctgggggacgctggctcttgtttccgt


 



gtcccgcagcacagggccagctcgctgggcctatgct


 



taccttgatgtctggggccggggcgtcagggtcgtcg


 



tctcctcaggggagagtcccctgaggctacgctgggg


 



*ggggactatggcagctccaccaggggcctggggacc


 



aggggcctggaccaggctgcagcccggaggacgggca


 



gggctctggctctccagcatctggccctcggaaatgg


 



cagaacccctggcgggtgagcgagctgagagcgggtc


 



agacagacaggggccggccggaaaggagaagttgggg


 



gcagagcccgccaggggccaggcccaaggttctgtgt


 



gccagggcctgggtgggcacattggtgtggccatggc


 



tacttagattcgtggggccagggcatcctggtcaccg


 



tctcctcaggtgagcctggtgtctgatgtccagctag


 



gcgctggtgggccgcgggtgggcctgtctcaggctag


 



ggcaggggctgggatgtgtatttgtcaaggaggggca


 



acagggtgcagactgtgcccctggaaacttgaccact


 



ggggcaggggcgtcctggtcacgtctcctcaggtaag


 



acggccctgtgcccctctctcgcgggactggaaaagg


 



aattttccaagattccttggtctgtgtggggccctct


 



ggggcccccgggggtggctcccctcctgcccagatgg


 



ggcctcggcctgtggagcacgggctgggcacacagct


 



cgagtctagggccacagaggcccgggctcagggctct


 



gtgtggcccggcgactggcagggggctcgggtttttg


 



gacaccccctaatgggggccacagcactgtgaccatc


 



ttcacagctggggccgaggagtcgaggtcaccgtctc


 



ctcaggtgagtcctcgtcagccctctctcactctctg


 



gggggttttgctgcattttgtgggggaaagaggatgc


 



ctgggtctcaggtctaaaggtctagggccagcgccgg


 



ggcccaggaaggggccgaggggccaggctcggctcgg


 



ccaggagcagagcttccagacatctcgcctcctggcg


 



gctgcagtcaggcctttggccgggggggtctcagcac


 



caccaggcctcttggctcccgaggtccccggccccgg


 



ctgcctcaccaggcaccgtgcgcggtgggcccgggct


 



cttggtcggccaccctttcttaactgggatccgggct


 



tagttgtcgcaatgtgacaacgggctcgaaagctggg


 



gccaggggaccctagtctacgacgcctcgggtgggtg


 



tcccgcacccctccccactttcacggcactcggcgag


 



acctggggagtcaggtgttggggacactttggaggtc


 



aggaacgggagctggggagagggctctgtcagcgggg


 



tccagagatgggccgccctccaaggacgccctgcgcg


 



gggacaagggcttcttggcctggcctggccgcttcac


 



ttgggcgtcagggggggcttcccggggcaggcggtca


 



gtcgaggcgggttggaattctgagtctgggttcgggg


 



ttcggggttcggccttcatgaacagacagcccaggcg


 



ggccgttgtttggcccctgggggcctggttggaatgc


 



gaggtctcgggaagtcaggagggagcctggccagcag


 



agggttcccagccctgcggccgagggacctggagacg


 



ggcagggcattggccgtcgcagggccaggccacaccc


 



cccaGGTTTTTGTggggcgagcctggagattgcacCA


 



CTGTGATTACTATGCTATGGATCTCTGGGGCCCAGGC


 



GTTGAAGTCGTCGTGTCCTCAGgtaagaacggccctc


 



cagggcctttaatttctgctctcgtctgtgggctttt


 



ctgactctgatcctcgggaggcgtctgtgcccccccc


 



ggggatgaggccggcttgccaggaggggtcagggacc


 



aggagcctgtgggaagttctgacgggggctgcaggcg


 



ggaagggccccaccggggggcgagccccaggccgctg


 



ggcggcaggagacccgtgagagtgcgccttgaggagg


 



gtgtctgcggaaccacgaacgcccgccgggaagggct


 



tgctgcaatgcggtcttcagacgggaggcgtcttctg


 



ccctcaccgtctttcaagcccttgtgggtctgaaaga


 



gccatgtcggagagagaagggacaggcctgtcccgac


 



ctggccgagagcgggcagccccgggggagagcggggc


 



gatcggcctgggctctgtgaggccaggtccaagggag


 



gacgtgtggtcctcgtgacaggtgcacttgcgaaacc


 



ttagaagacggggtatgttggaagcggctcctgatgt


 



ttaagaaaagggagactgtaaagtgagcagagtcctc


 



aagtgtgttaaggttttaaaggtcaaagtgttttaaa


 



cctttgtgactgcagttagcaagcgtgcggggagtga


 



atggggtgccagggtggccgagaggcagtacgagggc


 



cgtgccgtcctctaattcagggcttagttttgcagaa


 



taaagtcggcctgttttctaaaagcattggtggtgct


 



gagctggtggaggaggccgcgggcagccctggccacc


 



tgcagcaggtggcaggaagcaggtcggccaagaggct


 



attttaggaagccagaaaacacggtcgatgaatttat


 



agcttctggtttccaggaggtggttgggcatggcttt


 



gcgcagcgccacagaaccgaaagtgcccactgagaaa


 



aaacaactcctgcttaatttgcatttttctaaaagaa


 



gaaacagaggctgacggaaactggaaagttcctgttt


 



taactactcgaattgagttttcggtcttagcttatca


 



actgctcacttagattcattttcaaagtaaacgttta


 



agagccgaggcattcctatcctcttctaaggcgttat


 



tcctggaggctcattcaccgccagcacctccgctgcc


 



tgcaggcattgctgtcaccgtcaccgtgacggcgcgc


 



acgattttcagttggcccgcttcccctcgtgattagg


 



acagacgcgggcactctggcccagccgtcttggctca


 



gtatctgcaggcgtccgtctcgggacggagctcaggg


 



gaagagcgtgactccagttgaacgtgatagtcggtgc


 



gttgagaggagacccagtcgggtgtcgagtcagaagg


 



ggcccggggcccgaggccctgggcaggacggcccgtg


 



ccctgcatcacgggcccagcgtcctagaggcaggact


 



ctggtggagagtgtgagggtgcctggggcccctccgg


 



agctggggccgtgcggtgcaggttgggctctcggcgc


 



ggtgttggctgtttctgcgggatttggaggaattctt


 



ccagtgatgggagtcgccagtgaccgggcaccaggct


 



ggtaagagggaggccgccgtcgtggccagagcagctg


 



ggagggttcggtaaaaggctcgcccgtttcctttaat


 



gaggacttttcctggagggcatttagtctagtcggga


 



ccgttttcgactcgggaagagggatgcggaggagggc


 



atgtgcccaggagccgaaggcgccgcggggagaagcc


 



cagggctctcctgtccccacagaggcgacgccactgc


 



cgcagacagacagggcctttccctctgatgacggcaa


 



aggcgcctcggctcttgcggggtgctgggggggagtc


 



gccccgaagccgctcacccagaggcctgaggggtgag


 



actgaccgatgcctcttggccgggcctggggccggac


 



cgagggggactccgtggaggcagggcgatggtggctg


 



cgggagggaaccgaccctgggccgagcccggcttggc


 



gattcccgggcgagggccctcagccgaggcgagtggg


 



tccggcggaaccaccctttctggccagcgccacaggg


 



ctctcgggactgtccggggcgacgctgggctgcccgt


 



ggcaggccTGGGCTGACCTGGACTTCACGAGACAGAA


 



CAGGGCTTTCAGGGCTGAGCTGAGCCAGGTTTAGCGA


 



GGCCAAGTGGGGCTGAACCAGGCTCAACTGGCCTGAG


 



CTGGGTTGAGCTGGGCTGACCTGGGCTGAGCTGAGCT


 



GGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGG


 



ACTGGCTGAGCTGAGCTGGGTTGAGCTGAGCTGAGCT


 



GGCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGG


 



GTTGAGCTGGGTTGAGCTGGGTTGATCTGAGCTGAGC


 



TGGGCTGAGCTGAGCTAGGCTGGGGTGAGCTGGGCTG


 



AGCTGGTTTGAGTTGGGTTGAGCTGAGCTGAGCTGGG


 



CTGTGCTGGCTGAGCTAGGCTGAGCTAGGCTAGGTTG


 



AGCTGGGCTGGGCTGAGCTGAGCTAGGCTGGGCTGAT


 



TTGGGCTGAGCTGAGCTGAGCTAGGCTGCGTTGAGCT


 



GGCTGGGCTGGATTGAGCTGGCTGAGCTGGCTGAGCT


 



GGGCTGAGCTGGCCTGGGTTGAGCTGAGCTGGACTGG


 



TTTGAGCTGGGTCGATCTGGGTTGAGCTGTCCTGGGT


 



TGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTG


 



GGCTCAGCAGAGCTGGGTTGGGCTGAGCTGGGTTGAG


 



CTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCT


 



GAGCTGAGCTGGGCTGAGCTGGCCTGTGTTGAGCTGG


 



GCTGGGTTGAGCTGGGCTGAGCTGGATTGAGCTGGGT


 



TGAGCTGAGCTGGGCTGGGCTGTGCTGACTGAGCTGG


 



GCTGAGCTAGGCTGGGGTGAGCTGGGCTGAGCTGATC


 



CGAGCTAGGCTGGGCTGGTTTGGGCTGAGCTGAGCTG


 



AGCTAGGCTGGATTGATCTGGCTGAGCTGGGTTGAGC


 



TGAGCTGGGCTGAGCTGGTCTGAGCTGGCCTGGGTCG


 



AGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGG


 



CTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCT


 



GAGCTGGGTTGAGCTGGGCTGAGCTGAGGGCTGGGGT


 



GAGCTGGGCTGAACTAGCCTAGCTAGGTTGGGCTGAG


 



CTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTAGGCT


 



GCATTGAGCAGGCTGAGCTGGGCTGAGCAGGCCTGGG


 



GTGAGCTGGGCTAGGTGGAGCTGAGCTGGGTCGAGCT


 



GAGTTGGGCTGAGCTGGCCTGGGTTGAGGTAGGCTGA


 



GCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCT


 



GGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGG


 



GTTGAGCTGGGCTCGGTTGAGCTGGGCTGAGCTGAGC


 



CGACCTAGGCTGGGATGAGCTGGGCTGATTTGGGCTG


 



AGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGC


 



TGGGCCTGGAGCCTGGCCTGGGGTGAGCTGGGCTGAG


 



CTGCGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTG


 



GTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGG


 



ATGAGCTGGGCCGGTTTGGGCTGAGCTGAGCTGAGCT


 



AGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGC


 



CTGGGGTGAGCTGGGCTGAGCTAAGCTGAGCTGGGCT


 



GGTTTGGGCTGAGCTGGCTGAGCTGGGTCCTGCTGAG


 



CTGGGCTGAGCTGACCAGGGGTGAGCTGGGCTGAGTT


 



AGGCTGGGCTCAGCTAGGCTGGGTTGATCTGGCAGGG


 



CTGGTTTGCGCTGGGTCAAGCTCCCGGGAGATGGCCT


 



GGGATGAGCTGGGCTGGTTTGGGCTGAGCTGAGCTGA


 



GCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCT


 



GAGCTGGCCTGGGGTGAGCTGGGCTGGGTGGAGCTGA


 



GCTGGGCTGAACTGGGCTAAGCTGGCTGAGCTGGATC


 



GAGCTGAGCTGGGCTGAGCTGGCCTGGGGTTAGCTGG


 



GCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCT


 



GGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGG


 



CCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAGGC


 



TGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAGGCTG


 



CATTGAGCTGGCTGGGATGGATTGAGCTGGCTGAGCT


 



GGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGT


 



TGAGCTGGGCTGGGTTGAGCTGAGCTGGGCTGAGCTG


 



GGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAG


 



CTGGGGTGAGCTGGGCTGAGCAGAGCTGGGTTGAGCT


 



GAGCTGGGTTGAGCTGGGCTCGAGCAGAGCTGGGTTG


 



AGCTGAGCTGGGTTGAGCTGGGCTCAGCAGAGCTGGG


 



TTGAGCTGAGCTGGGTTGAGCTGGGCTGAGCTAGCTG


 



GGCTCAGCTAGGCTGGGTTGAGCTGAGCTGGGCTGAA


 



CTGGGCTGAGCTGGGCTGAACTGGGCTGAGCTGGGCT


 



GAGCTGGGCTGAGCAGAGCTGGGCTGAGCAGAGCTGG


 



GTTGGTCTGAGCTGGGTTGAGCTGGGCTGAGCTGGGC


 



TGAGCAGAGTTGGGTTGAGCTGAGCTGGGTTCAGCTG


 



GGCTGAGCTAGGCTGGGTTGAGCTGGGTTGAGTTGGG


 



CTGAGCTGGGCTGGGTTGAGCGGAGCTGGGCTGAACT


 



GGGCTGAGCTGGGCTGAGCGGAACTGGGTTGATCTGA


 



ATTGAGCTGGGCTGAGCCGGGCTGAGCCGGGCTGAGC


 



TGGGCTAGGTTGAGCTTGGGTGAGCTTGCCTCAGCTG


 



GTCTGAGCTAGGTTGGGTGGAGCTAGGCTGGATTGAG


 



CTGGGCTGAGCTGAGCTGATCTGGCCTCAGCTGGGCT


 



GAGGTAGGCTGAACTGGGCTGTGCTGGGCTGAGCTGA


 



GCTGAGCCAGTTTGAGCTGGGTTGAGCTGGGCTGAGC


 



TGGGCTGTGTTGATCTTTCCTGAACTGGGCTGAGCTG


 



GGCTGAGCTGGCCTAGCTGGATTGAACGGGGGTAAGC


 



TGGGCCAGGCTGGACTGGGCTGAGCTGAGCTAGGCTG


 



AGCTGAGTTGAATTGGGTTAAGCTGGGCTGAGATGGG


 



CTGAGCTGGGCTGAGCTGGGTTGAGCCAGGTCGGACT


 



GGGTTACCCTGGGCCACACTGGGCTGAGCTGGGCGGA


 



GCTCGattaacctggtcaggctgagtcgggtccagca


 



gacatgcgctggccaggctggcttgacctggacacgt


 



tcgatgagctgccttgggatggttcacctcagctgag


 



ccaggtggctccagctgggctgagctggtgaccctgg


 



gtgacctcggtgaccaggttgtcctgagtccgggcca


 



agccgaggctgcatcagactcgccagacccaaggcct


 



gggccccggctggcaagccaggggcggtgaaggctgg


 



gctggcaggactgtcccggaaggaggtgcacgtggag


 



ccgcccggaccccgaccggcaggacctggaaagacgc


 



ctctcactcccctttctcttctgtcccctctcgggtc


 



ctcagAGAGCCAGTCTGCCCCGAATCTCTACCCCCTC


 



GTCTCCTGCGTCAGCCCCCCGTCCGATGAGAGCCTGG


 



TGGCCCTGGGCTGCCTGGCCCGGGACTTCCTGCCCAG


 



CTCCGTCACCTTCTCCTGGAACTACAAGAACAGCAGC


 



AAGGTCAGCAGCCAGAACATCCAGGACTTCCCGTCCG


 



TCCTGAGAGGCGGCAAGTACTTGGCCTCCTCCCGGGT


 



GCTCCTACCCTCTGTGAGCATCCCCCAGGACCCAGAG


 



GCCTTCCTGGTGTGCGAGGTCCAGCACCCCAGTGGCA


 



CCAAGTCCGTGTCCATCTCTGGGCCAGgtgagctggg


 



ctccccctgtggctgtggcgggggcggggccgggtgc


 



cgccggcacagtgacgccccgttcctgcctgcagTCG


 



TAGAGGAGCAGCCCCCCGTCTTGAACATCTTCGTCCC


 



CACCCGGGAGTCCTTCTCCAGTACTCCCCAGCGCACG


 



TCCAAGCTCATCTGCCAGGCCTCAGACTTCAGCCCCA


 



AGCAGATCTCCATGGCCTGGTTCCGTGATGGGAAACG


 



GGTGGTGTCTGGCGTCAGCACAGGCCCCGTGGAGACC


 



CTACAGTCCAGTCCGGTGACCTACAGGCTCCACAGCA


 



TGCTGACCGTCACGGAGTCCGAGTGGCTCAGCCAGAG


 



CGTCTTCACCTGCCAGGTGGAGCACAAAGGGCTGAAC


 



TACGAGAAGAACGCGTCCTCTCTGTGCACCTCCAgtg


 



agtgcagcccctcgggccgggcggcggggcggcggga


 



gccacacacacaccagctgctccctgagccttggctt


 



ccgggagtggccaaggcggggaggggctgtgcagggc


 



agctggagggcactgtcagctggggcccagcaccccc


 



tcaccccggcagggcccgggctccgaggggccccgca


 



gtcgcaggccctgctcttgggggaagccctacttggc


 



cccttcagggcgctgacgctccccccacccacccccg


 



cctagATCCCAACTCTCCCATCACCGTCTTCGCCATC


 



GCCCCCTCCTTCGCTGGCATCTTCCTCACCAAGTCGG


 



CCAAGCTTTCCTGCGTGGTCACGGGCCTCGTCACCAG


 



GGAGAGCCTCAACATCTCCTGGACCCGCCAGGACGGC


 



GAGGTTCTGAAGACCAGTATCGTCTTCTCTGAGATCT


 



ACGCCAACGGCACCTTCGGCGCCAGGGGCGAAGCCTC


 



CGTCTGCGTGGAGGACTGGGAGTCGGGCGACAGGTTC


 



ACGTGCACGGTGACCCACACGGACCTGCCCTCGCCGC


 



TGAAGCAGAGCGTCTCCAAGCCCAGAGgtaggccctg


 



ccctgcccctgcctccgcccggcctgtgccttggccg


 



ccggggcgggagccgagcctggccgaggagcgccctc


 



ggccccccgcggtcccgacccacacccctcctgctct


 



cctccccagGGATCGCCAGGCACATGCCGTCCGTGTA


 



CGTGCTGCCGCCGGCCCCGGAGGAGCTGAGCCTGCAG


 



GAGTGGGCCTCGGTCACCTGCCTGGTGAAGGGCTTCT


 



CCCCGGCGGACGTGTTCGTGCAGTGGCTGCAGAAGGG


 



GGAGCCCGTGTCCGCCGACAAGTACGTGACCAGCGCG


 



CCGGTGCCCGAGCCCGAGCCCAAGGCCCCCGCCTCCT


 



ACTTCGTGCAGAGCGTCCTGACGGTGAGCGCCAAGGA


 



CTGGAGCGACGGGGAGACCTACACCTGCGTCGTGGGC


 



CACGAGGCCCTGCCCCACACGGTGACCGAGAGGACCG


 



TGGACAAGTCCACCGGTAAACCCACCCTGTACAACGT


 



CTCCCTGGTCCTGTCCGACACGGCCAGCACCTGCTAC


 



TGACCCCCTGGCTGCCCGCCGCGGCCGGGGCCAGAGC


 



CCCCGGGCGACCATCGCTCTGTGTGGGCCTGTGTGCA


 



ACCCGACCCTGTCGGGGTGAGCGGTCGCATTTCTGAA


 



AATTAGAaataaaAGATCTCGTGCCG


 


Seq ID No. 1
TCTAgAAGACGCTGGAGAGAGGCCagACTTCCTCGGA


 



ACAGCTCAAAGAGCTCTGTCAAAGCCAGATCCCATCA


 



CACGTGGGCACCAATAGGCCATGCCAGCCTCCAAGGG


 



CCGAACTGGGTTCTCCACGGCGCACATGAAGCCTGCA


 



GCCTGGCTTATCCTCTTCCGTGGTGAAGAGGCAGGCC


 



CGGGACTGGACGAGGGGCTAGCAGGGTGTGGTAGGCA


 



CCTTGCGCCCCCCACCCCGGCAGGAACCAGAGACCCT


 



GGGGCTGAGAGTGAGCCTCCAAACAGGATGCCCCACC


 



CTTCAGGCCACCTTTCAATCCAGCTACACTCCACCTG


 



CCATTCTCCTCTGGGCACAGGGCCCAGCCCCTGGATC


 



TTGGCCTTGGCTCGACTTGCACCGACGCGCACACACA


 



CACTTCCTAACGTGCTGTCCGCTCACCCCTCCCCAGC


 



GTGGTCCATGGGCAGCACGGCAGTGCGCGTCCGGCGG


 



TAGTGAGTGCAGAGGTCCCTTCCCCTCCCCCAGGAGC


 



CCCAGGGGTGTGTGCAGATCTGGGGGCTCCTGTCCCT


 



TACACCTTCATGCCCCTCCCCTCATACCCACCCTCCA


 



GGCGGGAGGCAGCGAGACCTTTGCCCAGGGACTCAGC


 



CAACGGGCACACGGGAGGCCA GCCCTCAGCAGCTGG


 



G


 


Seq ID No. 4
GGCCAGACTTCCTCGGAACAGCTCAAAGAGCTCTGTC


 



AAAGCCAGATCCCATCACACGTGGGCACCAATAGGCC


 



ATGCCAGCCTCCAAGGGCCGAACTGGGTTCTCCACGG


 



CGCACATGAAGCCTGCAGCCTGGCTTATCCTCTTCCG


 



TGGTGAAGAGGCAGGCCCGGGACTGGACGAGGGGCTA


 



GCAGGGTGTGGTAGGCACCTTGCGCCCCCCACCCCGG


 



CAGGAACCAGAGACCCTGGGGCTGAGAGTGAGCCTCC


 



AAACAGGATGCCCCACCCTTCAGGCCACCTTTCAATC


 



CAGCTACACTCCACCTGCCATTCTCCTCTGGGCACAG


 



GGCCCAGCCCCTGGATCTTGGCCTTGGCTCGACTTGC


 



ACCCACGCGCACACACACACTTCGTAACGTGCTGTCC


 



GCTCACCCCTCCCCAGCGTGGTCCATGGGCAGCACGG


 



CAGTGCGCGTCCGGCGGTAGTGAGTGCAGAGGTCCCT


 



TCCCCTCCCCCAGGAGCCCCAGGGGTGTGTGCAGATC


 



TGGGGGCTCCTGTCCCTTACACCTTCATGCCCCTCCC


 



CTCATACCCACCCTCCAGGCGGGAGGCAGCGAGACCT


 



TTGCCCAGGGACTCAGCCAACGGGCACACGGGAGGCC


 



AGCCCTCAGCAGCTGGCTCCCAAAGAGGAGGTGGGAG


 



GTAGGTCCACAGCTGCCACAGAGAGAAACCCTGACGG


 



ACCCCACAGGGGCCACGCCAGCCGGAACCAGCTCCCT


 



CGTGGGTGAGCAATGGCCAGGGCCCCGCCGGCCACCA


 



CGGCTGGCCTTGCGCCAGCTGAGAACTCACGTCCAGT


 



GCAGGGAGACTCAAGACAGCCTGTGCACACAGCCTCG


 



GATCTGCTCCCATTTCAAGCAGAAAAAGGAAACCGTG


 



CAGGCAGCCCTCAGCATTTCAAGGATTGTAGCAGCGG


 



CCAACTATTCGTCGGCAGTGGCCGATTAGAATGACCG


 



TGGAGAAGGGCGGAAGGGTCTCTCGTGGGCTCTGCGG


 



CCAACAGGCCCTGGCTCCACCTGCCCGCTGCCAGCCC


 



GAGGGGCTTGGGCCGAGCCAGGAACCACAGTGCTCAC


 



CGGGACCACAGTGACTGACCAAACTCCCGGCCAGAGC


 



AGCCCCAGGCCAGCCGGGCTCTCGCCCTGGAGGACTC


 



ACCATCAGATGCACAAGGGGGCGAGTGTGGAAGAGAC


 



GTGTCGCCCGGGCCATTTGGGAAGGCGAAGGGACCTT


 



CCAGGTGGACAGGAGGTGGGACGCACTCCAGGCAAGG


 



GACTGGGTCCCCAAGGCCTGGGGAAGGGGTACTGGCT


 



TGGGGGTTAGCCTGGCCAGGGAACGGGGAGCGGGGCG


 



GGGGGCTGAGCAGGGAGGACCTGACCTCGTGGGAGCG


 



AGGCAAGTCAGGCTTCAGGCAGCAGCCGCACATCCCA


 



GACCAGGAGGCTGAGGCAGGAGGGGCTTGCAGCGGGG


 



CGGGGGCCTGCCTGGCTCCGGGGGCTCCTGGGGGACG


 



CTGGCTCTTGTTTCCGTGTCCCGCAGCACAGGGCCAG


 



CTCGCTGGGCCTATGCTTACCTTGATGTCTGGGGCCG


 



GGGCGTCAGGGTCGTCGTCTCCTCAGGGGAGAGTCCC


 



CTGAGGCTACGCTGGGG*GGGGACTATGGCAGCTCCA


 



CCAGGGGCCTGGGGACCAGGGGCCTGGACCAGGCTGC


 



AGCCCGGAGGACGGGCAGGGCTCTGGCTCTCCAGCAT


 



CTGGCCCTCGGAAATGGCAGAACCCCTGGCGGGTGAG


 



CGAGCTGAGAGCGGGTCAGACAGACAGGGGCCGGCCG


 



GAAAGGAGAAGTTGGGGGCAGAGCCCGCCAGGGGCCA


 



GGCCCAAGGTTCTGTGTGCCAGGGCCTGGGTGGGCAC


 



ATTGGTGTGGCCATGGCTACTTAGATTCGTGGGGCCA


 



GGGCATCCTGGTCAGCGTCTCCTCAGGTGAGCCTGGT


 



GTCTGATGTCCAGCTAGGCGCTGGTGGGCCGCGGGTG


 



GGCCTGTCTCAGGCTAGGGCAGGGGCTGGGATGTGTA


 



TTTGTCAAGGAGGGGCAACAGGGTGCAGACTGTGCCC


 



CTGGAAACTTGACCACTGGGGCAGGGGCGTCCTGGTC


 



ACGTCTCCTCAGGTAAGACGGCCCTGTGCCCCTCTCT


 



CGCGGGACTGGAAAAGGAATTTTCCAAGATTCCTTGG


 



TCTGTGTGGGGCCCTCTGGGGCCCCCGGGGGTGGCTC


 



CCCTCCTGCCCAGATGGGGCCTCGGCCTGTGGAGCAC


 



GGGCTGGGCACACAGCTCGAGTCTAGGGCCACAGAGG


 



CCCGGGCTCAGGGCTCTGTGTGGCCCGGCGACTGGCA


 



GGGGGCTCGGGTTTTTGGACACCCCCTAATGGGGGCC


 



ACAGCACTGTGACCATCTTCACAGCTGGGGCCGAGGA


 



GTCGAGGTCACCGTCTCCTCAGGTGAGTCCTCGTCAG


 



CCCTCTCTCACTCTCTGGGGGGTTTTGCTGCATTTTG


 



TGGGGGAAAGAGGATGCCTGGGTCTCAGGTCTAAAGG


 



TCTAGGGCCAGCGCCGGGGCCCAGGAAGGGGCCGAGG


 



GGCCAGGCTCGGCTCGGCCAGGAGCAGAGCTTCCAGA


 



CATCTCGCCTCCTGGCGGCTGCAGTCAGGCCTTTGGC


 



CGGGGGGGTCTCAGCACCACCAGGCCTCTTGGCTCCC


 



GAGGTCCCCGGCCCCGGCTGCCTCACCAGGCACCGTG


 



CGCGGTGGGCCCGGGCTCTTGGTCGGCCACCCTTTCT


 



TAACTGGGATCCGGGCTTAGTTGTCGCAATGTGACAA


 



CGGGCTCGAAAGCTGGGGCCAGGGGACCCTAGT*TAC


 



GACGCCTCGGGTGGGTGTCCCGCACCCCTCCCCACTT


 



TCACGGCAGTCGGCGAGACCTGGGGAGTCAGGTGTTG


 



GGGACACTTTGGAGGTCAGGAACGGGAGCTGGGGAGA


 



GGGCTCTGTCAGCGGGGTCCAGAGATGGGCCGCCCTC


 



CAAGGACGCCCTGCGCGGGGACAAGGGCTTCTTGGCC


 



TGGCCTGGCCGCTTCACTTGGGCGTCAGGGGGGGCTT


 



CCCGGGGCAGGCGGTCAGTCGAGGCGGGTTGGAATTC


 



TGAGTCTGGGTTCGGGGTTCGGGGTTCGGCCTTCATG


 



AACAGACAGCCCAGGCGGGCCGTTGTTTGGCCCCTGG


 



GGGCCTGGTTGGAATGCGAGGTCTCGGGAAGTCAGGA


 



GGGAGCCTGGCCAGCAGAGGGTTCCCAGCCCTGCGGC


 



CGAGGGACCTGGAGACGGGCAGGGCATTGGCCGTCGC


 



AGGGCCAGGCCACACCCCCCAGGTTTTTGTGGGGCGA


 



GCCTGGAGATTGCACCACTGTGATTACTATGCTATGG


 



ATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTC


 



AGGTAAGAACGGCCCTCCAGGGCCTTTAATTTCTGCT


 



CTCGTCTGTGGGCTTTTCTGACTCTGATCCTCGGGAG


 



GCGTCTGTGCCCCCCCCGGGGATGAGGCCGGCTTGCC


 



AGGAGGGGTCAGGGACCAGGAGCCTGTGGGAAGTTCT


 



GACGGGGGCTGCAGGCGGGAAGGGCCCCACCGGGGGG


 



CGAGCCCCAGGCCGCTGGGCGGCAGGAGACCCGTGAG


 



AGTGCGCCTTGAGGAGGGTGTCTGCGGAACCACGAAC


 



GCCCGCCGGGAAGGGCTTGCTGCAATGCGGTCTTCAG


 



ACGGGAGGCGTCTTCTGCCCTCACCGTCTTTCAAGCC


 



CTTGTGGGTCTGAAAGAGCCATGTCGGAGAGAGAAGG


 



GACAGGCCTGTCCCGACCTGGCCGAGAGCGGGCAGCC


 



CCGGGGGAGAGCGGGGCGATCGGCCTGGGCTCTGTGA


 



GGCCAGGTCCAAGGGAGGACGTGTGGTCCTCGTGACA


 



GGTGCACTTGCGAAACCTTAGAAGACGGGGTATGTTG


 



GAAGCGGCTCCTGATGTTTAAGAAAAGGGAGACTGTA


 



AAGTGAGCAGAGTCCTCAAGTGTGTTAAGGTTTTAAA


 



GGTCAAAGTGTTTTAAACCTTTGTGACTGCAGTTAGC


 



AAGCGTGCGGGGAGTGAATGGGGTGCCAGGGTGGCCG


 



AGAGGCAGTACGAGGGCCGTGCCGTCCTCTAATTCAG


 



GGCTTAGTTTTGCAGAATAAAGTCGGCCTGTTTTCTA


 



AAAGCATTGGTGGTGCTGAGCTGGTGGAGGAGGCCGC


 



GGGCAGCCCTGGCCACCTGCAGCAGGTGGCAGGAAGC


 



AGGTCGGCCAAGAGGCTATTTTAGGAAGCCAGAAAAC


 



ACGGTCGATGAATTTATAGCTTCTGGTTTCCAGGAGG


 



TGGTTGGGCATGGCTTTGCGCAGCGCCACAGAACCGA


 



AAGTGCCCACTGAGAAAAAACAACTCCTGCTTAATTT


 



GCATTTTTCTAAAAGAAGAAACAGAGGCTGACGGAAA


 



CTGGAAAGTTCCTGTTTTAACTACTCGAATTGAGTTT


 



TCGGTCTTAGCTTATCAACTGCTCACTTAGATTCATT


 



TTCAAAGTAAACGTTTAAGAGCCGAGGCATTCCTATC


 



CTCTTCTAAGGCGTTATTCCTGGAGGCTCATTCACCG


 



CCAGCACCTCCGCTGCCTGCAGGCATTGCTGTCACCG


 



TCACCGTGACGGCGCGCACGATTTTCAGTTGGCCCGC


 



TTCCCCTCGTGATTAGGACAGACGCGGGCACTCTGGC


 



CCAGCCGTCTTGGCTCAGTATCTGCAGGCGTCCGTCT


 



CGGGACGGAGCTCAGGGGAAGAGCGTGACTCCAGTTG


 



AACGTGATAGTCGGTGCGTTGAGAGGAGACCCAGTCG


 



GGTGTCGAGTCAGAAGGGGCCCGGGGCCCGAGGCCCT


 



GGGCAGGACGGCCCGTGCCCTGCATCACGGGCCCAGC


 



GTCCTAGAGGCAGGACTCTGGTGGAGAGTGTGAGGGT


 



GCCTGGGGCCCCTCCGGAGCTGGGGCCGTGCGGTGCA


 



GGTTGGGCTCTCGGCGCGGTGTTGGCTGTTTCTGCGG


 



GATTTGGAGGAATTCTTCCAGTGATGGGAGTCGCCAG


 



TGACCGGGCACCAGGCTGGTAAGAGGGAGGCCGCCGT


 



CGTGGCCAGAGCAGCTGGGAGGGTTCGGTAAAAGGCT


 



CGCCCGTTTCCTTTAATGAGGACTTTTCCTGGAGGGC


 



ATTTAGTCTAGTCGGGACCGTTTTCGACTCGGGAAGA


 



GGGATGCGGAGGAGGGCATGTGCCCAGGAGCCGAAGG


 



CGCCGCGGGGAGAAGCCCAGGGCTCTCCTGTCCCCAC


 



AGAGGCGACGCCACTGCCGCAGACAGACAGGGCCTTT


 



CCCTCTGATGACGGCAAAGGCGCCTCGGCTCTTGCGG


 



GGTGCTGGGGGGGAGTCGCCCCGAAGCCGCTCACCCA


 



GAGGCCTGAGGGGTGAGACTGACCGATGCCTCTTGGC


 



CGGGCCTGGGGCCGGACCGAGGGGGACTCCGTGGAGG


 



CAGGGCGATGGTGGCTGCGGGAGGGAACCGACCCTGG


 



GCCGAGCCCGGCTTGGCGATTCCCGGGCGAGGGCCCT


 



CAGCCGAGGCGAGTGGGTCCGGCGGAACCACCCTTTC


 



TGGCCAGCGCCACAGGGCTCTCGGGACTGTCCGGGGC


 



GACGCTGGGCTGCCCGTGGCAGGCCTGGGCTGACCTG


 



GACTTCACCAGACAGAACAGGGCTTTCAGGGCTGAGC


 



TGAGCCAGGTTTAGCGAGGCCAAGTGGGGCTGAACCA


 



GGCTCAACTGGCCTGAGCTGGGTTGAGCTGGGCTGAC


 



CTGGGCTGAGCTGAGCTGGGCTGGGCTGGGCTGGGCT


 



GGGCTGGGCTGGGCTGGACTGGCTGAGCTGAGCTGGG


 



TTGAGCTGAGCTGAGCTGGCCTGGGTTGAGCTGGGCT


 



GGGTTGAGCTGAGCTGGGTTGAGCTGGGTTGAGCTGG


 



GTTGATCTGAGCTGAGCTGGGCTGAGCTGAGCTAGGC


 



TGGGGTGAGCTGGGCTGAGCTGGTTTGAGTTGGGTTG


 



AGCTGAGCTGAGCTGGGCTGTGCTGGCTGAGCTAGGC


 



TGAGCTAGGCTAGGTTGAGCTGGGCTGGGCTGAGCTG


 



AGCTAGGCTGGGCTGATTTGGGCTGAGCTGAGCTGAG


 



CTAGGCTGCGTTGAGCTGGCTGGGCTGGATTGAGCTG


 



GCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGTT


 



GAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGG


 



GTTGAGCTGTCCTGGGTTGAGCTGGGCTGGGTTGAGC


 



TGAGCTGGGTTGAGCTGGGCTCAGCAGAGCTGGGTTG


 



GGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCTGGC


 



CTGGGTTGAGCTGGGCTGAGCTGAGCTGGGCTGAGCT


 



GGCCTGTGTTGAGCTGGGCTGGGTTGAGCTGGGCTGA


 



GCTGGATTGAGCTGGGTTGAGCTGAGCTGGGCTGGGC


 



TGTGCTGACTGAGCTGGGCTGAGCTAGGCTGGGGTGA


 



GCTGGGCTGAGCTGATCCGAGCTAGGCTGGGCTGGTT


 



TGGGCTGAGCTGAGCTGAGCTAGGCTGGATTGATCTG


 



GCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCTGGTC


 



TGAGCTGGCCTGGGTCGAGCTGAGCTGGACTGGTTTG


 



AGCTGGGTCGATCTGGGCTGAGCTGGCCTGGGTTGAG


 



CTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCT


 



GAGCTGAGGGCTGGGGTGAGCTGGGCTGAACTAGCCT


 



AGCTAGGTTGGGCTGAGCTGGGCTGGTTTGGGCTGAG


 



CTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTG


 



GGCTGAGCAGGCCTGGGGTGAGCTGGGCTAGGTGGAG


 



CTGAGCTGGGTCGAGCTGAGTTGGGCTGAGCTGGCCT


 



GGGTTGAGGTAGGCTGAGCTGAGCTGAGCTAGGCTGG


 



GTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAAGCT


 



GGGCCGAGCTGGCCTGGGTTGAGCTGGGCTCGGTTGA


 



GCTGGGCTGAGCTGAGCCGACCTAGGCTGGGATGAGC


 



TGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTG


 



CATTGAGCAGGCTGAGCTGGGCCTGGAGCCTGGCCTG


 



GGGTGAGCTGGGCTGAGCTGCGCTGAGCTAGGCTGGG


 



TTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAAGCTG


 



GGCCGAGCTGGCCTGGGATGAGCTGGGCCGGTTTGGG


 



CTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTG


 



AGCTGGGCTGAGCTGGCCTGGGGTGAGCTGGGCTGAG


 



CTAAGCTGAGCTGGGCTGGTTTGGGCTGAGCTGGCTG


 



AGCTGGGTCCTGCTGAGCTGGGCTGAGCTGACCAGGG


 



GTGAGCTGGGCTGAGTTAGGCTGGGCTCAGCTAGGCT


 



GGGTTGATCTGGCAGGGCTGGTTTGCGCTGGGTCAAG


 



CTCCCGGGAGATGGCCTGGGATGAGCTGGGCTGGTTT


 



GGGCTGAGCTGAGCTGAGCTGAGCTAGGCTGCATTGA


 



GCAGGCTGAGCTGGGCTGAGCTGGCCTGGGGTGAGCT


 



GGGCTGGGTGGAGCTGAGCTGGGCTGAACTGGGCTAA


 



GCTGGCTGAGCTGGATCGAGCTGAGCTGGGCTGAGCT


 



