Tlr8 Transgenic Animals

Abstract

Provided herein are human Toll-like receptor 8 (TLR8)-expressing transgenic animals and methods of use thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 13/434,805, filed Mar. 29, 2012, which claims the benefit of U.S. Provisional Patent Application No. 61/469,055, filed Mar. 29, 2011, both of which are incorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 377882005000SeqList.txt, date recorded: Mar. 29, 2012, size: 184 KB).

FIELD OF THE INVENTION

[0003] This application relates to human Toll-like receptor 8 (TLR8)-expressing transgenic animals and methods of use thereof.

BACKGROUND OF THE INVENTION

[0004] Immunity can generally be classified as innate immunity or as adaptive immunity. Innate immune responses typically occur immediately upon infection to provide an early barrier to infectious disease, whereas adaptive immune responses occur later with the generation of antigen-specific effector cells and immunological memory. Innate immune responses do not generate lasting protective immunity, but appear to play a role in the generation of later arising adaptive immune responses.

[0005] Toll-like receptors (TLRs) are essential for innate immune responses as they recognize several different antigens and initiate immune responses (e.g., activation of dendritic cells and macrophages, and cytokine production). TLRs are type-I transmembrane proteins that recognize a variety of pathogen-associated molecular patterns (PAMPs) from bacteria, viruses and fungi. In this way PAMPs serve as a first-line of defense against invading pathogens. Human TLRs can elicit overlapping yet distinct biological responses due to differences in cellular expression and activation of downstream signal transduction pathways (Akira et al., Adv. Immunol. 78: 1-56, 2001).

[0006] TLRs are characterized by an ectodomain composed of leucine-rich repeats and a cytoplasmic domain, known as a Toll/interleukin-1 receptor domain. The ectodomain is responsible for recognition of PAMPs, while the cytoplasmic domain is required for downstream signaling. TLRs usually undergo dimer formation and/or a conformation change to activate downstream signal transduction pathways. Studies have shown that LRR8 is involved in DNA and RNA recognition, whereas LRR17 is involved in nucleic acid binding (Smits et al., Oncologist, 13: 859-875, 2008).

[0007] The family of TLRs consists of ten members in human (TLR1-TLR10) and twelve members in mice (TLR1-TLR9 and TLR11-TLR13). The TLRs that are located in the plasma membrane recognize bacterial membrane components, whereas the TLRs that detect nucleic acid-based ligands are predominately located within endosomal compartments. The nucleic acid-sensing TLRs include TLR3, TLR7, TLR8, and TLR9. Upon ligand-binding, TLRs initiate a signal transduction cascade leading to activation of NF.kappa.B through the adapter protein myeloid differentiation primary response gene 88 (MyD88) and recruitment of the IL-1 receptor associated kinase (IRAK). Phosphorylation of IRAK in turn leads to the recruitment of TNF-receptor associated factor 6 (TRAF6), which results in the phosphorylation and degradation of the NF-.kappa.B inhibitor, I-.kappa.B, thereby releasing NF-.kappa.B. NF-.kappa.B enters the cell nucleus and initiates gene transcription, leading to production of proflammatory cytokines, chemokines, and type I interferons (IFNs), as well as the upregulation of costimulatory molecules.

[0008] TLR8 belongs to the same subfamily as the TLR7 and TLR9 endosomal receptors and is highly homologous to TLR7 (Liu et al., Mol Immunol. 47:1083-90, 2010). The role of TLR8, and of its close homologue TLR7, is to detect the presence of "foreign" single-stranded RNA within a cell, as a means to respond to viral invasion (Heil et al., Science 303:1526, 2004; and Diebold et al., Science 303:1529, 2004). While the TLR8 gene in humans is closely related to TLR7, TLR8 has distinct, but overlapping specificity for RNA and synthetic small molecules with a structure related to nucleic acids (Medzhitov et al., Immunol. Rev. 173:89-97, 2000). Some ssRNA synthetic sequences containing repetitive A/U motifs are able to specifically activate TLR8 but not TLR7 (Gorden et al. J Immunol. 174:1259-68, 2005). Further, in humans, TLR8 is highly expressed in monocytes, macrophages, myeloid dendritic cells (mDC) and neutrophils, whereas TLR7 in blood cells is principally expressed in pDCs, B-cells, and neutrophils. Because of this difference in cellular expression, triggering by RNA through TLR7 in blood leads to a response dominated by Type I IFN production, whereas activation through TLR8 induces multiple pro-inflammatory cytokines: TNF, IL-12, IL-6, IL-8 and IL-1 (Barrat et al., J Exp Med. 2202:1131-9, 2005; and Gorden et al., J Immunol. 174:1259-68, 2005).

[0009] Although TLR8 polymorphisms have been associated with some autoimmune diseases, the role of TLR8 and its specific ligands has not been clearly defined. One key limitation in elucidating TLR8 biology is the lack of an animal model. For example, mouse TLR8 has a very different specificity than human TLR8. Mouse TLR8 lacks the ability to respond to ssRNA ligands, RNA viruses or small molecules; all of which have been shown to activate human TLR8 (Heil et al. Science 303:1526-9, 2004; Jurk et al. Nat Immunol 3:499, 2002; Hemmi et al. Nat. Immunol 3:196-200, 2002; and Lund et al. PNAS 101:5598-603, 2004). Further, by comparing the amino acid sequence, TLR8 of mice and rats lack a five amino-acid sequence required for ligand recognition in man (Liu et al. Mol Immunol. 47:1083-90, 2010). The lack of useful animal models and the very different ligand specificities of human TLR8 and its rodent orthologs have proven to be major limitations to the study of TLR8 biology.

SUMMARY OF THE INVENTION

[0010] Provided herein are transgenic animals comprising a nucleotide sequence encoding human Toll-like receptor 8 (TLR8), wherein the human TLR8 is expressed in the transgenic animal. In some embodiments, the transgenic animal is a nonhuman mammal. In some embodiments, the transgenic animal is a small mammal selected from the group consisting of a mouse, hamster, rat, guineas pig, and rabbit. In some embodiments, the transgenic animal is a mouse. In some embodiments, the transgenic animal is a chimeric transgenic animal. In some embodiments, both germ cells and somatic cells of the transgenic animal comprise the nucleotide sequence encoding human TLR8. In some embodiments, the nucleotide sequence encoding human TLR8 is stably integrated into the genome of the transgenic animal. In some embodiments, the nucleotide sequence encoding human TLR8 is present at a copy number of from 1 to 15 (e.g., from 1 to 5, from 6 to 10, from 11 to 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15). In some embodiments, the nucleotide sequence encoding human TLR8 is operatively linked to a promoter and/or other regulatory regions. In some embodiments, the promoter and/or other regulatory regions are those of human TLR8. In some embodiments, the human TLR8 is expressed in a similar expression pattern in the transgenic animal as human TLR8 is expressed in humans. In some embodiments, the levels of expression of human TLR8 in the transgenic animal is similar to the level of expression of human TLR8 in humans. In some embodiments, the transgenic animal is predisposed to development of inflammation in one or more organs (e.g., pancreas, kidney, liver, joints, reproductive tissue, etc.). In some embodiments, the inflammation comprises an autoimmune disease (e.g., pancreatitis, nephritis, hepatitis, rheumatoid arthritis, diabetes, diabetes-related disorder, reproductive disease, etc.). In some embodiments, the present disclosure provides a cell obtained from the transgenic animal described herein, wherein the human TLR8 is expressed in the cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the hematopoeitic cell is a monocyte. Provided herein are also methods of screening candidate agents, the methods comprising: administering a candidate agent to the transgenic animal; and determining the effect of the candidate agent on the animal (e.g., as compared to a control such as untreated animal or an animal receiving a placebo). Some methods comprise administering a candidate agent to the transgenic animal; and determining the effect of the candidate agent on a TLR8-mediated response of the animal (e.g., as compared to a control such as an untreated animal or an animal receiving a placebo). Additionally, provided herein are methods of screening candidate agents, the method comprising: contacting the cell obtained from the transgenic animal with a candidate agent; and determining the effect of the candidate agent on the cell (e.g., as compared to a control such as an untreated or mock-treated cell). Some methods comprise contacting the cell obtained from the transgenic animal with a candidate agent; and determining the effect of the candidate agent on the cell (e.g., as compared to a control such as untreated or mock-treated cell). In some embodiments, the effect comprises inhibition of the TLR8-mediated response (e.g., reduction of the response to less than 50, 60, 70, 75, 80, 85, 90 or 95% of that observed in the absence of the candidate agent). In alternative embodiments, the effect comprises stimulation of the TLR8-mediated response (e.g., elevation of the response to at least 150, 160, 170, 180, 190 or 200% of that observed in the absence of the candidate agent). In some, the TLR8-mediated response is evidenced by a change in TLR8-mediated cytokine production, cell proliferation, and/or cell surface marker expression. A TLR8-mediated response is one that is stimulable by a TLR7/8 agonist such as R848 and CL075 or a TLR8 agonist such as a TLR8 ligand stabilized immunomodulatory RNA (e.g., SEQ ID NO:3). TLR8-mediated responses are assessed by measuring expression of a cytokine or a cell surface marker. Suitable cytokines are selected from but not limited to TNF-.alpha., IFN-.alpha., IFN-.beta., IFN-.gamma., IL-1.alpha., IL-1.beta., IL-6, IL-8, IL-10, IL-12, IL-23, IP-10, MIP-1, and MCP-1. Suitable cell surface molecules are selected from but not limited CD40, CD80, CD86, ICAM-1, ICAM-2, ICAM-3, and CCR7. In some embodiments, the cytokine comprises one or more of the group consisting of TNF, IL-12, IL-6, MIP-1.alpha., IFN.gamma., IP-10, IL-1.alpha., and IL-1.beta.. In some embodiments, the candidate agent is an antibody. In some embodiments, the candidate agent is a small molecule. In some preferred embodiments, the candidate agent is a polynucleotide. In some preferred embodiments, the TLR8-mediated cytokine production comprises production of one or more of the group consisting of TNF, IL-12, IL-6, MIP-1.alpha., IFN.gamma., IP-10, IL-1.alpha., and IL-1.beta..