GGCCTGGGGTTAGCTGGGCTGAGCTGAGCTGAGCTAG


 



GCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTC


 



AAGCTGGGCCGAGCTGGCCTGGGTTGAGCTGGGCTGG


 



GCTGAGCTGAGCTAGGCTGGGTTGAGCTGGGCTGGGC


 



TGAGCTGAGCTAGGCTGCATTGAGCTGGCTGGGATGG


 



ATTGAGCTGGCTGAGCTGGCTGAGCTGGCTGAGCTGG


 



GCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGC


 



TGAGCTGGGCTGAGCTGGGCTCAGCAGAGCTGGGTTG


 



AGCTGAGCTGGGTTGAGCTGGGGTGAGCTGGGCTGAG


 



CAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCT


 



CGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTG


 



GGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAG


 



CTGGGCTGAGCTAGCTGGGCTCAGCTAGGCTGGGTTG


 



AGCTGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAA


 



CTGGGCTGAGCTGGGCTGAGCTGGGCTGAGCAGAGCT


 



GGGCTGAGCAGAGCTGGGTTGGTCTGAGCTGGGTTGA


 



GCTGGGCTGAGCTGGGCTGAGCAGAGTTGGGTTGAGC


 



TGAGCTGGGTTCAGCTGGGCTGAGCTAGGCTGGGTTG


 



AGCTGGGTTGAGTTGGGCTGAGCTGGGCTGGGTTGAG


 



CGGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCG


 



GAACTGGGTTGATCTGAATTGAGCTGGGCTGAGCCGG


 



GCTGAGCCGGGCTGAGCTGGGCTAGGTTGAGCTTGGG


 



TGAGCTTGCCTCAGCTGGTCTGAGCTAGGTTGGGTGG


 



AGCTAGGCTGGATTGAGCTGGGCTGAGCTGAGCTGAT


 



CTGGCCTCAGCTGGGCTGAGGTAGGCTGAACTGGGCT


 



GTGCTGGGCTGAGCTGAGCTGAGCCAGTTTGAGCTGG


 



GTTGAGCTGGGCTGAGCTGGGCTGTGTTGATCTTTCC


 



TGAACTGGGCTGAGCTGGGCTGAGCTGGCCTAGCTGG


 



ATTGAACGGGGGTAAGCTGGGCCAGGCTGGACTGGGC


 



TGAGCTGAGCTAGGCTGAGCTGAGTTGAATTGGGTTA


 



AGCTGGGCTGAGATGGGCTGAGCTGGGCTGAGCTGGG


 



TTGAGCCAGGTCGGACTGGGTTACCCTGGGCCACACT


 



GGGCTGAGCTGGGCGGAGCTCGATTAACCTGGTCAGG


 



CTGAGTCGGGTCCAGCAGACATGCGCTGGCCAGGCTG


 



GCTTGACCTGGACACGTTCGATGAGCTGCCTTGGGAT


 



GGTTCACCTCAGCTGAGCCAGGTGGCTCCAGCTGGGC


 



TGAGCTGGTGACCCTGGGTGACCTCGGTGACCAGGTT


 



GTCCTGAGTCCGGGCCAAGCCGAGGCTGCATCAGACT


 



CGCCAGACCCAAGGCCTGGGCCCCGGCTGGCAAGCCA


 



GGGGCGGTGAAGGCTGGGCTGGCAGGACTGTCCCGGA


 



AGGAGGTGCACGTGGAGCCGCCCGGACCCCGACCGGC


 



AGGACCTGGAAAGACGCCTCTCACTCCCCTTCTCTTC


 



TGTCCCCTCTCGGGTCCTCAGAGAGCCAGTCTGCCCC


 



GAATCTCTACCCCCTCGTCTCCTGCGTCAGCCCCCCG


 



TCCGATGAGAGCCTGGTGGCCCTGGGCTGCCTGGCCC


 



GGGACTTCCTGCCCAGCTCCGTCACCTTCTCCTGGAA






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
GCGTCCGAAGTCAAAAATATCTGCAGCCTTCATGTAT


 



TCATAGAAACAAGGAATGTCTACATTTTCCAAAGTGG


 



GACCAGAATCTTGGGTCATGTCTAAGGCATGTGCATT


 



TGCACATGGTAGGCAAAGGACTTTGCTTCTCCCAGCA


 



CATCTTTCTGCAGAGATCCATGGAAACAAGACTCAAC


 



TCCAAAGCAGCAAAGAAGCAGCAAGTTCTCAAGTGAT


 



CTCCTCTGACTCCCTCCTCCCAGGCTAATGAAGCCAT


 



GTTGCCCCTGGGGGATTAAGGGCAGGTGTCCATTGTG


 



GCACCCAGCCCGAAGACAAGCAATTTGATCAGGTTCT


 



GAGCACTCCTGAATGTGGACTCTGGAATTTTCTCCTC


 



ACCTTGTGGCATATCAGCTTAAGTCAAGTACAAGTGA


 



CAAACAACATAATCCTAAGAAGAGAGGAATCAAGCTG


 



AAGTCAAAGGATCACTGCCTTGGATTCTACTGTGAAT


 



GATGACCTGGAAAATATCCTGAACAACAGCTTCAGGG


 



TGATCATCAGAGACAAAAGTTCCAGAGCCAGgtaggg


 



aaaccctcaagccttgcaaagagcaaaatcatgccat


 



tgggttcttaacctgctgagtgatttactatatgtta


 



ctgtgggaggcaaagcgctcaaatagcctgggtaagt


 



atgtcaaataaaaagcaaaagtggtgtttcttgaaat


 



gttagacctgaggaaggaatattgataacttaccaat


 



aattttcagaatgatttatagatgtgcacttagtcag


 



tgtctctccaccccgcacctgacaagcagtttagaat


 



ttattctaagaatctaggtttgctgggggctacatgg


 



gaatcagcttcagtgaagagtttgttggaatgattca


 



ctaaattttctatttccagcataaatccaagaacctc


 



tcagactagtttattgacactgcttttcctccataat


 



ccatctcatctccgtccatcatggacactttgtagaa


 



tgacaggtcctggcagagactcacagatgcttctgaa


 



acatcctttgccttcaaagaatgaacagcacacatac


 



taaggatctcagtgatccacaaattagtttttgccac


 



aatggttcttatgataaaagtctttcattaacagcaa


 



attgttttataatagttgttctgctttataataattg


 



catgcttcactttcttttcttttctttttttttcttt


 



ttttgctttttagtgccgcaggtgcagcatatgaaat


 



ttcccaggctaggggtcaaatcagaactacacctact


 



ggcctacgccacagccacagcaactcaggatctaagc


 



catgtcggtgacctacactacagctcatggcaatgcc


 



agatccttaacccaatgagcgaggccagggatcgaac


 



ccatgtcctcatggatactagtcaggctcattatccg


 



ctgagccataacaggaactcccgagtttgctttttat


 



caaaattggtacagccttattgtttctgaaaaccaca


 



aaatgaatgtattcacataattttaaaaggttaaata


 



atttatgatatacaagacaatagaaagagaaaacgtc


 



attgcctctttcttccacgacaacacgcctccttaat


 



tgatttgaagaaataactactgagcatggtttagtgt


 



acttctttcagcaattagcctgtattcatagccatac


 



atattcaattaaaatgagatcatgatatcacacaata


 



cataccatacagcctatagggatttttacaatcatct


 



tccacatgactacataaaaacctacctaaaaaaaaaa


 



aaaaccctacttcatcctcctattggctgctttgtgc


 



tccattaaaaagctctatcataattaggttatgatga


 



ggatttccattttctacctttcaagcaacatttcaat


 



gcacagtcttatatacacatttgagcctacttttctt


 



tttctttctttttttggtttttttttttttttttttt


 



ttggtctttttgtcttttctaaggctgcatatggagg


 



ttcccaggctagctgtctaatcagaactatagctgct


 



ggcctacgccacatccacagcaatacaagatctgagc


 



catgtctgcaacttacaccacagctcacagcaacggt


 



ggatccttaaaccactgagcaaggccagggatcaaac


 



ccataacttcatggctcctagttggatttgttaacca


 



ctgagccatgatggcaactcctgagcctacttttcta


 



atcatttccaaccctaggacacttttttaagtttcat


 



ttttctccccccaccccctgttttctgaagtgtgttt


 



gcttccactgggtgacttcactcccaggatctcatct


 



gcaggatactgcagctaagtgtatgagctctgaattt


 



gaatcccaactctgccactcaaagggataggagtttc


 



cgatgtggcccaatgggatcagtggcatctctgcagt


 



gccaggacgcaggttccatccctggcccagcacagtg


 



ggttaagaatctggcattgctgcagctgaggcataga


 



tttcaattgtgcctcagatctgatccttggcccaagg


 



actgcatatgcctcagggcaaccaaaaaagagaaaag


 



gggggtgatagcattagtttctagatttgggggataa


 



ttaaataaagtgatccatgtacaatgtatggcatttt


 



gtaaatgctcaacaaatttcaactattatggagttcc


 



catcatggctcagtggaagggaatctgattagcatcc


 



atgaggacacaggtccaaccccgaccttgctcagtgg


 



gcattgctgtgagctgtggcatgggttacagacgaag


 



ctcggatctggcattgctgtggctgtggtgtaagcca


 



gcaactacagctctcattcagcccctagcctgggaac


 



ctccatatgcctaaaagacaaaaaataaaatttaaat


 



taaaaataaagaaatgttaactattatgattggtact


 



gcttgcattactgcaaagaaagtcactttctatactc


 



tttaatatcttagttgactgtgtgctcagtgaactat


 



tttggacacttaatttccactctcttctatctccaac


 



ttgacaactctctttcctctcttctggtgagatccac


 



tgctgactttgctctttaaggcaactagaaaagtgct


 



cagtgacaaaatcaaagaaagttaccttaatcttcag


 



aattacaatcttaagttctcttgtaaagcttactatt


 



tcagtggttagtattattccttggtcccttacaactt


 



atcagctctgatctattgctgattttcaactatttat


 



tgttggagttttttccttttttccctgttcattctgc


 



aaatgtttgctgagcatttgtcaagtgaagatactgg


 



actgggccttccaaatataagacaatgaaacatcggg


 



agttctcattatggtgcagcagaaacgaatccaacta


 



ggaaatgtgaggttgcaggttcgatccctgcccttgc


 



tcagtgggttaaggatccagcattaccgtgagctgtg


 



gtgtaggttgcagacgtggctcagatcctgcgttgct


 



gtggctgtggcataggctggcagctctagctctgatt


 



cgaccgctagcctgggaacctccatgcgccccgagtg


 



cagcccttaaaaagcaaaaaaaaaagaaagaaagaaa


 



aagacaatgaaacatcaaacagctaacaatccagtag


 



ggtagaaagaatctggcaacagataagagcgattaaa


 



tgttctaggtccagtgaccttgcctctgtgctctaca


 



cagtcgtgccacttgctgagggagaaggtctctcttg


 



agttgagtcctgaaagacattagttgttcacaaacta


 



atgccagtgagtgaaggtgtttccaagcagagggaga


 



gtttggtaaaaagctggaagtcacagaaagactctaa


 



agagtttaggatggtgggagcaacatacgctgagatg


 



gggctggaaggttaagagggaaacaactatagtaagt


 



gaagctggactcacagcaaagtgaggacctcagcatc


 



cttgatggggttaccatggaaacaccaaggcacacct


 



tgatttccaaaacagcaggcacctgattcagcccaat


 



gtgacatggtgggtacccctctagctctacctgttct


 



gtgacaactgacaaccaacgaagttaagtctggattt


 



tctactctgctgatccttgtttttgtttcacacgtca


 



tctatagcttcatgccaaaatagagttcaaggtaaga


 



cgcgggccttggtttgatatacatgtagtctatcttg


 



tttgagacaatatggtggcaaggaagaggttcaaaca


 



ggaaaatactctctaattatgattaactgagaaaagc


 



taaagagtcccataatgacactgaatgaagttcatca


 



tttgcaaaagccttcccccccccccaggagactataa


 



aaaagtgcaattttttaaatgaacttatttacaaaac


 



agaaatagactcacagacataggaaacgaacagatgg


 



ttaccaagggtgaaagggagtaggagggataaataag


 



gagtctggggttagcagatacaccccagtgtacacaa


 



aataaacaacagggacctactatatagcacagggaac


 



tatatgcagtagcttacaataacctataatggaaaag


 



aatgtgaaaaagaatatatgtatgcgtgtgtgtgtaa


 



ctgaatcactttgctgtaacctgaatctaacataaca


 



ttgtaaatcaactacagttttttttttttttaagtgc


 



agggttttggtgttttttttttttcatttttgttttt


 



gtttttgttttttgctttttagggccacacccagaca


 



tatgggggttcccaggctaggggtctaattagagcta


 



cagttgccggcttgcaccacagccacagcaacatcag


 



atccgagccgcacttgcgacttacaccacagctcatg


 



gcaataccagatccttaacccactgagcaaggcccag


 



ggatcgtacccgcaacctcatggttcctagtcagatt


 



catttctgctgcgctacaatgggaactccaagtgcag


 



ttttttgtaatgtgcttgtctttctttgtaattcata


 



ttcatcctacttcccaataaataaataaatacataaa


 



taataaacataccattgtaaatcaactacaatttttt


 



ttaaatgcagggtttttgttttttgttttttgttttg


 



tctttttgccttttctagggccgctcccatggcatat


 



ggaggttcccaggctaggggtcgaatcggagctgtag


 



ccaccggcctacgccagagccacagcaacgcgggatc


 



cgagccgcgtctgcaacctacaccacagctcacggca


 



acgccggatcgttaacccactgagcaagggcagggat


 



cgaacctgcaacctcatggttcctagtcagattcgtt


 



aactactgagccacaacggaaactcctaaagtgcagt


 



ttttaaatgtgcttgtctttctttgtaatttacactc


 



aacctacttcccaataaataaataaataaacaaataa


 



atcatagacatggttgaattctaaaggaagggaccat


 



caggccttagacagaaatacgtcatcttctagtattt


 



taaaacacactaaagaagacaaacatgctctgccaga


 



gaagcccagggcctccacagctgcttgcaaagggagt


 



taggcttcagtagctgacccaaggctctgttcctctt


 



cagggaaaagggtttttgttcagtgagacagcagaca


 



gctgtcactgtgGTGGACGTTCGGCCAAGGAACCAAG


 



CTGGAACTCAAACgtaagtcaatccaaacgttccttc


 



cttggctgtctgtgtcttacggtctctgtggctctga


 



aatgattcatgtgctgactctctgaaaccagactgac


 



attctccagggcaaaactaaagcctgtcatcaaactg


 



gaaaactgagggcacattttctgggcagaactaagag


 



tcaggcactgggtgaggaaaaacttgttagaatgata


 



gtttcagaaacttactgggaagcaaagcccatgttct


 



gaacagagctctgctcaagggtcaggaggggaaccag


 



tttttgtacaggagggaagttgagacgaacccctgtg


 



TATATGGTTTCGGCGCGGGGACCAAGCTGGAGCTCAA


 



ACgtaagtggctttttccgactgattctttgctgttt


 



ctaattgttggttggctttttgtccatttttcagtgt


 



tttcatcgaattagttgtcagggaccaaacaaattgc


 



cttcccagattaggtaccagggaggggacattgctgc


 



atgggagaccagagggtggctaatttttaacgtttcc


 



aagccaaaataactggggaagggggcttgctgtcctg


 



tgagggtaggtttttatagaagtggaagttaagggga


 



aatcgctatgGTTCACTTTTGGCTCGGGGACCAAAGT


 



GGAGCCCAAAAttgagtacattttccatcaattattt


 



gtgagatttttgtcctgttgtgtcatttgtgcaagtt


 



tttgacattttggttgaatgagccattcccagggacc


 



caaaaggatgagaccgaaaagtagaaaagagccaact


 



tttaagctgagcagacagaccgaattgttgagtttgt


 



gaggagagtagggtttgtagggagaaaggggaacaga


 



tcgctggctttttctctgaattagcctttctcatggg


 



actggcttcagagggggtttttgatgagggaagtgtt


 



ctagagccttaactgtgGGTTGTGTTCGGTAGCGGGA


 



CCAAGCTGGAAATCAAACgtaagtgcacttttctact


 



cctttttctttcttatacgggtgtgaaattggggact


 



tttcatgtttggagtatgagttgaggtcagttctgaa


 



gagagtgggactcatccaaaaatctgaggagtaaggg


 



tcagaacagagttgtctcatggaagaacaaagaccta


 



gttagttgatgaggcagctaaatgagtcagttgactt


 



gggatccaaatggccagacttcgtctgtaaccaacaa


 



tctaatgagatgtagcagcaaaaagagatttccattg


 



aggggaaagtaaaattgttaatattgtgGATCACCTT


 



TGGTGAAGGGACATCCGTGGAGATTGAACgtaagtat


 



tttttctctactaccttctgaaatttgtctaaatgcc


 



agtgttgacttttagaggcttaagtgtcagttttgtg


 



aaaaatgggtaaacaagagcatttcatatttattatc


 



agtttcaaaagttaaactcagctccaaaaatgaattt


 



gtagacaaaaagattaatttaagccaaattgaatgat


 



tcaaaggaaaaaaaaattagtgtagatgaaaaaggaa


 



ttcttacagctccaaagagcaaaagcgaattaatttt


 



ctttgaactttgccaaatcttgtaaatgatttttgtt


 



ctttacaatttaaaaaggttagagaaatgtatttctt


 



agtctgttttctctcttctgtctgataaattattata


 



tgagataaaaatgaaaattaataggatgtgctaaaaa


 



atcagtaagaagttagaaaaatatatgtttatgttaa


 



agttgccacttaattgagaatcagaagcaatgttatt


 



tttaaagtctaaaatgagagataaactgtcaatactt


 



aaattctgcagagattctatatcttgacagatatctc


 



ctttttcaaaaatccaatttctatggtagactaaatt


 



tgaaatgatcttcctcataatggagggaaaagatgga


 



ctgaccccaaaagctcagatttaaagaaatctgttta


 



agtgaaagaaaataaaagaactgcattttttaaaggc


 



ccatgaatttgtagaaaaataggaaatattttaataa


 



gtgtattcttttattttcctgttattacttgatggtg


 



tttttataccgccaaggaggccgtggcaccgtcagtg


 



tgatctgtagaccccatggcggccttttttcgcgatt


 



gaatgaccttggcggtgggtccccagggctctggtgg


 



cagcgcaccagccgctaaaagccgctaaaaactgccg


 



ctaaaggccacagcaaccccgcgaccgcccgttcaac


 



tgtgctgacacagtgatacagataatgtcgctaacag


 



aggagaatagaaatatgacgggcacacgctaatgtgg


 



ggaaaagagggagaagcctgatttttattttttagag


 



attctagagataaaattcccagtattatatcctttta


 



ataaaaaatttctattaggagattataaagaatttaa


 



agctatttttttaagtggggtgtaattctttcagtag


 



tctcttgtcaaatggatttaagtaatagaggcttaat


 



ccaaatgagagaaatagacgcataaccctttcaaggc


 



aaaagctacaagagcaaaaattgaacacagcagccag


 



ccatctagccactcagattttgatcagttttactgag


 



tttgaagtaaatatcatgaaggtataattgctgataa


 



aaaaataagatacaggtgtgacacatctttaagtttc


 



agaaatttaatggcttcagtaggattatatttcacgt


 



atacaaagtatctaagcagataaaaatgccattaatg


 



gaaacttaatagaaatatatttttaaattccttcatt


 



ctgtgacagaaattttctaatctgggtcttttaatca


 



cctaccctttgaaagagtttagtaatttgctatttgc


 



catcgctgtttactccagctaatttcaaaagtgatac


 



ttgagaaagattatttttggtttgcaaccacctggca


 



ggactattttagggccattttaaaactcttttcaaac


 



taagtattttaaactgttctaaaccatttagggcctt


 



ttaaaaatcttttcatgaatttcaaacttcgttaaaa


 



gttattaaggtgtctggcaagaacttccttatcaaat


 



atgctaatagtttaatctgttaatgcaggatataaaa


 



ttaaagtgatcaaggcttgacccaaacaggagtatct


 



tcatagcatatttcccctcctttttttctagaattca


 



tatgattttgctgccaaggctattttatataatctct


 



ggaaaaaaaatagtaatgaaggttaaaagagaagaaa


 



atatcagaacattaagaattcggtattttactaactg


 



cttggttaacatgaaggtttttattttattaaggttt


 



ctatctttataaaaatctgttcccttttctgctgatt


 



tctccaagcaaaagattcttgatttgttttttaactc


 



ttactctcccacccaagggcctgaatgcccacaaagg


 



ggacttccaggaggccatctggcagctgctcaccgtc


 



agaagtgaagccagccagttcctcctgggcaggtggc


 



caaaattacagttgacccctcctggtctggctgaacc


 



ttgccccatatggtgacagccatctggccagggccca


 



ggtctccctctgaagcctttgggaggagagggagagt


 



ggctggcccgatcacagatgcggaaggggctgactcc


 



tcaaccggggtgcagactctgcagggtgggtctgggc


 



ccaacacacccaaagcacgcccaggaaggaaaggcag


 



cttggtatcactgcccagagctaggagaggcaccggg


 



aaaatgatctgtccaagacccgttcttgcttctaaac


 



tccgagggggtcagatgaagtggttttgtttcttggc


 



ctgaagcatcgtgttccctgcaagaagcggggaacac


 



agaggaaggagagaaaagatgaactgaacaaagcatg


 



caaggcaaaaaaggccttaggatggctgcaggaagtt


 



agttcttctgcattggctccttactggctcgtcgatc


 



gcccacaaacaacgcacccagtggagaacttccctgt


 



tacttaaacaccattctctgtgcttgcttcctcagGG


 



GCTGATGCCAAGCCATCCGTCTTCATCTTCCCGCCAT


 



CGAAGGAGCAGTTAGCGACCCCAACTGTCTCTGTGGT


 



GTGCTTGATCAATAACTTCTTCCCCAGAGAAATCAGT


 



GTCAAGTGGAAAGTGGATGGGGTGGTCCAAAGCAGTG


 



GTCATCCGGATAGTGTCACAGAGCAGGACAGCAAGGA


 



CAGCACCTACAGCCTCAGCAGCACCCTCTCGCTGCCC


 



ACGTCACAGTACCTAAGTCATAATTTATATTCCTGTG


 



AGGTCACCCACAAGACCCTGGCCTCCCCTCTGGTCAC


 



AAGCTTCAACAGGAACGAGTGTGAGGCTtagAGGCCC


 



ACAGGCCCCTGGCCTGCCCCCAGCCCCAGCCCCCCTC


 



CCCACCTCAAGCCTCAGGCCCTTGCCCCAGAGGATCC


 



TTGGCAATCCCCCAGCCCCTCTTCCCTCCTCATCCCC


 



TCCCCCTCTTTGGCTTTAACCGTGTTAATACTGGGGG


 



GTGGGGGAATGAATAaataaaGTGAACCTTTGCACCT


 



GTGAtttctctctcctgtctgattttaaggttgttaa


 



atgttgttttccccattatagttaatcttttaaggaa


 



ctacatactgagttgctaaaaactacaccatcactta


 



taaaattcacgccttctcagttctcccctcccctcct


 



gtcctccgtaagacaggcctccgtgaaacccataagc


 



acttctctttacaccctctcctgggccggggtaggag


 



actttttgatgtcccctcttcagcaagcctcagaacc


 



attttgagggggacagttcttacagtcacat*tcctg


 



tgatctaatgactttagttaccgaaaagccagtctct


 



caaaaagaagggaacggctagaaaccaagtcatagaa


 



atatatatgtataaaatatatatatatccatatatgt


 



aaaataacaaaataatgataacagcataggtcaacag


 



gcaacagggaatgttgaagtccattctggcacttcaa


 



tttaagggaataggatgccttcattacattttaaata


 



caatacacatggagagcttcctatctgccaaagacca


 



tcctgaatgccttccacactcactacaaggttaaaag


 



cattcattacaatgttgatcgaggagttcccgttgtg


 



gctcagcaggttaagaacgtgactggtatccaggagg


 



atgcgggtttggtccccagcctcgctcagtggattaa


 



ggatccagtgttgctgcaagatcacgggctcagatcc


 



cgtgttctatggctatggtgtaggctggtagctgcat


 



gcagccctaatttgacccctagcctgggaactgccat


 



atgccacatgtgaggcccttaaaacctaaaagaaaaa


 



aaaagaaaagaaatatcttacacccaatttatagata


 



agagagaagctaaggtggcaggcccaggatcaaagcc


 



ctacctgcctatcttgacacctgatacaaattctgtc


 



ttctagggtttccaacactgcatagaacagagggtca


 



aacatgctaccctcccagggactcctcccttcaaatg


 



acataaattttgttgcccatctctgggggcaaaactc


 



aacaatcaatggcatctctagtaccaagcaaggctct


 



tctcatgaagcaaaactctgaagccagatccatcatg


 



acccaaggaagtaaagacaggtgttactggttgaact


 



gtatccttcaattcaatatgctcaatttccaactccc


 



agtccccgtaaatacaaccccctttgggaagagagtc


 



cttgcagatgtagccacgttaaaaagagattatacag


 



aaaggctagtgaggatgcagtgaaacgggatctttca


 



tacattgctggtggaaatgtaaaatgctgcaggcact


 



ctagaaaataatttgccagttttttgaaaagctaaac


 



aaaatagtttagttgcattctgggttatttatccccc


 



agaaattaaaaattatgtccgcacaaaaacgtgtaca


 



taatcattcataacagccttgtac


 


Seq ID No. 12
caaggaaccaagctggaactcaaacgtaagtcaatcc


 



aaacgttccttccttggctgtctgtgtcttacggtct


 



ctgtggctctgaaatgattcatgtgctgactctctga


 



aaccagactgacattctccagggcaaaactaaagcct


 



gtcatcaaactggaaaactgagggcacattttctggg


 



cagaactaagagtcaggcactgggtgaggaaaaactt


 



gttagaatgatagtttcagaaacttactgggaagcaa


 



agcccatgttctgaacagagctctgctcaagggtcag


 



gaggggaaccagtttttgtacaggagggaagttgaga


 



cgaacccctgtgtatatggtttcggcgcggggaccaa


 



gctggagctcaaacgtaagtggctttttccgactgat


 



tctttgctgtttctaattgttggttggctttttgtcc


 



atttttcagtgttttcatcgaattagttgtcagggac


 



caaacaaattgccttcccagattaggtaccagggagg


 



ggacattgctgcatgggagaccagagggtggctaatt


 



tttaacgtttccaagccaaaataactggggaaggggg


 



cttgctgtcctgtgagggtaggtttttatagaagtgg


 



aagttaaggggaaatcgctatggttcacttttggctc


 



ggggaccaaagtggagcccaaaattgagtacattttc


 



catcaattatttgtgagatttttgtcctgttgtgtca


 



tttgtgcaagtttttgacattttggttgaatgagcca


 



ttcccagggacccaaaaggatgagaccgaaaagtaga


 



aaagagccaacttttaagctgagcagacagaccgaat


 



tgttgagtttgtgaggagagtagggtttgtagggaga


 



aaggggaacagatcgctggctttttctctgaattagc


 



ctttctcatgggactggcttcagagggggtttttgat


 



gagggaagtgttctagagccttaactgtgggttgtgt


 



tcggtagcgggaccaagctggaaatcaaacgtaagtg


 



cacttttctactcctttttctttcttatacgggtgtg


 



aaattggggacttttcatgtttggagtatgagttgag


 



gtcagttctgaagagagtgggactcatccaaaaatct


 



gaggagtaagggtcagaacagagttgtctcatggaag


 



aacaaagacctagttagttgatgaggcagctaaatga


 



gtcagttgacttgggatccaaatggccagacttcgtc


 



tgtaaccaacaatctaatgagatgtagcagcaaaaag


 



agatttccattgaggggaaagtaaaattgttaatatt


 



gtggatcacctttggtgaagggacatccgtggagatt


 



gaacgtaagtattttttctctactaccttctgaaatt


 



tgtctaaatgccagtgttgacttttagaggcttaagt


 



gtcagttttgtgaaaaatgggtaaacaagagcatttc


 



atatttattatcagtttcaaaagttaaactcagctcc


 



aaaaatgaatttgtagacaaaaagattaatttaagcc


 



aaattgaatgattcaaaggaaaaaaaaattagtgtag


 



atgaaaaaggaattcttacagctccaaagagcaaaag


 



cgaattaattttctttgaactttgccaaatcttgtaa


 



atgatttttgttctttacaatttaaaaaggttagaga


 



aatgtatttcttagtctgttttctctcttctgtctga


 



taaattattatatgagataaaaatgaaaattaatagg


 



atgtgctaaaaaatcagtaagaagttagaaaaatata


 



tgtttatgttaaagttgccacttaattgagaatcaga


 



agcaatgttatttttaaagtctaaaatgagagataaa


 



ctgtcaatacttaaattctgcagagattctatatctt


 



gacagatatctcctttttcaaaaatccaatttctatg


 



gtagactaaatttgaaatgatcttcctcataatggag


 



ggaaaagatggactgaccccaaaagctcagattt*aa


 



gaaaacctgtttaag*gaaagaaaataaaagaactgc


 



attttttaaaggcccatgaatttgtagaaaaatagga


 



aatattttaataagtgtattcttttattttcctgtta


 



ttacttgatggtgtttttataccgccaaggaggccgt


 



ggcaccgtcagtgtgatctgtagaccccatggcggcc


 



ttttttcgcgattgaatgaccttggcggtgggtcccc


 



agggctctggtggcagcgcaccagccgctaaaagccg


 



ctaaaaactgccgctaaaggccacagcaaccccgcga


 



ccgcccgttcaactgtgctgacacagtgatacagata


 



atgtcgctaacagaggagaatagaaatatgacgggca


 



cacgctaatgtggggaaaagagggagaagcctgattt


 



ttattttttagagattctagagataaaattcccagta


 



ttatatccttttaataaaaaatttctattaggagatt


 



ataaagaatttaaagctatttttttaagtggggtgta


 



attctttcagtagtctcttgtcaaatggatttaagta


 



atagaggcttaatccaaatgagagaaatagacgcata


 



accctttcaaggcaaaagctacaagagcaaaaattga


 



acacagcagccagccatctagccactcagattttgat


 



cagttttactgagtttgaagtaaatatcatgaaggta


 



taattgctgataaaaaaataagatacaggtgtgacac


 



atctttaagtttcagaaatttaatggcttcagtagga


 



ttatatttcacgtatacaaagtatctaagcagataaa


 



aatgccattaatggaaacttaatagaaatatattttt


 



aaattccttcattctgtgacagaaattttctaatctg


 



ggtcttttaatcacctaccctttgaaagagtttagta


 



atttgctatttgccatcgctgtttactccagctaatt


 



tcaaaagtgatacttgagaaagattatttttggtttg


 



caaccacctggcaggactattttagggccattttaaa


 



actcttttcaaactaagtattttaaactgttctaaac


 



catttagggccttttaaaaatcttttcatgaatttca


 



aacttcgttaaaagttattaaggtgtctggcaagaac


 



ttccttatcaaatatgctaatagtttaatctgttaat


 



gcaggatataaaattaaagtgatcaaggcttgaccca


 



aacaggagtatcttcatagcatatttcccctcctttt


 



tttctagaattcatatgattttgctgccaaggctatt


 



ttatataatctctggaaaaaaaatagtaatgaaggtt


 



aaaagagaagaaaatatcagaacattaagaattcggt


 



attttactaactgcttggttaacatgaaggtttttat


 



tttattaaggtttctatctttataaaaatctgttccc


 



ttttctgctgatttctccaagcaaaagattcttgatt


 



tgttttttaactcttactctcccacccaagggcctga


 



atgcccacaaaggggacttccaggaggccatctggca


 



gctgctcaccgtcagaagtgaagccagccagttcctc


 



ctgggcaggtggccaaaattacagttgacccctcctg


 



gtctggctgaaccttgccccatatggtgacagccatc


 



tggccagggcccaggtctccctctgaagcctttggga


 



ggagagggagagtggctggcccgatcacagatgcgga


 



aggggctgactcctcaaccggggtgcagactctgcag


 



ggtgggtctgggcccaacacacccaaagcacgcccag


 



gaaggaaaggcagcttggtatcactgcccagagctag


 



gagaggcaccgggaaaatgatctgtccaagacccgtt


 



cttgcttctaaactccgagggggtcagatgaagtggt


 



tttgtttcttggcctgaagcatcgtgttccctgcaag


 



aagcggggaacacagaggaaggagagaaaagatgaac


 



tgaacaaagcatgcaaggcaaaaaaggccttaggatg


 



gctgcaggaagttagttcttctgcattggctccttac


 



tggctcgtcgatcgcccacaaacaacgcacccagtgg


 



agaacttccctgttacttaaacaccattctctgtgct


 



tgcttcctcaggggctgatgccaagccatccgtcttc


 



atcttcccgccatcgaaggagcagttagcgaccccaa


 



ctgtctctgtggtgtgcttgatca


 


Seq ID No. 15
gatgccaagccatccgtcttcatcttcccgccatcga


 



aggagcagttagcgaccccaactgtctctgtggtgtg


 



cttgatcaataacttcttccccagagaaatcagtgtc


 



aagtggaaagtggatggggtggtccaaagcagtggtc


 



atccggatagtgtcacagagcaggacagcaaggacag


 



cacctacagcctcagcagcaccctctcgctgcccacg


 



tcacagtacctaagtcataatttatattcctgtgagg


 



tcacccacaagaccctggcctcccctctggtcacAAG


 



CTTCAACAGGAACGAGTGTGAGGCTTAGAGGCCCACA


 



GGCCCCTGGCCTGCCCCCAGCCCCAGCCCCCCTCCCC


 



ACCTCAAGCCTCAGGCCCTTGCCCCAGAGGATCCTTG


 



GCAATCCCCCAGCCCCTCTTCCCTCCTCATCCCCTCC


 



CCCTCTTTGGCTTTAACCGTGTTAATACTGGGGGGTG


 



GGGGAATGAATAAATAAAGTGAACCTTTGCACCTGTG


 



ATTTCTCTCTCCTGTCTGATTTTAAGGTTGTTAAATG


 



TTGTTTTCCCCATTATAGTTAATCTTTTAAGGAACTA


 



CATACTGAGTTGCTAAAAACTACACCATCACTTATAA


 



AATTCAcgCCTTCTCAGTTCTCCCCTCCCCTCCTGTC


 



CTCCGTAAGACAGGCCTCCGTGAAACCCATAAGCACT


 



TCTCTTTACACCCTCTCCTGGGCCGGGGTAGGAGACT


 



TTTTGATGTCCCCTcTTCAGCAAGCCTCAGAACCATT


 



TTGAGGGGGACAGTTCTTACAGTCACAT*TCCtGtGA


 



TCTAATGACTTTAGTTaCCGAAAAGCCAGTCTCTCAA


 



AAAGAAGGGAACGGCTAGAAACCAAGTCATAGAAATA


 



TATATGTATAAAATATATATATATCCATATATGTAAA


 



ATAACAAAATAATGATAACAGCATAGGTCAACAGGCA


 



ACAGGGAATGTTGAAGTCCATTCTGGCACTTCAATTT


 



AAGGGAATAGGATGCCTTCATTACATTTTAAATACAA


 



TACACATGGAGAGCTTCCTATCTGCCAAAGACCATCC


 



TGAATGCCTTCCACACTCACTACAAGGTTAAAAGCAT


 



TCATTACAATGTTGATCGAGGAGTTCCCGTTGTGGCT


 



CAGCAGGTTAAGAACGTGACTGGTATCCAGGAGGATG


 



CGGGTTTGGTCCCCAGCCTCGCTCAGTGGATTAAGGA


 



TCCAGTGTTGCTGCAAGATCACGGGCTCAGATCCCGT


 



GTTCTATGGCTATGGTGTAGGCTGGTAGCTGCATGCA


 



GCCCTAATTTGACCCCTAGCCTGGGAACTGCCATAtG


 



CCACATGTGAGGCCCTTAAAACCTAAAAGAAAAAaAA


 



AGAAAAGAAATATCTTACACCCAATTTATAGATAAGA


 



GAGAAGCTAAGGTGGCAGGCCCAGGATCAAAGCCCTA


 



CCTGCCTATCTTGACACCTGAtACAAATTCTGTCTTC


 



TAGGGtTTCCAACACTGCATAGAACAGAGGGTCAAAC


 



ATGCTACCCTCCCAGGGACTCCTCCCTTCAAATGACA


 



TAAATTTTGTTGCCCATCTCTGGGGGCAAAACTCAAC


 



AATCAATGGCATCTCTAGTACCAAGCAAGGCTCTTCT


 



CATGAAGCAAAACTCTGAAGCCAGATCCATCATGACC


 



CAAGGAAGTAAAGACAGGTGTTACTGGTTGAACTGTA


 



TCCTTCAATTCAATATGCTCAATTTCCAACTCCCAGT


 



CCCCGTAAATACAACCCCCTTTGGGAAGAGAGTCCTT


 



GCAGATGTAGCCACGTTAAAAAGAGATTATACAGAAA


 



GGCTAGTGAGGATGCAGTGAAACGGGATCTTTCATAC


 



ATTGCTGGTGGAAATGTAAAATGCTGCAGGCACTCTA


 



GAAAATAATTTGCCAGTTTTTTGAAAAGCTAAACAAA


 



ATAGTTTAGTTGCATTCTGGGTTATTTATCCCCCAGA


 



AATTAAAAATTATGTCCGCACAAAAACGTGTACATAA


 



TCATTCATAACAGCCTTGTACGAAAAGCTT


 


Seq ID No. 16
GGATCCTTAACCCACTAATCGAGGATCAAACACGCAT


 



CCTCATGGACAATATGTTGGGTTCTTAGCCTGCTGAG


 



ACACAACAGGAACTCCCCTGGCACCACTTTAGAGGCC


 



AGAGAAACAGCACAGATAAAATTCCCTGCCCTCATGA


 



AGCTTATAGTCTAGCTGGGGAGATATCATAGGCAAGA


 



TAAACACATACAAATACATCATCTTAGGTAATAATAT


 



ATACTAAGGAGAAAATTACAGGGGAGAAAGAGGACAG


 



GAATTGCTAGGGTAGGATTATAAGTTCAGATAGTTCA


 



TCAGGAACACTGTTGCTGAGAAGATAACATTTAGGTA


 



AAGACCGAAGTAGTAAGGAAATGGACCGTGTGCCTAA


 



GTGGGTAAGACCATTCTAGGCAGCAGGAACAGCGATG


 



AAAGCACTGAGGTGGGTGTTCACTGCACAGAGTTGTT


 