[0011] Moreover, the present disclosure provides methods for screening, and/or identifying, TLR8 modulators, the method comprising: providing a candidate agent to a cell culture, wherein cells of the cell culture comprises cells derived from a TLR8 transgenic animal which comprises a nucleotide sequence encoding human TLR8; and determining the effect of the candidate agent on the cell culture (as compared to a control such as untreated or mock-treated cell culture). Further, provided are methods of screening for, and/or identifying, TLR8 modulators, the method comprising: providing a candidate agent to a cell culture, wherein cells of the cell culture are obtained or prepared from a transgenic animal described herein which comprise a nucleotide sequence encoding human TLR8; and determining the effect of the candidate agent on the cell culture (as compared to a control such as untreated or mock-treated cell culture). In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are monocytes. In some embodiments, the effect is inhibition of cytokine production, cell proliferation and/or cell surface molecule expression. In some embodiments, the effect is stimulation of cytokine production, cell proliferation and/or cell surface molecule expression. Suitable cytokines are selected from but not limited to TNF-.alpha., IFN-.alpha., IFN-.beta., IFN-.gamma., IL-1.alpha., IL-1.beta., IL-6, IL-8, IL-10, IL-12, IL-23, IP-10, MIP-1, and MCP-1. Suitable cell surface molecules are selected from but not limited CD40, CD80, CD86, ICAM-1, ICAM-2, ICAM-3, and CCR7. In some embodiments, the cytokine comprises one or more of the group consisting of TNF, IL-12, IL-6, MIP-1.alpha., IFN.gamma., IP-10, IL-1.alpha., and IL-1.beta.. In some embodiments, the candidate agent is an antibody (e.g., antibody fragment). In some embodiments, the candidate agent is a small molecule. In some embodiments, the candidate agent is a polynucleotide. In some embodiments, the polynucleotide comprises a TLR8 immunoregulatory sequence (IRS). In some embodiments, the polynucleotide comprises a TLR8 immunostimulatory sequence (ISS). In some embodiments, the candidate agent is an antagonist. In some embodiments, the candidate agent is an agonist.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows the level of expression of human TLR8 in purified human subsets: [0013] plasmacytoid dendritic cells (PDC), monocytes, myeloid dendritic cells (MDC), CD4+ T-cells, CD8+ T-cells, and neutrophils.

[0014] FIG. 2A-B shows the effect of 200, 100, and 20 .mu.g/mL of TLR7 or TLR8-stimulating RNA polynucleotides (PN) (SEQ ID NO:2 and SEQ ID NO:3, respectively) or medium alone on hIL-6 and hTNF production (pg/mL) by human peripheral blood mononuclear cells (PBMC) and monocytes.

[0015] FIG. 3A-C shows the effect of 100 .mu.g/mL of TLR7-stimulating RNA PN (SEQ ID NO:2), 100 .mu.g/mL of TLR8-stimulating RNA PN (SEQ ID NO:3), or medium alone on hIL-6, hTNF and hIFN-.alpha. production (pg/mL) by human PDC.

[0016] FIG. 4A-D shows the effect of 100 .mu.g/mL of TLR7-stimulating RNA PN (SEQ ID NO:2), 100 .mu.g/mL of TLR8-stimulating RNA PN (SEQ ID NO:3), or medium alone on mIL-12 production (pg/mL) and mTNF production (pg/mL) by mouse splenocytes and mouse PBMC, respectively.

[0017] FIG. 5 shows the TLR8 BAC construct used to produce the TLR8 transgenic mice.

[0018] FIG. 6A-C shows the correlation between survival and the level of human TLR8 gene expression in chimeric mice, and the elevated level of expression of TNF, IL-6, IL-17, IL-12p40, CCL-2 and IP10 in sera of TLR8Tg mice. FIG. 6A shows the survival curve of human TLR8 chimeric mice from clone 6 (number of mice=14), clone 23 (number of mice=8), clone 12 (number of mice=18), and clone 8 (number of mice=18). FIG. 6B shows the TLR8 expression levels as relative CT in these mice. FIG. 6C shows the elevated level of inflammatory cytokines in serum of TLR8Tg mice from clone 12 (n=15) as compared to control, wild-type C57BL/6 mice (n=18).

[0019] FIG. 7A-F shows upregulation of expression of inflammatory cytokines IFN-.gamma., IL12-p40, TNF-.alpha., IP-10, IL-1.alpha., IL-1.beta. by relative CT in the pancreas from chimeric mice from clone 6, 12 and 23 (total number of mice=6) compared to wild-type mice C57BL/6 (total number of mice=6). FIG. 7G-7I shows the titers of anti-nuclear antibody (ANA), anti-double stranded DNA (dsDNA) antibody and anti-ribonucleoprotein (RNP) antibody in the serum of TLR8TgCL12 (n=27) and age-matched controls (n=37).

[0020] FIG. 8A-D shows the levels of CD80, GITRL, CD40, and OX40L as determined by FACS analyses of myeloid dendritic cells (MDC) from chimeric TLR8TGCL23 mice as compared to wild-type C57BL/6 mice.

[0021] FIG. 9 shows the percentage survival per day post-bone marrow transplant of mice receiving bone marrow from TLR8 chimeric mice from clones 6, 12 and 23.

[0022] FIG. 10A-B shows example of dot blot results of FACS analyses of T-cells from the spleen and blood of mice transplanted with bone marrow from chimeric TLR8TGCL12 mice. Examples of dot blots for both CD4 T-cells (FIG. 10A) and for CD8 T-cells (FIG. 10B) are provided.

[0023] FIG. 11A-L shows cumulative data of FACS analyses of T-cells from the spleen and blood of mice transplanted with bone marrow from chimeric TLR8TGCL12 mice.

[0024] FIG. 12A-E shows expression of MHC Class II, CD80, GITRL, OX40L, and PDL-1, respectively, as determined by FACS analyses of dendritic cells of mice transplanted with human TLR8 transgenic mouse bone marrow.