CACTGCACAGAGTTGTGTGGGGAGGGGTAGGTCTTGC


 



AGGCTCTTATGGTCACAGGAAGAATTGTTTTACTCCC


 



ACCGAGATGAAGGTTGGTGGATTTTGAGCAGAAGAAT


 



AATTCTGCCTGGTTTATATATAACAGGATTTCCCTGG


 



GTGCTCTGATGAGAATAATCTGTCAGGGGTGGGATAG


 



GGAGAGATATGGCAATAGGAGCCTTGGCTAGGAGCCC


 



ACGACAATAATTCCAAGTGAGAGGTGGTGCTGCATTG


 



AAAGCAGGACTAACAAGACCTGCTGACAGTGTGGATG


 



TAGAAAAAGATAGAGGAGACGAAGGTGCATCTAGGGT


 



TTTCTGCCTGAGGAATTAGAAAGATAAAGCTAAAGCT


 



TATAGAAGATGCAGCGCTCTGGGGAGAAAGACCAGCA


 



GCTCAGTTTTGATCCATCTGGAATTAATTTTGGCATA


 



AAGTATGAGGTATGTGGGTTAACATTATTTGTTTTTT


 



TTTTTTCCATGTAGCTATCCAACTGTCCCAGCATCAT


 



TTATTTTAAAAGACTTTCCTTTCCCCTATTGGATTGT


 



TTTGGCACCTTCACTGAAGATCAACTGAGCATAAAAT


 



TGGGTCTATTTCTAAGCTCTTGATTCCATTCCATGAC


 



CTATTTGTTCATCTTTACCCCAGTAGACACTGCCTTG


 



ATGATTAAAGCCCCTGTTACCATGTCTGTTTTGGACA


 



TGGTAAATCTGAGATGCCTATTAGCCAACCAAGCAAG


 



CACGGCCCTTAGAGAGCTAGATATGAGAGCCTGGAAT


 



TCAGACGAGAAAGGTCAGTCCTAGAGACATACATGTA


 



GTGCCATCACCATGCGGATGGTGTTAAAAGCCATCAG


 



ACTGCAACAGACTGTGAGAGGGTACCAAGCTAGAGAG


 



CATGGATAGAGAAACCCAAGCACTGAGCTGGGAGGTG


 



CTCCTACATTAAGAGATTAGTGAGATGAAGGACTGAG


 



AAGATTGATCAGAGAAGAAGGAaAATCAGGAAAATGG


 



TGCTGTCcTGAAAATCCAAGGGAAGAGATGTTCCAAA


 



GAGGAGAaAACTGATCAGTTGTCAGCTAGCGTCAATT


 



GGGATGAAAATGGACCATTGGACAGAGGGATGTAGTG


 



GGTCATGGGTGAATAGATAAGAGCAGCTTCTATAGAA


 



TGGCAGGGGCAAAATTCTCATCTGATCGGCATGGGTT


 



cTAAAGAAAACGGGAAGAAAAAATTGAGTGCATGACC


 



AGTCCCTTCAAGTAGAGAGGTgGAAAAGGGAAGGAGG


 



AAAATGAGGCCACGACAACATGAGAGAAATGACAGCA


 



TTTTTAAAAATTTTTTATTTTATTTtATTTATTTATT


 



TTTGCTTTTTAGGGCTGCCCCTGCAAcatatggaggt


 



tcccaggttaggggtctaatcagagctatagctgcca


 



gcctacaccacagccatagcaatgccagatctacatg


 



acctacaccacagctcacagcaacgccggatccttaa


 



cccactgagtgaggccagagatcaaacccatatcctt


 



atggatactagtcaggttcattaccactgagccaaaa


 



tgggaaATCCTGAGTAATGACAGCATTTTTTAATGTG


 



CCAGGAAGCAAAACTTGCCACCCCGAAATGTCTCTCA


 



GGCATGTGGATTATTTTGAGCTGAAAACGATTAAGGC


 



CCAAAAAACACAAGAAGAAATGTGGACCTTCCCCCAA


 



CAGCCTAAAAAATTTAGATTGAGGGCCTGTTCCCAGA


 



ATAGAGCTATTGCCAGACTTGTCTACAGAGGCTAAGG


 



GCTAGGTGTGGTGGGGAAACCCTCAGAGATCAGAGGG


 



ACGTTTATGTACCAAGCATTGACATTTCCATCTCCAT


 



GCGAATGGCCTTCTTCCCCTCTGTAGCCCCAAACCAC


 



CACCCCCAAAATCTTCTTCTGTCTTTAGCTGAAGATG


 



GTGTTGAAGGTGATAGTTTCAGCCACTTTGGCGAGTT


 



CCTCAGTTGTTCTGGGTCTTTCCTCCGGATCCACATT


 



ATTCGACTGTGTTTGATTTTCTCCTGTTTATCTGTCT


 



CATTGGCACCCATTTCATTCTTAGACCAGCCCAAAGA


 



ACCTAGAAGAGTGAAGGAAAATTTCTTCCACCCTGAC


 



AAATGCTAAATGAGAATCACCgCAGTAGAGGAAAATG


 



ATCTGGTgCTGCGGGAGATAGAAGAGAAAATcGCTGG


 



AGAGATGTCACTGAGTAGGTGAGATGGGAAAGGGGGG


 



GCACAGGTGGAGGTGTTGCCCTCAGCTAGGAAGACAG


 



ACAGTTcacagaagagaagcgggtgtccgtGGACATC


 



TTGCCTCATGGATGAGGAAACCGAGGCTAAGAAAGAC


 



TGCAAAAGAAAGGTAAGGATTGCAGAGAGGTCGATCC


 



ATGACTAAAATCACAGTAACCAACCCCAAACCACCAT


 



GTTTTCTCCTAGTCTGGCACGTGGCAGGTACTGTGTA


 



GGTTTTCAATATTATTGGTTTGTAACAGTACCTATTA


 



GGCCTCCATCcCCTCCTCTAATACTAACAAAAGTGTG


 



AGACTGGTCAGTGAAAAATGGTCTTCTTTCTCTATGC


 



AATCTTTCTCAAGAAGATACATAACTTTTTATTTTAT


 



CATaGGCTTGAAGAGCAAATGAGAAACAgCCTCCAAC


 



CTATGACACCGTAACAAAGTGTTTATGATCAGTGAAG


 



GGCAAGAAACAAAACATACACaGTAAAGACCCTCCAT


 



AATATTGtGGGCTGGCCCAaCACAGGCCAGGTTGTAA


 



AAGCTTTTTATTCTTTGATAGAGGAATGGATAGTAAT


 



GTTTCAACCTGGACAGAGAT*CATGTTCACTGAATCC


 



TTCCAAAAATTCATGGGTAGTTTGAAtTATAAGGAAA


 



ATAAGACTTAGGATAAATACTTTgTCCA*GATCCCAG


 



AGTTAATgCCAAAATCAGTTTTCAGACTCCAGGCAGC


 



CTGATCAAGAGCCTAAACTTTAAAGACACAGTCCCTT


 



AATAACTACTATTCACAGTTGCACTTTCAgGGCGCAA


 



AGACTCATTGAATCCTACAATAGAATGAGTTTAGATA


 



TCAAATCTCTCAGTAATAGATGAGGAGACTAAATAGC


 



GGGCATGACCTGGTCACTTAAAGACAGAATTGAGATT


 



CAAGGCTAGTGTTCTTTCTACCTGTTTTGTTTCTACA


 



AGATGTAGCAATGCGCTAATTACAGACCTCTCAGGGA


 



AGGAATTCACAACCCTCAGCAAAAACCAAAGACAAAT


 



CTAAGACAACTAAGAGTGTTGGTTTAATTTGGAAAAA


 



TAACTCACTAACCAAACGCCCCTCTTAGCACCCCAAT


 



GTCTTCCACCATCACAGTGCTCAGGCCTCAACCATGC


 



CCCAATGACCCCAGCCCCAGACTGGTTATTACCAAGT


 



TTCATGATGACTGGCCTGAGAAGATCAAAAAAGCAAT


 



GACATCTTACAGGGGACTACCCCGAGGACCAAGATAG


 



CAACTGTCATAGCAACCGTCACACTGCTTTGGTCA


 


Seq ID No. 19
ggatcaaacacgcatcctcatggacaatatgttgggt


 



tcttagcctgctgagacacaacaggaactcccctggc


 



accactttagaggccagagaaacagcacagataaaat


 



tccctgccctcatgaagcttatagtctagctggggag


 



atatcataggcaagataaacacatacaaatacatcat


 



cttaggtaataatatatactaaggagaaaattacagg


 



ggagaaagaggacaggaattgctagggtaggattata


 



agttcagatagttcatcaggaacactgttgctgagaa


 



gataacatttaggtaaagaccgaagtagtaaggaaat


 



ggaccgtgtgcctaagtgggtaagaccattctaggca


 



gcaggaacagcgatgaaagcactgaggtgggtgttca


 



ctgcacagagttgttcactgcacagagttgtgtgggg


 



aggggtaggtcttgcaggctcttatggtcacaggaag


 



aattgttttactcccaccgagatgaaggttggtggat


 



tttgagcagaagaataattctgcctggtttatatata


 



acaggatttccctgggtgctctgatgagaataatctg


 



tcaggggtgggatagggagagatatggcaataggagc


 



cttggctaggagcccacgacaataattccaagtgaga


 



ggtggtgctgcattgaaagcaggactaacaagacctg


 



ctgacagtgtggatgtagaaaaagatagaggagacga


 



aggtgcatctagggttttctgcctgaggaattagaaa


 



gataaagctaaagcttatagaagatgcagcgctctgg


 



ggagaaagaccagcagctcagttttgatccatctgga


 



attaattttggcataaagtatgaggtatgtgggttaa


 



cattatttgttttttttttttccatgtagctatccaa


 



ctgtcccagcatcatttattttaaaagactttccttt


 



cccctattggattgttttggcaccttcactgaagatc


 



aactgagcataaaattgggtctatttctaagctcttg


 



attccattccatgacctatttgttcatctttacccca


 



gtagacactgccttgatgattaaagcccctgttacca


 



tgtctgttttggacatggtaaatctgagatgcctatt


 



agccaaccaagcaagcacggcccttagagagctagat


 



atgagagcctggaattcagacgagaaaggtcagtcct


 



agagacatacatgtagtgccatcaccatgcggatggt


 



gttaaaagccatcagactgcaacagactgtgagaggg


 



taccaagctagagagcatggatagagaaacccaagca


 



ctgagctgggaggtgctcctacattaagagattagtg


 



agatgaaggactgagaagattgatcagagaagaagga


 



aaatcaggaaaatggtgctgtcctgaaaatccaaggg


 



aagagatgttccaaagaggagaaaactgatcagttgt


 



cagctagcgtcaattgggatgaaaatggaccattgga


 



cagagggatgtagtgggtcatgggtgaatagataaga


 



gcagcttctatagaatggcaggggcaaaattctcatc


 



tgatcggcatgggttctaaagaaaacgggaagaaaaa


 



attgagtgcatgaccagtcccttcaagtagagaggtg


 



gaaaagggaaggaggaaaatgaggccacgacaacatg


 



agagaaatgacagcatttttaaaaattttttatttta


 



ttttatttatttatttttgctttttagggctgcccct


 



gcaacatatggaggttcccaggttaggggtctaatca


 



gagctatagctgccagcctacaccacagccatagcaa


 



tgccagatctacatgacctacaccacagctcacagca


 



acgccggatccttaacccactgagtgaggccagagat


 



caaacccatatccttatggatactagtcaggttcatt


 



accactgagccaaaatgggaaatcctgagtaatgaca


 



gcattttttaatgtgccaggaagcaaaacttgccacc


 



ccgaaatgtctctcaggcatgtggattattttgagct


 



gaaaacgattaaggcccaaaaaacacaagaagaaatg


 



tggaccttcccccaacagcctaaaaaatttagattga


 



gggcctgttcccagaatagagctattgccagacttgt


 



ctacagaggctaagggctaggtgtggtggggaaaccc


 



tcagagatcagagggacgtttatgtaccaagcattga


 



catttccatctccatgcgaatggccttcttcccctct


 



gtagccccaaaccaccacccccaaaatcttcttctgt


 



ctttagctgaagatggtgttgaaggtgatagtttcag


 



ccactttggcgagttcctcagttgttctgggtctttc


 



ctccTgatccacattattcgactgtgtttgattttct


 



cctgtttatctgtctcattggcacccatttcattctt


 



agaccagcccaaagaacctagaagagtgaaggaaaat


 



ttcttccaccctgacaaatgctaaatgagaatcaccg


 



cagtagaggaaaatgatctggtgctgcgggagataga


 



agagaaaatcgctggagagatgtcactgagtaggtga


 



gatgggaaaggggtgacacaggtggaggtgttgccct


 



cagctaggaagacagacagttcacagaagagaagcgg


 



gtgtccgtggacatcttgcctcatggatgaggaaacc


 



gaggctaagaaagactgcaaaagaaaggtaaggattg


 



cagagaggtcgatccatgactaaaatcacagtaacca


 



accccaaaccaccatgttttctcctagtctggcacgt


 



ggcaggtactgtgtaggttttcaatattattggtttg


 



taacagtacctattaggcctccatcccctcctctaat


 



actaacaaaagtgtgagactggtcagtgaaaaatggt


 



cttctttctctatgaatctttctcaagaagatacata


 



actttttattttatcataggcttgaagagcaaatgag


 



aaacagcctccaacctatgacaccgtaacaaaatgtt


 



tatgatcagtgaagggcaagaaacaaaacatacacag


 



taaagaccctccataatattgtgggtggcccaacaca


 



ggccaggttgtaaaagctttttattctttgatagagg


 



aatggatagtaatgtttcaacctggacagagatcatg


 



ttcactgaatccttccaaaaattcatgggtagtttga


 



attataaggaaaataagacttaggataaatactttgt


 



ccaagatcccagagttaatgccaaaatcagttttcag


 



actccaggcagcctgatcaagagcctaaactttaaag


 



acacagtcccttaataactactattcacagttgcact


 



ttcagggcgcaaagactcattgaatcctacaatagaa


 



tgagtttagatatcaaatctctcagtaatagatgagg


 



agactaaatagcgggcatgacctggtcacttaaagac


 



agaattgagattcaaggctagtgttctttctacctgt


 



tttgtttctacaagatgtagcaatgcgctaattacag


 



acctctcagggaaggaattcacaaccctcagcaaaaa


 



ccaaagacaaatctaagacaactaagagtgttggttt


 



aatttggaaaaataactcactaaccaaacgcccctct


 



tagcaccccaatgtcttccaccatcacagtgctcagg


 



cctcaaccatgccccaatcacc


 


Seq ID No.25
GCACATGGTAGGCAAAGGACTTTGCTTCTCCCAGCAC


 



ATCTTTCTGCAGAGATCCATGGAAACAAGACTCAACT


 



CCAAAGCAGCAAAGAAGCAGCAAGTTCTCAAGTGATC


 



TCCTCTGACTCCCTCCTCCCAGGCTAATGAAGCCATG


 



TTGCCCCTGGGGGATTAAGGGCAGGTGTCCATTGTGG


 



CACCCAGCCCGAAGACAAGCAATTTGATCAGGTTCTG


 



AGCACTCCTGAATGTGGACTCTGGAATTTTCTCCTCA


 



CCTTGTGGCATATCAGCTTAAGTCAAGTACAAGTGAC


 



AAACAACATAATCCTAAGAAGAGAGGAATCAAGCTGA


 



AGTCAAAGGATCACTGCCTTGGATTCTACTGTGAATG


 



ATGACCTGGAAAATATCCTGAACAACAGCTTCAGGGT


 



GATCATCAGAGACAAAAGTTCCAGAGCCAGGTAGGGA


 



AACCCTCAAGCCTTGCAAAGAGCAAAATCATGCCATT


 



GGGTTCTTAACCTGCTGAGTGATTTACTATATGTTAC


 



TGTGGGAGGCAAAGCGCTCAAATAGCCTGGGTAAGTA


 



TGTCAAATAAAAAGCAAAAGTGGTGTTTCTTGAAATG


 



TTAGACCTGAGGAAGGAATATTGATAACTTACCAATA


 



ATTTTCAGAATGATTTATAGATGTGCACTTAGTCAGT


 



GTCTCTCCACCCCGCACCTGACAAGCAGTTTAGAATT


 



TATTCTAAGAATCTAGGTTTGCTGGGGGCTACATGGG


 



AATCAGCTTCAGTGAAGAGTTTGTTGGAATGATTCAC


 



TAAATTTTCTATTTCCAGCATAAATCCAAGAACCTCT


 



CAGACTAGTTTATTGACACTGCTTTTCCTCCATAATC


 



CATCTCATCTCCGTCCATCATGGACACTTTGTAGAAT


 



GACAGGTCCTGGCAgAGACTCaCAGATGCTTCTGAAA


 



CATCCTTTGCCTTCAAAGAATGAACAGCACACATACT


 



AAGGATCTCAGTGATCCACAAATTAGTTTTTGCCACA


 



ATGGTTCTTATGATAAAAGTCTTTCATTAACAGCAAA


 



TTGTTTTATAATAGTTGTTCTGCTTTATAATAATTGC


 



ATGCTTCACTTTCTTTTCTTTTCTTTTTTTTTCTTTT


 



TTTGCTTTTTAGTGCCGCAGGTgcagcatatgaaatt


 



tcccaggctaggggtcaaatcagaactacacctactg


 



gcctacgccacagccacagcaactcaggatctaagcc


 



atgtcggtgacctacactacagctcatggcaatgcca


 



gatccttaacccaatgagcgaggccagggatcgaacc


 



catgtcctcatggatactagtcaggctcattatccgc


 



tgagccataacaggaactcccGAGTTTGCTTTTTATC


 



AAAATTGGTACAGCCTTATTGTTTCTGAAAACCACAA


 



AATGAATGTATTCACATAATTTTAAAAGGTTAAATAA


 



TTTATGATATACAAGACAATAGAAAGAGAAAACGTCA


 



TTGCCTCTTTCTTCCACGACAACACGCCTCCTTAATT


 



GATTTGAAGAAATAACTACTGAGCATGGTTTAGTGTA


 



CTTCTTTCAGCAATTAGCCTGTATTCATAGCCATACA


 



TATTCAATTAAAATGAGATCATGATATCACACAATAC


 



ATACCATACAGCCTATAGGGATTTTTACAATCATCTT


 



CCACATGACTACATAAAAACCTACCTAAAAAAAAAAA


 



AAACCCTACTTCATCCTCCTATTGGCTGCTTTGTGCT


 



CCATTAAAAAGCTCTATCATAATTAGGTTATGATGAG


 



GATTTCCATTTTCTACCTTTCAAGCAACATTTCAATG


 



CACAGTCTTATATACACATTTGAGCCTACTTTTCTTT


 



TTCTTTCTTTTTTTGGTTTTTTTTTTTTTTTTTTTTT


 



TGGTCTTTTTGTCTTTTCTAAGgctgcatatggaggt


 



tcccaggctagctgtctaatcagaactatagctgctg


 



gcctacgccacatccacagcaatacaagatctgagcc


 



atgtctgcaacttacaccacagctcacagcaacggtg


 



gatccttaaaccactgagcaaggccagggatcaaacc


 



catAACTTCATGGCTCCTAGTTGGATTTGTTAACCAC


 



TGAGCCATGATGGCAACTCCTGAGCCTACTTTTCTAA


 



TCATTTCCAACCCTAGGACACTTTTTTAAGTTTCATT


 



TTTCTCCCCCCACCCCCTGTTTTCTGAAGtGTGTTTG


 



CTTCCACTGGGTGACTTCACtCCCAGGATCTCATCTG


 



CAGGATACTGCAGCTAAGTGTATGAGCTCTGAATTTG


 



AATCCCAACTCTGCCACTCAAAGGGATAGGAGTTTCC


 



GATGTGGCCCAATGGGATCAGTGGCATCTCTGCAGTG


 



CCAGGACGCaggttccatccctggcccagcacagtgg


 



gttaagaatctggCATTGCTGCAGCTGAGGCATAGAT


 



TTCAATTGTGCCTCAgATCTGATCCTTGGCCCAAGGA


 



CTGCATATGCCTCAGGGCAACCAAAAAAGAGAAAAGG


 



GGGGTGATAGCATTAGTTTCTAGATTTGGGGGATAAT


 



TAAATAAAGTGATCCATGTACAATGTATGGCATTTTG


 



TAAATGCTCAACAAATTTCAACTATTATggagttccc


 



atcatggctcagtggaagggaatctgattagcatcca


 



tgaggacacaggtCCAACCCCGACCTTGCTCAGTGGG


 



CATTGCTGTGAGCTGTGGCATGGGTTACAGACGAAGC


 



TCGGATCTGGCATTGCTGTGGCTGTGGTGTAAGCCAg


 



CAActacagctctcattcagcccctagcctgggaacc


 



tccatatgccTAAAAGACAAAAAATAAAATTTAAATT


 



AAAAATAAAGAAATGTTAACTATTATGATTGgTACTG


 



CTTGCATTACTGCAAAGAAAGTCACTTTCTATACTCT


 



TTAATATCTTAGTTGACTGTGTGCTCAGTGAACTATT


 



TTGGACACTTAATTTCCACTCTCTTCTATCTCCAACT


 



TGACAACTCTCTTTCCTCTCTTCTGGTGAGATCCACT


 



GCTGACTTTGCTCTTTAAGGCAACTAGAAAAGTGCTC


 



AGTGACAAAATCAAAGAAAGTTACCTTAATCTTCAGA


 



ATTACAATCTTAAGTTCTCTTGTAAAGCTTACTATTT


 



CAGTGGTTAGTATTATTCCTTGGTCCCTTACAACTTA


 



TCAGCTCTGATCTATTGCTGATTTTCAACTATTTATT


 



GTTGGAGTTTTTTCCTTTTTTCCCTGTTCATTCTGCA


 



AATGTTTGCTGAGCATTTGTCAAGTGAAGATACTGGA


 



CTGGGCCTTCCAAATATAAGACAATGAAACATCGGGA


 



GTTCTCATTATGGTGCAGCAGAaacgaatccaactag


 



gaaatgtgaggttgcaggttcgatccctgcccttgct


 



cagtgggttaaggatccagcattaccgtgagctgtgg


 



tgtaggttgcagacgtggctcagatcctgcgttgctg


 



tggctgtggcataggctggcagctctagctctgattc


 



gaccgctagcctgggaacctccatGCGCCCCGAGTGC


 



AGCCCTTAAAAAGCAAAAAAAAAAGAAAGAAAGAAAA


 



AGACAATGAAACATCAAACAGCTAACAATCCAGTAGG


 



GTAGAAAGAATCTGGCAACAGATAAGAGCGATTAAAT


 



GTTCTAGGTCCAGTGACCTTGCCTCTGTGCTCTACAC


 



AGTCGTGCCACTTGCTGAGGGAGAAGGTCTCTCTTGA


 



GTTGAGTCCTGAAAGACATTAGTTGTTCACAAACTAA


 



TGCCAGTGAGTGAAGGTGTTTCCAAGCAGAGGGAGAG


 



TTTGGTAAAAAGCTGGAAGTCACAGAAAGACTCTAAA


 



GAGTTTAGGATGGTGGGAGCAACATACGCTGAGATGG


 



GGCTGGAAGGTTAAGAGGGAAACAACTATAGTAAGTG


 



AAGCTGGACTCACAGCAAAGTGAGGACCTCAGCATCC


 



TTGATGGGGTTACCATGGAAACACCAAGGCACACCTT


 



GATTTCCAAAACAGCAGGCACCTGATTCAGCCCAATG


 



TGACATGGTGGGTACCCCTCTAGCTCTACCTGTTCTG


 



TGACAACTGACAACCAACGAAGTTAAGTCTGGATTTT


 



CTACTCTGCTGATCCTTGTTTTTGTTTCACACGTCAT


 



CTATAGCTTCATGCCAAAATAGAGTTCAAGGTAAGAC


 



GCGGGCCTTGGTTTGATATACATGTAGTCTATCTTGT


 



TTGAGACAATATGGTGGCAAGGAAGAGGTTCAAACAG


 



GAAAATACTCTCTAATTATGATTAACTGAGAAAAGCT


 



AAAGAGTCCCATAATGACACTGAATGAAGTTCATCAT


 



TTGCAAAAGCCTTCCCCCCCCCCCAGGAGACTATAAA


 



AAAGTGCAATTTTTTAAATGAACTTATTTACAAAACA


 



GAAATAGACTCACAGACATAGGAAACGAACAGATGGT


 



TACCAAGGGTGAAAGGGAGTAGGAGGGATAAATAAGG


 



AGTCTGGGGTTAGCAGATACACCCCAGTGTACACAAA


 



ATAAACAACAGGGACCTACTATATAGCACAGGGAACT


 



ATATGCAGTAGCTTACAATAACCTATAATGGAAAAGA


 



ATGTGAAAAAGAATATATGTATGCGTGTGTGTGTAAC


 



TGAATCACTTTGCTGTAACCTGAATCTAACATAACAT


 



TGTAAATCAACTACAGTTTTTTTTTTTTTTAAGTGCA


 



GGGTTTTGGTGTTTTTTTTTTTTCATTTTTGTTTTTGT


 



TTTTGTTTTTTGCTTTTTAGGGCCACACCCAGACAT


 



ATGGGGGTTCCCAGGctAGGGGTcTAaTTAGAGcTAC


 



AGtTGCCGGCTTGCAccacagccacagcaacatcaga


 



tccgagccgcacttgcgacttacaccacagctcatgg


 



caataccagatccttaacccactgagcaaggcccagg


 



gatcgtacccgcaacctcatggttcctagtcagattc


 



attTCTGCTGCGCTACAATGGGAACTCCAAGTGCAGT


 



TTTTTGTAATGTGCTtGTCTTTCTTTGTAATTCATAT


 



TCATCCTACTTCCCAATAAATAAATAAATACATAAAT


 



AATAAACATACCATTGTAAATCAACTACAATTTTTTT


 



TAAATGCAGGGTTTTTGTTTTTTGTTTTTTGTTTTGT


 



CTTTTTGCCTTTTCTAgggccgctcccatggcatatg


 



gaggttcccaggctaggggtcgaatcggagctgtagc


 



caccggcctacgccagagccacagcaacgcgggatcc


 



gagccgcgtctgcaacctacaccacagctcacggcaa


 



cgccggatcgttaacccactgagcaagggcagggatc


 



gaacctgcaacctcatggttcctagtcagattcgtta


 



actactgagccacaacggaaacTCCTAAAGTGCAGTT


 



TTTAAATGTGCTTGTCTTTCTTTGTAATTTACACTCA


 



ACCTACTTCCCAATAAATAAATAAATAAACAAATAAA


 



TCATAGACATGGTTGAATTCTAAAGGAAGGGACCATC


 



AGGCCTTAGACAGAAATACGTCATCTTCTAGTATTTT


 



AAAACACACTAAAGAAGACAAACATGCTCTGCCAGAG


 



AAGCCCAGGGCCTCCACAGCTGCTTGCAAAGGGAGTT


 



AGGCTTCAGTAGCTGACCCAAGGCTCTGTTCCTCTTC


 



AGGGAAAAGGGTTTTTGTTCAGTGAGACAGCAGACAG


 



CTGTCACTGTGgtggacgttcggccaaggaaccaagc


 



tggaactcaaacGTAAGTCAATCCAAACGTTCCTTCC


 



TTGGCTGTCTGTGTCTTACGGTCTCTGTGGCTCTGAA


 



ATGATTCATGTGCTGACTCTCTGAAACCAGACTGACA


 



TTCTCCAGGGCAAAACTAAAGCCTGTCATCAAACcGG


 



AAAACTGAGGGCACATTTTCTGGGCAGAACTAAGAGT


 



CAGGCACTGGGTGAGGAAAAACTTGTTAGAATGATAG


 



TTTCAGAAACTTACTGGGAAGCAAAGCCCATGTTCTG


 



AACAGAGCTCTGCTCAAGGGTCAGGAGGGGAACCAGT


 



TTTTGTACAGGAGGGAAGTTGAGACGAACCCCTGTGT


 



Atatggtttcggcgcggggaccaagctggagctcaaa


 



cGTAAGTGGCTTTTTCCGACTGATTCTTTGCTGTTTC


 



TAATTGTTGGTTGGCTTTTTGTCCATTTTTCAGTGTT


 



TTCATCGAATTAGTTGTCAGGGACCAAACAAATTGCC


 



TTCCCAGATTAGGTACCAGGGAGGGGACATTGCTGCA


 



TGGGAGACCAGAGGGTGGCTAATTTTTAACGTTTCCA


 



AGCCAAAATAACTGGGGAAGGGGGCTTGCTGTCCTGT


 



GAGGGTAGGTTTTTATAGAAGTGGAAGTTAAGGGGAA


 



ATCGCTATGGTtcacttttggctcggggaccaaagtg


 



gagcccaaaattgaGTACATTTTCCATCAATTATTTG


 



TGAGATTTTTGTCCTGTTGTGTCATTTGTGCAAGTTT


 



TTGACATTTTGGTTGAATGAGCCATTCCCAGGGACCC


 



AAAAGGATGAGACCGAAAAGTAGAAAAGAGCCAACTT


 



TTAAGCTGAGCAGACAGACCGAATTGTTGAGTTTGTG


 



AGGAGAGTAGGGTTTGTAGGGAGAAAGGGGAACAGAT


 



CGCTGGCTTTTTCTCTGAATTAGCCTTTCTCATGGGA


 



CTGGCTTCAGAGGGGGTTTTTGATGAGGGAAGTGTTC


 



TAGAGCCTTAACTGTGGgttgtgttcggtagcgggac


 



caagctggaaatcaaaCGTAAGTGCACTTTTCTACTC


 



C






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
CCTTCCTCCTGCACCTGTCAACTCCCAATAAACCGTC


No. 28



 



CTCCTTGTCATTCAGAAATCATGCTCTCCGCTCACTT


 



GTGTCTACCCATTTTCGGGCTTGCATGGGGTCATCCT


 



CGAAGGTGGAGAGAGTCCCCCTTGGCCTTGGGGAAGT


 



CGAGGGGGGCGGGGGGAGGCCTGAGGCATGTGCCAGC


 



GAGGGGGGTCACCTCCACGCCCCTGAGGACCTTCTAG


 



AACCAGGGGCGTGGGGCCACCGCCTGAGTGGAAGGCT


 



GTCCACTTTTCCCCCGGGCCCCCAGGCTCCCTCCTCC


 



GTGTGGACCTTGTCCACCTCTGACTGGCCCAGCCACT


 



CATGCATTGTTTCCCCGAAACCCCAGGACGATAGCTC


 



AGCACGCGACAGTGTCCCCCTCTGAGGGCCTCTGTCC


 



ATTTCAGGACGACCCGCATGTACAGCGTGACCACTCT


 



GCTCACGCCCACTCACCACGTCCTAGAGCCCCACCCC


 



CAGCCCCATCCTTAGGGGCACAGCCAGcTCCGACCGC


 



CCCGGGGACACCACCCTCTGCCCCTTcCCCAGGCCCT


 



CCCTGTCACACGCACCACAGGGCCCTCCGTCCCGAGA


 



CCCTGCTCCCTCATCCCTCGGTCCCCTCAGGTAGCCT


 



TCCACCCGCGTGTGTCCCGAGGTCCCAGATGCAGCAA


 



GGCCCCTGGGACAACGCCAGATCTCTGCTCTcCCCGA


 



CCCCTCAGAAGCCAGCCCACGCCTGGCCCCACCACCA


 



CTGCCTAACgTCCAAGTGTCCATAGGCCTCGGGACCT


 



CCAAGTCCAGGTTCTGCCTCTGGGATTCCGCCATGGG


 



TCTGCCTGGGAAATGATGCACTTGGAGGAGCTCAGCA


 



TGGGATGCGGGACCTTGTCTCTAGGCGCTcCCTCAGG


 



ATCCCACAGCTGCCCTGTGAGACACACACACACACAC


 



ACACACACACACACACACACACACACACACAAACACG


 



CATGCACGCACGCCGGCACACACGCTATTGCAGAGAT


 



GGCCACGGTAGCTGTGCCTCGAGGCCGAGTGGAGTGT


 



CTAGAACTCTCGGGGGTCCCCTCTGCAGACGACACTG


 



CTCCATCCCCCCCGTGCCCTGAAGGGCTCCTCACTCT


 



CCCATCAGGATCTCTCCAAGCTGCTGACCTGGAGAGG


 



AAGGGGCCTGGGACAGGCGGGGACACTCAGACCTCCC


 



TGCTGCCCCTCCTCTGCCTGGGCTTGGACGGCTCCCC


 



CCTTCCCACGGGTGAAGGTGCAGGTGGGGAGAGGGCA


 



CCCCCCTCAGCCTCCCAGACCCAGACCAGCCCCCGTG


 



GCAGGGGCAGCCTGTGAGCCTCCAGCCAGATGCAGGT


 



GGCCTGGGGTGGGGGGTGGAGGGGGCGGGAGGTTTAT


 



GTTTGAGGCTGTATCACTGTGTAATATTTTCGGCGGT


 



GGGACCCATCTGACCGTCCTCGGTGAGTCTCCCCTTT


 



TCTCTCCTCCTTGGGGATCCGAGTGAAATCTGGGTCG


 



ATCTTCTCTCCGTTCTCCTCCGACTGGGGCTGAGGTC


 



TGAACCTCGGTGGGGTCCGAAGAGGAGGCCCCTAGGC


 



CAGGCTCCTCAGCCCCTCCAGCCCGACcgGCCCTCTT


 



GACACAGGGTCCAGCTAAGGGCAGACATGGAGGCTGC


 



TAGTCCAGGGCCAGGCTCTGAGACCCAAGGGCGCTGC


 



CCAAGGAACCCTTGCCCCAGGGACCCTGGGAGCAAAG


 



CTCCTCACTCAGAGCCTGCAGCCCTGGGGTCTGAGGA


 



CAAGGAGGGACTGAGGACTGGGCGTGGGGAGTTCAGG


 



CGGGGACACCAGGTCCAGGGAGGTGACAAAGGCGCTG


 



GGAGGGGGCGGACGGTGCCGGGGACTCCTCCTGGGCC


 



CTGTGGGCTCGGGGTCCTTGTGAGGACCCTGAGGGAC


 



TGAGGGGCCCCTGGGCCTAGGGACTTGCAgTgAGGGA


 



GGCAGGGAGTGTCCCTTGAGAACGTGGCCTCCGCGGG


 



CTGGGTCCCCCTCGTGCTCCCAGCC*GGGAGGACACC


 



CCAGAGCAAGCGCCCCAGGTGGGCGGGGAGGGTCTCC


 



TCACAGGGGCAGCTGACAGATAGAGGCCCCCGCCAGG


 



CAGATGCTTGATCCTGGCAgTTATACTGGGTTC**GC


 



ACAACTTTCCCTGAACAAGGGGCCCTCCGAACAGACA


 



CAGACGCAACCCAGTCGACCcaggCTCAGCACAgAAA


 



ATGCACTGACACCCAAAACCCTCATCTggggGCCTGG


 



CCGGcAtCCCGCCCCAGGACCCAAGGCCCCTGCCCCC


 



TGGCAGCCCTGGACACGGTCCTCTGTGGGCGGTGGGG


 



TCgGGGCTGTGGTGACGGTGGCATCGGGGAGCCTGTG


 



CCCCCTCCCTGAAAGGGCGGAGAGGCTCAAGAGGGGA


 



GAGAAATGTCCTCCCCTAGGAAGACCTCGGACGGGGG


 



CGGGGGGGTGGTCTCCGACAGACAGATGCCCGGGACC


 



GACAGACCTGCCGAGGGAAGAGGGCACCTCGGTCGGG


 



TTAGGCTCCAGGCAGCACGAGGGAGCGAGGCTGGGAG


 



GGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAGACT


 



TCAGCAGGCCCCCAGCTCCGGGCTTCGGGCTCTGAGA


 



TGCTCGGACGCAAGGTGAGTGACCCCACCTGTGGCTG


 



ACCTGACCTCAgGGgGACAAGGCTCAGCCTGGGACTC


 



TGTGTCCCCATCGCCTGcACAGGGGATTCCCCTGATG


 



GACACTGAGCCAACGACCTCCCGTCTCTCCCCGACCC


 



CCAGGTCAGCCCAAgGCCaCTCCCACGGTCAACCTCT


 



TCCCGCCCTCCTCTGAGGAGCTCGGCACCAACAAGGC


 



CACCCTGGTGTGTCTAATAAGTGACTTCTACCCGGGC


 



GCCGTGACGGTGACCTGGAAGGCAGGCGGCACCACCG


 



TCACCCAGGGCGTGGAGACCACCAAGCCCTCGAAACA


 



GAGCAACAACAAGTACGCGGCCAGCAGCTACCTGGCC


 



CTGTCCGCCAGTGACTGGAAATCTTCCAGCGGCTTCA


 



CCTGCCAGGTCACCCACGAGGGGACCATTGTGGAGAA


 



GACAGTGACGCCCTCCGAGTGCGCCTAGGTCCCTGGG


 



CCCCCACCCTCAGGGGCCTGGAGCCACAGGACCCCCG


 



CGAGGGTCTCCCCGCGACCCTGGTCCAGCCCAGCCCT


 



TCCTCCTGCACCTGTCAACTCCCAATAAACCGTCCTC


 



CTTGTCATTCAGAAATCATGCTCTCCGCTCACTTGTG


 



TCTACCCATTTTCGGGCTTGCATGGGGTCATCCTCGA


 



AGGTGGAGAGAGTCCCCCTTGGCCTTGGGgAAATCGA


 



GGGGGGCGGGGGGAGGCCTGAGGCATGTGCCAGCGAG


 



GGGGGTCACCTCCACGCCCCTGAGGACCTTCTAGAAC


 



CAGGGGCGTGGGGCCACCGCCAGAGTGGAAGGCTGTC


 



CACTTTTCCCCCGGGCCCCCAGGCTCCCTCCTCCGTG


 



TGGACCTTGTCCACCTCTGACTGGCCCAGCCACTCAT


 



GCATTGTTTCCCCGAAACCCCAGGACGATAGCTCAGC


 



ACGCGACAGTGTCCCCCTCTGAGGGCCTCTGTCCATT


 



TCAGGACGACCCGCATGTACAGCGTGACCACTCTGCT


 



CACGCCCACTCACCACGTCCTAGAGCCCCACCCCCAG


 



CCCCATCCTTAGGGGCACAGCCAGCTCCGACCGCCCC


 



GGGGACACCACCCTCTGCCCCTTCCCCAGGCCCTCCC


 