[0025] FIG. 13A-C shows activation of TLR8 expressed from the human TLR8 transgene in blood cells in TLR8TGCL8 mice as compared to C56BL/6 wild-type mice upon activation with 200 .mu.g/mL or 100 .mu.g/mL of TLR8-stimulating RNA PN (SEQ ID NO:3), or media alone as assayed by measuring levels of mIL-12 (pg/mL), mTNF-.alpha. (pg/mL), and mIL-6 (pg/mL), respectively.

[0026] FIG. 14A-B shows activation of TLR8 expressed from the human TLR8 transgene in bone marrow (BM) cells from TLR8TGCL8 mice upon activation with 100 .mu.g/mL or 50 .mu.g/mL of a TLR8-specific ligand (SEQ ID NO:3) as assayed by measuring levels of mTNF-.alpha. (pg/mL) and mIL-6 (pg/mL), respectively.

[0027] FIG. 15A-C shows that mice over expressing human TLR8 spontaneously develop arthritis. Chimeric mice from clone 12 (n=6) and C57BL/6 CTLR animals (n=6) were euthanized 90 days after birth. All the hTLR8 animals exhibited signs of swelling in forelimbs and hindlimbs. Paws and joints were fixed, sectioned and stained with toluene blue. FIG. 15A provides a representative section of a joint from WT (C57BL/6 CTLR) mice. FIG. 15B provides a representative section of a joint from a TLR8Tg mouse. The histological changes, degree of inflammation and cartilage damage were evaluated by a pathologist in a blinded fashion and scored 1 to 5 as follows: 1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe. Two paws and two ankles were evaluated for each mouse, and the scores were summed to obtain the histological disease score shown in FIG. 15C.

[0028] FIG. 16A-B shows the cumulative score and the percentage incidence of symptoms with a clinical score of greater than or equal to four, respectively, using a collagen-induced rheumatoid arthritis (CIA) model for wild-type mice (C57BL/6) and TLR8 transgenic mice (TLR8TGCL8) in days after first collagen injection. FIG. 16C-E shows that hTLR8, F4/80 and TNF were significantly elevated in joints of hTLR8 transgenic mice (TLR8Tg clone 8) as compared to wild-type mice (C57BL/6). FIG. 16F-H shows hTLR8, F4/80 and TNF expression relative to the disease score or level of hTLR8 expression respectively. Clearly, the levels of F4/80 and TNF were directly correlated with the level of hTLR8 in the transgenic mice. Likewise, the levels of proinflammatory cytokines (IL-1beta, IL-1alpha, IL-6, IL-18, IP-10, and MMP-9) were found to be directly correlated with hTLR8 expression.

[0029] FIG. 17 shows a significant increase in blood glucose level (mg/dL) in mice over-expressing human TLR8 (C57BL/6-SJL WT mice transplanted with TLR8TGCL12 bone marrow cells) compared to C57BL/6-SJL WT mice transplanted with WT C57BL/6 bone marrow cells.

[0030] FIG. 18A shows that human TLR8 is expressed in monocytes, neutrophils and dendritic cells of hTLR8Tg mice. The expression pattern of huTLR8 in leukocyte subsets from these mice is very similar to its expression in human leukocytes. FIG. 18B-D shows that hTLR8 is functional in hTLR8Tg mice. Briefly, PBMC from hTLR8Tg chimeras, but not from wild-type mice produced IL-12 and TNF when stimulated with an RNA-based TLR8 agonist, ORN-8L (FIG. 18B-C). Similarly, elevated levels of IL-12 were detected in the serum from hTLR8Tg mice, but not from wild-type mice, which were injected intravenously with an RNA-based TLR8 agonist (ORN-8L) or a small molecule-based TLR8 agonist (CL075).

[0031] FIG. 19A-C shows that hTLR8Tg mice develop lymphopenia (decrease in number of circulating lymphocytes), granulocytosis (increase in number of granulocytes, which are primarily neutrophils) and monocytosis (increase in number of monocytes). Cell number was assessed in peripheral blood (FIG. 19A) and in spleens of hTLR8Tg chimeras or age-matched control C57BL/6 mice (FIG. 19B). There is a significant decrease in circulating lymphocytes paralleled by an increase in granulocytes and monocytes in the blood of the hTLR8-expressing mice as compared to WT mice CTRL (FIG. 19C).

[0032] FIG. 20A-C shows that T lymphocytes from mice over-expressing human TLR8 produce more TNF and IFN-gamma as compared to T lymphocytes from wild type mice.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Provided herein are hTLR8 transgenic animals, and cells therefrom that express human TLR8. Also provided are methods of screening for and/or identifying TLR8 modulators using the hTLR8 transgenic animals or cells expressing human TLR8.

[0034] Unless otherwise indicated, reference to an agent can include the compound in any pharmaceutically acceptable form, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, and the like. In particular, if a compound is optically active, reference to the compound can include each of the compounds enantiomers as well as racemic mixtures of the enantiomers.

DEFINITIONS

[0035] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), modified polynucleotides and polynucleosides or combinations thereof. The polynucleotide can be linearly, branched, or circularly configured, or the polynucleotide can contain one or more linear, branched, and/or circular segments. Polynucleotides are polymers of nucleosides joined, generally, through phosphodiester linkages, although alternate linkages, such as phosphorothioate esters may also be used in polynucleotides. A nucleoside consists of a purine (adenine (A) or guanine (G) or derivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil (U), or derivative thereof) base bonded to a sugar. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine. A nucleotide is a phosphate ester of a nucleoside.

[0036] The term "peptide" includes polypeptides that are of any length and composition to affect a biological response, e.g., antibody production or cytokine activity whether or not the peptide is a hapten. The term "peptide" further includes modified amino acids (whether or not naturally or non-naturally occurring), such modifications including, but not limited to, phosphorylation, glycosylation, pegylation, lipidization and methylation.

[0037] The terms "promoter element" or "promoter" refer to a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences.

[0038] The term "vector" refers to a nucleic acid assembly capable of transferring gene sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). The term "expression vector" refers to a nucleic acid assembly containing a promoter that is capable of directing the expression of a sequence or gene of interest in a cell. Vectors typically contain nucleic acid sequences encoding selectable markers for selection of cells that have been transfected by the vector. Generally, "vector construct," "expression vector," and "gene transfer vector," refer to any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0039] The term "wild-type" refers to a nucleic acid, protein, and/or animal (e.g., mouse) that has the characteristics of that nucleic acid, protein, and/or animal (e.g., mouse) when isolated from a naturally occurring source. A wild-type nucleic acid, protein, and/or animal (e.g., mouse) is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of that molecule. In contrast, the term "modified" or "mutant" refers to a nucleic acid, protein, and/or animal (e.g., mouse) that displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type nucleic acid, protein, and/or animal (e.g., mouse).

[0040] The term "agonist" is used in the broadest sense and refers to a molecule that can elevate or stimulate an induced cellular activity

[0041] The term "antagonist" or "inhibitor" is used in the broadest sense, and refers to a composition that can reduce or inhibit an induced cellular activity.

[0042] The term "antibody" is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain antibodies, and fragments of antibodies as long as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0043] The term "small molecule" as used herein refers to a low molecular weight organic compound (e.g., not a polymer). In some embodiments, the small molecule has a molecular weight of less than 2000, 1600, 800, or 400 daltons.

[0044] The term "immunostimulatory" or "stimulating an immune response" as used herein includes stimulation of cell types that participate in immune reactions and enhancement of an immune response to a specific antigenic substance. An immune response that is stimulated by an immunostimulatory nucleic acid is generally a "Th1-type" immune response, as opposed to a "Th2-type" immune response. Th1-type immune responses are normally characterized by "delayed-type hypersensitivity" reactions to an antigen and activated macrophage function and can be detected at the biochemical level by increased levels of Th1-associated cytokines such as IFN-.gamma., IL-2, IL-12, and TNF-.alpha.. Th2-type immune responses are generally associated with high levels of antibody production, especially IgE antibody production and enhanced eosinophils numbers and activation, as well as expression of Th2-associated cytokines such as IL-4, IL-5 and IL-13.