TGTCACACGCACCACAGGGCCCTCCGTCCCGAGACCC


 



TGCTCCCTCATCCCTCGGTCCCCTCAGGTAGCCTTCC


 



ACCCGCGTGTGTCCCGAGGTCCCAGATGCAGCAAGGC


 



CCCTGGGACAACGCCAGATCTCTGCTCTCCCCGACCC


 



TCAGAAGCCAGCCCACGCCTGGCCCACCACCACTGCC


 



TAACGTCCAAGTGTCCATAGGCTCGGGAcCTCcAaGT


 



CCAGGTTCTGCCTCTGGGATTCCGCCATGGGTCTGCC


 



TGGAATGATGCACTTGGAGgAgCTCAGcATGGGATGc


 



GGAACTTGTCTAGcGCTCCTCAGATCCAcAGcTGCCT


 



GtGAgAcacacacacacacacacacacaccAAAcaCG


 



cATGCACGCACGCCGGCACACACGCTATTACAGAGAT


 



GGCCACGGTAGCTGTGCCTCGAGGCCGAGTGGAGTGT


 



CTAGAACTCTCGGGGGTCCCCTCTGCAGACGACACTG


 



CTCCATCCCCCCCGTGCCCTGAAGGGCTCCTCACTCT


 



CCCATCAGGATCTCTCCAAGCTGCTGACCTGGAGAGG


 



AAGGGGCCTGGGACAGGCGGGGACACTCAGACCTCCC


 



TGCTGCCCCTCCTCTGCCTGGGCTTGGACGGCTCCCC


 



CCTTCCCACGGGTGAAGGTGCAGGTGGGGAGAGGGCA


 



CCCCCCTCACCCTCCCAGACCCAGACCAGCCCCCGTG


 



GCAGGGGCAGCCTGTGAGCCTCCAGCCAGATGCAGGT


 



GGCCTGGGGTGGGGGGTGGAGGGGGCGGGAGGTTTAT


 



GTTTGAGGCTGTATTCATCTGTGTAATATttTCGGCG


 



GTGGGACCCATCTGACCGTCCTCGGTGAGTCTCCCCT


 



tttctttcctccttggggatccgagtgaaATcTGGGT


 



CGATCTTCTCTCCGTTCTCCTCCGACTGGGGCTGAGG


 



TCTGAACCTCGGTgGGGTCCGAAGAGGAGGCCCCTAG


 



GCC*GGCTCcTCAGCCCCTCCAGCCCGACCCGCCCTC


 



TTGACACAGGGTCCAGCTAAGGGCAGACAT***GGCT


 



GCTAGTCCAGGGCCAGGCTcTGAGACCCAAGGGCGCT


 



GCCCAAGGAACCCTTGCCCCAGGGACCCTGGGAGCAA


 



AGCTCCTCACTCAGAGCCTGCAGCCCTGGgGTGTGAG


 



GACAAGGAGGGACTGAGGACTGGGCGTGGGGAGTTCA


 



CGGCgGGGACACCGGGTCAGGGAGGTGACAAAGGCGC


 



TGGGAGGGGGCGGACGGTGCCGGAGACTCCTCCTGGG


 



CCCTGTGGGCTCGTGGTCCTTGTGAGGACCCTGAGGG


 



*CTGAGGGGCCCCTGGGCCTAGGGACTTGCAGTGAGG


 



GAGGCAGGGAGTGTCCCTTGAGAACGTGGCCTCCGCG


 



GGCTGGGTCCCCCTCGTGCTCCCAGCAGGGAGGACAC


 



CCCAGAGCAAGCGCCCCAGGTGGGCGGGGAGGGTCTC


 



CTCACAGGGGCAGCTGACAGATAGAC*GgccCCCGCC


 



AGACAGATGCTTGATCCTGGTCag***TACTGGGTTC


 



GCcACTTCCCTGAACAGGGGCCCTCCGAACAGACACA


 



GACGCAGACCaggCTCAGCACAgAAAATGCACTGACA


 



CCCAAAACCCTCATCTGggGGCCTGGCCGGCATCCCG


 



CCCCAGGACCCAAGGCCCCTGCCCCCTGGCAGCCCTG


 



GACACGGTCCTCTGTGGGCGGTGGGGTCgGGGCTGTG


 



GTGACGGTGGCATCGGGGAGCCTGTGCCCCCTCCCTG


 



AAAGGGCGGAGAGGCTCAAGAGGGGACAGAAATGTCC


 



TCCCCTAGGAAGACCTCGGACGGGGGCGGGGGGGTGG


 



TCTCCGACAGACAGATGCCCGGGACCGACAGACCTGC


 



CGAGGGAAGAGGGCACCTCGGTCGGGTTAGGCTCCAG


 



GCAGCACGAGGGAGCGAGGCTGGGAGGGTGAGGACAT


 



GGGAGCCTGAGGAGGAGCTGGAGACTTCAGCAGGCCC


 



CCAGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACGC


 



AAGGTGAGTGACCCCACCTGTGGCTGACCTGACCTGA


 



CCtCAGGGGGACAAGGCTCAGCCTGGGACTCTgTGTC


 



CCCATCGCCTGCACAGGGGATTCCCCTGATGGACACT


 



GAGCCAACGACCTCCCGTCTCTCCCCGACCCCCAGGT


 



CAGCCCAAGGCCACTCCCACGGTCAACCTCTTCCCGC


 



CCTCCTCTGAGGAGCTCGGCACCAACAAGGCCACCCT


 



GGTGTGTCTA


 


Seq ID 
GCCACGCCCACTCCATCATGCGGGGAGGGGATGGGCA


No. 32



 



GACCCTCCAGAAAGAAGCTCCCTGGGGTGCAGGTTAA


 



CAGCTTTCCCAGACACAGCCAGTACTAGAGTGAGGTG


 



AATAAGACATCCTCCTTGCTTGTGAAATTTAGGAAGT


 



GCCCCCAAACATCAGTCATTAAGATAAATAATATTGA


 



ATGCACTTTTTTTTTTTTATTTTTTTTTTTTGCTTTT


 



TAGGGCCTAATCTGCAGCatatggaagttcccaggct


 



acaagtcgaaccagagctgcagctgccagcctacatc


 



acagccacagcaacaccagatccgagccacatctgtg


 



actaacactgcagttcacagcaacgccagatccttaa


 



cccattgagtgaggccagggatcaaacccacatcctc


 



atggatactagtctggttcgtaaaccactgagccaCA


 



AGGGGAACTCCTGAATGCAATATTTTTGAAAATTGAA


 



ATTAAATCTGTCACTCTTTCACTTAAGAGTCCCCTTA


 



GATTGGGGAAAATTTAAATATCTGTCATCTTAGTGCA


 



TCTTTGCTCATATGATGTGAATAAAATCCCAAAATCC


 



ATATGAATGAAGCATCAAAATGTACATGAAGTCAGCC


 



TGACCCTGCACTGCCCTCACTTGCCTCATGTACCCCC


 



CACCTCAAAGGAAGATGCAGAAAGGAGTCCAGCCCCT


 



ACACCGCCACCTGCCCCCACCACTGGAGCCCCTCAGG


 



TCTCCCACCTCCTTTTCTGAGCTTCAGTCTTCCTGTG


 



GCATTGCCTACCTCTACAGCTGCCCCCTACTAGGCCC


 



TCCCCCTGGGGCTGAGCTCCAGGCACTGGACTGGGAA


 



AGTTAGAGGTTAAAGCATGGAAAATTCCCAAAGCCAC


 



CAGTTCCAGGCTGCCCCCCACCCCACCGCCACGTCCA


 



AAAAGGGGCATCTTCCCAGATCTCTGGCTGGTATTGG


 



TAGGACCCAGGACATAGTCTTTATACCAATTCTGCTG


 



TGTGTCTTAGGAAAGAaactctccctctctgtgcttc


 



agtttcctcatcaataaaAGGAGCAGGCCAGGTTGGA


 



GGGTCTGTGACGTCTGCTGAAGCAGCAGGATTCTCTC


 



TCCTTTTGCTGGAGGAGAACTGATCCTTCACCCCCAG


 



GATCAACAGAGAAGCCAAGGTCTTCAGCCTTCCTGGG


 



GACCCCTCAGAGGGAACTCAGGGCCACAGAGCCAGAC


 



CCTGATGCCAGAACCTTTGTCATATGCCCAGACGGAG


 



ACTTCATCCCCCTCCTCCTCAGACCCTCCAGGCCCCA


 



ACAGTGAGATGCTGAAGATATTAAGAGAAGGGCAAGT


 



CAGcTTAAGTTTGGGGGTAGAGGGGAACAGGGAGTGA


 



GGAGATCTGGCCTGAGAGATAGGAGCCCTGGTGGCCA


 



CAGGAGGACTCTTTGGGTCCTGTCGGATGGACACAGG


 



GCGGCCCGGGGGCATGTTGGAGCCCGGCTGGTTCTTA


 



CCAGAGGCAGGGGGCACCCTCTGACACGGGAGCAGGG


 



CATGTTCCATACATGACACACCCCTCTGCTCCAGGGC


 



AGGTGGGTGGCGGCACAGAGGAGCCAGGGACTCTGAG


 



CAAGGGGTCCACCAGTGGGGCAGTTGGATCCAGACTT


 



CTCTGGGCCAGCGAGAGTCTAGCCCTCAGCCGTTCTC


 



TGTCCAGGAGGGGGGTGGGGCAGGCCTGGGCGGCCAG


 



AGCTCATCCCTCAAGGGTTCCCAGGGTCCTGCCAGAC


 



CCAGATTTCCGACCGCAGCCACCACAAGAGGATGTGG


 



TCTGCTGTGGCAGCTGCCAAGACCTTGCAGCAGGTGC


 



AGGGTGGGGGGGTGGGGGCACCTGGGGGCAGCTGGGG


 



TCACTGAGTTCAGGGAAAACCCCTTTTTTCCCCTAAA


 



CCTGGGGCCATCCCTAGGGGAAACCACAACTTCTGAG


 



CCCTGGGCAGTGGCTGCTGGGAGGGAAGAGCTTCATC


 



CTGGACCCTGGGGGGGAACCCAGCTCCAAAGGTGCAA


 



GGGGCCCAGGTCCAAGGCTAGAGTGGGCCAAGCACCG


 



CAATGGCCAGGGAGTGGGGGAGGTGGAGCTGGACTGG


 



ATCAGGGCCTCCTTGGGACTCCCTACACCCTGTGTGA


 



CATGTTAGGGTACCCACACCCCATCACCAGTCAGGGC


 



CTGGCCCATCTCCAGGGCCAGGGATGTGCATGTAAGT


 



GTGTGTGAGTGTGTGTGTGTGGTGTAGTACACCCCTT


 



GGCATCCGGTTCCGAGGCCTTGGGTTCCTCCAAAGTT


 



GCTCTCTGAATTAGGTCAAACTGTGAGGTCCTGATCG


 



CCATCATCAACTTCGTTCTCCCCACCTCCCATCATTA


 



TCAAGAGCTGGGGAGGGTCTGGGATTTCTTCCCACCC


 



ACAAGCCAAAAGATAAGCCTGCTGGTGATGGCAGAAG


 



ACACAGGATCCTGGGTCAGAGACAAAGGCCAGTGTGT


 



CACAGCGAGAGAGGCAGCCGGACTATCAGCTGTCACA


 



GAGAGGCCTTAGTCCGCTGAACTCAGGCCCCAGTGAC


 



TCCTGTTCCACTGGGCACTGGCCCCCCTCCACAGCGC


 



CCCCAGGCCCCAGGGAGAGGCGTCACAGCTTAGAGAT


 



GGCCCTGCTGAACAGGGAACAAGAACAGGTGTGCCCC


 



ATCCAGCGCCCCAGGGGTGGGACAGGTGGGCTGGATT


 



TGGTGTGAAGCCCTTGAGCCCTGgAACCCAAcCACAG


 



CAgGGCAGTTGGTAGATGCCATTTGGGGAGAGGCCCC


 



AGGAGTAAGGGCCATGGGCCCTTGAGGGGGCCAGGAG


 



CTGAGGACAGGGACAGAGACGGCCCAGGCAGAGGACA


 



GGGCCATGAGGGGTGCACTGAGATGGCCACTGCCAGC


 



AGGGGCAGCTGCCAACCCGTCCAGGGAACTTATTCAG


 



CAGTCAGCTGGAGGTGCCATTGACCCTGAGGGCAGAT


 



GAAGCCCAGGCCAGGCTAGGTGGGCTGTGAAGACCCC


 



AGGGGACAGAGCTCTGTCCCTGGGCAGCACTGGCCTC


 



TCATTCTGCAGGGCTTGACGGGATCCCAAGGCCTGCT


 



GCCCCTGATGGTAGTGGCAGTACCGCCCAGAGCAGGA


 



CCCCAGCATGGAAACCCCAACGGGACGCAGCCTGCGG


 



AGCCCACAAAACCAGTAAGGAGCCGAAGCAGTCATGG


 



CACGGGGAGTGTGGACTTCCCTTTGATGGGGCCCAGG


 



CATGAAGGACAGAATGGGACAGCGGCCATGAGCAGAA


 



AATCAGCCGGAGGGGATGGGCCTAGGCAGACGCTGGC


 



TTTATTTGAAGTGTTGGCATTTTGTCTGGTGTGTATT


 



GTTGGTATTGATTTTATTTTAGTATGTCAGTGACATA


 



CTGACATATTATGTAACGACATATTATTATGTGTTTT


 



AAGAAGCACTCCAAGGGAACAGGCTGTCTGTAATGTG


 



TCCAGAGAAGAGAGCAAGAGCTTGGCTCAGTCTCCCC


 



CAAGGAGGTCAGTTCCTCAACAGGGGTCCTAAATGTT


 



TCCTGGAGCCAGGCCTGAATCAAGGGGgTCATATCTA


 



CACGTGGGGCAGACCCATGGACCATTTTCGGAGCAAT


 



AAGATGGCAGGGAGGATACCAAGCTGGTCTTACAGAT


 



CCAGGGCTTTGACCTGTGACGCGGGCGCTCCTCCAGG


 



CAAAGGGAGAAGCCAGCAGGAAGCTTTCAGAACTGGG


 



GAGAACAGGGTGCAGACCTCCAGGGTCTTGTACAACG


 



CACCCTTTATCCTGGGGTCCAGGAGGGGTCACTGAGG


 



GATTTAAGTGGGGGACCATCAGAACCAGGTTTGTGTT


 



TTGGAAAAATGGCTCCAAAGCAGAGACCAGTGTGAGG


 



CCAGATTAGATGATGAAGAAGAGGCAGTGGAAAGTCG


 



ATGGGTGGCCAGGTAGCAAGAGGGCCTATGGAGTTGG


 



CAAGTGAATTTAAAGTGGTGGCACCAGAGGGCAGATG


 



GGGAGGAGCAGGCACTGTCATGGACTGTCTATAGAAA


 



TCTAAAATGTATACCCTTTTTAGCAATATGCAGTGAG


 



TCATAAAAGAACACATATATATTTAAATTGTGTAATT


 



CCACTTCTAAGGATTCATCCCAAGGGGGGAAAATAAT


 



CAAAGATGTAACCAAAGGTTTACAAACAAGAACTCAT


 



CATTAATCTTCCTTGTTGTTATTTCAACGATATTATT


 



ATTATTACTATTATTATTATTATTATTttgtcttttt


 



gcattttctagggccactcccacggcatagagaggtt


 



cccaggctaggggtcaaatcggagctacagctgccgg


 



cctacgccagagccacagcaacgcaggatctgagcca


 



cagcaatgcaggatctacaccacagctcatggtaacg


 



ctggatccttaacccaatgagtgaggccagggatcga


 



acctgtaacttcatggttcctagtcggattcattaac


 



cactgagccacgacaggaactccAACATTATTAATGA


 



TGGGAGAAAACTGGAAGTAACCTAAATATCCAGCAGA


 



AAGGGTGTGGCCAAATACAGCATGGAGTAGCCATCAT


 



AAGGAATCTTACACAAGCCTCCAAAATTGTGTTTCTG


 



AAATTGGGTTTAAAGTACGTTTGCATTTTAAAAAGCC


 



TGCCAGAAAATACAGAAAAATGTCTGTGATATGTCTC


 



TGGCTGATAGGATTTTGCTTAGTTTTAATTTTGGCTT


 



TATAATTTTCTATAGTTATGAAAATGTTCACAAGAAG


 



ATATATTTCATTTTAGCTTCTAAAATAATTATAACAC


 



AGAAGTAATTTGTGCTTTAAAAAAATATTCAACACAG


 



AAGTATATAAAGTAAAAATTGaggagttcccatcgtg


 



gctcagtgattaacaaacccaactagtatccatgagg


 



atatggatttgatccctggccttgctcagtgggttga


 



ggatccagtgttgctgtgagctgtggtgtaggttgca


 



gacacagcactctggcgttgctgtgactctggcgtag


 



gccggcagctacagctccatttggacccttagcctgg


 



gaacctccatatgcctgagatacggcccTAAAAAGTC


 



AAAAGCCAAAAAAATAGTAAAAATTGAGTGTTTCTAC


 



TTACCACCCCTGCCCACATCTTATGCTAAAACCCGTT


 



CTCCAGAGACAAACATCGTCAGGTGGGTCTATATATT


 



TCCAGCCCTCCTCCTGTGTGTGTATGTCCGTAAAACA


 



CACACACACACACACACACGCACACACACACACACGT


 



ATCTAATTAGCATTGGTATTAGTTTTTCAAAAGGGAG


 



GTCATGCTCTACCTTTTAGGCGGCAAATAGATTATTT


 



AAACAAATCTGTTGACATTTTCTATATCAACCCATAA


 



GATCTCCCATGTTCTTGGAAAGGCTTTGTAAGACATC


 



AACATCTGGGTAAACCAGCATGGTTTTTAGGGGGTTG


 



TGTGGATTTTTTTCATATTTTTTAGGGCACACCTGCA


 



gcatatggaggttcccaggctaggggttgaatcagag


 



ctgtagctgccggcctacaccacagccacagcaacgc


 



cagatccttaacccactgagaaaggccagggattgaa


 



cctgcatcctcatggATGCTGGTCAGATTTATTTCTG


 



CTGAGCCACAACAGGAACTCCCTGAACCAGAATGCTT


 



TTAACCATTCCACTTTGCATGGACATTTAGATTGTTT


 



CCATTTAAAAATACAAATTACAaggagttcccgtcgt


 



ggctcagtggtaacgaattggactaggaaccatgagg


 



tttcgggttcgatccctggccttgctcggtgggttaa


 



ggatccagcattgatgtgagatatggtgtaggtcgca


 



gacgtggctcggatcccacgttgctgtggctctggcg


 



taggccggcaacaacagctccgattcgacccctagcc


 



TGggaacctccatgtgccacaggagcagccctaGAAA


 



AGGCAAAAAGACAAAAAAATAAAAAATTAAAATGAAA


 



AAATAAAATAAAAATACAAATTACAAGAGACGGCTAC


 



AAGGAAATCCCCAAGTGTGTGCAAATGCCATATATGT


 



ATAAAATGTACTAGTGTCTCCTCGCGGGAAAGTTGCC


 



TAAAAGTGGGTTGGCTGGACAGAGAGGACAGGCTTTG


 



ACATTCTCATAGGTAGTAGCAATGGGCTTCTCAAAAT


 



GCTGTTCCAGTTTACACTCACCATAGCAAATGACAGT


 



GCCTCTTCCTCTCCACCCTTGCCAATAATGTGACAGG


 



TGGATCTTTTTCTATTTTGTGTATCTGACAAGCAAAA


 



AATGAGAACAggagttcctgtcgtggtgcagtggaga


 



caaatctgactaggaaccatgaaatttcgggttcaat


 



ccctggcctcactcagtaggtaaaggatccagggttg


 



cagtgagctgtggggtaggtcgcagacacagtgcaaa


 



tttggccctgttgtggctgtggtgtaggccggcagct


 



atagctccaattggacccctagcctgggaacctcctt


 



atgccgtgggtgaggccctAAAAAAAAGAGTGCAAAA


 



AAAAAAAATAAGAACAAAAATGATCATCGTTTAATTC


 



TTTATTTGATCATTGGTGAAACTTATTTTCCTTTTAT


 



ATTTTTATTGACTGATTTTATTTCTCCTATGAATTTA


 



CCGGTCATAGTTTTGCCTGGGTGTTTTTACTCCGGTT


 



TTAGTTTTGGTTGGTTGTATTTTCTTAGAGAGCTATA


 



GAAACTCTTCATCTATTTGGAATAGTAATTCCTCATT


 



AAGTATTTGTGCTGCAAAAAATTTTCCCTGATCTGTT


 



TTATGCTTTTGTTTGTGGGGTCTTTCACGAGAAAGCC


 



TTTTTAGTTTTTACACCTCAGCTTGGTTGTTTTTCTT


 



GATTGTGTCTGTAATCTGCGGCCAACATAGGAAACAC


 



ATTTTTACTTTAGTGTTTTTTTCCTATTTTCTTCAAG


 



TACGTCCATTGTTTTGGTGTCTGATTTTACTTTGCCT


 



GGGGTTTGTTTTTGTGTGGCAGGAATATAAACTTATG


 



TATTTTCCAAATGGAGAGCCAATGGTTGTATATTTGT


 



TGAATTCAAATGCAACTTTATCAAACACCAAATCATC


 



GATTTATCACAACTCTTCTCTGGTTTATTGATCTAAT


 



GATCAATTCCTGTTCCACGCTGTTTTAATTATTTTAG


 



CTTTGTGGATTTTGGTGCCTGGTAGAGAACAAAGCCT


 



CCATTATTTTCATTCAAAATAGTCCCGTCTATTATCT


 



GCCATTGTTGTAGTATTAGACTTTAAAATCAATTTAC


 



TGATTTTCAAAAGTTATTCCTTTGGTGATGTGGAATA


 



CTTTATACTTCATAAGGTACATGGATTCATTTGTGGG


 



GAATTGATGTCTTTGCTATTGTGGCCATTTGTCAAGT


 



TGTGTAATATTTTACCCATGCCAACTTTGCATATTGT


 



ATGTGAGTTTATTCCCAGGGTTTTTAATAGGATGTTT


 



ATTGAAGTTGTCAGTGTTTCCACAATTTCATCGCCTC


 



AGTGCTTACTGTTTGCATAAAAGGAAACCTACTCACT


 



TTTGCCTATTGCTCTTGTATTCAATCATTTTAGTTAA


 



CTCTTGTGTTAATTTTGAGAGTTTTTCAGCTGACTGT


 



CTGGGGTTTTCTTTAATAGACTAGCCCTTTGTCTGTA


 



AAGAATAATTTTATCGAATTTTTCTTAACACTCACAC


 



TCTCCCCACCCCCACCCCCGCTCATCTCCTTTCATTG


 



GGTCAAATCTGTAGAATACAATAAAAGTAAGAGTGGG


 



AACCTTAGCCTTTAAGTCGATTTTGCCTTTAAATGTG


 



AATGTTGCTATGTTTCGGGACATTCTCTTTATCAAGT


 



TGCGGATGTTTCCTTAGATAATTAACTTAATAAAAGA


 



CTGGATGTTTGCTTTCTTCAAATCAGAATTGTGTTGA


 



ATTTATATTGCTATTCTGTTTAATTTTGTTTCAAAAA


 



ATTTACATGCACACCTTAAAGATAACCATGACCAAAT


 



AGTCCTCCTGCTGAGAGAAAATGTTGGCCCCAATGCC


 



ACAGGTTACCTCCCGACTCAGATAAACTACAATGGGA


 



GATAAAATCAGATTTGGCAAAGCCTGTGGATTCTTGC


 



CATAACTCTCAGAGCATGACTTGGGTGTTTTTTCCTT


 



TTCTAAGTATTTTAATGGTATTTTTGTGTTACAATAG


 



GAAATCTAGGACACAGAGAGTGATTCAATGAGGGGAA


 



CGCATTCTGGGATGACTCTAGGCCTCTGGTTTGGGGA


 



GAGCTCTATTGAAGTAAAGACAATGAGAGGAAGCAAG


 



TTTGCAGGGAACTGTGAGGAATTTAGATGGGGAATGT


 



TGGGTTTGAGGTTTCTATAGGGCACGCAAGCAGAGAT


 



GCACTCAGGAGGAAGAAGGAGCATAAATCTAGAGGCA


 



AAAAGAGAGGTCAGGACTGGAAATAGAGATGCGAGAC


 



ACCAGGGTGGCAGTCAGAGAGCACAGTGTGGGTCAGA


 



AGACAGTGGAAGAACACAAGGGACAGAGAGGGATCTC


 



CAACTTCACTGGGATGAGGGCCTTGTTGGCCTTGACC


 



TGAGAGATTTCCAGGAGTTGAGGGTGGGAAGGAGAGG


 



GCTCCTGCACATGTCCTGACATGAAACGGTGCCCAGC


 



ATATGGGTGCTTGGAAGACATTGTTGGACAGATGGAT


 



GGATGATGGATGATGGATGAATGGATGGATGGAAGAT


 



GATGGATAAATGGATGATGGATGGATGGACAGAAGGA


 



CAAAGAGATGGACAGAAAGACAGTGATCTGAGAGAGC


 



AGAGAAGGCTTCATGAAAGGACAGGAACTGAACTGTC


 



TCAGTGGGTGGAGACAATGGTGTAGGGGGTTTCCACA


 



TGGAGGCACCAGGGGTCAGGAATAATCTAGTGTCCAC


 



AGGCCCAGGAAGGAAGCTGTCTGCAGGAAATTGTGGG


 



GAAGAACCTCAGAGTCCTTAAATGAGGTCAGGAGTGG


 



TCAGGAGGGTCTGATCAGGTAAGGACTCATGTCCATC


 



ATCACATGGTCACCTAAGGGCATGTAGCTCTCAGCAT


 



CTCCATCAGGACAGTCTCAGAATGGGGGCGGGGTCAC


 



ACACTGGGTGACTCAAGGCGTGGGTCATGCCTGCCTC


 



GGACGTGGGCCTGGGCATGGGGACACCTCCAGACCAT


 



GGGCCCGCCCAGGGCTGCACTGGcctctggtgggcta


 



gctacccgtccaagcaacacaggacacagccctacct


 



gctgcaaccctgtgcccgaaacgcccatctggttcct


 



gctccagcccggccccagggaacaggactcaggtgct


 



agcccaatggggttttgttcgagcctcagtcagcgtg


 



gTATTTCTCCGGCAGCGAGACTCAGTTCACCGCCTTA


 



GGttaagtggttctcatgaatttcctagcagtcctgc


 



actctgctatgccgggaaagtcacttttgtcgctggg


 



ggctgtttccccgtgcccttggagaatcaaggattgc


 



ccaactttctctgtgggggaggtggctggtcttgggg


 



tgaccagcaggaagggccccaaaagcaggagcagctg


 



cctccagAATACAACTGTCGGCTACAGCTCAAACAGG


 



AGGCCTGGACTGGGGTTTAACCACCAGGGCGGCACGA


 



AGGAGCGAGGCTGGGAGGGTGAGGACATGGGAGCCTG


 



AGGAGGAGCTGGAGACTTCAGCAGGCCCCCAGCTCCG


 



GGCTTCGGGCTCTGAGATGCTCGGACGCAAGGTGAGT


 



GACCCCACCTGTGGCTGACCTGACCTCAGGGGGACAA


 



GGCTCAGCCTGAGACTCTGTGTCCCCATCGCCTGCAC


 



AGgggattcccctgatggacactgagccaacgacctc


 



ccgtctctccccgacccccaggtcagcccaaggccgc


 



ccccacggtcaacctcttcccgccctcctctgaggag


 



ctcggcaccaacaaggccaccctggtgtgtctaataa


 



gtgacttctacccgAAGGGCGAATTCCAGCACACTGG


 



CGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCT


 



TGATGCATAGCTTGAGTATCTA


 


Seq ID
agatctttaaaccaccgagcaaggccagggatcgaac


No. 33



 



ccgcatcctcatgaatcctagttgggttcgttaaccg


 



ctgaaccacaatgggaactcctGTCTTTCACATTTAA


 



TTCACAACCTCTCCAGGATTCTGGGGGTGGGTGGGGA


 



ATCCTAGGTACCCACTGGGAAAGTAATCCAAGGGGAG


 



AGGCTCACGGACTcTAGGGATCGGCGGAGGAGGGAAG


 



GTATCTCCCAGGAAACTGGCCAGGACACATTGGTCCT


 



CCGCCCTCCCCTTCCTCCCACTCCTCCTCCAGACAGG


 



ACTGTGCCCACCCCCTGCCACCTTTCTGGCCAGAACT


 



GTCCATGGCAGGTGACCTTCACATGAGCCCTTCCTCC


 



CTGCCTGCCCTAGTGGGACCCTCCATACCTCCCCCTG


 



GACCCCGTTGTCCTTTCTTTCCAGTGTGGCCCTGAGC


 



ATAACTGATGCCATCATGGGCTGCTGACCCACCCGGG


 



ACTGTGTTGTGCAGTGAGTCACTTCTCTGTCATCAGG


 



GCTTTGTAATTGATAGATAGTGTTTCATCATCATTAG


 



GACCGGGTGGCCTCTATGCTCTGTTAGTCTCCAAACA


 



CTGATGAAAACCTTCGTTGGCATAGTCCCAGCTTCCT


 



GTTGCCCATCCATAAATCTTGACTTAGGGATGCACAT


 



CCTGTCTCCAAGCAACCACCCCTCCCCTAGGCTAACT


 



ATAAAACTGTCCCAATGGCCCTTGTGTGGTGCAGAGT


 



TCATGCTTCCAGATCATTTCTCTGCTAGATCCATATC


 



TCACCTTGTAAGTCATCCTATAATAAACTGATCCATT


 



GATTATTTGCTTCTGTTTTTTCCATCTCAAAACAGCT


 



TCTCAGTTCAGTTCGAATTTTTTATTCCCTCCATCCA


 



CCCATACTTTCCTCAGCCTGGGGAACCCTTGCCCCCA


 



GTCCCATGCCCTTCCTCCCTCTCTGCCCAGCTCAGCA


 



CCTGCCCACCCTCACCCTTCCTGTCACTCCCTAGGAC


 



TGGACCATCCACTGGGGCCAGGACACTCCAGCAGCCT


 



TGGCTTCATGGGCTCTGAAATCCATGGCCCATCTCTA


 



TTCCTCACTGGATGGCAGGTTCAGAGATGTGAAAGGT


 



CTAGGAGGAAGCCAGGAAGGAAACTGTTGCATGAAAG


 



GCCGGCCTGATGGTTCAGTACTTAAATAATATGAGCT


 



CTGAGCTCCCCAGGAACCAAAGCATGGAGGGAGTATG


 



TGCCTCAGAATCTCTCTGAGATTCAGCAAAGCCTTTG


 



CTAGAGGGAAAATAGTGGCTCAACCTTGAGGGCCAGC


 



ATCTTGCACCACAGTTAAAAGTGGGTATTTGTTTTAC


 



CTGAGGCCTCAGCATTATGGGAACCGGGCTCTGACAC


 



AAACACAGGTGCAGCCCGGCAGCCTCAGAACACAGCA


 



ACGACCACAAGCTGGGACAGCTGCCCCTGAACGGGGA


 



GTCCACCATGCTTCTGTCTCGGGTACCACCAGGTCAC


 



CATCCCTGGGGGAGGTAGTTCCATAGCAGTAGTCCCC


 



TGATTTCGCCCCTCGGGCGTGTAGCCAGGCAAGCTCC


 



TGCCTCTGGACCCAGGGTGGACCCTTGCTCCCCACTA


 



CCCTGCACATGCCAGACAGTCAAGACCACTCCCACCT


 



CTGTCTGAGGCCCCCTTGGGTGTCCCAGGGCCCCCGA


 



GCTGTCCTCTACTCATGGTTCTTCCACCTGGGTACAA


 



AAGAGGCGAGGGACACTTTTCTCAGGTTTGCGGCTCA


 



GAAAGGTACCTTCCTAGGGTTTGTCCACTGGGAGTCA


 



CCTCCCTTGCATCTCAATGTCAGTGGGGAAAACTGGG


 



TCCCATGGGGGGATTAGTGCCACTGTGAGGCCCCTGA


 



AGTCTGGGGCCTCTAGACACTATGATGATGAGGGATG


 



TGGTGAAAAACCCCACCCCAGCCCTTCTTGCCGGGAC


 



CCTGGGCTGTGGCTCCCCCATTGCACTTGGGGTCAGA


 



GGGGTGGATGGTGGCTATGGTCAGGCATGTTTCCCAT


 



GAGCTGGGGGCACCCTGGGTGACTTTCTCCTGTGAAT


 



CCTGAATTAGCAGCTATAACAAATTGCCCAAACTCTT


 



AGGCTTAAAACAACACACATTTATTCCTCTGGGTCCC


 



AGGGTCAGAAGTCCAAAATGAGTCCTATAGGCTAAAT


 



TTGAGGTGTCTCTGGGTTGAGCTCCTCCTGGAAGCCT


 



TTTCCAGCCTCTAGAGTCCCAAGTCCTTGGCTCTGGG


 



CCCCTCCCTCAAGCTTCAAAGCCACAGAAGCTTCTAA


 



TCTCTCTCCCTTCCCCTCTGACCTCTGCTCCCATCCT


 



CATACCCTGTCCCCTCACTCTGACCCTCCTGCCTCCC


 



TCTTTCCCTTATAAAGACCCTGCATGGGGCCAGGGAG


 



ATAATCCAGGGTAATCGCCCCTCTTCCAGCCCTTAAC


 



TCCATCCCATCTGCAAAATCCCTGTCACCCCATAATG


 



GACCTACTGATGGTCTGGGGGTTAGGACGTGGACAAC


 



TTGGGGCCTTATTCATCTGATCACAACTCCAGTTCCC


 



AGACCCCCAGACCCCCGGGCATTAGGGAAACTTCTCC


 



CAGTTCCTCTCCCTCTGTGTCCTGCCCAGTCTCCAGG


 



ATGGGCCACTCCCGAGGGCCCTTCAGCTCAGGCTCCC


 



CCTCCTTTCTCCCTGGCCTCTTGTGGCCCCATCTCCT


 



CCTCCGCTCACAGGGAGAGAACTTTGATTTCAGCTTT


 



GGCTCTGGGGCTTTGCTTCCTTCTGGCCATTGGCTGA


 



AGGGCGGGTTTCTCCAGGTCTTACCTGTCAGTCATCA


 



AACCGCCCTTGGAGGAAGACCCTAATATGATCCTTAC


 



CCTACAGATGGAGACTCGAGGCCCAGAGATCCTGAGT


 



GACCTGCTCACATTCACAGCAGGGACTGAACCCCAGT


 



CACCTACCCAACTCCAGGGCTCAGCGCTTTTTTTTTT


 



TTTTTTCTTTTTgccttttcgagggccgctcccgcaa


 



catatggagatttccaggctaggggtctaattggagc


 



agtcgacactggcctaagccaaagccacagcaacaag


 



ggcaagccgcttctgcagcctataccacagctcacgg


 



caatgccggatccttaacccactgagcaaagccaggg


 



attgaacctgcaacctcatgtttcctagtcaaatttg


 



ttaaccactgacccatgacgggaactcccAGGGCTCA


 



GCTCTTGACTCCAGGTTCGCAGCTGCCCTCAAAGCAA


 



TGCAACCCTGGCTGGCCCCGCCTCATGCATCCGGCCT


 



CCTCCCCAAAGAGCTCTGAGCCCACCTGGGCCTAGGT


 



CCTCCTCCCTGGGACTCATGGCCTAAGGGTACAGAGT


 



TACTGGGGCTGATGAAGGGACCAATGGGGACAGGGGC


 



CTCAAATCAAAGTGGCTGTCTCTCTCATGTCCCTTCC


 



TCTCCTCAGGGTCCAAAATCAGGGTCAGGGCCCCAGG


 



GCAGGGGCTGAGAGGGCCTCTTTCTGAAGGCCCTGTC


 



TCAGTGCAGGTTATGGGGGTCTGGGGGAGGGTCAATG


 



CAGGGCTCACCCTTCAGTGCCCCAAAGCCTAGAGAGT


 



GAGTGCCTGCCAGTGGCTTCCCAGGCCCAATCCCTTG


 



ACTGCCTGGGAATGCTCAAATGCAGGAACTGTCACAA


 



CACCTTCAGTCAGGGGCTGCTCTGGGAGGAAAAACAC


 



TCAGAATTGGGGGTTCAGGGAAGGCCCAGTGCCAAGC


 



ATAGCAGGAGCTCAGGTGGCTGCAGATGGTGTGAACC


 



CCAGGAGCAGGATGGCCGGCACTCCCCCCAGACCCTC


 



CAGAGCCCCAGGTTGGCTGCCCTCTTCACTGCCGACA


 



CCCCTGGGTCCACTTCTGCCCTTTCCCACCTAAAACC


 



TTTAGGGCTCCCACTTTCTCCCAAATGTGAGACATCA


 



CCACGGCTCCCAGGGAGTGTCCAGAAGGGCATCTGGC


 



TGAGAGGTCCTGACATCTGGGAGCCTCAGGCCCCACA


 



ATGGACAGACGCCCTGCCAGGATGCTGCTGCAGGGCT


 



GTTAGCTAGGCGGGGTGGAGATGGGGTACTTTGCCTC


 



TCAGAGGCCCCGGCCCCACCATGAAACCTCAGTGACA


 



CCCCATTTCCCTGAGTTCACATACCTGTATCCTACTC


 



CAGTCACCTTCCCCACGAACCCCTGGGAGCCCAGGAT


 



GATGCTGGGGCTGGAGCCACGACCAGCCCACGAGTGA


 



TCCAGCTCTGCCAATCAGCAGTCATTTCCCAAGTGTT


 



CCAGCCCTGCCAGGTCCCACTACAGCAGTAATGGAGG


 



CCCCAGACACCAGTCCAGCAGTTAGAGGGCTGGACTA


 



GCACCAGCTTTCAAGCCTCAGCATCTCAAGGTGAATG


 



GCCAGTGCCCCTCCCCGTGGCCATCACAGGATCGCAG


 



ATATGACCCTAGGGGAAGAAATATCCTGGGAGTAAGG


 



AAGTGCCCATACTCAAGGATGGCCCCTCTGTGACCTA


 



ACCTGTCCCTGAGGATTGTACTTCCAGGCGTTAAAAC


 



AGTAGAACGCCTGCCTGTGAACCCCCGCCAAGGGACT


 



GCTTGGGGAGGCCCCCTAAACCAGAACACAGGCACTC


 



CAGCAGGACCTCTGAACTCTGACCACCCTCAGCAAGT


 



GGCACCCCCCGCAGCTTCCAAGGCAC


 


Seq ID
AACAAGATGCTACCCCACCAACAAAATTCACCGGAGA


No. 34



 



AGACAAGGACAGGGGGTTCCTGGGGTCCTGACAGGGT


 



CACCAAAGAGGGTTCTGGGGCAGCAGCAACTCCAGCC


 



GCCTCAGAACAGAGCCTGGAAGCTGTACCCTCAGAGC


 



AGAGGCGGAGAGAGAAAGGGCCTCTTGGTGGGTCAGC


 



AGGAGCAGAGGCTCAGAGGTGGGGGTTGCAGCCCCCC


 



CTTCAACAGGCCAACACAGTGAAGCAGCTGACCCCTC


 



CACCTTGGAGACCCCAGACTCCTGTCTCCCACGCCAC


 



CTTGGTTTTTAAGGTAATTTTTATTTTATATCAGAGT


 



ATGGTTGACTTACAATGTTGTGTTGGTTTCAGGTGTA


 



CAGCAGAGTGATTCACTTCTACATAGACTCATATCTA


 



TTCTTTCTCAGATTCTTTTCCCATATAGGTTATTACA


 



GAATATTGAGTAGATCCCTGCTGATTACCCATTTTTA


 



TAATTGTATATGTTAATCCCAAACTCCTAATTTATCC


 



CTCCCCAGACTATGATTCTTTATATCTCTATCTGTTT


 



CCTAATCTGTCTCCTCTAAGTCACCCTAGGAGAGCAG


 



AGGGGTCACGTCTGTCCTGTCCTGGCCCAGCCACCTC


 



TCTCCACCCAGGAATCCCTTGCATTTGGTGCCAAGGG


 



CCCGGCCCCGCCCTAAAGAGAAAGGAGAACGGGATGT


 



GGACAGGACACCGGGCAGAGAGGGACAAGCAGAGGAT


 



GCCAGGGTAGGGAGGTCTCCAGGGTGGATGGTGGTCT


 



GTCCGCAGGCAGGATGAGGCAGGAAGGGTGTGGATGT


 



ACTCGGTGAGGCTGGCGCATGGCCTGGAGTGTCCTGA


 



GCCCTGGGAGGCCTCAGCCCTGGATCAGATCTGTGAT


 



TCCAAAGGGCCACTGCATCCAGAGACCGTTGAGTGGC


 



CCATTGTCCTGAACCATTTATAGAACACAGGACAAGC


 



GGTACCTGACTAAGCTGCTCACAGATTCCATGAGGCT


 



GATGCCAGGGTTGTCACCCCATCTCACAGGCAGGGAA


 



ACTGATGCATATACTGCAGAGCCAGGCAGAGGCCCTC


 



CCAGTGCCCCCTCCCAGCCTGTGGCCCCCCTCCAGTG


 



GCTGGACACTGAGGCCACACTGGGGCACCCTGTGGAG


 



ATCt


 


Seq ID
AGATCTGGCCAGGCCAGAGAAGCCCATGTGGTGACCT


No. 35



 



CCCTCCATCACTCCACGCCCTGACCTGCCAGGGAGCA


 



GAAAGTAGGCCCAGGGTGGACCCGGTGGCCACCTGCC


 



ACCCCATGGCTGGGAGAAGGGAGGGCCTGGGCAAAGG


 



GCCTGGGAAGCCTGTGGTGGGACCCCAGACCCCAGGG


 



TGGACAGGGAGGGTCCCACACCCACAGCCATTTGCTT


 



CCCTCTGTGGGTTCAGTGTCCTCATCTCATCTGTGGG


 



GAGGGGGCTGATAATGAATCTCCCCCATTGGGGTGGG


 



CTTGGGGATTAAAGGGCCAGTGTCTGTGATATGCCTG


 



GACCATAGTGACCCTCACCCTCCCCAGCCATTGCTGT


 



CACCTTCCGGGCTCTTGCCCAGGCCTGCCTGACATGC


 



TGTGTGACCCTGGGCAAGATGATCCCCCTTTCTGGGC


 



CCCAGCCTTCCTCTCTGCTCCGGAAGTGCTTCCTGGG


 



GAAACCTGTGGGCTGGATCCTATAGGAAACCTGTCCA


 



ATTCCTGGATGCACAGAGGGGCAGGGAGGCCCTGGGC


 



CTGGAGGGGCAGGGAGGCTCGAGGTGGGAGCAGGGTA


 



GGGGCCAGTCCAGGGCAAGGAGGTGGGTGGGTAGGGT


 



G


 


Seq ID
GATCTGTGTTCCATCTCAGAGCTATCTTAGCAGAGAG


No. 36:



 



GTGCAGGGGCCTCCAGGGCCACCAAAGTCCAGGCTCA


 



GCCAGAGGCAATGGGGTATCGATGAGCTACAGGACAC


 



AGGCGTCAGCCCAGTGTCAGGGAGAATCACCTTGTTT


 



GTTTTCTGAGTTCCTCTTAAAATAGAGTTAATTGGTC


 



TTGGCCTTACGGTTTACAATAACAACTGCACCCTGTA


 



AACAACGTGAAGAGTACAGAACAACAAATGGGGGAAA


 



ACATATTTCACCTGAAAGAGCCACCGCTCATATTTTG


 



ATGGATTTCCTTCTAGTTTAATCCTGTTTTAATTGTA


 



AACTGTTAAAACAAACATAAATAAAGAAAATGCATCT


 



GTAAAGTTTAAAAGTCATATCTATGGTGATGGTTGCA


 



AAACACTGTGAATGTTCACTTTGAAATCGTGAACTCT


 



ACGTGATATGCATGTCCCGTTAATTAACCTCACAGGC


 



TCAGAATGTGGTTCATTATTTCTTTAATTTTCCTTTA


 



ATTTTATGTCCTGTGTGTGTGCCCTTAAACCAACTAC


 



TTTTCAGCTCTGCCTGTTTTTGACCTTCACATAGATG


 



GACATTTGTAGTGTTTTCTTTCTCAACACTGGGTCTG


 



ATACCCACCCACGCTGTCTGCTGTCACTGCGGACGTG


 



GAGGGCCACCACCCAGCTATGGCCCCAGCCAGGCCAA


 



CACTGGATGAATCTGCCCCCAGAGCAGGGCCACCAAC


 



ACTGGAGGTGCAGAGAGGGTTTCTTCAGGGCCATCAT


 



TATCCAAGGCATTGTTTCTACTGTAAGCTTTCAAAAT


 



GCTTCCCCTGATTATTAAAAGAAATAATAAGATGGGG


 



GGAAAGTACAAGAAGGGAAGTTTCCAGCCCAGCCTGA


 



AGATCGTGCTGGTTGTATCTGGAGCCTGTCTTCCTGA


 



CAGGCCTCTATTCCCAGAGTTA


 


Seq ID
GGATCCTAGGGAAGGGAGGGCGGGGGCCTGGACAAAG


No. 37:



 



GGGGCCTAAAGGACATTCTCACCTATCCCACTGGACC


 



cctgctgtgctctgagggagggagcagagagggggtc


 



tgaggccttttcccagCTCCTCTGAGTCCCTCCTCCG


 



AGCACCTGGACGGAAGCCCCTCCTCAGGGAGTCCTCA


 



GACCCCTCCCCTCCAGCCAGGTTGGCCTGTGTGGAGT


 



CCCCAGTAAGAATAGAATGCTCAGGGCTTCGAGCTGA


 



GCCCTGGCTACTTGGGGGGGTGCTGGGGATTGGGGGT


 



GCTGGGCGGGGAGCTGGGGTGTCACTAGATGCCAGTA


 



GGCTGTGGGCTCGGGTCTGGGGGGTCTGCACATGTGC


 



AGCTGTGGGAAGGCCCTATTGGTGGTACCCTCAGACA


 



CATATGGCCCCTCAATTTCTGAGACCAGAGACCCCAG


 



TCTGGCCTTCCCAGAACAGCTGCCCCTGGTGGGGGAG


 



ATGTAGGGGGGCCTTCAGCCCAGGACCCCCAACGGCA


 



GGGCCTGAGGCCCCCATCCCCTTGTCCTGGGCCCAGA


 



GCCTCAGCTATCAGGCCTATCAGAGATCCTGGCTGCC


 



CAGCTCAGGTTCCCCAGGAGCCAGAGGGAGGCCAGGG


 



GTTACTAGGAAATCCGGAAAGGGTCTTTGAGGCTGGG


 



CCCCACCCTCTCAGCTTTCACAGGAGAAACAGAGGCC


 



CACAGGGGGCAAAGGACTTGCCAGACTCACAATGAGC


 



CCAGCAGCTGGACTCAAGGCCCAGTGTTCGGCCCCAC


 



AACAGCACTCACGTGCCCTTGATCGTGAGGGGCCCCC


 



TCTCAGCCAGGCATTCAGACCTGTGACCTGCATCTAA


 



GATTCAGCATCAGCCATTCTGAGCTGAAGAGCCCTCA


 



GGGTCTGCAGTCAAGGCCACAGGGCCAGACCTCCAAC


 



GGCCAGACATCCCAGCCAGATTCCTTTCTGGTCAATG


 



GGCCCCAGTCTGGCTTGGCTCCTGCAGGCCCAGTGCC


 



GCCTTCTTCCCCTGGGCCTGTGGAGTCCAGCCTTTCA


 



GTTTCCCACCCACATCCTCAGCCACAATCCAGGCTCA


 



GAGGCAATGTCCGTGGGCAGCCCCTGTGTGACCCCTC


 



TGTGGGTGATCCTCAGTCCTACCCTTAGCAGACAGCG


 



CATGAGGGGCCCTCTTGAACCTGAGGGATACTCCATG


 



TCGGAGGGGAGAAGCTGGCCTTCCCCACCCCCACTTC


 



CAGGCCTTGGGGAGCAGAGAAAGACCCCAGACCTGGG


 



TCCCTTCTAACAGGCCAGGCCCCAGCCCAGCTCTCCA


 



CCAGCCCCAGGGGCCTCGGGTCCACGCCTGGGGACTG


 



GAGGGTGGGCCTGTCAGGCGCTGACCCAGAGGCAGGA


 



CAGCCAAGTTCAGGATCCCAGCCAGGTGGTCCCCGTG


 



CACCATGCAGGGGTGTCACCCACACAGGGGTGTTGCC


 



ACCCTCACCTGACTGTCCTCATGGGCCACATGGAGGT


 



ATCCTGGGTTCATTACTGGTCAACATACCCGTGTCCC


 



TGCAGTGCCCCCTCTGGcgcacgcgtgcacgcgcaca


 



cgcacacactcatacaGAGGCTCCAGCCAACAGTGCC


 



CTCTAGTAGGCACTGCTGTCACTTCTCTAAAAGGTCG


 



CAATCATACTTGTAAAGACCCAAGATTGTTCAGAAAT


 



CCCAGATGGAGAAGTCTGGAAAGATCtTTTTCTCCTT


 



TCACGGGCTGGGGAAATGTGACCTGGCCAAGGTCACA


 



CAGCAAGTGGTGGAACCCTGGCCCCTGATTCCAGCTC


 



ATTCCAGTTCCCAAGGCCCTGCCAGAGCCCAGAGGCT


 



GGGCCCTCTGGGGCAGAGGAGCTGGGGTCCTCCCCCC


 



TACACAGAGCACACAGCCCCGCAAGAGAGAAGAGACA


 



CCTTGGGGAGAGGAATCTCCAGACCAGAGATCCCAGT


 



ATGGGTCTCCTCTATGCTGACGGGATGGGATGTCAAG


 



AGGGGAGGGGGCTGGGCTTTAGGGAAACACACAAAAA


 



TCGCTGAGAACACTGACAGGTGCGACACACCCACCCC


 



TAATGCTAACCTGTGGCCCATTACTCAgatct


 


Seq ID
GATCTTCTCCTAAGACCAAGGAAAACTGGTCATACCA


No. 38



 



GGTCCACTTGTCCCCTGTGGCCATTGTCCCTCCTTCC


 



CCAGAAGAAACAAGCACTTTCCACTCCACAAGTAGCT


 



CCTGATCAGCTTGGAAGCCCGGTGCTGCTCTGGGCCC


 



TGGGGACACGGCAGGGGCATCAGAGACCAAATCCTGG


 



AACAAAGTTCCAGTGGGTGAGGCAGGCCGGACAAGCA


 



ACACGTTATACCATAATATGAGGCAAAATATAATGTG


 



AGTTCTTTATGAAAGGAAGGGGTTGCAGGTGCAACTG


 



TTGGCTTAGGTGGATGGTCACCCCTGAATGGAGGAGG


 



GGGTTCCCAGGGCATGTGCCTGGGGAGAAGGGCTCCT


 



GGCAGGAGGGACAGCAAGTGCAAGGGCCCTGTGATCA


 



AATGTGCCTGGCAAGTTGCAGGAACAGCTAGAAGGCC


 



AGCAAGGTTGGAACCAAGGAAGGGGTGAGGGGAGGGG


 



CAGGGCCCTCAGGGCCTTGCCCAGCAGCCTGAGCATC


 



TGGAGATTTGTCCAAAGTTTCAAATGTACCTGGGCAA


 



CCTCATGCCCATATACCATTCCTAACTTCTGCACTTA


 



ACATCTCTAGGACTGGGACCCAGCCAGTCAAGCGGGG


 



GGACCCAGAGAGCTCCGGTGTGAACACCGAGGTGCTG


 



GTGGGTCTGCGTGTGTGGACATAGGGCAGTCCCGGTC


 



CTTCCTTCACTAACACGGCCCGGGAAGCCCTGTGCCT


 



CCCTGGTGCGCGGGTCGGCGCTTCCGGAGGGTACAGG


 



CCCACCTGGAGCCCGGGCACAGTGCATGCAAGTCGGG


 



TTCACGGCAACCTGAGCTGGCTCTGCAGGGCAGTGGG


 



ACTCACAGCCAGGGGTACAGGGCAGACCGGTCCTGCC


 



TCTGCGCCCCTCCCTGGCCTGTGGCCCCTGGACGTGA


 



TCCCCAACAGTTAGCATGCCCCGCCGGTGCTGAGAAC


 



CTGGACGAGGTCCGCAGGCGTCACTGGGCGGTCACTG


 



AGCCCGCCCCAGGCCCCCTCTGCCCCTTCCTGGGGTG


 



ACCGTGGACTCCTGGATGACCCTGGACCCTAGACTTC


 



CCAGGGTGTCTCGCGGAGGTTCCTCAGCCAGGATCTC


 



TGCGTCTCCTCCTTCCATAGAGGGGACGGCGCCCCCT


 



TGTGGCCAAGGAGGGGACGGTGGGTCCCGGAGCTGGG


 



GCGGAGAACACAGGGAGCCCCTCCCAGACCCCGCTCT


 



GGGCAGAACCTGGGAAGGGATGTGGCCATCGGGGGAT


 



CCCTCCAGGCCATCTCCTCAGATGGGGGCTGGTCGAC


 



TAGCTTCTGAGTCCTCCAAGGAACCGGGTCCTTCTAG


 



TCATGACTCTGCCCAGATGAAGAAGGAGAGCACTTCT


 



CTCCATCAGGAGGATCTGAGCTTCTCTTAATTAGAAT


 



CAGCTCCTTGGCTTCTACCCCTTAAAAAAAGGTACAG


 



AAACTTTGCACCTTGATCCAGTATCAGGGGAATTTAT


 



CAATCAATGTGGGAGAAATTGGCATCTTTACCACACT


 



GAATCTTTCAATCCATGAATATCCTCTCTCTCTTCCA


 



TGCATAGGTTTTAATAATTCTCAATGGAGTTTAATGT


 



AAGTTTTCCTCATAGACAATTGCCTTTGGACATCTCT


 



TTAGACTCATCTCTAGTAAACTGATATTCTTAATGCA


 



ATTATAAAATGTATCCTGCTTAATGTTATTTTCTATT


 



CATTTGCTGTTATATAGAGATACAATGAGTTTCCACA


 



TTTGAAACTGGATCTGGTAAATTGGCTACCCTTTTTT


 



TATAGATTCTATTAATTTTTATACATTCTGTGGGACT


 



TGCTACATACTTAATCATGTCACCTGTGAAGAATGAC


 



AATTTGGTTGCTACCCTCCCAATTCTTATATGTCTCA


 



TTTCTTTCCCTCTGCTGGTACTCTGGCAGCAGCAGGG


 



AAGATAATGGGCCTCCTTATCTTGTCACAAAAGGATG


 



TTTTTAAAGATTTCGTTATAAAACATAACGCTTTCTG


 



GTTTTCTTTAAAGATTCTCTCACCAGCTTAAGAAAAT


 



TTTCTTATACTCTGTATGATAAATGGGTTTTTGACAA


 



TCATTTGTTGCATTTTACCTAGTGTTTTCTCTGCATC


 



TTTATATGCTTTTTCTCCTTTAATCCTGAAAATTGTT


 



TCGATTTTTCTAACATTGAACCAATCTTACATTCCTG


 



GAATGGATGGACCAGACTAGTCCACATGTTTATTCTG


 



CCCAATGGCTAGATTTTGTGTTCaatattttgttcag


 



aatgtttgcatctatattcttGAGTGAGACAGAGCTG


 



CCCTTGTTAGGTTTCACAACCGAGGTTGTGTTAGCTT


 



CATAAAATGAGACGTTTATTCTCTAAAAGAATTGTTT


 



CGCTTCTCTGGATGAATTTGTGTAAGGTTAGAATTGC


 



TTACCAGTGAagatctCGGGgCCAGTTCTTCTTTAGG


 



GGAAGATTTTCAACAATTAAGCTCAATGCCTTTAGAA


 



GAACTGAGAGTTTCTATTATTTCTTGAGTTAAATATA


 



TGTATTTAATTAGACTTTCTAGGAATAGTCTCATTTC


 



ATCTCAAATAATTGACATATGCTATTAAAGCAGATTC


 



TCATGAACCATTGTAGGTATTCCAGGTCTAGAAAAAT


 



GTTCCCCTTTGCATCCCTAATGTGTTTAATTTTCACC


 



TTCTTTCTTTTGTTCTTGAGAAATTCACCAAATCATT


 



TTCAATTTCAGTCATATCCCAAAGCAACCAACTCTCT


 



ACCTTCTTGTTTTATCATCCCTGCTGGATTTTTGTTA


 



TCTACTTCTTCAGTATTTGTTCTTCCCTTTCTTCTAT


 



TCCTCATTCCATTTTTCCCTTGTTTTCTAACTTTCTG


 



AGATATATGCTTAGTTCCTTCATTTGAAGCCTTTTTA


 



TTTTCTTTTTTTTTTTTTGGTCTTTTTGTCTTTtGTT


 



GTTGTTGTTGTGCTATTtCTTGGGCCGCTCCCGCGGC


 



ATATGGAGGTTCCCAGGCTAGGAGTCGAATCGGAGCT


 



GTAGCCACCGGCCTACGCCAGAGCCACAGCAATGCGG


 



GATCCGAGCCGCGTCTGCAACCTACACCACAGCTCAT


 



GGCAACGCCGGATCGTTAACCCACTGAGCAAGGGCAG


 



GAACCGAACCCGCAACCTCATGGTTCCTAGTCGGATT


 



CGTAACCACTGTGCCACAACAGGAACTCCGCCTTTTT


 



ATTTTCTATAAAAATTTCTATGTACATTTTAAGGTTA


 



TAGGTTTCCTTCTATGTACCCCATTGGCTGTATCCTC


 



AGGGTTCTGTGGAGTGATTTCATTATTGTTCAAGTTC


 



AATATGTCTTCTGATTTTCCAATTTGAATACCTCTCT


 



AAATCAGTAGGTGAATATTTCTTTTTCTTTTTCTTTT


 



CTTTTCTTCTTTTTTTTTTTCTTTCAGCCAGGTCCAT


 



GGCATGCAGAAATTCCCAGGCCAGGAATCAAACTCTC


 



ACCATGGCAGTGACAATGTCGGATCCTTTACCCACTA


 



GGCCACCAGGGAACTCTGGGAGCATATGTTTTTATTT


 



CCCGACATCTGAGGATGCCTAGTATGTCTTCATTATT


 



GATTTCTAGTTTGCCACTGATTTCTAGTATTTTGCTC


 



ATAGAGTGTATGCTCAATGGTTTTGGTCATTTGAAAT


 



GTATTTAGTCCTGCTTTATGACCCAGTATGTGGTCAG


 



TTTTGTCAATGTTCCTTTTCTGCTTGAAGAGAACCTA


 



CATGCTGTAACTCTGGGTGCATGTTCTGTATATAAGT


 



CTATAGGCTGAGCCGGGGGAGCCTTCTAATCTGCCGT


 



TATCTTCTTCGAGTTATTCTAGGTACTATTTCTTAGC


 



CATAAACCTTTAAATTCTGATATCAATATAATGACCC


 



CAGCCCGCTTAGGGTCGGCACTTCATGTTATCTTTTT


 



CCATCCATTTAATCCCTCCCCACTGTTTTGGCCACAC


 



CCGTGGGATATGGGAGTTCCTGGGCCAAGGATCaGAT


 



CTGAGCCGCAGCTGCCACCTATGCCACAGCAgcagca


 



atgatggatctttaacccactgcaccacactggggat


 



tgaacccaagcctcagcagcaacccaagctactgcag


 



agacaacaccagatccttaacctgctgtgccatagcg


 



ggaaTTTCCATCCATTTACTTTCAAGCCAGCTGAATA


 



ACCTAGCCCACCATGCCTGGACATGGGTGCTCTGCTT


 



CAAATGATTTTGTTCAGTCAGCATCCATCTCTGAAAT


 



GTGTGCCAAGCATTTATATGCATGCAAGAGTCATGTT


 



GGCACTTCTATCATTTCCAACAGTTCAGTAGCCTTTG


 



TATCATGACATTTCTTGGCCTTTTCTCTACAATATTT


 



GAGGCTGAGCAGACTGGCCGTGCCCCTGTCCATGCTT


 



CCAGAGCCTGTGTGCAGACTTCTGCTCTAGACAGAGA


 



CAGCTAACCATCCTGCAGTGCCCAGAAAACCCAACTC


 



AAAGACCCTCAAGTAAGGAAGGATTTATTGGCTCACG


 



TAATCTGGAATCCAGGCATGGGGTATTCAGGGCCACC


 



TGAACCAGAGGCCCTGGCCCTGTTCTCTAAGCTTCTT


 



CCTGCCCTGCCCTCGTTCTGGAAGTGACCCTGAAGGA


 



CAGCAATGAAGGGCAGCTCCCCCAGGGACAGATGACT


 



GAGAGGTCCATTTCAAGTCCAACTTGGCCTAGATTGA


 



GAGGCAGCAAGAAATATGGACCTACAGTGAGTCACAG


 



GATTTACCAGTGGTTTGGCTGGGTTGTCAGTGTTACA


 



GGCTAAACATTTGGGTCCCTCCAAAATTAACATGTTG


 



CCACTCTAACCACCAAAATCatggtatttgggggtgg


 



ggcccttggaggtaattaggtttagaaAGAATGAAGA


 



GGGGGCCCTTGTGATGGGACTAGTGCCTTTATAGAGA


 



GAGAAGAGAGAGGG


 


Seq ID 
CACTCATCCCCAACCACCTGGATGGTGGCAAGTGGC


No. 39



 



AGGCTGAGAGGCTGCATATGAGCTCATCAAGAGGGTC


 



CCCACCCCACAGAGGCTGACCCAGCTGCCACTGCCAC


 



CTAGTGGCTGATCGGCCAAGAGCAGGAGCCCCAGGGG


 



CAGGTCCATTCCCTGGGGCGGCCAGGGAACCACCTGG


 



TGGTAGGACAATTCCATTGCACCTCATCCATCAGGAA


 



AAGGTTTGCCTTCCCTGGCAGTAATGCATCTTCCCAT


 



AACATGGTCCCTGGCCTCTTGGAATGGCTTGGCCACC


 



GTCATGGCCTCACCCACAAAGCCTTGTGTCTCAGCAA


 



GGAACTTATTCCACAGCAAAGGACTTGCAGCCTGGAA


 



TGAACTGGTCTGACTACATACCCCATTGCCCAGAAGT


 



AGGTGGTCTATTGCAAAGTGGAGTGGCTTACCCAAGA


 



CTCAGTTGTGCCCAAGTTGAGAGATAGCATCCTAAAA


 



TATGGGCTTATGTCTCACTGGCTGAGGTTTATTCTTT


 



GAATCAAAGACAATTATATGGTGTGGTCCCCCCAGAG


 



ATAGAATACATGAGTCTGGGAATCAAGGGATAGAAGT


 



AAGAAGAGATTTTGTCACCATTAATCCCAATAACTCG


 



CCCAAAGAATATTTGCTTTCTGTCCTGGCAGCTCTGC


 



TGCTTTGGCAATAACTTCCTAGAATATAATGTCTCCA


 



CCAGGGGACTCCACAACGGTTCCATTGATTTGAAGCC


 



AATGGGCAGAGGAGGGGCTGCCTTACTGGTCGGACTG


 



GTCAGCCCTGATTACTAAGGAGAAATCAGGCAACTTC


 



AACAAAACTAAGGCAGGGGGGACTTTGTCTAGAACCC


 



AAAGCACTAAGCATCTTAGTACTTTTTAGTTCTCAGA


 



GCCTCCAAGAACAAAGATTTAGCCCCTCAGCACCACC


 



AGGTAAAGAACAGGTAAATCCAGCTGAGGACAAGAGA


 



AATATTGAATGGATAGAGGAAGAAAGAAATTATAGAT


 



ATCAACTATGGCCTCATGACTAGAGTCTCCAGATTAA


 



GCGGAATAAAAATACAGATGATTaGATCTGAACATCA


 



GGCCAAACAACGAACAACAGTTTAAGTGCGACCTAGG


 



CAATATTTGGGACATACTTATACTAAAATTTTTTCGC


 



TATTTGAGCATCCTGTATTTTATCTGGCAACTTTATT


 



CATCCCTAGCGAAAAAGGAACTGTGGTAACTTAGTGT


 



ATTTTTACTTTGCTCATTATTGTGTATATACCTACTT


 



GTATTTATCAATCATATTTACTCTGTTCTCAGTATTA


 



CTTTATATAGCAGTTGGTGGTGATGGTTAGCAACATA


 



TTCAGTGGAACTGTGACTGAATTTGAGGAGAAATTAA


 



CAGAGTTGGCTGTGGCTACAATAACCCTTCGGGACAT


 



GTGTCCCCTCATTTTGGGGAGATGGTTagatctCTGG


 



GTAAATGTTAGGGCATCTGAGCCAGAAACCAAGATTT


 



TGCCAGCTGGTGCAATGTCAGATTTTACCAGCAGAGG


 



GTGCCAGAGGAATGCGGCAAAACCCGAGTGCCAGAAA


 



GCACCTCCCTGTTTTCCAGCTTTTCTTCCTTTTTATT


 



TATTTTATTTACGGCCCAGGAGTCCGTAATAGCGCTG


 



AGGATGGCCCAGGCTCTTCTCAGCAGCCCTGACTGAC


 



TAGTTCAGCAATGCGCTCAGGCCCCATCTGGCCACCG


 



GGCAGCCTCTTCTGTGGTAGCTCCAGCCTCAGCCAGT


 



GCAAAAGGCTACCCTACACTGGCGCCACTTCTACAAT


 



CAGCACTGGCCACACCCTCCACGCCATCCGGCACGGA


 



GCCAGGTGATCTGCCGGCCAGATTGCAGTTCGTGCTG


 



CCTGAGTCCAGGTGATTACACTGGCTGCATCTTTTCT


 



TTCTGGACCAtTCattccattttttt






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 
     1
tgggttctat gccacccagc ttggtctctg atggtcactt gaggccccca tctcatggca



No 31




 



    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 nnnnnnnnnn 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 tgcncnnncn 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 tctgagtttg



 



 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
GGCCAGACTTCCTCGGAACAGCTCAAAGAGCTCTGTC


 



AAAGCCAGATCCCATCACACGTGGGCACCAATAGGCC


 



ATGCCAGCCTGCAAGGGCCGAACTGGGTTCTCCACGG


 



CGCACATGAAGCCTGCAGCCTGGCTTATCCTCTTCCG


 



TGGTGAAGAGGCAGGCCCGGGACTGGACGAGGGGCTA


 



GCAGGGTGTGGTAGGCACCTTGCGCCCCCCACCCCGG


 



CAGGAACCAGAGACCGTGGGGCTGAGAGTGAGCCTCC


 



AAACAGGATGCGCCACCCTTCAGGCCACCTTTCAATC


 



CAGCTACACTCCACCTGCCATTCTGCTCTGGGCACAG


 



GGCCCAGCCCCTGGATCTTGGCCTTGGCTCGACTTGC


 



ACCCACGCGCACACACACACTTCCTAACGTGCTGTGC


 



GCTCACGCCTCCCCAGCGTGGTCCATGGGGAGCACGG


 



GAGTGCGCGTCCGGCGGTAGTGAGTGCAGAGGTCCCT


 



TCCCCTCCCCCAGGAGCCCCAGGGGTGTGTGCAGATC


 



TGGGGGCTCCTGTCCCTTACACCTTCATGCCCCTCCC


 



CTCATAGGCACCCTCCAGGCGGGAGGGAGCGAGACCT


 



TTGCGGAGGGACTCAGCCAACGGGGACACGGGAGGCC


 



AGCCCTGAGGAGCTGGCTCGCAAAGAGGAGGTGGGAG


 



GTAGGTCCACAGCTGCCACAGAGAGAAACCCTGACGG


 



ACCCCACAGGGGCCACGGCAGCCGGAACCAGCTCCCT


 



CGTGGGTGAGCAATGGCCAGGGCCCCGCCGGCCACCA


 



CGGCTGGCCTTGCGCCAGCTGAGAACTCACGTCCAGT


 



GCAGGGAGACTCAAGACAGCCTGTGCACACAGCCTCG


 



GATCTGCTCCCATTTCAAGCAGAAAAAGGAAACCGTG


 



CAGGCAGCCCTCAGCATTTCAAGGATTGTAGCAGCGG


 



CCAACTATTCGTCGGCAGTGGCCGATTAGAATGACCG


 



TGGAGAAGGGCGGAAGGGTGTCTCGTGGGCTCTGCGG


 



CCAACAGGCCCTGGCTCCACCTGCCCGCTGCCAGCCC


 



GAGGGGCTTGGGCCGAGCCAGGAACCAGAGTGCTCAC


 



CGGGAGCACAGTGACTGACCAAACTCCCGGCCAGAGC


 



AGCCCCAGGCCAGCCGGGCTCTCGCCCTGGAGGACTC


 



ACCATCAGATGCACAAGGGGGCGAGTGTGGAAGAGAC


 



GTGTCGCCCGGGCCATTTGGGAAGGCGAAGGGACCTT


 



CCAGGTGGACAGGAGGTGGGACGCACTCCAGGCAAGG


 



GACTGGGTCCCCAAGGCCTGGGGAAGGGGTACTGGCT


 



TGGGGGTTAGCCTGGCCAGGGAACGGGGAGCGGGGCG


 



GGGGGCTGAGCAGGGAGGACCTGACCTCGTGGGAGCG


 



AGGCAAGTCAGGCTTCAGGCAGCAGCCGCACATCCCA


 



GACCAGGAGGCTGAGGCAGGAGGGGCTTGCAGCGGGG


 



CGGGGGCCTGCCTGGCTCCGGGGGCTCCTGGGGGACG


 



CTGGCTCTTGTTTCCGTGTCCCGCAGCAGAGGGCGAG


 



CTCGCTGGGCCTATGCTTACCTTGATGTCTGGGGCCG


 



GGGCGTCAGGGTCGTCGTCTCCTCAGGGGAGAGTCCC


 



CTGAGGCTACGCTGGGG*GGGGACTATGGCAGGTCCA


 



CGAGGGGCCTGGGGACGAGGGGCCTGGACCAGGCTGC


 



AGCCCGGAGGACGGGGAGGGCTCTGGCTCTCCAGCAT


 



CTGGCCCTCGGAAATGGCAGAACCCCTGGCGGGTGAG


 



CGAGCTGAGAGCGGGTCAGACAGACAGGGGCCGGCCG


 



GAAAGGAGAAGTTGGGGGCAGAGCCCGCCAGGGGCCA


 



GGCCCAAGGTTCTGTGTGCCAGGGCCTGGGTGGGGAC


 



ATTGGTGTGGCCATGGCTACTTAGACGCGTGATCAAG


 



GGCGAATTCCAGCACACTGGCGGCCGTTACTAGTgga


 



tcccggcgcgccctaccgggtaggggaggcgcttttc


 



ccaaggcagtctggagcatgcgctttagcagccccgc


 



tgggcacttggcgctacacaagtggcctctggcctcg


 



cacacattccacatccaccggtaggcgccaaccggct


 



ccgttctttggtggccccttcgcgccaccttctactc


 



ctcccctagtcaggaagttcccccccgccccgcagct


 



cgcgtcgtgcaggacgtgacaaatggaagtagcacgt


 



ctcactagtctcgtgcagatggacagcaccgctgagc


 



aatggaagcgggtaggcctttggggcagcggccaata


 



gcagctttggctccttcgctttctgggctcagaggct


 



gggaaggggtgggtccgggggcgggctcaggggcggg


 



ctcaggggcggggcgggcgcccgaaggtcctccggaa


 



gcccggcattctgcacgcttcaaaagcgcacgtctgc


 



cgcgctgttctcctcttcctcatctccgggcctttcg


 



acctgcagccaatatgggatcggccattgaacaagat


 



ggattgcacgcaggttctccggccgcttgggtggaga


 



ggctattcggctatgactgggcacaacagacaatcgg


 



ctgctctgatgccgccgtgttccggctgtcagcgcag


 



gggcgcccggttctttttgtcaagaccgacctgtccg


 



gtgccctgaatgaactgcaggacgaggcagcgcggct


 



atcgtggctggccacgacgggcgttccttgcgcagct


 



gtgctcgacgttgtcactgaagcgggaagggactggc


 



tgctattgggcgaagtgccggggcaggatctcctgtc


 



atctcaccttgctcctgccgagaaagtatccatcatg


 



gctgatgcaatgcggcggctgcatacgcttgatccgg


 



ctacctgcccattcgaccaccaagcgaaacatcgcat


 



cgagcgagcacgtactcggatggaagccggtcttgtc


 



aatcaggatgatctggacgaagagcatcaggggctcg


 



cgccagccgaactgttcgccaggctcaaggcgcgcat


 



gcccgacggcgaggatctcgtcgtgacccatggcgat


 



gcctgcttgccgaatatcatggtggaaaatggccgct


 



tttctggattcatcgactgtggccggctgggtgtggc


 



ggatcgctatcaggacatagcgttggctacccgtgat


 



attgctgaagagcttggcggcgaatgggctgaccgct


 



tcctcgtgctttacggtatcgccgctcccgattcgca


 



gcgcatcgccttctatcgccttcttgacgagttcttc


 



tgaggggatcaattcTCTAGATGCATGCTCGAGCGGC


 



CGCCAGTGTGATGGATATCTGCAGAATTGGCCCTtCC


 



AGGCGTTGAAGTCGTGGTGTCCTCAGGTAAGAACGGC


 



CCTCCAGGGCCTTTAATTTCTGCTCTCGTCTGTGGGC


 



TTTTCTGACTCTGATCCTCGGGAGGCGTCTGTGCCCC


 



CCCCGGGGATGAGGCCGGCTTGCCAGGAGGGGTCAGG


 



GACCAGGAGCCTGTGGGAAGTTCTGACGGGGGCTGCA


 



GGCGGGAAGGGCCCCACCGGGGGGCGAGCCCCAGGGC


 



GCTGGGCGGCAGGAGACCCGTGAGAGTGCGCCTTGAG


 



GAGGGTGTCTGCGGAAGCACGAACGCCGGCCGGGAAG


 



GGCTTGGTGCAATGCGGTCTTCAGACGGGAGGCGTCT


 



TCTGCCCTCACCGTCTTTCAAGCCCTTGTGGGTCTGA


 



AAGAGCCATGTCGGAGAGAGAAGGGACAGGCCTGTCC


 



CGACCTGGCCGAGAGCGGGCAGCCCCGGGGGAGAGGG


 



GGGCGATCGGGGTGGGCTCTGTGAGGCCAGGTCCAAG


 



GGAGGACGTGTGGTCCTCGTGACAGGTGCACTTGCGA


 



AACCTTAGAAGACGGGGTATGTTGGAAGCGGCTCCTG


 



ATGTTTAAGAAAAGGGAGACTGTAAAGTGAGCAGAGT


 



CCTCAAGTGTGTTAAGGTTTTAAAGGTCAAAGTGTTT


 



TAAACCTTTGTGACTGCAGTTAGCAAGCGTGCGGGGA


 



GTGAATGGGGTGCCAGGGTGGCCGAGAGGCAGTACGA


 



GGGCCGTGCCGTCCTCTAATTCAGGGCTTAGTTTTGG


 



AGAATAAAGTCGGCCTGTTTTCTAAAAGCATTGGTGG


 



TGCTGAGCTGGTGGAGGAGGCCGCGGGCAGCGCTGGC


 



CACCTGCAGCAGGTGGCAGGAAGCAGGTCGGCCAAGA


 



GGCTATTTTAGGAAGCCAGAAAACACGGTCGATGAAT


 



TTATAGCTTCTGGTTTCCAGGAGGTGGTTGGGCATGG


 



CTTTGCGCAGCGCCACAGAACCGAAAGTGCCCACTGA


 



GAAAAAACAACTCCTGCTTAATTTGCATTTTTGTAAA


 



AGAAGAAACAGAGGCTGAGGGAAACTGGAAAGTTCCT


 



GTTTTAACTACTCGAATTGAGTTTTCGGTCTTAGCTT


 



ATCAAGTGCTCACTTAGATTCATTTTCAAAGTAAACG


 



TTTAAGAGCCGAGGCATTCCTATCCTCTTCTAAGGCG


 



TTATTCCTGGAGGCTCATTCACCGCCAGCACCTCCGC


 



TGCCTGCAGGCATTGCTGTCACCGTCACCGTGACGGC


 



GCGCACGATTTTCAGTTGGCCCGCTTCCCCTCGTGAT


 



TAGGACAGACGCGGGCACTCTGGCCCAGCCGTCTTGG


 



CTCAGTATGTGCAGGCGTGCGTCTCGGGACGGAGCTC


 



AGGGGAAGAGCGTGACTCCAGTTGAACGTGATAGTCG


 