[0045] The term "immunoregulatory compound" or "IRC", as used herein, refers to a molecule which has immunoregulatory activity and which comprises a nucleic acid moiety comprising an IRS. The IRC may consist of a nucleic acid moiety that comprises more than one IRS or consists of an IRS. The IRC may comprise a modified and/or unmodified IRS. The IRC may consist of a polynucleotide (a "polynucleotide IRC") or it may comprise additional moieties. Accordingly, the term IRC includes compounds which incorporate one or more nucleic acid moieties, at least one of which comprises an IRS, covalently linked to a non-nucleotide spacer moiety.

[0046] The term "immunoregulatory sequence" or "IRS", as used herein, refers to a nucleic acid sequence that inhibits and/or suppresses a measurable immune response as measured in vitro, in vivo, and/or ex vivo. The term "immunoregulatory sequence" or "IRS", as used herein, refers to both nucleic acid sequences that comprise a modification (i.e., modified IRS) as well as nucleic acids which do not comprise a modification (i.e., unmodified IRS).

[0047] The term "TLR8 modulator" is used in the broadest sense, and includes TLR8 agonists and antagonists.

[0048] The term "cells," as used herein, is understood to refer not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term as used herein.

[0049] The term "transfection" refers to the uptake of DNA by a cell. A cell has been "transfected" when exogenous (i.e., foreign) DNA has been introduced inside the cell membrane. Transfection can be either transient (i.e., the introduced DNA remains extrachromosomal and is diluted out during cell division) or stable (i.e., the introduced DNA integrates into the cell genome or is maintained as a stable episomal element).

[0050] "Stimulation" of a response or parameter includes eliciting and/or enhancing that response or parameter when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, "stimulation" of an immune response means an increase in the response, which can arise from eliciting and/or enhancement of a response. Similarly, "stimulation" of production of a cytokine (such as IL-1.alpha., IL-1.beta., IL-6, and/or TNF-.alpha.) or "stimulation" of cell type (such as CTLs) means an increase in the amount or level of cytokine or cell type.

[0051] "Suppression" or "inhibition" of a response or parameter includes decreasing that response or parameter when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.

[0052] "Correlate" or "correlating" as used herein refer to comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of gene expression analysis or protocol, one may use the results of the gene expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.

[0053] The term "innate immune response" or "innate immunity" as used herein includes a variety of innate resistance mechanisms by which a cell or individual recognizes and responds to the presence of a pathogen. As used herein, an "innate immune response" includes the intracellular and intercellular events and reactions that occur when the cell recognizes pathogen associated molecular patterns or signals. Cellular receptors active in an innate immune response include a family of Toll-like receptors (TLRs) and microbial ligands have been identified for several TLRs, as described herein.

[0054] The term "individual" refers to a mammal, including humans. An individual includes, but is not limited to, human, bovine, horse, feline, canine, rodent, or primate.

[0055] The term "animal" is used herein to include all vertebrate and invertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A "transgenic animal" is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule may be integrated within a chromosome, or it may be extra-chromosomally replicating DNA.

[0056] The term "germ cell-line transgenic animal" refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too, are transgenic animals.

[0057] "Adjuvant" refers to a substance which, when added to an immunogenic agent such as antigen, nonspecifically enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture.

[0058] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0059] "Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration refers to treatment that is not consecutively and/or continuously done without interruption, but rather is cyclic in nature.

[0060] An "effective amount" of an agent disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" may be determined empirically and in a routine manner, in relation to the stated purpose.

[0061] A "growth inhibitory amount" as used herein is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "growth inhibitory amount" for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.

[0062] The term "therapeutically effective amount" refers to an agent effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the agent may reduce the number of cancer cells: reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of "treating". To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.

[0063] As used herein, "sample" refers to a composition which contains a molecule which is to be characterized and/or identified, for example, based on physical, biochemical, chemical, physiological, and/or genetic characteristics.

[0064] As used herein, and as well-understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Thus, "treating" or "treatment" does not require complete alleviation of signs or symptoms, and does not require a cure.

[0065] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0066] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0067] It is understood that aspects and embodiments described herein include "consisting" and/or "consisting essentially of" aspects and embodiments.