GTGCGTTGAGAGGAGACCCAGTCGGGTGTCGAGTCAG


 



AAGGGGCCCGGGGCCCGAGGCCCTGGGCAGGACGGCC


 



CGTGCCCTGCATCACGGGCCCAGCGTCCTAGAGGCAG


 



GACTCTGGTGGAGAGTGTGAGGGTGCCTGGGGCCCCT


 



CCGGAGCTGGGGCCGTGCGGTGCAGGTTGGGCTCTCG


 



GCGCGGTGTTGGCTGTTTCTGCGGGATTTGGAGGAAT


 



TCTTCCAGTGATGGGAGTCGCCAGTGACCGGGCAGCA


 



GGCTGGTAAGAGGGAGGCCGGCGTCGTGGCCAGAGCA


 



GCTGGGAGGGTTCGGTAAAAGGCTCGCCCGTTTCGTT


 



TAATGAGGACTTTTCCTGGAGGGCATTTAGTCTAGTC


 



GGGACCGTTTTCGACTCGGGAAGAGGGATGCGGAGGA


 



GGGCATGTGCCCAGGAGCCGAAGGCGCCGCGGGGAGA


 



AGCCCAGGGCTCTCCTGTCCCCACAGAGGCGACGCCA


 



CTGCCGCAGACAGACAGGGGCTTTCCCTCTGATGACG


 



GGAAAGGCGCCTGGGGTCTTGCGGGGTGCTGGGGGGG


 



AGTCGCCCCGAAGCCGCTCAGCCAGAGGCCTGAGGGG


 



TGAGACTGACCGATGCCTCTTGGCCGGGCCTGGGGCC


 



GGACCGAGGGGGACTCCGTGGAGGCAGGGCGATGGTG


 



GCTGCGGGAGGGAACCGACCCTGGGCCGAGCCCGGCT


 



TGGCGATTCCCGGGCGAGGGCCCTCAGCCGAGGCGAG


 



TGGGTCCGGCGGAACCACCCTTTCTGGCCAGCGGGAG


 



AGGGCTCTCGGGACTGTCCGGGGCGACGCTGGGCTGC


 



CCGTGGCAGGCCTGGGCTGACCTGGACTTCACCAGAC


 



AGAACAGGGCTTTCAGGGCTGAGCTGAGCCAGGTTTA


 



GCGAGGCCAAGTGGGGCTGAACCAGGCTCAACTGGCC


 



TGAGCTGGGTTGAGCTGGGCTGACCTGGGCTGAGCTG


 



AGCTGGGCTGGGCTGGGCTGGGCTGGGGTGGGCTGGG


 



CTGGACTGGCTGAGCTGAGCTGGGTTGAGCTGAGCTG


 



AGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAG


 



CTGGGTTGAGCTGGGTTGAGCTGGGTTGATCTGAGCT


 



GAGCTGGGCTGAGCTGAGCTAGGCTGGGGTGAGCTGG


 



GCTGAGCTGGTTTGAGTTGGGTTGAGCTGAGCTGAGC


 



TGGGCTGTGCTGGCTGAGCTAGGCTGAGCTAGGCTAG


 



GTTGAGCTGGGCTGGGCTGAGCTGAGGTAGGCTGGGC


 



TGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCGTTG


 



AGCTGGCTGGGCTGGATTGAGCTGGCTGAGCTGGCTG


 



AGCTGGGCTGAGCTGGCCTGGGTTGAGCTGAGCTGGA


 



CTGGTTTGAGCTGGGTCGATCTGGGTTGAGCTGTCCT


 



GGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGA


 



GCTGGGCTCAGCAGAGCTGGGTTGGGCTGAGCTGGGT


 



TGAGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTG


 



GGCTGAGCTGAGCTGGGCTGAGCTGGCCTGTGTTGAG


 



CTGGGCTGGGTTGAGCTGGGCTGAGCTGGATTGAGCT


 



GGGTTGAGCTGAGCTGGGCTGGGCTGTGCTGACTGAG


 



CTGGGGTGAGCTAGGGTGGGGTGAGCTGGGCTGAGCT


 



GATCCGAGCTAGGCTGGGCTGGTTTGGGCTGAGCTGA


 



GCTGAGCTAGGCTGGATTGATCTGGCTGAGCTGGGTT


 



GAGCTGAGCTGGGCTGAGGTGGTCTGAGCTGGGGTGG


 



GTCGAGCTGAGGTGGACTGGTTTGAGCTGGGTCGATC


 



TGGGCTGAGCTGGCGTGGGTTGAGCTGGGCTGGGTTG


 



AGCTGAGCTGGGTTGAGCTGGGCTGAGCTGAGGGCTG


 



GGGTGAGCTGGGCTGAACTAGGGTAGCTAGGTTGGGC


 



TGAGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTA


 



GGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCAGGCC


 



TGGGGTGAGCTGGGCTAGGTGGAGCTGAGCTGGGTCG


 



AGCTGAGTTGGGGTGAGCTGGCCTGGGTTGAGGTAGG


 



CTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCTG


 



GGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGC


 



CTGGGTTGAGCTGGGCTCGGTTGAGCTGGGCTGAGCT


 



GAGCCGACCTAGGCTGGGATGAGCTGGGCTGATTTGG


 



GCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCT


 



GAGCTGGGCCTGGAGCCTGGCCTGGGGTGAGCTGGGC


 



TGAGCTGCGCTGAGCTAGGCTGGGTTGAGCTGGCTGG


 



GCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCC


 



TGGGATGAGCTGGGCCGGTTTGGGCTGAGCTGAGCTG


 



AGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGC


 



TGGCCTGGGGTGAGCTGGGCTGAGCTAAGCTGAGCTG


 



GGCTGGTTTGGGGTGAGGTGGGTGAGCTGGGTCCTGC


 



TGAGCTGGGCTGAGCTGACCAGGGGTGAGCTGGGCTG


 



AGTTAGGCTGGGCTCAGCTAGGCTGGGTTGATCTGGC


 



AGGGCTGGTTTGCGCTGGGTCAAGCTCCCGGGAGATG


 



GGCTGGGATGAGCTGGGCTGGTTTGGGCTGAGCTGAG


 



CTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTG


 



GGCTGAGCTGGCCTGGGGTGAGCTGGGCTGGGTGGAG


 



CTGAGCTGGGCTGAACTGGGGTAAGCTGGCTGAGGTG


 



GATCGAGCTGAGCTGGGCTGAGCTGGCCTGGGGTTAG


 



CTGGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCT


 



GGCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAG


 



CTGGCCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCT


 



AGGCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAG


 



GCTGCATTGAGCTGGCTGGGATGGATTGAGCTGGCTG


 



AGCTGGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCT


 



GGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGCTGA


 



GCTGGGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGT


 



TGAGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGTTG


 



AGCTGAGCTGGGTTGAGCTGGGCTCGAGCAGAGCTGG


 



GTTGAGCTGAGCTGGGTTGAGCTGGGCTCAGCAGAGC


 



TGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTGAGCTA


 



GCTGGGCTCAGCTAGGCTGGGTTGAGCTGAGCTGGGC


 



TGAACTGGGCTGAGCTGGGCTGAACTGGGCTGAGCTG


 



GGCTGAGCTGGGCTGAGCAGAGCTGGGCTGAGCAGAG


 



CTGGGTTGGTCTGAGCTGGGTTGAGCTGGGCTGAGCT


 



GGGCTGAGCAGAGTTGGGTTGAGCTGAGCTGGGTTCA


 



GCTGGGCTGAGCTAGGCTGGGTTGAGGTGGGTTGAGT


 



TGGGCTGAGCTGGGCTGGGTTGAGCGGAGCTGGGCTG


 



AACTGGGCTGAGCTGGGCTGAGCGGAACTGGGTTGAT


 



CTGAATTGAGCTGGGCTGAGCCGGGCTGAGCCGGGCT


 



GAGCTGGGCTAGGTTGAGCTTGGGTGAGCTTGCCTCA


 



GCTGGTCTGAGCTAGGTTGGGTGGAGCTAGGCTGGAT


 



TGAGCTGGGCTGAGGTGAGCTGATCTGGCCTCAGCTG


 



GGCTGAGGTAGGCTGAACTGGGCTGTGCTGGGCTGAG


 



CTGAGCTGAGCCAGTTTGAGCTGGGTTGAGCTGGGCT


 



GAGCTGGGCTGTGTTGATCTTTCCTGAACTGGGCTGA


 



GCTGGGCTGAGCTOGCCTAGCTGGATTGAACGGGGGT


 



AAGCTGGGCCAGGCTGGACTGGGCTGAGGTGAGCTAG


 



GCTGAGCTGAGTTGAATTGGGTTAAGCTGGGCTGAGA


 



TGGGCTGAGCTGGGCTGAGCTGGGTTGAGCCAGGTCG


 



GACTGGGTTACCCTGGGCCACACTGGGCTGAGCTGGG


 



GGGAGCTCGATTAACCTGGTCAGGCTGAGTCGGGTCC


 



AGCAGACATGCGCTGGGCAGGCTGGCTTGACCTGGAC


 



ACGTTGGATGAGCTGCCTTGGGATGGTTCACCTCAGC


 



TGAGCCAGGTGGCTCCAGCTGGGCTGAGCTGGTGACC


 



CTGGGTGACCTCGGTGACCAGGTTGTCCTGAGTCCGG


 



GCCAAGGGGAGGCTGCATCAGACTCGCCAGACCCAAG


 



GCCTGGGCCCCGGCTGGCAAGCCAGGGGCGGTGAAGG


 



CTGGGCTGGCAGGACTGTCCCGGAAGGAGGTGCACGT


 



GGAGCCGCCCGGACCCCGACCGGCAGGACCTGGAAAG


 



ACGCCTCTCACTCCCCTTTCTCTTCTGTCCCCTCTGG


 



GGTCCTCAGAGAGCCAGTCTGCCCCGAATCTCTACCC


 



CCTCGTCTCCTGCGTGAGCCCCCCGTGCGATGAGAGC


 



CTGGTGGCCCTGGGCTGCCTGGCCCGGGACTTCCTGC


 



CCAGCTCCGTCACCTTCTCCTGGAA






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
ctcaaacgtaagtggctttttccgactgattctttgc


 



tgtttctaattgttggttggctttttgtccatttttc


 



agtgttttcatcgaattagttgtcagggaccaaacaa


 



attgccttcccagattaggtaccagggaggggacatt


 



gctgcatgggagaccagagggtggctaatttttaacg


 



tttccaagccaaaataactggggaagggggcttgctg


 



tcctgtgagggtaggtttttatagaagtggaagttaa


 



ggggaaatcgctatggttcacttttggctcggggacc


 



aaagtggagcccaaaattgagtacattttccatcaat


 



tatttgtgagatttttgtcctgttgtgtcatttgtgc


 



aagtttttgacattttggttgaatgagccattcccag


 



ggacccaaaaggatgagaccgaaaagtagaaaagagc


 



caacttttaagctgagcagacagaccgaattgttgag


 



tttgtgaggagagtagggtttgtagggagaaagggga


 



acagatcgctggctttttctctgaattagcctttctc


 



atgggactggcttcagagggggtttttgatgagggaa


 



gtgttctagagccttaactgtgggttgtgttcggtag


 



cgggaccaagctggaaatcaaacgtaagtgcactttt


 



ctactcctttttctttcttatacgggtgtgaaattgg


 



ggacttttcatgtttggagtatgagttgaggtcagtt


 



ctgaagagagtgggactcatccaaaaatctgaggagt


 



aagggtcagaacagagttgtctcatggaagaacaaag


 



acctagttagttgatgaggcagctaaatgagtcagtt


 



gacttgggatccaaatggccagacttcgtctgtaacc


 



aacaatctaatgagatgtagcagcaaaaagagatttc


 



cattgaggggaaagtaaaattgttaatattgtggatc


 



acctttggtgaagggacatccgtggagattgaacgta


 



agtattttttctctactaccttctgaaatttgtctaa


 



atgccagtgttgacttttagaggcttaagtgtcagtt


 



ttgtgaaaaatgggtaaacaagagcatttcatattta


 



ttatcagtttcaaaagttaaactcagctccaaaaatg


 



aatttgtagacaaaaagattaatttaagccaaattga


 



atgattcaaaggaaaaaaaaattagtgtagatgaaaa


 



aggaattcttacagctccaaagagcaaaagcgaatta


 



attttctttgaactttgccaaatcttgtaaatgattt


 



ttgttctttacaatttaaaaaggttagagaaatgtat


 



ttcttagtctgttttctctcttctgtctgataaatta


 



ttatatgagataaaaatgaaaattaataggatgtgct


 



aaaaaatcagtaagaagttagaaaaatatatgtttat


 



gttaaagttgccacttaattgagaatcagaagcaatg


 



ttatttttaaagtctaaaatgagagataaactgtcaa


 



tacttaaattctgcagagattctatatcttgacagat


 



atctcctttttcaaaaatccaatttctatggtagact


 



aaatttgaaatgatcttcctcataatggagggaaaag


 



atggactgaccccaaaagctcagattt*aagaaaacc


 



tgtttaag*gaaagaaaataaaagaactgcatttttt


 



aaaggcccatgaatttgtagaaaaataggaaatattt


 



taataagtgtattcttttattttcctgttattacttg


 



atggtgtttttataccgccaaggaggccgtggcaccg


 



tcagtgtgatctgtagaccccatggcggccttttttc


 



gcgattgaatgaccttggcggtgggtccccagggctc


 



tggtggcagcgcaccagccgctaaaagccgctaaaaa


 



ctgccgctaaaggccacagcaaccccgcgaccgcccg


 



ttcaactgtgctgacacagtgatacagataatgtcgc


 



taacagaggagaatagaaatatgacgggcacacgcta


 



atgtggggaaaagagggagaagcctgatttttatttt


 



ttagagattctagagataaaattcccagtattatatc


 



cttttaataaaaaatttctattaggagattataaaga


 



atttaaagctatttttttaagtggggtgtaattcttt


 



cagtagtctcttgtcaaatggatttaagtaatagagg


 



cttaatccaaatgagagaaatagacgcataacccttt


 



caaggcaaaagctacaagagcaaaaattgaacacagc


 



agccagccatctagccactcagattttgatcagtttt


 



actgagtttgaagtaaatatcatgaaggtataattgc


 



tgataaaaaaataagatacaggtgtgacacatcttta


 



agtttcagaaatttaatggcttcagtaggattatatt


 



tcacgtatacaaagtatctaagcagataaaaatgcca


 



ttaatggaaacttaatagaaatatatttttaaattcc


 



ttcattctgtgacagaaattttctaatctgggtcttt


 



taatcacctaccctttgaaagagtttagtaatttgct


 



atttgccatcgctgtttactccagctaatttcaaaag


 



tgatacttgagaaagattatttttggtttgcaaccac


 



ctggcaggactattttagggccattttaaaactcttt


 



tcaaactaagtattttaaactgttctaaaccatttag


 



ggccttttaaaaatcttttcatgaatttcaaacttcg


 



ttaaaagttattaaggtgtctggcaagaacttcctta


 



tcaaatatgctaatagtttaatctgttaatgcaggat


 



ataaaattaaagtgatcaaggcttgacccaaacagga


 



gtatcttcatagcatatttcccctcctttttttctag


 



aattcatatgattttgctgccaaggctattttatata


 



atctctggaaaaaaaatagtaatgaaggttaaaagag


 



aagaaaatatcagaacattaagaattcggtattttac


 



taactgcttggttaacatgaaggtttttattttatta


 



aggtttctatctttataaaaatctgttcccttttctg


 



ctgatttctccaagcaaaagattcttgatttgttttt


 



taactcttactctcccacccaagggcctgaatgccca


 



caaaggggacttccaggaggccatctggcagctgctc


 



accgtcagaagtgaagccagccagttcctcctgggca


 



ggtggccaaaattacagttgacccctcctggtctggc


 



tgaaccttgccccatatggtgacagccatctggccag


 



ggcccaggtctccctctgaagcctttgggaggagagg


 



gagagtggctggcccgatcacagatgcggaaggggct


 



gactcctcaaccggggtgcagactctgcagggtgggt


 



ctgggcccaacacacccaaagcacgcccaggaaggaa


 



aggcagcttggtatcactgcccagagctaggagaggc


 



accgggaaaatgatctgtccaagacccgttcttgctt


 



ctaaactccgagggggtcagatgaagtggttttgttt


 



cttggcctgaagcatcgtgttccctgcaagaagcggg


 



gaacacagaggaaggagagaaaagatgaactgaacaa


 



agcatgcaaggcaaaaaaggGGGTCTAGCCGCGGTCT


 



AGGAAGCTTTCTAGGGTACCTCTAGGGATCCCGGCGC


 



GCCCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAG


 



TCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTT


 



GGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTC


 



CACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTT


 



GGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAG


 



TCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTG


 



CAGGACGTGACAAATGGAAGTAGCACGTGTCACTAGT


 



CTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGC


 



GGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTG


 



GCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGG


 



TGGGTCCGGGGGCGGGGTCAGGGGCGGGCTCAGGGGC


 



GGGGCGGGCGCCCGAAGGTCCTCCGGAAGCCCGGCAT


 



TCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTT


 



CTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGC


 



CAATATGGGATCGGCCATTGAACAAGATGGATTGCAC


 



GCAGGTTCTCCGGCCGCTTGGGTGGAGAGGGTATTCG


 



GCTATGACTGGGCAGAACAGACAATCGGCTGCTCTGA


 



TGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCG


 



GTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGA


 



ATGAACTGCAGGACGAGGCAGCGCGGCTATGGTGGCT


 



GGCCAGGAGGGGCGTTCGTTGGGCAGCTGTGCTCGAC


 



GTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGG


 



GCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCT


 



TGCTCCTGCCGAGAAAGTATCCATGATGGCTGATGCA


 



ATGGGGCGGCTGCATACGGTTGATGGGGCTACCTGCC


 



CATTCGACCACCAAGCGAAACATCGCATCGAGCGAGC


 



ACGTACTCGGATGGAAGCCGGTCTTGTCAATCAGGAT


 



GATCTGGACGAAGAGCATCAGGGGCTCGCGCGAGCCG


 



AACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGG


 



CGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTG


 



CCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGAT


 



TCATCGACTGTGGCCGGCTGGGTGTGGGGGATCGCTA


 



TGAGGACATAGCGTTGGCTACCGGTGATATTGCTGAA


 



GAGCTTGGCGGCGAATGGGCTGACCGGTTCCTCGTGC


 



TTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGC


 



CTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGAT


 



CAATTCTCTAGAGCTCGCTGATCAGCCTCGACTGTGC


 



CTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCC


 



CGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT


 



GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT


 



GTGTGAGTAGGTGTCATTCTATTGTGGGGGGTGGGGT


 



GGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAAT


 



AGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTT


 



CTGAGGCGGAAAGAACCAGCTGGGGGCGCGGCCctcg


 



agcggccgccagtgtgatggatatctgcagaattcgc


 



ccttggatcaaacacgcatcctcatggacaatatgtt


 



gggttcttagcctgctgagacacaacaggaactcccc


 



tggcaccactttagaggccagagaaacagcacagata


 



aaattccctgccctcatgaagcttatagtctagctgg


 



ggagatatcataggcaagataaacacatacaaataca


 



tcatcttaggtaataatatatactaaggagaaaatta


 



caggggagaaagaggacaggaattgctagggtaggat


 



tataagttcagatagttcatcaggaacactgttgctg


 



agaagataacatttaggtaaagaccgaagtagtaagg


 



aaatggaccgtgtgcctaagtgggtaagaccattcta


 



ggcagcaggaacagcgatgaaagcactgaggtgggtg


 



ttcactgcacagagttgttcactgcacagagttgtgt


 



ggggaggggtaggtcttgcaggctcttatggtcacag


 



gaagaattgttttactcccaccgagatgaaggttggt


 



ggattttgagcagaagaataattctgcctggtttata


 



tataacaggatttccctgggtgctctgatgagaataa


 



tctgtcaggggtgggatagggagagatatggcaatag


 



gagccttggctaggagcccacgacaataattccaagt


 



gagaggtggtgctgcattgaaagcaggactaacaaga


 



cctgctgacagtgtggatgtagaaaaagatagaggag


 



acgaaggtgcatctagggttttctgcctgaggaatta


 



gaaagataaagctaaagcttatagaagatgcagcgct


 



ctggggagaaagaccagcagctcagttttgatccatc


 



tggaattaattttggcataaagtatgaggtatgtggg


 



ttaacattatttgttttttttttttccatgtagctat


 



ccaactgtcccagcatcatttattttaaaagactttc


 



ctttcccctattggattgttttggcaccttcactgaa


 



gatcaactgagcataaaattgggtctatttctaagct


 



cttgattccattccatgacctatttgttcatctttac


 



cccagtagacactgccttgatgattaaagcccctgtt


 



accatgtctgttttggacatggtaaatctgagatgcc


 



tattagccaaccaagcaagcacggcccttagagagct


 



agatatgagagcctggaattcagacgagaaaggtcag


 



tcctagagacatacatgtagtgccatcaccatgcgga


 



tggtgttaaaagccatcagactgcaacagactgtgag


 



agggtaccaagctagagagcatggatagagaaaccca


 



agcactgagctgggaggtgctcctacattaagagatt


 



agtgagatgaaggactgagaagattgatcagagaaga


 



aggaaaatcaggaaaatggtgctgtcctgaaaatcca


 



agggaagagatgttccaaagaggagaaaactgatcag


 



ttgtcagctagcgtcaattgggatgaaaatggaccat


 



tggacagagggatgtagtgggtcatgggtgaatagat


 



aagagcagcttctatagaatggcaggggcaaaattct


 



catctgatcggcatgggttctaaagaaaacgggaaga


 



aaaaattgagtgcatgaccagtcccttcaagtagaga


 



ggtggaaaagggaaggaggaaaatgaggccacgacaa


 



catgagagaaatgacagcatttttaaaaattttttat


 



tttattttatttatttatttttgctttttagggctgc


 



ccctgcaacatatggaggttcccaggttaggggtcta


 



atcagagctatagctgccagcctacaccacagccata


 



gcaatgccagatctacatgacctacaccacagctcac


 



agcaacgccggatccttaacccactgagtgaggccag


 



agatcaaacccatatccttatggatactagtcaggtt


 



cattaccactgagccaaaatgggaaatcctgagtaat


 



gacagcattttttaatgtgccaggaagcaaaacttgc


 



caccccgaaatgtctctcaggcatgtggattattttg


 



agctgaaaacgattaaggcccaaaaaacacaagaaga


 



aatgtggaccttcccccaacagcctaaaaaatttaga


 



ttgagggcctgttcccagaatagagctattgccagac


 



ttgtctacagaggctaagggctaggtgtggtggggaa


 



accctcagagatcagagggacgtttatgtaccaagca


 



ttgacatttccatctccatgcgaatggccttcttccc


 



ctctgtagccccaaaccaccacccccaaaatcttctt


 



ctgtctttagctgaagatggtgttgaaggtgatagtt


 



tcagccactttggcgagttcctcagttgttctgggtc


 



tttcctccTgatccacattattcgactgtgtttgatt


 



ttctcctgtttatctgtctcattggcacccatttcat


 



tcttagaccagcccaaagaacctagaagagtgaagga


 



aaatttcttccaccctgacaaatgctaaatgagaatc


 



accgcagtagaggaaaatgatctggtgctgcgggaga


 



tagaagagaaaatcgctggagagatgtcactgagtag


 



gtgagatgggaaaggggtgacacaggtggaggtgttg


 



ccctcagctaggaagacagacagttcacagaagagaa


 



gcgggtgtccgtggacatcttgcctcatggatgagga


 



aaccgaggctaagaaagactgcaaaagaaaggtaagg


 



attgcagagaggtcgatccatgactaaaatcacagta


 



accaaccccaaaccaccatgttttctcctagtctggc


 



acgtggcaggtactgtgtaggttttcaatattattgg


 



tttgtaacagtacctattaggcctccatcccctcctc


 



taatactaacaaaagtgtgagactggtcagtgaaaaa


 



tggtcttctttctctatgaatctttctcaagaagata


 



cataactttttattttatcataggcttgaagagcaaa


 



tgagaaacagcctccaacctatgacaccgtaacaaaa


 



tgtttatgatcagtgaagggcaagaaacaaaacatac


 



acagtaaagaccctccataatattgtgggtggcccaa


 



cacaggccaggttgtaaaagctttttattctttgata


 



gaggaatggatagtaatgtttcaacctggacagagat


 



catgttcactgaatccttccaaaaattcatgggtagt


 



ttgaattataaggaaaataagacttaggataaatact


 



ttgtccaagatcccagagttaatgccaaaatcagttt


 



tcagactccaggcagcctgatcaagagcctaaacttt


 



aaagacacagtcccttaataactactattcacagttg


 



cactttcagggcgcaaagactcattgaatcctacaat


 



agaatgagtttagatatcaaatctctcagtaatagat


 



gaggagactaaatagcgggcatgacctggtcacttaa


 



agacagaattgagattcaaggctagtgttctttctac


 



ctgttttgtttctacaagatgtagcaatgcgctaatt


 



acagacctctcagggaaggaa






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 44
taaacaaataggggttccgcgcacatttccccgaaaagtgc


 



cacctgacgtcgctgagcaggccctggcctccctggccgag


 



ggcggtttgcgtattagaggcctaaatggccgaattcagcg


 



gataacaatttcacacaggaaacagctatgaccatgattat


 



ctagtaactataacggtcctaaggtagcgagcgatcgctta


 



attaacctgcagggatatcccatgggggccgccagtgtgat


 



ggatatctgcagaattcgcccttgatattaagagaagggca


 



agtcagcttaagtttgggggtagaggggaacagggagtgag


 



gagatctggcctgagagataggagccctggtggccacagga


 



ggactctttgggtcctgtcggatggacacagggcggcccgg


 



gggcatgttggagcccggctggttcttaccagaggcagggg


 



gcaccctctgacacgggagcagggcatgttccatacatgac


 



acacccctctgctccagggcaggtgggtggcggcacagagg


 



agccagggactctgagcaaggggtccaccagtggggcagtt


 



ggatccagacttctctgggccagcgagagtctagccctcag


 



ccgttctctgtccaggaggggggtggggcaggcctgggcgg


 



ccagagctcatccctcaagggttcccagggtcctgccagac


 



ccagatttccgaccgcagccaccacaagaggatgtggtctg


 



ctgtggcagctgccaagaccttgcagcaggtgcagggtggg


 



ggggtgggggcacctgggggcagctggggtcactgagttca


 



gggaaaaccccttttttcccctaaacctggggccatcccta


 



ggggaaaccacaacttctgagccctgggcagtggctgctgg


 



gagggaagagcttcatcctggaccctgggggggaacccagc


 



tccaaaggtgcaaggggcccaggtccaaggctagagtgggc


 



caagcaccgcaatggccagggagtgggggaggtggagctgg


 



actggatcagggcctccttgggactccctacaccctgtgtg


 



acatgttagggtacccacaccccatcaccagtcagggcctg


 



gcccatctccagggccagggatgtgcatgtaagtgtgtgtg


 



agtgtgtgtgtgtggtgtagtacaccccttggcatccggtt


 



ccgaggccttgggttcctccaaagttgctctctgaattagg


 



tcaaactgtgaggtcctgatcgccatcatcaacttcgttct


 



ccccacctcccatcattatcaagagctggggagggtctggg


 



atttcttcccacccacaagccaaaagataagcctgctggtg


 



atggcagaagacacaggatcctgggtcagagacaaaggcca


 



gtgtgtcacagcgagagaggcagccggactatcagctgtca


 



cagagaggccttagtccgctgaactcaggccccagtgactc


 



ctgttccactgggcactggcccccctccacagcgcccccag


 



gccccagggagaggcgtcacagcttagagatggccctgctg


 



aacagggaacaagaacaggtgtgccccatccagcgccccag


 



gggtgggacaggtgggctggatttggtgtgaagcccttgag


 



ccctggaacccaaccacagcagggcagttggtagatgccat


 



ttggggagaggccccaggagtaagggccatgggcccttgag


 



ggggccaggagctgaggacagggacagagacggcccaggca


 



gaggacagggccatgaggggtgcactgagatggccactgcc


 



agcaggggcagctgccaacccgtccagggaacttattcagc


 



agtcagctggaggtgccattgaccctgagggcagatgaagc


 



ccaggccaggctaggtgggctgtgaagaccccaggggacag


 



agctctgtccctgggcagcactggcctctcattctgcaggg


 



cttgacgggatcccaaggcctgctgcccctgatggtagtgg


 



cagtaccgcccagagcaggaccccagcatggaaaccccaac


 



gggacgcagcctgcggagcccacaaaaccagtaaggagccg


 



aagcagtcatggcacggggagtgtggacttccctttgatgg


 



ggcccaggcatgaaggacagaatgggacagcggccatgagc


 



agaaaatcagccggaggggatgggcctaggcagacgctggc


 



tttatttgaagtgttggcattttgtctggtgtgtattgttg


 



gtattgattttattttagtatgtcagtgacatactgacata


 



ttatgtaacgacatattattatgtgttttaagaagcactcc


 



aagggaacaggctgtctgtaatgtgtccagagaagagagca


 



agagcttggctcagtctcccccaaggaggtcagttcctcaa


 



caggggtcctaaatgtttcctggagccaggcctgaatcaag


 



ggggtcatatctacacgtggggcagacccatggaccatttt


 



cggagcaataagatggcagggaggataccaagctggtctta


 



cagatccagggctttgacctgtgacgcgggcgctcctccag


 



gcaaagggagaagccagcaggaagctttcagaactggggag


 



aacagggtgcagacctccagggtcttgtacaacgcaccctt


 



tatcctggggtccaggaggggtcactgagggatttaagtgg


 



gggaccatcagaaccaggtttgtgttttggaaaaatggctc


 



caaagcagagaccagtgtgaggccagattagatgatgaaga


 



agaggcagtggaaagtcgatgggtggccaggtagcaagagg


 



gcctatggagttggcaagtgaatttaaagtggtggcaccag


 



agggcagatggggaggagcaggcactgtcatggactgtcta


 



tagaaatctaaaatgtataccctttttagcaatatgcagtg


 



agtcataaaagaacacatatatatttcctttggccggccgg


 



cgcgccacgcgtataacttcgtatagcatacattatacgaa


 



gttatcttaagggctatggcagggcctgccgccccgacgtt


 



ggctgcgagccctgggccttcacccgaacttggggggtggg


 



gtggggaaaaggaagaaacgcgggcgtattggccccaatgg


 



ggtctcggtggggtatcgacagagtgccagccctgggaccg


 



aaccccgcgtttatgaacaaacgacccaacaccgtgcgttt


 



tattctgtctttttattgccgtcatagcgcgggttccttcc


 



ggtattgtctccttccgtgtttcactcgagttagaagaact


 



cgtcaagaaggcgatagaaggcgatgcgctgcgaatcggga


 



gcggcgataccgtaaagcacgaggaagcggtcagcccattc


 



gccgccaagctcttcagcaatatcacgggtagccaacgcta


 



tgtcctgatagcggtccgccacacccagccggccacagtcg


 



atgaatccagaaaagcggccattttccaccatgatattcgg


 



caagcaggcatcgccatgggtcacgacgagatcctcgccgt


 



cgggcatgcgcgccttgagcctggcgaacagttcggctggc


 



gcgagcccctgatgctcttcgtccagatcatcctgatcgac


 



aagaccggcttccatccgagtacgtgctcgctcgatgcgat


 



gtttcgcttggtggtcgaatgggcaggtagccggatcaagc


 



gtatgcagccgccgcattgcatcagccatgatggatacttt


 



ctcggcaggagcaaggtgagatgacaggagatcctgccccg


 



gcacttcgcccaatagcagccagtcccttcccgcttcagtg


 



acaacgtcgagcacagctgcgcaaggaacgcccgtcgtggc


 



cagccacgatagccgcgctgcctcgtcctgcagttcattca


 



gggcaccggacaggtcggtcttgacaaaaagaaccgggcgc


 



ccctgcgctgacagccggaacacggcggcatcagagcagcc


 



gattgtctgttgtgcccagtcatagccgaatagcctctcca


 



cccaagcggccggagaacctgcgtgcaatccatcttgttca


 



atggccgatcccattccagatctgttagcctcccccatctc


 



ccgtgcaaacgtgcgcgccaggtcgcagatcgtcggtatgg


 



agcctggggtggtgacgtgggtctggatcatcccggaggta


 



agttgcagcagggcgtcccggcagccggcgggcgattggtc


 



gtaatccaggataaagacgtgcatgggacggaggcgtttgg


 



tcaagacgtccaaggcccaggcaaacacgttgtacaggtcg


 



ccgttgggggccagcaactcgggggcccgaaacagggtaaa


 



taacgtgtccccgatatggggtcgtgggcccgcgttgctct


 



ggggctcggcaccctggggcggcacggccgtccccgaaagc


 



tgtccccaatcctcccgccacgacccgccgccctgcagata


 



ccgcaccgtattggcaagcagcccgtaaacgcggcgaatcg


 



cggccagcatagccaggtcaagccgctcgccggggcgctgg


 



cgtttggccaggcggtcgatgtgtctgtcctccggaagggc


 



ccccaacacgatgtttgtgccgggcaaggtcggcgggatga


 



gggccacgaacgccagcacggcctggggggtcatgctgccc


 



ataaggtatcgcgcggccgggtagcacaggagggcggcgat


 



gggatggcggtcgaagatgagggtgagggccgggggcgggg


 



catgtgagctcccagcctcccccccgatatgaggagccaga


 



acggcgtcggtcacggcataaggcatgcccattgttatctg


 



ggcgcttgtcattaccaccgccgcgtccccggccgatatct


 



caccctggtcaaggcggtgttgtgtggtgtagatgttcgcg


 



attgtctcggaagcccccagcacccgccagtaagtcatcgg


 



ctcgggtacgtagacgatatcgtcgcgcgaacccagggcca


 



ccagcagttgcgtggtggtggttttccccatcccgtgggga


 



ccgtctatataaacccgcagtagcgtgggcattttctgctc


 



cgggcggacttccgtggcttcttgctgccggcgagggcgca


 



acgccgtacgtcggttgctatggccgcgagaacgcgcagcc


 



tggtcgaacgcagacgcgtgctgatggccggggtacgaagc


 



catggtggctctagaggtcgaaaggcccggagatgaggaag


 



aggagaacagcgcggcagacgtgcgcttttgaagcgtgcag


 



aatgccgggcttccggaggaccttcgggcgcccgccccgcc


 



cctgagcccgcccctgagcccgcccccggacccaccccttc


 



ccagcctctgagcccagaaagcgaaggagccaaagctgcta


 



ttggccgctgccccaaaggcctacccgcttccattgctcag


 



cggtgctgtccatctgcacgagactagtgagacgtgctact


 



tccatttgtcacgtcctgcacgacgcgagctgcggggcggg


 



ggggaacttcctgactaggggaggagtagaaggtggcgcga


 



aggggccaccaaagaacggagccggttggcgcctaccggtg


 



gatgtggaatgtgtgcgaggccagaggccacttgtgtagcg


 



ccaagtgcccagcggggctgctaaagcgcatgctccagact


 



gccttgggaaaagcgcctcccctacccggtagggatccgcg


 



ttacataacttacggtaaatggcccgcctggctgaccgccc


 



aacgacccccgcccattgacgtcaataatgacgtatgttcc


 



catagtaacgccaatagggactttccattgacgtcaatggg


 



tggagtatttacggtaaactgcccacttggcagtacatcaa


 



gtgtatcatatgccaagtacgccccctattgacgtcaatga


 



cggtaaatggcccgcctggcattatgcccagtacatgacct


 



tatgggactttcctacttggcagtacatctacgtattagtc


 



atcgctattaccatggtgatgcggttttggcagtacatcaa


 



tgggcgtggatagcggtttgactcacggggatttccaagtc


 



tccaccccattgacgtcaatgggagtttgttttggcaccaa


 



aatcaacggttaacaagcttataacttcgtatagcatacat


 



tatacgaagttattacgtagcggccgcgtcgacgataaatt


 



gtgtaattccacttctaaggattcatcccaaggggggaaaa


 



taatcaaagatgtaaccaaaggtttacaaacaagaactcat


 



cattaatcttccttgttgttatttcaacgatattattatta


 



ttactattattattattattattttgtctttttgcattttc


 



tagggccactcccacggcatagagaggttcccaggctaggg


 



gtcaaatcggagctacagctgccggcctacgccagagccac


 



agcaacgcaggatctgagccacagcaatgcaggatctacac


 



cacagctcatggtaacgctggatccttaacccaatgagtga


 



ggccagggatcgaacctgtaacttcatggttcctagtcgga


 



ttcattaaccactgagccacgacaggaactccaacattatt


 



aatgatgggagaaaactggaagtaacctaaatatccagcag


 



aaagggtgtggccaaatacagcatggagtagccatcataag


 



gaatcttacacaagcctccaaaattgtgtttctgaaattgg


 



gtttaaagtacgtttgcattttaaaaagcctgccagaaaat


 



acagaaaaatgtctgtgatatgtctctggctgataggattt


 



tgcttagttttaattttggctttataattttctatagttat


 



gaaaatgttcacaagaagatatatttcattttagcttctaa


 



aataattataacacagaagtaatttgtgctttaaaaaaata


 



ttcaacacagaagtatataaagtaaaaattgaggagttccc


 



atcgtggctcagtgattaacaaacccaactagtatccatga


 



ggatatggatttgatccctggccttgctcagtgggttgagg


 



atccagtgttgctgtgagctgtggtgtaggttgcagacaca


 



gcactctggcgttgctgtgactctggcgtaggccggcagct


 



acagctccatttggacccttagcctgggaacctccatatgc


 



ctgagatacggccctaaaaagtcaaaagccaaaaaaatagt


 



aaaaattgagtgtttctacttaccacccctgcccacatctt


 



atgctaaaacccgttctccagagacaaacatcgtcaggtgg


 



gtctatatatttccagccctcctcctgtgtgtgtatgtccg


 



taaaacacacacacacacacacacacgcacacacacacaca


 



cgtatctaattagcattggtattagtttttcaaaagggagg


 



tcatgctctaccttttaggcggcaaatagattatttaaaca


 



aatctgttgacattttctatatcaacccataagatctccca


 



tgttcttggaaaggctttgtaagacatcaacatctgggtaa


 



accagcatggtttttagggggttgtgtggatttttttcata


 



ttttttagggcacacctgcagcatatggaggttcccaggct


 



aggggttgaatcagagctgtagctgccggcctacaccacag


 



ccacagcaacgccagatccttaacccactgagaaaggccag


 



ggattgaacctgcatcctcatggatgctggtcagatttatt


 



tctgctgagccacaacaggaactccctgaaccagaatgctt


 



ttaaccattccactttgcatggacatttagattgtttccat


 



ttaaaaatacaaattacaaggagttcccgtcgtggctcagt


 



ggtaacgaattggactaggaaccatgaggtttcgggttcga


 



tccctggccttgctcggtgggttaaggatccagcattgatg


 



tgagatatggtgtaggtcgcagacgtggctcggatcccacg


 



ttgctgtggctctggcgtaggccggcaacaacagctccgat


 



tcgacccctagcctgggaacctccatgtgccacaggagcag


 



ccctagaaaaggcaaaaagacaaaaaaataaaaaattaaaa


 



tgaaaaaataaaataaaaatacaaattacaagagacggcta


 



caaggaaatccccaagtgtgtgcaaatgccatatatgtata


 



aaatgtactagtgtctcctcgcgggaaagttgcctaaaagt


 



gggttggctggacagagaggacaggctttgacattctcata


 



ggtagtagcaatgggcttctcaaaatgctgttccagtttac


 



actcaccatagcaaatgacagtgcctcttcctctccaccct


 



tgccaataatgtgacaggtggatctttttctattttgtgta


 



tctgacaagcaaaaaatgagaacaggagttcctgtcgtggt


 



gcagtggagacaaatctgactaggaaccatgaaatttcggg


 



ttcaatccctggcctcactcagtaggtaaaggatccagggt


 



tgcagtgagctgtggggtaggtcgcagacacagtgcaaatt


 



tggccctgttgtggctgtggtgtaggccggcagctatagct


 



ccaattggacccctagcctgggaacctccttatgccgtggg


 



tgaggccctaaaaaaaagagtgcaaaaaaaaaaaataagaa


 



caaaaatgatcatcgtttaattctttatttgatcattggtg


 



aaacttattttccttttatatttttattgactgattttatt


 



tctcctatgaatttaccggtcatagttttgcctgggtgttt


 



ttactccggttttagttttggttggttgtattttcttagag


 



agctatagaaactcttcatctatttggaatagtaattcctc


 



attaagtatttgtgctgcaaaaaattttccctgatctgttt


 



tatgcttttgtttgtggggtctttcacgagaaagccttttt


 



agtttttacacctcagcttggttgtttttcttgattgtgtc


 



tgtaatctgcggccaacataggaaacacatttttactttag


 



tgtttttttcctattttcttcaagtacgtccattgttttgg


 



tgtctgattttactttgcctggggtttgtttttgtgtggca


 



ggaatataaacttatgtattttccaaatggagagccaatgg


 



ttgtatatttgttgaattcaaatgcaactttatcaaacacc


 



aaatcatcgatttatcacaactcttctctggtttattgatc


 



taatgatcaattcctgttccacgctgttttaattattttag


 



ctttgtggattttggtgcctggtagagaacaaagcctccat


 



tattttcattcaaaatagtcccgtctattatctgccattgt


 



tgtagtattagactttaaaatcaatttactgattttcaaaa


 



gttattcctttggtgatgtggaatactttatacttcataag


 



gtacatggattcatttgtggggaattgatgtctttgctatt


 



gtggccatttgtcaagttgtgtaatattttacccatgccaa


 



ctttgcatattgtatgtgagtttattcccagggtttttaat


 



aggatgtttattgaagttgtcagtgtttccacaatttcatc


 



gcctcagtgcttactgtttgcataaaaggaaacctactcac


 



ttttgcctattgctcttgtattcaatcattttagttaactc


 



ttgtgttaattttgagagtttttcagctgactgtctggggt


 



tttctttaatagactagccctttgtctgtaaagaataattt


 



tatcgaatttttcttaacactcacactctccccacccccac


 



ccccgctcatctcctttcattgggtcaaatctgtagaatac


 



aataaaagtaagagtgggaaccttagcctttaagtcgattt


 



tgcctttaaatgtgaatgttgctatgtttcgggacattctc


 



tttatcaagttgcggatgtttccttagataattaacttaat


 



aaaagactggatgtttgctttcttcaaatcagaattgtgtt


 



gaatttatattgctattctgtttaattttgtttcaaaaaat


 



ttacatgcacaccttaaagataaccatgaccaaatagtcct


 



cctgctgagagaaaatgttggccccaatgccacaggttacc


 



tcccgactcagataaactacaatgggagataaaatcagatt


 



tggcaaagcctgtggattcttgccataactctcagagcatg


 



acttgggtgttttttccttttctaagtattttaatggtatt


 



tttgtgttacaataggaaatctaggacacagagagtgattc


 



aatgaggggaacgcattctgggatgactctaggcctctggt


 



ttggggagagctctattgaagtaaagacaatgagaggaagc


 



aagtttgcagggaactgtgaggaatttagatggggaatgtt


 



gggtttgaggtttctatagggcacgcaagcagagatgcact


 



caggaggaagaaggagcataaatctagtggcgctgccggca


 



agcttgctggaggaggccaattgggagctgctggaatgcat


 



ggaggcggcgctctcgaggctggaggaggccagctgattta


 



aatcggtccgcgtacgatgcatattaccctgttatccctac


 



cgcggttactggccgtcgttttacaacgtcgtgactgggaa


 



aaccctggcgatgctcttctcccggtgaaaacctctgacac


 



atggctcttctaaatccggagtttaaacgcttccttcatgt


 



gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggcc


 



gcgttgctggcgtttttccataggctccgcccccctgacga


 



gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacc


 



cgacaggactataaagataccaggcgtttccccctggaagc


 



tccctcgtgcgctctcctgttccgaccctgccgcttaccgg


 



atacctgtccgcctttctcccttcgggaagcgtggcgcttt


 



ctcatagctcacgctgtaggtatctcagttcggtgtaggtc


 



gttcgctccaagctgggctgtgtgcacgaaccccccgttca


 



gcccgaccgctgcgccttatccggtaactatcgtcttgagt


 



ccaacccggtaagacacgacttatcgccactggcagcagcc


 



actggtaacaggattagcagagcgaggtatgtaggcggtgc


 



tacagagttcttgaagtggtggcctaactacggctacacta


 



gaaggacagtatttggtatctgcgctctgctgaagccagtt


 



accttcggaaaaagagttggtagctcttgatccggcaaaca


 



aaccaccgctggtagcggtggtttttttgtttgcaagcagc


 



agattacgcgcagaaaaaaaggatctcaagaagatcctttg


 



atcttttctacggggtctgacgctcagtggaacgaaaactc


 



acgttaagggattttggtcatgcctaggtggcaaacagcta


 



ttatgggtattatgggtctaccggtgcatgagattatcaaa


 



aaggatcttcacctagatccttttaaattaaaaatgaagtt


 



ttaaatcaatctaaagtatatatgagtaaacttggtctgac


 



agttaccaatgcttaatcagtgaggcacctatctcagcgat


 



ctgtctatttcgttcatccatagttgcctgactccccgtcg


 



tgtagataactacgatacgggagggcttaccatctggcccc


 



agtgctgcaatgataccgcgagacccacgctcaccggctcc


 



agatttatcagcaataaaccagccagccggaagggccgagc


 



gcagaagtggtcctgcaactttatccgcctccatccagtct


 



attaattgttgccgggaagctagagtaagtagttcgccagt


 



taatagtttgcgcaacgttgttgccattgctacaggcatcg


 



tggtgtcacgctcgtcgtttggtatggcttcattcagctcc


 



ggttcccaacgatcaaggcgagttacatgatcccccatgtt


 



gtgcaaaaaagcggttagctccttcggtcctccgatcgttg


 



tcagaagtaagttggccgcagtgttatcactcatggttatg


 



gcagcactgcataattctcttactgtcatgccatccgtaag


 



atgcttttctgtgactggtgagtactcaaccaagtcattct


 



gagaatagtgtatgcggcgaccgagttgctcttgcccggcg


 



tcaatacgggataataccgcgccacatagcagaactttaaa


 



agtgctcatcattggaaaacgttcttcggggcgaaaactct


 



caaggatcttaccgctgttgagatccagttcgatgtaaccc


 



actcgtgcacccaactgatcttcagcatcttttactttcac


 



cagcgtttctgggtgagcaaaaacaggaaggcaaaatgccg


 



caaaaaagggaataagggcgacacggaaatgttgaatactc


 



atactcttcctttttcaatattattgaagcatttatcaggg


 



ttattgtctcgggagcggatacatatttgaatgtatttaga


 



aaaa


 


SEQ ID 45
taaacaaataggggttccgcgcacatttccccgaaaagtgc


 



cacctgacgtcgctgagcaggccctggcctccctggccgag


 



ggcggtttgcgtattagaggcctaaatggccgaattcagcg


 



gataacaatttcacacaggaaacagctatgaccatgattat


 



ctagtaactataacggtcctaaggtagcgagcgatcgctta


 



attaacctgcagggataaccactgacccatgacgggaactc


 



ccagggctcagctcttgactccaggttcgcagctgccctca


 



aagcaatgcaaccctggctggccccgcctcatgcatccggc


 



ctcctccccaaagagctctgagcccacctgggcctaggtcc


 



tcctccctgggactcatggcctaagggtacagagttactgg


 



ggctgatgaagggaccaatggggacaggggcctcaaatcaa


 



agtggctgtctctctcatgtcccttcctctcctcagggtcc


 



aaaatcagggtcagggccccagggcaggggctgagagggcc


 



tctttctgaaggccctgtctcagtgcaggttatgggggtct


 



gggggagggtcaatgcagggctcacccttcagtgccccaaa


 



gcctagagagtgagtgcctgccagtggcttcccaggcccaa


 



tcccttgactgcctgggaatgctcaaatgcaggaactgtca


 



caacaccttcagtcaggggctgctctgggaggaaaaacact


 



cagaattgggggttcagggaaggcccagtgccaagcatagc


 



aggagctcaggtggctgcagatggtgtgaaccccaggagca


 



ggatggccggcactccccccagaccctccagagccccaggt


 



tggctgccctcttcactgccgacacccctgggtccacttct


 



gccctttcccacctaaaacctttagggctcccactttctcc


 



caaatgtgagacatcaccacggctcccagggagtgtccaga


 



agggcatctggctgagaggtcctgacatctgggagcctcag


 



gccccacaatggacagacgccctgccaggatgctgctgcag


 



ggctgttagctaggcggggtggagatggggtactttgcctc


 



tcagaggccccggccccaccatgaaacctcagtgacacccc


 



atttccctgagttcacatacctgtatcctactccagtcacc


 



ttccccacgaacccctgggagcccaggatgatgctggggct


 



ggagccacgaccagcccacgagtgatccagctctgccaatc


 



agcagtcatttcccaagtgttccagccctgccaggtcccac


 



tacagcagtaatggaggccccagacaccagtccagcagtta


 



gagggctggactagcaccagctttcaagcctcagcatctca


 



aggtgaatggccagtgcccctccccgtggccatcacaggat


 



cgcagatatgaccctaggggaagaaatatcctgggagtaag


 



gaagtgcccatactcaaggatggcccctctgtgacctaacc


 



tgtccctgaggattgtacttccaggcgttaaaacagtagaa


 



cgcctgcctgtgaacccccgccaagggactgcttggggagg


 



ccccctaaaccagaacacaggcactccagcaggacctctga


 



actctgaccaccctcagcaagtgggcaccccccgcagcttc


 



caaggcaccccagggctcaccacagcggcccctcctggcag


 



cccctcacccaggcccagaccctctaagatggcacatctaa


 



gccaatccacctccttgtcattcctcctgtccccacccagg


 



acccttctcagatgaaaccttcgctccagccgctgggccct


 



ctctcctgcccctctggcagttctccagggactccgcctcc


 



cactctctgtctctccctgcactcctaggaacaagcgacct


 



ccaggaagcccagtccaattatcccctctgtgtcctcccca


 



atctctgcctctgggtggatttgagcaccacatcctgttct


 



cttcgacctgaaactccttggccccggtgtccgctctcctg


 



ggccctcttttctctcctcccctcttccgtgccccgtttgt


 



ttggtgttacaggcaggccccggggagccgtccctccagct


 



gctcttccttgtctgtctcaggagccagaaactggcagcat


 



ctaaaaagggctcctgtttcttcatctgcccagcctcctag


 



cccaaccagggctctggcctcactccagagggtgggctcca


 



gagggcaggggttgcaccctcttagtgcctcagaggctcag


 



ctgggtgcaggatgggggggccctcagggagcccctcagtg


 



actgctgatcacttactgcaggactgttcccagctcttccc


 



aatcattggaatgacaatacctagttctgctccatcatagt


 



gatgcaggaaaaatgttactgaaatcctggttcttgtttag


 



caatcgaagaatgaattccgcgaacacacaggcagcaagca


 



agcgaagcctttattaaaggaaagcagatagctcccagggc


 



tgcagggagcggggagaagagctccccactctctattgtcc


 



tatagggctttttaccccttaaagttggggggatacaaaaa


 



aaatagaagaaaaagggagttcccgtcagggcacagcagaa


 



acaaatccaactaggaaccatgaggttgggggttcgattcc


 



tggcctctctcagtgggttaaggatgcagcgttgccgtgag


 



ctatgatacaggtcacagatgcagctcagatctactagtca


 



attgacaggcgccggagcaggagctaggcctttggccggcc


 



ggcgcgccagatctcttaagggctatggcagggcctgccgc


 



cccgacgttggctgcgagccctgggccttcacccgaacttg


 



gggggtggggtggggaaaaggaagaaacgcgggcgtattgg


 



ccccaatggggtctcggtggggtatcgacagagtgccagcc


 



ctgggaccgaaccccgcgtttatgaacaaacgacccaacac


 



cgtgcgttttattctgtctttttattgccgtcatagcgcgg


 



gttccttccggtattgtctccttccgtgtttcactcgagtt


 



agaagaactcgtcaagaaggcgatagaaggcgatgcgctgc


 



gaatcgggagcggcgataccgtaaagcacgaggaagcggtc


 



agcccattcgccgccaagctcttcagcaatatcacgggtag


 



ccaacgctatgtcctgatagcggtccgccacacccagccgg


 



ccacagtcgatgaatccagaaaagcggccattttccaccat


 



gatattcggcaagcaggcatcgccatgggtcacgacgagat


 



cctcgccgtcgggcatgcgcgccttgagcctggcgaacagt


 



tcggctggcgcgagcccctgatgctcttcgtccagatcatc


 



ctgatcgacaagaccggcttccatccgagtacgtgctcgct


 



cgatgcgatgtttcgcttggtggtcgaatgggcaggtagcc


 



ggatcaagcgtatgcagccgccgcattgcatcagccatgat


 



ggatactttctcggcaggagcaaggtgagatgacaggagat


 



cctgccccggcacttcgcccaatagcagccagtcccttccc


 



gcttcagtgacaacgtcgagcacagctgcgcaaggaacgcc


 



cgtcgtggccagccacgatagccgcgctgcctcgtcctgca


 



gttcattcagggcaccggacaggtcggtcttgacaaaaaga


 



accgggcgcccctgcgctgacagccggaacacggcggcatc


 



agagcagccgattgtctgttgtgcccagtcatagccgaata


 



gcctctccacccaagcggccggagaacctgcgtgcaatcca


 



tcttgttcaatggccgatcccattccagatctgttagcctc


 



ccccatctcccgtgcaaacgtgcgcgccaggtcgcagatcg


 



tcggtatggagcctggggtggtgacgtgggtctggatcatc


 



ccggaggtaagttgcagcagggcgtcccggcagccggcggg


 



cgattggtcgtaatccaggataaagacgtgcatgggacgga


 



ggcgtttggtcaagacgtccaaggcccaggcaaacacgttg


 



tacaggtcgccgttgggggccagcaactcgggggcccgaaa


 



cagggtaaataacgtgtccccgatatggggtcgtgggcccg


 



cgttgctctggggctcggcaccctggggcggcacggccgtc


 



cccgaaagctgtccccaatcctcccgccacgacccgccgcc


 



ctgcagataccgcaccgtattggcaagcagcccgtaaacgc


 



ggcgaatcgcggccagcatagccaggtcaagccgctcgccg


 



gggcgctggcgtttggccaggcggtcgatgtgtctgtcctc


 



cggaagggcccccaacacgatgtttgtgccgggcaaggtcg


 



gcgggatgagggccacgaacgccagcacggcctggggggtc


 



atgctgcccataaggtatcgcgcggccgggtagcacaggag


 



ggcggcgatgggatggcggtcgaagatgagggtgagggccg


 



ggggcggggcatgtgagctcccagcctcccccccgatatga


 



ggagccagaacggcgtcggtcacggcataaggcatgcccat


 



tgttatctgggcgcttgtcattaccaccgccgcgtccccgg


 



ccgatatctcaccctggtcaaggcggtgttgtgtggtgtag


 



atgttcgcgattgtctcggaagcccccagcacccgccagta


 



agtcatcggctcgggtacgtagacgatatcgtcgcgcgaac


 



ccagggccaccagcagttgcgtggtggtggttttccccatc


 



ccgtggggaccgtctatataaacccgcagtagcgtgggcat


 



tttctgctccgggcggacttccgtggcttcttgctgccggc


 



gagggcgcaacgccgtacgtcggttgctatggccgcgagaa


 



cgcgcagcctggtcgaacgcagacgcgtgctgatggccggg


 



gtacgaagccatggtggctctagaggtcgaaaggcccggag


 



atgaggaagaggagaacagcgcggcagacgtgcgcttttga


 



agcgtgcagaatgccgggcttccggaggaccttcgggcgcc


 



cgccccgcccctgagcccgcccctgagcccgcccccggacc


 



caccccttcccagcctctgagcccagaaagcgaaggagcca


 



aagctgctattggccgctgccccaaaggcctacccgcttcc


 



attgctcagcggtgctgtccatctgcacgagactagtgaga


 



cgtgctacttccatttgtcacgtcctgcacgacgcgagctg


 



cggggcgggggggaacttcctgactaggggaggagtagaag


 



gtggcgcgaaggggccaccaaagaacggagccggttggcgc


 



ctaccggtggatgtggaatgtgtgcgaggccagaggccact


 



tgtgtagcgccaagtgcccagcggggctgctaaagcgcatg


 



ctccagactgccttgggaaaagcgcctcccctacccggtag


 



ggatccgcgttacataacttacggtaaatggcccgcctggc


 



tgaccgcccaacgacccccgcccattgacgtcaataatgac


 



gtatgttcccatagtaacgccaatagggactttccattgac


 



gtcaatgggtggagtatttacggtaaactgcccacttggca


 



gtacatcaagtgtatcatatgccaagtacgccccctattga


 



cgtcaatgacggtaaatggcccgcctggcattatgcccagt


 



acatgaccttatgggactttcctacttggcagtacatctac


 



gtattagtcatcgctattaccatggtgatgcggttttggca


 



gtacatcaatgggcgtggatagcggtttgactcacggggat


 



ttccaagtctccaccccattgacgtcaatgggagtttgttt


 



tggcaccaaaatcaacggttaacaagcttataacttcgtat


 



agcatacattatacgaagttattacgtagcggccgcgtcga


 



cgatatcgctgccggagcccccggggccgctgccggaagat


 



ctggcattgctgtgactgtggtgtaggccggcagctggagc


 



tctgattagacccctcacctgggaatctccatatgctgcac


 



gtgcggccctaaaaagacaaaagacaaaaaaaaaaaaaaaa


 



aaaaaaaatcaaaaaaaaacatagggggttaccaacgtggg


 



gtccagaaagatgtggttttctcccattggccttgcccagt


 



tacctatatcagtccttgtccaacaggggttttaggggtgg


 



aaatgccccataaattttacggtttctttgcccttctcttc


 



ctttagactgagtcaccattgctctcattccttttctatca


 



gttgaggagtgggttagagattaaggtccatgtggtggagg


 



tacacttcttatagtaaacaaggcctatggggaattactct


 



ctggagcccttaaaccacaaatgataatccatgccacatca


 



aagatgcatcgaagcccatgctcctacactgactacctgag


 



ttagcattctgcctcaacaggactgaccatccccagctctg


 



gggcagatatcctctctctgccacaagggcagtgaccccca


 



tgctgtctgagggtcacgctttaccccccccccacccctgc


 



cgtgaccccccagaccaccccaggaggtgggcactaatatc


 



cctcattaccccatagatgaggaaacagaggttcccccggg


 



gtcccacaggtgctcagggtcacatgcaccgtgggcaccca


 



ggccccatcccaaggccaccctccctcctcaggaagctgtg


 



ctgcgctgggccagaaggtactgcacacgactcctcagcct


 



ccggtggtgggaggcagcctcaagcctctgagtgggggggc


 



acccgggctcctcaatctatactgactcctgggggtgggag


 



aaggggagggggagctgtggcctctgagtccactaagcaaa


 



tcagggtgggcaatgcgggcccatttcaaggaggagagaac


 



cgaggctctgacagcaggccgggggtccagggacctgccca


 



gggtcataggctgaactgctggctgacctgccttgggttct


 



ttccttggctcctcagccctgtgtgatgtgacaggtcattc


 



attcactcactcgctcattcattcagcaaaccctcagtgag


 



ccctgctgggagcaggtgctaggggcaaggagacaggacct


 



cttgccctggaacagctgaagcactgggggacaggcagtgg


 



cagggaggtgcgtgatcaccgctgaccccattccatcctcc


 



agcccccaggtcagtttccacccaccattgaccccaccatg


 



tcctccatccccaaggtcagtttcccgcccaaggagcatct


 



ccttacacactagggacaaaatttcacggctgtcactgggc


 



atctctccacgctcatcacagccctctagcagccttgaagt


 



cctgtagagcccttcccatttcacagaagggacaagactat


 



gagggccacaccgtgagccatgagccttaggctgtgagccg


 



ggacagcccctgcaggactggtggcctcagggcactgggtg


 



gggagggtgcacagtgggtgggccccttgtggaatagagag


 



gagtgtcaggtcaggggagggggcttggcctggccctggcc


 



tgcctggtgtgcaaccctaggcagcccctccttcccaggcc


 



tcctacttcctggaggccaagcctcagggaggtaattgagt


 



caggtgggggagggggggttgtggctttcttcacagcagaa


 



aaacagagcccacaatagtgtccactgagacagaggggtcc


 



tgggggaggggaggggtgggaggtgactgctgagccctgtg


 



ggagggagggagcaactactgagctgagctgggtgactctc


 



ccatctgccccgccccctgtggggccagcagagtcaccgag


 



agaacatgacccagccaggcctggacagggggacacccatg


 



tcctttaccccacagggttcactgagcctatctgccccaag


 



cctgtgtctccctgggacggagaccctcactcccaaccaca


 



aaggtctaaactcaagttcccaacagccttgaaaatacagc


 



ttccgggggcctccaaggagcagtcagccgtccactgccag


 



gctcgctggctcagtgacacaggacacatcctgatgacggt


 



ccacctgtctccaagcaggttctcctctgccgatggggcaa


 



cgagctcctcctgtggctccctggctggatgcgtgggaggc


 



ggggtgggggggcaggcggtgttcctggccgcacacaagga


 



gcacccccaccagcatccgaagacgggggcccggtctttcc


 



ccaaaacactgcttgcgggagactttgtgacgtttccaggg


 



gccatgctcccttcgggcagcttgggggacttctgctccta


 



tgtggtcacctgcagggactccccccaggccttggggacaa


 



acaaagtgatgagagggagggttagtgggtcggggcagggc


 



cagtctttggaccggtttatctgaaaagccagttggtcacc


 



gggaaccacagcaaacctaaacccatttggccaggcatctc


 



ccagggacagtctcccccaggatgcggggcccaggggggct


 



ccaggggtgacctgcgtcctggatttccctgatgctcccag


 



ttcgtgcctctgtccaagcatgatttttaatagtgcccctt


 



ccactcccagaaatgtccaagtgtgggcaataaattctggt


 



cacctgagctcagtgtaactgtttgctgaatgacacttact


 



gtaacaggttaaaatgggaggcccaaggccacgcagagcca


 



tcgaaggctctgtgtgtcccagccctgatagaagcatcagg


 



atggggactgtggcctcaccaggggccacatccaggcggtc


 



accatggggttcctggtctccgtgggccttgactggagccc


 



ctggtgtgagctcaccccatcccagcctgtgagaggcctgg


 



atgtgggcctgacatcatttcccacccagtgacagcactgc


 



atgtgatggggcctctgggcagcctttttcccgggggaaac


 



tggcaggaatcaggaccaccaggacaggggtcaggggagag


 



gcgatgctgggcaccagagcctggaccaccctcgggttctc


 



agcgatgggcaacccctgccacccagggccccgccttcctg


 



gggagacatcggggtttccaggccatcctgggaggagggtg


 



ggagcctcagctagaccccagctggcttgcccccccatgcc


 



ccggccaagagagggtcttggagggaagggggaccccagac


 



cagcctggcgagcccatcctcagggtctctggtcagacagg


 



ggctcagctgagctccagggtagaccaaggccctgcgtgga


 



tgaggccagtgtggtcactgcccagagcaaagccacctctc


 



agcagccctttcctgagcaccttctgtgtgcggggacatca


 



gcagtggcaacacagccatgctggggactcagggctagaga


 



caggggaccagcctatggagagtgggtagtgtcctgcaggg


 



caggcttgtgccctggagaaaacaaaccagggtgaggccag


 



ggacgctggccgggttcacagggtgatggctgagcacagag


 



tgccaggggctggactgtcctgactctgggttggtggctga


 



gggcctgtgtccctctatgcctctgggttggtgataatgga


 



aacttgctccctggagagacaggacgaatggttgatgggaa


 



atgaatgtttgcttgtcacttggttgactgttgttgccgtt


 



agcattgggcttcttgggccaggcagcctcaggccagcact


 



gctgggctccccacaggcccgacaccctcagccctgtgcag


 



ctggcctggcgaaaccaagaggccctgatgcccaaaatagc


 



cgggaaaccccaaccagcccagccctggcagcaggtgcctc


 



ccatttgcctgggctgggggaggggtggctctggttctgga


 



agtttctgccagtccagctggagaagggacctgtatcccag


 



cacccaggccgcccaagcccctgcaccagggcctgggccag


 



gcagagttgacatcaatcaattgggagctgctggaatgcat


 



ggaggcggcgctctcgaggctggaggaggccagctgattta


 



aatcggtccgcgtacgatgcatattaccctgttatccctac


 



cgcggttactggccgtcgttttacaacgtcgtgactgggaa


 



aaccctggcgatgctcttctcccggtgaaaacctctgacac


 



atggctcttctaaatccggagtttaaacgcttccttcatgt


 



gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggcc


 



gcgttgctggcgtttttccataggctccgcccccctgacga


 



gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacc


 



cgacaggactataaagataccaggcgtttccccctggaagc


 



tccctcgtgcgctctcctgttccgaccctgccgcttaccgg


 



atacctgtccgcctttctcccttcgggaagcgtggcgcttt


 



ctcatagctcacgctgtaggtatctcagttcggtgtaggtc


 



gttcgctccaagctgggctgtgtgcacgaaccccccgttca


 



gcccgaccgctgcgccttatccggtaactatcgtcttgagt


 



ccaacccggtaagacacgacttatcgccactggcagcagcc


 



actggtaacaggattagcagagcgaggtatgtaggcggtgc


 



tacagagttcttgaagtggtggcctaactacggctacacta


 



gaaggacagtatttggtatctgcgctctgctgaagccagtt


 



accttcggaaaaagagttggtagctcttgatccggcaaaca


 



aaccaccgctggtagcggtggtttttttgtttgcaagcagc


 



agattacgcgcagaaaaaaaggatctcaagaagatcctttg


 



atcttttctacggggtctgacgctcagtggaacgaaaactc


 



acgttaagggattttggtcatgcctaggtggcaaacagcta


 



ttatgggtattatgggtctaccggtgcatgagattatcaaa


 



aaggatcttcacctagatccttttaaattaaaaatgaagtt


 



ttaaatcaatctaaagtatatatgagtaaacttggtctgac


 



agttaccaatgcttaatcagtgaggcacctatctcagcgat


 



ctgtctatttcgttcatccatagttgcctgactccccgtcg


 



tgtagataactacgatacgggagggcttaccatctggcccc


 



agtgctgcaatgataccgcgagacccacgctcaccggctcc


 



agatttatcagcaataaaccagccagccggaagggccgagc


 



gcagaagtggtcctgcaactttatccgcctccatccagtct


 



attaattgttgccgggaagctagagtaagtagttcgccagt


 



taatagtttgcgcaacgttgttgccattgctacaggcatcg


 



tggtgtcacgctcgtcgtttggtatggcttcattcagctcc


 



ggttcccaacgatcaaggcgagttacatgatcccccatgtt


 



gtgcaaaaaagcggttagctccttcggtcctccgatcgttg


 



tcagaagtaagttggccgcagtgttatcactcatggttatg


 



gcagcactgcataattctcttactgtcatgccatccgtaag


 



atgcttttctgtgactggtgagtactcaaccaagtcattct


 



gagaatagtgtatgcggcgaccgagttgctcttgcccggcg


 



tcaatacgggataataccgcgccacatagcagaactttaaa


 



agtgctcatcattggaaaacgttcttcggggcgaaaactct


 



caaggatcttaccgctgttgagatccagttcgatgtaaccc


 



actcgtgcacccaactgatcttcagcatcttttactttcac


 



cagcgtttctgggtgagcaaaaacaggaaggcaaaatgccg


 



caaaaaagggaataagggcgacacggaaatgttgaatactc


 



atactcttcctttttcaatattattgaagcatttatcaggg


 



ttattgtctcgggagcggatacatatttgaatgtatttaga


 



aaaa


 


SEQ ID 46
taaacaaataggggttccgcgcacatttccccgaaaagtgc


 



cacctgacgtcgctgagcaggccctggcctccctggccgag


 



ggcggtttgcgtattagaggcctaaatggccgaattcagcg


 



gataacaatttcacacaggaaacagctatgaccatgattat


 



ctagtaactataacggtcctaaggtagcgagcgatcgctta


 



attaacctgcagggatatcccatgggggccgccagtgtgat


 



ggatatctgcagaattcgcccttgatattaagagaagggca


 



agtcagcttaagtttgggggtagaggggaacagggagtgag


 



gagatctggcctgagagataggagccctggtggccacagga


 



ggactctttgggtcctgtcggatggacacagggcggcccgg


 



gggcatgttggagcccggctggttcttaccagaggcagggg


 



gcaccctctgacacgggagcagggcatgttccatacatgac


 



acacccctctgctccagggcaggtgggtggcggcacagagg


 



agccagggactctgagcaaggggtccaccagtggggcagtt


 



ggatccagacttctctgggccagcgagagtctagccctcag


 



ccgttctctgtccaggaggggggtggggcaggcctgggcgg


 



ccagagctcatccctcaagggttcccagggtcctgccagac


 



ccagatttccgaccgcagccaccacaagaggatgtggctgc


 



tgtggcagctgccaagaccttgcagcaggtgcagggtgggg


 



gggtgggggcacctgggggcagctggggtcactgagttcag


 



ggaaaaccccttttttcccctaaacctggggccatccctag


 



gggaaaccacaacttctgagccctgggcagtggctgctggg


 



agggaagagcttcatcctggaccctgggggggaacccagct


 



ccaaaggtgcaaggggcccaggtccaaggctagagtgggcc


 



aagcaccgcaatggccagggagtgggggaggtggagctgga


 



ctggatcagggcctccttgggactccctacaccctgtgtga


 



catgttagggtacccacaccccatcaccagtcagggcctgg


 



cccatctccagggccagggatgtgcatgtaagtgtgtgtga


 



gtgtgtgtgtgtggtgtagtacaccccttggcatccggttc


 



cgaggccttgggttcctccaaagttgctctctgaattaggt


 



caaactgtgaggtcctgatcgccatcatcaacttcgttctc


 



cccacctcccatcattatcaagagctggggagggtctggga


 



tttcttcccacccacaagccaaaagataagcctgctggtga


 



tggcagaagacacaggatcctgggtcagagacaaaggccag


 



tgtgtcacagcgagagaggcagccggactatcagctgtcac


 



agagaggccttagtccgctgaactcaggccccagtgactcc


 



tgttccactgggcactggcccccctccacagcgcccccagg


 



ccccagggagaggcgtcacagcttagagatggccctgctga


 



acagggaacaagaacaggtgtgccccatccagcgccccagg


 



ggtgggacaggtgggctggatttggtgtgaagcccttgagc


 



cctggaacccaaccacagcagggcagttggtagatgccatt


 



tggggagaggccccaggagtaagggccatgggcccttgagg


 



gggccaggagctgaggacagggacagagacggcccaggcag


 



aggacagggccatgaggggtgcactgagatggccactgcca


 



gcaggggcagctgccaacccgtccagggaacttattcagca


 



gtcagctggaggtgccattgaccctgagggcagatgaagcc


 



caggccaggctaggtgggctgtgaagaccccaggggacaga


 



gctctgtccctgggcagcactggcctctcattctgcagggc


 



ttgacgggatcccaaggcctgctgcccctgatggtagtggc


 



agtaccgcccagagcaggaccccagcatggaaaccccaacg


 



ggacgcagcctgcggagcccacaaaaccagtaaggagccga


 



agcagtcatggcacggggagtgtggacttccctttgatggg


 



gcccaggcatgaaggacagaatgggacagcggccatgagca


 



gaaaatcagccggaggggatgggcctaggcagacgctggct


 



ttatttgaagtgttggcattttgtctggtgtgtattgttgg


 



tattgattttattttagtatgtcagtgacatactgacatat


 



tatgtaacgacatattattatgtgttttaagaagcactcca


 



agggaacaggctgtctgtaatgtgtccagagaagagagcaa


 



gagcttggctcagtctcccccaaggaggtcagttcctcaac


 



aggggtcctaaatgtttcctggagccaggcctgaatcaagg


 



gggtcatatctacacgtggggcagacccatggaccattttc


 



ggagcaataagatggcagggaggataccaagctggtcttac


 



agatccagggctttgacctgtgacgcgggcgctcctccagg


 



caaagggagaagccagcaggaagctttcagaactggggaga


 



acagggtgcagacctccagggtcttgtacaacgcacccttt


 



atcctggggtccaggaggggtcactgagggatttaagtggg


 



ggaccatcagaaccaggtttgtgttttggaaaaatggctcc


 



aaagcagagaccagtgtgaggccagattagatgatgaagaa


 



gaggcagtggaaagtcgatgggtggccaggtagcaagaggg


 



cctatggagttggcaagtgaatttaaagtggtggcaccaga


 



gggcagatggggaggagcaggcactgtcatggactgtctat


 



agaaatctaaaatgtataccctttttagcaatatgcagtga


 



gtcataaaagaacacatatatatttcctttggccggccggc


 



gcgccacgcgtataacttcgtatagcatacattatacgaag


 



ttatcttaagggctatggcagggcctgccgccccgacgttg


 



gctgcgagccctgggccttcacccgaacttggggggtgggg


 



tggggaaaaggaagaaacgcgggcgtattggccccaatggg


 



gtctcggtggggtatcgacagagtgccagccctgggaccga


 



accccgcgtttatgaacaaacgacccaacaccgtgcgtttt


 



attctgtctttttattgccgtcatagcgcgggttccttccg


 



gtattgtctccttccgtgtttcactcgagttagaagaactc


 



gtcaagaaggcgatagaaggcgatgcgctgcgaatcgggag


 



cggcgataccgtaaagcacgaggaagcggtcagcccattcg


 



ccgccaagctcttcagcaatatcacgggtagccaacgctat


 



gtcctgatagcggtccgccacacccagccggccacagtcga


 



tgaatccagaaaagcggccattttccaccatgatattcggc


 



aagcaggcatcgccatgggtcacgacgagatcctcgccgtc


 



gggcatgcgcgccttgagcctggcgaacagttcggctggcg


 



cgagcccctgatgctcttcgtccagatcatcctgatcgaca


 



agaccggcttccatccgagtacgtgctcgctcgatgcgatg


 



tttcgcttggtggtcgaatgggcaggtagccggatcaagcg


 



tatgcagccgccgcattgcatcagccatgatggatactttc


 



tcggcaggagcaaggtgagatgacaggagatcctgccccgg


 



cacttcgcccaatagcagccagtcccttcccgcttcagtga


 



caacgtcgagcacagctgcgcaaggaacgcccgtcgtggcc


 



agccacgatagccgcgctgcctcgtcctgcagttcattcag


 



ggcaccggacaggtcggtcttgacaaaaagaaccgggcgcc


 



cctgcgctgacagccggaacacggcggcatcagagcagccg


 



attgtctgttgtgcccagtcatagccgaatagcctctccac


 



ccaagcggccggagaacctgcgtgcaatccatcttgttcaa


 



tggccgatcccattccagatctgttagcctcccccatctcc


 



cgtgcaaacgtgcgcgccaggtcgcagatcgtcggtatgga


 



gcctggggtggtgacgtgggtctggatcatcccggaggtaa


 



gttgcagcagggcgtcccggcagccggcgggcgattggtcg


 



taatccaggataaagacgtgcatgggacggaggcgtttggt


 



caagacgtccaaggcccaggcaaacacgttgtacaggtcgc


 



cgttgggggccagcaactcgggggcccgaaacagggtaaat


 



aacgtgtccccgatatggggtcgtgggcccgcgttgctctg


 



gggctcggcaccctggggcggcacggccgtccccgaaagct


 



gtccccaatcctcccgccacgacccgccgccctgcagatac


 



cgcaccgtattggcaagcagcccgtaaacgcggcgaatcgc


 



ggccagcatagccaggtcaagccgctcgccggggcgctggc


 



gtttggccaggcggtcgatgtgtctgtcctccggaagggcc


 



cccaacacgatgtttgtgccgggcaaggtcggcgggatgag


 



ggccacgaacgccagcacggcctggggggtcatgctgccca


 



taaggtatcgcgcggccgggtagcacaggagggcggcgatg


 



ggatggcggtcgaagatgagggtgagggccgggggcggggc


 



atgtgagctcccagcctcccccccgatatgaggagccagaa


 



cggcgtcggtcacggcataaggcatgcccattgttatctgg


 



gcgcttgtcattaccaccgccgcgtccccggccgatatctc


 



accctggtcaaggcggtgttgtgtggtgtagatgttcgcga


 



ttgtctcggaagcccccagcacccgccagtaagtcatcggc


 



tcgggtacgtagacgatatcgtcgcgcgaacccagggccac


 



cagcagttgcgtggtggtggttttccccatcccgtggggac


 



cgtctatataaacccgcagtagcgtgggcattttctgctcc


 



gggcggacttccgtggcttcttgctgccggcgagggcgcaa


 



cgccgtacgtcggttgctatggccgcgagaacgcgcagcct


 



ggtcgaacgcagacgcgtgctgatggccggggtacgaagcc


 



atggtggctctagaggtcgaaaggcccggagatgaggaaga


 



ggagaacagcgcggcagacgtgcgcttttgaagcgtgcaga


 



atgccgggcttccggaggaccttcgggcgcccgccccgccc


 



ctgagcccgcccctgagcccgcccccggacccaccccttcc


 



cagcctctgagcccagaaagcgaaggagccaaagctgctat


 



tggccgctgccccaaaggcctacccgcttccattgctcagc


 



ggtgctgtccatctgcacgagactagtgagacgtgctactt


 



ccatttgtcacgtcctgcacgacgcgagctgcggggcgggg


 



gggaacttcctgactaggggaggagtagaaggtggcgcgaa


 



ggggccaccaaagaacggagccggttggcgcctaccggtgg


 



atgtggaatgtgtgcgaggccagaggccacttgtgtagcgc


 



caagtgcccagcggggctgctaaagcgcatgctccagactg


 



ccttgggaaaagcgcctcccctacccggtagggatccgcgt


 



tacataacttacggtaaatggcccgcctggctgaccgccca


 



acgacccccgcccattgacgtcaataatgacgtatgttccc


 



atagtaacgccaatagggactttccattgacgtcaatgggt


 



ggagtatttacggtaaactgcccacttggcagtacatcaag


 



tgtatcatatgccaagtacgccccctattgacgtcaatgac


 



ggtaaatggcccgcctggcattatgcccagtacatgacctt


 



atgggactttcctacttggcagtacatctacgtattagtca


 



tcgctattaccatggtgatgcggttttggcagtacatcaat


 



gggcgtggatagcggtttgactcacggggatttccaagtct


 



ccaccccattgacgtcaatgggagtttgttttggcaccaaa


 



atcaacggttaacaagcttagatctgcggccgcgtcgacga


 



taaattgtgtaattccacttctaaggattcatcccaagggg


 



ggaaaataatcaaagatgtaaccaaaggtttacaaacaaga


 



actcatcattaatcttccttgttgttatttcaacgatatta


 



ttattattactattattattattattattttgtctttttgc


 



attttctagggccactcccacggcatagagaggttcccagg


 



ctaggggtcaaatcggagctacagctgccggcctacgccag


 



agccacagcaacgcaggatctgagccacagcaatgcaggat


 



ctacaccacagctcatggtaacgctggatccttaacccaat


 



gagtgaggccagggatcgaacctgtaacttcatggttccta


 



gtcggattcattaaccactgagccacgacaggaactccaac


 



attattaatgatgggagaaaactggaagtaacctaaatatc


 



cagcagaaagggtgtggccaaatacagcatggagtagccat


 



cataaggaatcttacacaagcctccaaaattgtgtttctga


 



aattgggtttaaagtacgtttgcattttaaaaagcctgcca


 



gaaaatacagaaaaatgtctgtgatatgtctctggctgata


 



ggattttgcttagttttaattttggctttataattttctat


 



agttatgaaaatgttcacaagaagatatatttcattttagc


 



ttctaaaataattataacacagaagtaatttgtgctttaaa


 



aaatattcaacacagaagtatataaaaaaattgaggagttc


 



ccatcgtggctcagtgattaacaaacccaactagtatccat


 



gaggatatggatttgatccctggccttgctcagtgggttga


 



ggatccagtgttgctgtgagctgtggtgtaggttgcagaca


 



cagcactctggcgttgctgtgactctggcgtaggccggcag


 



ctacagctccatttggacccttagcctgggaacctccatat


 



gcctgagatacggccctaaaaagtcaaaagccaaaaaaata


 



gtaaaaattgagtgtttctacttaccacccctgcccacatc


 



ttatgctaaaacccgttctccagagacaaacatcgtcaggt


 



gggtctatatatttccagccctcctcctgtgtgtgtatgtc


 



cgtaaaacacacacacacacacacacacgcacacacacaca


 



cacgtatctaattagcattggtattagtttttcaaaaggga


 



ggtcatgctctaccttttaggcggcaaatagattatttaaa


 



caaatctgttgacattttctatatcaacccataagatctcc


 



catgttcttggaaaggctttgtaagacatcaacatctgggt


 



aaaccagcatggtttttagggggttgtgtggatttttttca


 



tattttttagggcacacctgcagcatatggaggttcccagg


 



ctaggggttgaatcagagctgtagctgccggcctacaccac


 



agccacagcaacgccagatccttaacccactgagaaaggcc


 



agggattgaacctgcatcctcatggatgctggtcagattta


 



tttctgctgagccacaacaggaactccctgaaccagaatgc


 



ttttaaccattccactttgcatggacatttagattgtttcc


 



atttaaaaatacaaattacaaggagttcccgtcgtggctca


 



gtggtaacgaattggactaggaaccatgaggtttcgggttc


 



gatccctggccttgctcggtgggttaaggatccagcattga


 



tgtgagatatggtgtaggtcgcagacgtggctcggatccca


 



cgttgctgtggctctggcgtaggccggcaacaacagctccg


 



attcgacccctagcctgggaacctccatgtgccacaggagc


 



agccctagaaaaggcaaaaagacaaaaaaataaaaaattaa


 



aatgaaaaaataaaataaaaatacaaattacaagagacggc


 



tacaaggaaatccccaagtgtgtgcaaatgccatatatgta


 



taaaatgtactagtgtctcctcgcgggaaagttgcctaaaa


 



gtgggttggctggacagagaggacaggctttgacattctca


 



taggtagtagcaatgggcttctcaaaatgctgttccagttt


 



acactcaccatagcaaatgacagtgcctcttcctctccacc


 



cttgccaataatgtgacaggtggatctttttctattttgtg


 



tatctgacaagcaaaaaatgagaacaggagttcctgtcgtg


 



gtgcagtggagacaaatctgactaggaaccatgaaatttcg


 



ggttcaatccctggcctcactcagtaggtaaaggatccagg


 



gttgcagtgagctgtggggtaggtcgcagacacagtgcaaa


 



tttggccctgttgtggctgtggtgtaggccggcagctatag


 



ctccaattggacccctagcctgggaacctccttatgccgtg


 



ggtgaggccctaaaaaaaagagtgcaaaaaaaaaaaataag


 



aacaaaaatgatcatcgtttaattctttatttgatcattgg


 



tgaaacttattttccttttatatttttattgactgatttta


 



tttctcctatgaatttaccggtcatagttttgcctgggtgt


 



ttttactccggttttagttttggttggttgtattttcttag


 



agagctatagaaactcttcatctatttggaatagtaattcc


 



tcattaagtatttgtgctgcaaaaaattttccctgatctgt


 



tttatgcttttgtttgtggggtctttcacgagaaagccttt


 



ttagtttttacacctcagcttggttgtttttcttgattgtg


 



tctgtaatctgcggccaacataggaaacacatttttacttt


 



agtgtttttttcctattttcttcaagtacgtccattgtttt


 



ggtgtctgattttactttgcctggggtttgtttttgtgtgg


 



caggaatataaacttatgtattttccaaatggagagccaat


 



ggttgtatatttgttgaattcaaatgcaactttatcaaaca


 



ccaaatcatcgatttatcacaactcttctctggtttattga


 



tctaatgatcaattcctgttccacgctgttttaattatttt


 



agctttgtggattttggtgcctggtagagaacaaagcctcc


 



attattttcattcaaaatagtcccgtctattatctgccatt


 



gttgtagtattagactttaaaatcaatttactgattttcaa


 



aagttattcctttggtgatgtggaatactttatacttcata


 



aggtacatggattcatttgtggggaattgatgtctttgcta


 



ttgtggccatttgtcaagttgtgtaatattttacccatgcc


 



aactttgcatattgtatgtgagtttattcccagggttttta


 



ataggatgtttattgaagttgtcagtgtttccacaatttca


 



tcgcctcagtgcttactgtttgcataaaaggaaacctactc


 



acttttgcctattgctcttgtattcaatcattttagttaac


 



tcttgtgttaattttgagagtttttcagctgactgtctggg


 



gttttctttaatagactagccctttgtctgtaaagaataat


 



tttatcgaatttttcttaacactcacactctccccaccccc


 



acccccgctcatctcctttcattgggtcaaatctgtagaat


 



acaataaaagtaagagtgggaaccttagcctttaagtcgat


 



tttgcctttaaatgtgaatgttgctatgtttcgggacattc


 



tctttatcaagttgcggatgtttccttagataattaactta


 



ataaaagactggatgtttgctttcttcaaatcagaattgtg


 



ttgaatttatattgctattctgtttaattttgtttcaaaaa


 



atttacatgcacaccttaaagataaccatgaccaaatagtc


 



ctcctgctgagagaaaatgttggccccaatgccacaggtta


 



cctcccgactcagataaactacaatgggagataaaatcaga


 



tttggcaaagcctgtggattcttgccataactctcagagca


 



tgacttgggtgttttttccttttctaagtattttaatggta


 



tttttgtgttacaataggaaatctaggacacagagagtgat


 



tcaatgaggggaacgcattctgggatgactctaggcctctg


 



gtttggggagagctctattgaagtaaagacaatgagaggaa


 



gcaagtttgcagggaactgtgaggaatttagatggggaatg


 



ttgggtttgaggtttctatagggcacgcaagcagagatgca


 



ctcaggaggaagaaggagcataaatctagtggcgctgccgg


 



caagcttgctggaggaggccaattgggagctgctggaatgc


 



atggaggcggcgctctcgaggctggaggaggccagctgatt


 



taaatcggtccgcgtacgatgcatattaccctgttatccct


 



accgcggttactggccgtcgttttacaacgtcgtgactggg


 



aaaaccctggcgatgctcttctcccggtgaaaacctctgac


 



acatggctcttctaaatccggagtttaaacgcttccttcat


 



gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaagg


 



ccgcgttgctggcgtttttccataggctccgcccccctgac


 



gagcatcacaaaaatcgacgctcaagtcagaggtggcgaaa


 



cccgacaggactataaagataccaggcgtttccccctggaa


 



gctccctcgtgcgctctcctgttccgaccctgccgcttacc


 



ggatacctgtccgcctttctcccttcgggaagcgtggcgct


 



ttctcatagctcacgctgtaggtatctcagttcggtgtagg


 



tcgttcgctccaagctgggctgtgtgcacgaaccccccgtt


 



cagcccgaccgctgcgccttatccggtaactatcgtcttga


 



gtccaacccggtaagacacgacttatcgccactggcagcag


 



ccactggtaacaggattagcagagcgaggtatgtaggcggt


 



gctacagagttcttgaagtggtggcctaactacggctacac


 



tagaaggacagtatttggtatctgcgctctgctgaagccag


 



ttaccttcggaaaaagagttggtagctcttgatccggcaaa


 



caaaccaccgctggtagcggtggtttttttgtttgcaagca


 



gcagattacgcgcagaaaaaaaggatctcaagaagatcctt


 



tgatcttttctacggggtctgacgctcagtggaacgaaaac


 



tcacgttaagggattttggtcatgcctaggtggcaaacagc


 



tattatgggtattatgggtctaccggtgcatgagattatca


 



aaaaggatcttcacctagatccttttaaattaaaaatgaag


 



ttttaaatcaatctaaagtatatatgagtaaacttggtctg


 



acagttaccaatgcttaatcagtgaggcacctatctcagcg


 



atctgtctatttcgttcatccatagttgcctgactccccgt


 



cgtgtagataactacgatacgggagggcttaccatctggcc


 



ccagtgctgcaatgataccgcgagacccacgctcaccggct


 



ccagatttatcagcaataaaccagccagccggaagggccga


 



gcgcagaagtggtcctgcaactttatccgcctccatccagt


 



ctattaattgttgccgggaagctagagtaagtagttcgcca


 



gttaatagtttgcgcaacgttgttgccattgctacaggcat


 



cgtggtgtcacgctcgtcgtttggtatggcttcattcagct


 



ccggttcccaacgatcaaggcgagttacatgatcccccatg


 



ttgtgcaaaaaagcggttagctccttcggtcctccgatcgt


 



tgtcagaagtaagttggccgcagtgttatcactcatggtta


 



tggcagcactgcataattctcttactgtcatgccatccgta


 



agatgcttttctgtgactggtgagtactcaaccaagtcatt


 



ctgagaatagtgtatgcggcgaccgagttgctcttgcccgg


 



cgtcaatacgggataataccgcgccacatagcagaacttta


 



aaagtgctcatcattggaaaacgttcttcggggcgaaaact


 



ctcaaggatcttaccgctgttgagatccagttcgatgtaac


 



ccactcgtgcacccaactgatcttcagcatcttttactttc


 



accagcgtttctgggtgagcaaaaacaggaaggcaaaatgc


 



cgcaaaaaagggaataagggcgacacggaaatgttgaatac


 



tcatactcttcctttttcaatattattgaagcatttatcag


 



ggttattgtctcgggagcggatacatatttgaatgtattta


 



gaaaaa


 


SEQ ID 47
taaacaaataggggttccgcgcacatttccccgaaaagtgc


 



cacctgacgtcgctgagcaggccctggcctccctggccgag


 



ggcggtttgcgtattagaggcctaaatggccgaattcagcg


 



gataacaatttcacacaggaaacagctatgaccatgattat


 



ctagtaactataacggtcctaaggtagcgagcgatcgctta


 



attaacctgcagggataaccactgacccatgacgggaactc


 



ccagggctcagctcttgactccaggttcgcagctgccctca


 



aagcaatgcaaccctggctggccccgcctcatgcatccggc


 



ctcctccccaaagagctctgagcccacctgggcctaggtcc


 



tcctccctgggactcatggcctaagggtacagagttactgg


 



ggctgatgaagggaccaatggggacaggggcctcaaatcaa


 



agtggctgtctctctcatgtcccttcctctcctcagggtcc


 



aaaatcagggtcagggccccagggcaggggctgagagggcc


 



tctttctgaaggccctgtctcagtgcaggttatgggggtct


 



gggggagggtcaatgcagggctcacccttcagtgccccaaa


 



gcctagagagtgagtgcctgccagtggcttcccaggcccaa


 



tcccttgactgcctgggaatgctcaaatgcaggaactgtca


 



caacaccttcagtcaggggctgctctgggaggaaaaacact


 



cagaattgggggttcagggaaggcccagtgccaagcatagc


 



aggagctcaggtggctgcagatggtgtgaaccccaggagca


 



ggatggccggcactccccccagaccctccagagccccaggt


 



tggctgccctcttcactgccgacacccctgggtccacttct


 



gccctttcccacctaaaacctttagggctcccactttctcc


 



caaatgtgagacatcaccacggctcccagggagtgtccaga


 



agggcatctggctgagaggtcctgacatctgggagcctcag


 



gccccacaatggacagacgccctgccaggatgctgctgcag


 



ggctgttagctaggcggggtggagatggggtactttgcctc


 



tcagaggccccggccccaccatgaaacctcagtgacacccc


 



atttccctgagttcacatacctgtatcctactccagtcacc


 



ttccccacgaacccctgggagcccaggatgatgctggggct


 



ggagccacgaccagcccacgagtgatccagctctgccaatc


 



agcagtcatttcccaagtgttccagccctgccaggtcccac


 



tacagcagtaatggaggccccagacaccagtccagcagtta


 



gagggctggactagcaccagctttcaagcctcagcatctca


 



aggtgaatggccagtgcccctccccgtggccatcacaggat


 



cgcagatatgaccctaggggaagaaatatcctgggagtaag


 



gaagtgcccatactcaaggatggcccctctgtgacctaacc


 



tgtccctgaggattgtacttccaggcgttaaaacagtagaa


 



cgcctgcctgtgaacccccgccaagggactgcttggggagg


 



ccccctaaaccagaacacaggcactccagcaggacctctga


 



actctgaccaccctcagcaagtgggcaccccccgcagcttc


 



caaggcaccccagggctcaccacagcggcccctcctggcag


 



cccctcacccaggcccagaccctctaagatggcacatctaa


 



gccaatccacctccttgtcattcctcctgtccccacccagg


 



acccttctcagatgaaaccttcgctccagccgctgggccct


 



ctctcctgcccctctggcagttctccagggactccgcctcc


 



cactctctgtctctccctgcactcctaggaacaagcgacct


 



ccaggaagcccagtccaattatcccctctgtgtcctcccca


 



atctctgcctctgggtggatttgagcaccacatcctgttct


 



cttcgacctgaaactccttggccccggtgtccgctctcctg


 



ggccctcttttctctcctcccctcttccgtgccccgtttgt


 



ttggtgttacaggcaggccccggggagccgtccctccagct


 



gctcttccttgtctgtctcaggagccagaaactggcagcat


 



ctaaaaagggctcctgtttcttcatctgcccagcctcctag


 



cccaaccagggctctggcctcactccagagggtgggctcca


 



gagggcaggggttgcaccctcttagtgcctcagaggctcag


 



ctgggtgcaggatgggggggccctcagggagcccctcagtg


 



actgctgatcacttactgcaggactgttcccagctcttccc


 



aatcattggaatgacaatacctagttctgctccatcatagt


 



gatgcaggaaaaatgttactgaaatcctggttcttgtttag


 



caatcgaagaatgaattccgcgaacacacaggcagcaagca


 



agcgaagcctttattaaaggaaagcagatagctcccagggc


 



tgcagggagcggggagaagagctccccactctctattgtcc


 



tatagggctttttaccccttaaagttggggggatacaaaaa


 



aaatagaagaaaaagggagttcccgtcagggcacagcagaa


 



acaaatccaactaggaaccatgaggttgggggttcgattcc


 



tggcctctctcagtgggttaaggatgcagcgttgccgtgag


 



ctatgatacaggtcacagatgcagctcagatctactagtca


 



attgacaggcgccggagcaggagctaggcctttggccggcc


 



ggcgcgccacgcgtataacttcgtatagcatacattatacg


 



aagttatcttaagggctatggcagggcctgccgccccgacg


 



ttggctgcgagccctgggccttcacccgaacttggggggtg


 



gggtggggaaaaggaagaaacgcgggcgtattggccccaat


 



ggggtctcggtggggtatcgacagagtgccagccctgggac


 



cgaaccccgcgtttatgaacaaacgacccaacaccgtgcgt


 



tttattctgtctttttattgccgtcatagcgcgggttcctt


 



ccggtattgtctccttccgtgtttcactcgagttagaagaa


 



ctcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgg


 



gagcggcgataccgtaaagcacgaggaagcggtcagcccat


 



tcgccgccaagctcttcagcaatatcacgggtagccaacgc


 



tatgtcctgatagcggtccgccacacccagccggccacagt


 



cgatgaatccagaaaagcggccattttccaccatgatattc


 



ggcaagcaggcatcgccatgggtcacgacgagatcctcgcc


 



gtcgggcatgcgcgccttgagcctggcgaacagttcggctg


 



gcgcgagcccctgatgctcttcgtccagatcatcctgatcg


 



acaagaccggcttccatccgagtacgtgctcgctcgatgcg


 



atgtttcgcttggtggtcgaatgggcaggtagccggatcaa


 



gcgtatgcagccgccgcattgcatcagccatgatggatact


 



ttctcggcaggagcaaggtgagatgacaggagatcctgccc


 



cggcacttcgcccaatagcagccagtcccttcccgcttcag


 



tgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtg


 



gccagccacgatagccgcgctgcctcgtcctgcagttcatt


 



cagggcaccggacaggtcggtcttgacaaaaagaaccgggc


 



gcccctgcgctgacagccggaacacggcggcatcagagcag


 



ccgattgtctgttgtgcccagtcatagccgaatagcctctc


 



cacccaagcggccggagaacctgcgtgcaatccatcttgtt


 



caatggccgatcccattccagatctgttagcctcccccatc


 



tcccgtgcaaacgtgcgcgccaggtcgcagatcgtcggtat


 



ggagcctggggtggtgacgtgggtctggatcatcccggagg


 



taagttgcagcagggcgtcccggcagccggcgggcgattgg


 



tcgtaatccaggataaagacgtgcatgggacggaggcgttt


 



ggtcaagacgtccaaggcccaggcaaacacgttgtacaggt


 



cgccgttgggggccagcaactcgggggcccgaaacagggta


 



aataacgtgtccccgatatggggtcgtgggcccgcgttgct


 



ctggggctcggcaccctggggcggcacggccgtccccgaaa


 



gctgtccccaatcctcccgccacgacccgccgccctgcaga


 



taccgcaccgtattggcaagcagcccgtaaacgcggcgaat


 



cgcggccagcatagccaggtcaagccgctcgccggggcgct


 



ggcgtttggccaggcggtcgatgtgtctgtcctccggaagg


 



gcccccaacacgatgtttgtgccgggcaaggtcggcgggat


 



gagggccacgaacgccagcacggcctggggggtcatgctgc


 



ccataaggtatcgcgcggccgggtagcacaggagggcggcg


 



atgggatggcggtcgaagatgagggtgagggccgggggcgg


 



ggcatgtgagctcccagcctcccccccgatatgaggagcca


 



gaacggcgtcggtcacggcataaggcatgcccattgttatc


 



tgggcgcttgtcattaccaccgccgcgtccccggccgatat


 



ctcaccctggtcaaggcggtgttgtgtggtgtagatgttcg


 



cgattgtctcggaagcccccagcacccgccagtaagtcatc


 



ggctcgggtacgtagacgatatcgtcgcgcgaacccagggc


 



caccagcagttgcgtggtggtggttttccccatcccgtggg


 



gaccgtctatataaacccgcagtagcgtgggcattttctgc


 



tccgggcggacttccgtggcttcttgctgccggcgagggcg


 



caacgccgtacgtcggttgctatggccgcgagaacgcgcag


 



cctggtcgaacgcagacgcgtgctgatggccggggtacgaa


 



gccatggtggctctagaggtcgaaaggcccggagatgagga


 



agaggagaacagcgcggcagacgtgcgcttttgaagcgtgc


 



agaatgccgggcttccggaggaccttcgggcgcccgccccg


 



cccctgagcccgcccctgagcccgcccccggacccacccct


 



tcccagcctctgagcccagaaagcgaaggagccaaagctgc


 



tattggccgctgccccaaaggcctacccgcttccattgctc


 



agcggtgctgtccatctgcacgagactagtgagacgtgcta


 



cttccatttgtcacgtcctgcacgacgcgagctgcggggcg


 



ggggggaacttcctgactaggggaggagtagaaggtggcgc


 



gaaggggccaccaaagaacggagccggttggcgcctaccgg


 



tggatgtggaatgtgtgcgaggccagaggccacttgtgtag


 



cgccaagtgcccagcggggctgctaaagcgcatgctccaga


 



ctgccttgggaaaagcgcctcccctacccggtagggatccg


 



cgttacataacttacggtaaatggcccgcctggctgaccgc


 



ccaacgacccccgcccattgacgtcaataatgacgtatgtt


 



cccatagtaacgccaatagggactttccattgacgtcaatg


 



ggtggagtatttacggtaaactgcccacttggcagtacatc


 



aagtgtatcatatgccaagtacgccccctattgacgtcaat


 



gacggtaaatggcccgcctggcattatgcccagtacatgac


 



cttatgggactttcctacttggcagtacatctacgtattag


 



tcatcgctattaccatggtgatgcggttttggcagtacatc


 



aatgggcgtggatagcggtttgactcacggggatttccaag


 



tctccaccccattgacgtcaatgggagtttgttttggcacc


 



aaaatcaacggttaacaagcttataacttcgtatagcatac


 



attatacgaagttattacgtagcggccgcgtcgacgatatc


 



gctgccggagcccccggggccgctgccggaagatctggcat


 



tgctgtgactgtggtgtaggccggcagctggagctctgatt


 



agacccctcacctgggaatctccatatgctgcacgtgcggc


 



cctaaaaagacaaaagacaaaaaaaaaaaaaaaaaaaaaaa


 



atcaaaaaaaaacatagggggttaccaacgtggggtccaga


 



aagatgtggttttctcccattggccttgcccagttacctat


 



atcagtccttgtccaacaggggttttaggggtggaaatgcc


 



ccataaattttacggtttctttgcccttctcttcctttaga


 



ctgagtcaccattgctctcattccttttctatcagttgagg


 



agtgggttagagattaaggtccatgtggtggaggtacactt


 



cttatagtaaacaaggcctatggggaattactctctggagc


 



ccttaaaccacaaatgataatccatgccacatcaaagatgc


 



atcgaagcccatgctcctacactgactacctgagttagcat


 



tctgcctcaacaggactgaccatccccagctctggggcaga


 



tatcctctctctgccacaagggcagtgacccccatgctgtc


 



tgagggtcacgctttaccccccccccacccctgccgtgacc


 



ccccagaccaccccaggaggtgggcactaatatccctcatt


 



accccatagatgaggaaacagaggttcccccggggtcccac


 



aggtgctcagggtcacatgcaccgtgggcacccaggcccca


 



tcccaaggccaccctccctcctcaggaagctgtgctgcgct


 



gggccagaaggtactgcacacgactcctcagcctccggtgg


 



tgggaggcagcctcaagcctctgagtgggggggcacccggg


 



ctcctcaatctatactgactcctgggggtgggagaagggga


 



gggggagctgtggcctctgagtccactaagcaaatcagggt


 



gggcaatgcgggcccatttcaaggaggagagaaccgaggct


 



ctgacagcaggccgggggtccagggacctgcccagggtcat


 



aggctgaactgctggctgacctgccttgggttctttccttg


 



gctcctcagccctgtgtgatgtgacaggtcattcattcact


 



cactcgctcattcattcagcaaaccctcagtgagccctgct


 



gggagcaggtgctaggggcaaggagacaggacctcttgccc


 



tggaacagctgaagcactgggggacaggcagtggcagggag


 



gtgcgtgatcaccgctgaccccattccatcctccagccccc


 



aggtcagtttccacccaccattgaccccaccatgtcctcca


 



tccccaaggtcagtttcccgcccaaggagcatctccttaca


 



cactagggacaaaatttcacggctgtcactgggcatctctc


 



cacgctcatcacagccctctagcagccttgaagtcctgtag


 



agcccttcccatttcacagaagggacaagactatgagggcc


 



acaccgtgagccatgagccttaggctgtgagccgggacagc


 



ccctgcaggactggtggcctcagggcactgggtggggaggg


 



tgcacagtgggtgggccccttgtggaatagagaggagtgtc


 



aggtcaggggagggggcttggcctggccctggcctgcctgg


 



tgtgcaaccctaggcagcccctccttcccaggcctcctact


 



tcctggaggccaagcctcagggaggtaattgagtcaggtgg


 



gggagggggggttgtggctttcttcacagcagaaaaacaga


 



gcccacaatagtgtccactgagacagaggggtcctggggga


 



ggggaggggtgggaggtgactgctgagccctgtgggaggga


 



gggagcaactactgagctgagctgggtgactctcccatctg


 



ccccgccccctgtggggccagcagagtcaccgagagaacat


 



gacccagccaggcctggacagggggacacccatgtccttta


 



ccccacagggttcactgagcctatctgccccaagcctgtgt


 



ctccctgggacggagaccctcactcccaaccacaaaggtct


 



aaactcaagttcccaacagccttgaaaatacagcttccggg


 



ggcctccaaggagcagtcagccgtccactgccaggctcgct


 



ggctcagtgacacaggacacatcctgatgacggtccacctg


 



tctccaagcaggttctcctctgccgatggggcaacgagctc


 



ctcctgtggctccctggctggatgcgtgggaggcggggtgg


 



gggggcaggcggtgttcctggccgcacacaaggagcacccc


 



caccagcatccgaagacgggggcccggtctttccccaaaac


 



actgcttgcgggagactttgtgacgtttccaggggccatgc


 



tcccttcgggcagcttgggggacttctgctcctatgtggtc


 



acctgcagggactccccccaggccttggggacaaacaaagt


 



gatgagagggagggttagtgggtcggggcagggccagtctt


 



tggaccggtttatctgaaaagccagttggtcaccgggaacc


 



acagcaaacctaaacccatttggccaggcatctcccaggga


 



cagtctcccccaggatgcggggcccaggggggctccagggg


 



tgacctgcgtcctggatttccctgatgctcccagttcgtgc


 



ctctgtccaagcatgatttttaatagtgccccttccactcc


 



cagaaatgtccaagtgtgggcaataaattctggtcacctga


 



gctcagtgtaactgtttgctgaatgacacttactgtaacag


 



gttaaaatgggaggcccaaggccacgcagagccatcgaagg


 



ctctgtgtgtcccagccctgatagaagcatcaggatgggga


 



ctgtggcctcaccaggggccacatccaggcggtcaccatgg


 



ggttcctggtctccgtgggccttgactggagcccctggtgt


 



gagctcaccccatcccagcctgtgagaggcctggatgtggg


 



cctgacatcatttcccacccagtgacagcactgcatgtgat


 



ggggcctctgggcagcctttttcccgggggaaactggcagg


 



aatcaggaccaccaggacaggggtcaggggagaggcgatgc


 



tgggcaccagagcctggaccaccctcgggttctcagcgatg


 



ggcaacccctgccacccagggccccgccttcctggggagac


 



atcggggtttccaggccatcctgggaggagggtgggagcct


 



cagctagaccccagctggcttgcccccccatgccccggcca


 



agagagggtcttggagggaagggggaccccagaccagcctg


 



gcgagcccatcctcagggtctctggtcagacaggggctcag


 



ctgagctccagggtagaccaaggccctgcgtggatgaggcc


 



agtgtggtcactgcccagagcaaagccacctctcagcagcc


 



ctttcctgagcaccttctgtgtgcggggacatcagcagtgg


 



caacacagccatgctggggactcagggctagagacagggga


 



ccagcctatggagagtgggtagtgtcctgcagggcaggctt


 



gtgccctggagaaaacaaaccagggtgaggccagggacgct


 



ggccgggttcacagggtgatggctgagcacagagtgccagg


 



ggctggactgtcctgactctgggttggtggctgagggcctg


 



tgtccctctatgcctctgggttggtgataatggaaacttgc


 



tccctggagagacaggacgaatggttgatgggaaatgaatg


 



tttgcttgtcacttggttgactgttgttgccgttagcattg


 



ggcttcttgggccaggcagcctcaggccagcactgctgggc


 



tccccacaggcccgacaccctcagccctgtgcagctggcct


 



ggcgaaaccaagaggccctgatgcccaaaatagccgggaaa


 



ccccaaccagcccagccctggcagcaggtgcctcccatttg


 



cctgggctgggggaggggtggctctggttctggaagtttct


 



gccagtccagctggagaagggacctgtatcccagcacccag


 



gccgcccaagcccctgcaccagggcctgggccaggcagagt


 



tgacatcaatcaattgggagctgctggaatgcatggaggcg


 



gcgctctcgaggctggaggaggccagctgatttaaatcggt


 



ccgcgtacgatgcatattaccctgttatccctaccgcggtt


 



actggccgtcgttttacaacgtcgtgactgggaaaaccctg


 



gcgatgctcttctcccggtgaaaacctctgacacatggctc


 



ttctaaatccggagtttaaacgcttccttcatgtgagcaaa


 



aggccagcaaaaggccaggaaccgtaaaaaggccgcgttgc


 



tggcgtttttccataggctccgcccccctgacgagcatcac


 



aaaaatcgacgctcaagtcagaggtggcgaaacccgacagg


 



actataaagataccaggcgtttccccctggaagctccctcg


 



tgcgctctcctgttccgaccctgccgcttaccggatacctg


 



tccgcctttctcccttcgggaagcgtggcgctttctcatag


 



ctcacgctgtaggtatctcagttcggtgtaggtcgttcgct


 



ccaagctgggctgtgtgcacgaaccccccgttcagcccgac


 



cgctgcgccttatccggtaactatcgtcttgagtccaaccc


 



ggtaagacacgacttatcgccactggcagcagccactggta


 



acaggattagcagagcgaggtatgtaggcggtgctacagag


 



ttcttgaagtggtggcctaactacggctacactagaaggac


 



agtatttggtatctgcgctctgctgaagccagttaccttcg


 



gaaaaagagttggtagctcttgatccggcaaacaaaccacc


 



gctggtagcggtggtttttttgtttgcaagcagcagattac


 



gcgcagaaaaaaaggatctcaagaagatcctttgatctttt


 



ctacggggtctgacgctcagtggaacgaaaactcacgttaa


 



gggattttggtcatgcctaggtggcaaacagctattatggg


 



tattatgggtctaccggtgcatgagattatcaaaaaggatc


 



ttcacctagatccttttaaattaaaaatgaagttttaaatc


 



aatctaaagtatatatgagtaaacttggtctgacagttacc


 



aatgcttaatcagtgaggcacctatctcagcgatctgtcta


 



tttcgttcatccatagttgcctgactccccgtcgtgtagat


 



aactacgatacgggagggcttaccatctggccccagtgctg


 



caatgataccgcgagacccacgctcaccggctccagattta


 



tcagcaataaaccagccagccggaagggccgagcgcagaag


 



tggtcctgcaactttatccgcctccatccagtctattaatt


 



gttgccgggaagctagagtaagtagttcgccagttaatagt


 



ttgcgcaacgttgttgccattgctacaggcatcgtggtgtc


 



acgctcgtcgtttggtatggcttcattcagctccggttccc


 



aacgatcaaggcgagttacatgatcccccatgttgtgcaaa


 



aaagcggttagctccttcggtcctccgatcgttgtcagaag


 



taagttggccgcagtgttatcactcatggttatggcagcac


 



tgcataattctcttactgtcatgccatccgtaagatgcttt


 



tctgtgactggtgagtactcaaccaagtcattctgagaata


 



gtgtatgcggcgaccgagttgctcttgcccggcgtcaatac


 



gggataataccgcgccacatagcagaactttaaaagtgctc


 



atcattggaaaacgttcttcggggcgaaaactctcaaggat


 



cttaccgctgttgagatccagttcgatgtaacccactcgtg


 



cacccaactgatcttcagcatcttttactttcaccagcgtt


 



tctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaa


 



gggaataagggcgacacggaaatgttgaatactcatactct


 



tcctttttcaatattattgaagcatttatcagggttattgt


 



ctcgggagcggatacatatttgaatgtatttagaaaaa






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, U.S. Ser. No. 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 Vλ gene segments, Jλ gene segments, and the single Cλ gene. In another embodiment, the AC can express at least one Vλ gene segment, at least one Jλ gene segment, and the Cλ 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 Vκ gene segments, Jκ gene segments and the single Cκ gene. In another embodiment, the AC can express at least one Vκ gene segment, at least one Jκ gene segment and the Cκ 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 VH, DH, JH and CH gene segments. In another embodiment, the AC can express at least one VH gene segment, at least one DH gene segment, at least one JH gene segment and at least one at least one CH gene 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−1 cytochalasin B (Sigma) and 7.5 μg ml−1 Hoechst 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


amplify J to C heavy-


chain (637Xba5′)


 


Seq ID 3: primer for C
ttccaggagaaggtgacggagct


to amplify J to C


heavychain (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 (10 mM 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−1 BSA 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−1 cytochalasin B (Sigma) and 7.5 μg ml−1 Hoechst 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 (10 mM 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


 



AAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGALAACTCT


 



CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT


 



CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT


 



TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGG


 



GAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT


 



TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAG


 



CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGG


 



TTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCAAACAG


 



CTATGACCATGGCGGCCGCgtcgacAGGGTGTGGCCAAATACAG


 



CATGGAGTAGCCATCATAAGGAATCTTACACAAGCCTCCAAAAT


 



TGTGTTTCTGAAATTGGGTTTAAAGTACGTTTGCATTTTAAAAA


 



GCCTGCCAGAAAATACAGAAAAATGTCTGTGATATGTCTCTGGC


 



TGATAGGATTTTGCTTAGTTTTAATTTTGGCTTTATAATTTTCT


 



ATAGTTATGAAAATGTTCACAAGAAGATATATTTCATTTTAGCT


 



TCTAAAATAATTATAACACAGAAGTAATTTGTGCTTTAAAAAAA


 



TATTCAACACAGAAGTATATAAAGTALAAATTGAGGAGTTCCCA


 



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


 



TTAACACTCACACTCTCCCCACCCCCACCCCCGCTGATCTCCTT


 



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 transgenic porcine animal whose genome comprises a homozygous disruption of nucleotides 3296-3352 of SEQ ID NO: 29 of the joining region (J region) of the endogenous porcine heavy chain gene, wherein the porcine lacks expression of the endogenous porcine heavy chain immunoglobulin gene.

2. The transgenic porcine of claim 1, wherein the porcine animal comprises a homozygous disruption of the constant region of the endogenous porcine kappa immunoglobulin gene, wherein the disruption results in a lack of expression of the endogenous kappa immunoglobulin gene.

3. The porcine of claim 1, wherein the porcine heavy chain gene is disrupted through substitution, deletion or insertion techniques.

4. The porcine of claim 3, wherein the porcine heavy chain gene is disrupted through site specific recombinase techniques.

5. The porcine of claim 1, wherein the homozygous disruption is produced by homologous recombination.

6. The porcine of claim 1, wherein the porcine expresses a xenogenous immunoglobulin locus or fragment thereof and wherein the immunoglobulin is expressed from an immunoglobulin locus that is integrated within an endogenous porcine chromosome.

7. The porcine of claim 6, wherein the xenogenous immunoglobulin comprises a human immunoglobulin or fragment thereof.

8. The porcine of claim 6, wherein the xenogenous immunoglobulin locus is inherited by offspring.

9. The porcine of claim 6, wherein the xenogenous immunoglobulin locus is inherited through the male germ line by offspring.

10. The porcine of claim 6, wherein the porcine is produced through nuclear transfer.

11. The porcine of claim 6, wherein the immunoglobulin locus is expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.

12. The porcine of claim 1, further comprising an additional genetic modification to eliminate the expression of a xenoantigen.

13. The porcine of claim 12, wherein the xenoantigen is alpha-1,3-galactosyltransferase.

14. The transgenic porcine of claim 2, wherein the porcine animal comprises a homozygous disruption of the constant region of the endogenous porcine lambda immunoglobulin gene, wherein the disruption results in the lack of expression of the endogenous porcine lambda immunoglobulin gene.

15. The porcine of claim 14, wherein the porcine heavy chain, kappa light chain and lambda light chain genes can be transcribed into RNA but not translated into protein.

16. The porcine of claim 14, wherein the porcine heavy chain, kappa light chain and lambda light chain genes are inactivated such that no transcription of the genes occurs.

17. The porcine of claim 14, wherein the porcine heavy chain, kappa light chain and lambda light chain genes are inactivated such that they are transcribed and then translated into non-functional proteins.

18. The porcine of any one of claims 1-14, wherein the porcine is produced by nuclear transfer.

19. Isolated cells derived from the porcine of claim 1.

20. Isolated A tissue derived from the porcine of claim 1.