Human TLR8 Transgenic Animals and Cells

[0068] Provided herein are TLR8 transgenic animals and transgenic cells, which comprise a nucleotide sequence encoding human TLR8. For example, provided herein are transgenic animals comprising a nucleotide sequence encoding human TLR8, wherein the human TLR8 is expressed in the transgenic animal.

[0069] In some embodiments, the TLR8 transgenic animal is a chimeric TLR8 transgenic animal. In some embodiments, the TLR8 transgenic animal is a TLR8 transgenic animal with germ cells and somatic cells containing a nucleotide sequence encoding human TLR8. In some embodiments, the nucleotide sequence encoding human TLR8 is stably integrated into the genome of the TLR8 transgenic animal. In some embodiments, the nucleotide sequence encoding human TLR8 is extrachromosomal. In some embodiments, the extrachromosomal nucleotide sequence encoding human TLR8 is provided as a minichromosome, yeast artificial chromosome, or bacterial artificial chromosome. In some embodiments, the TLR8 transgenic animal comprises one or more copies of the nucleotide sequence encoding human TLR8. In some embodiments, the TLR8 transgenic animal comprises more than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 copies of the nucleotide sequence encoding human TLR8. In some embodiments, the TLR8 transgenic animal comprises about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 copies of the nucleotide sequence encoding human TLR8. For example, provided herein are transgenic animals comprising a nucleotide sequence encoding human TLR8, wherein the human TLR8 is expressed in the transgenic animal, wherein the TLR8 transgenic animal comprises about any of 1 to 2 copies of the nucleotide sequence encoding human TLR8.

[0070] The terms "human Toll-like receptor 8," "human TLR8," "hTLR8," and "CD288" as used herein refer to a protein and functional variants thereof that bind to viral and synthetic single-stranded RNAs (e.g., ssRNA derived from viruses, synthetic guanosine-rich ssRNA, and uridine-rich ssRNA), as well as small molecules resembling nucleic acids. The amino acid sequence of a common isoform of hTLR8 is set forth as SEQ ID NO:4. Accordingly, hTLR8 comprises the amino acid sequence of SEQ ID NO:4 or variants having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:4. In particularly preferred embodiments, the "RQSYA" motif (SEQ ID NO:5) in the ectodomain of hTLR8 is retained.

TABLE-US-00001 Human TLR8 Sequence of GENBANK Accession No. NP_619542.1 (SEQ ID NO: 4): 1 menmflqssm ltcifllisg scelcaeenf srsypcdekk qndsviaecs nrrlqevpqt 61 vgkyvteldl sdnfithitn esfqglqnlt kinlnhnpnv qhqngnpgiq snglnitdga 121 flnlknlrel llednqlpqi psglpeslte lsliqnniyn itkegisrli nlknlylawn 181 cyfnkvcekt niedgvfetl tnlellslsf nslshvppkl psslrklfls ntqikyisee 241 dfkglinitl ldlsgncprc fnapfpcvpc dggasinidr fafqnitqlr ylnlsstslr 301 kinaawfknm phlkvldlef nylvgeiasg afltmlprle ildlsfnyik gsypqhinis 361 rnfskllslr alhlrgyvfq elreddfqpl mqlpnlstin lginfikqid fklfqnfsnl 421 eiiylsenri splvkdtrqs yansssfqrh irkrrstdfe fdphsnfyhf trplikpqca 481 aygkaldlsl nsiffigpnq fenlpdiacl nlsansnaqv lsgtefsaip hvkyldltnn 541 rldfdnasal telsdlevld lsynshyfri agvthhlefi qnftnlkvin lshnniytlt 601 dkynlesksl velvfsgnrl dilwndddnr yisifkglkn ltrldlslnr lkhipneafl 661 nlpasltelh indnmlkffn wtllqqfprl elldlrgnkl lfltdslsdf tsslrtllls 721 hnrishlpsg flsevsslkh ldlssnllkt inksaletkt ttklsmlelh gnpfectcdi 781 gdfrrwmdeh lnvkiprlvd vicaspgdqr gksivslelt tcvsdvtavi lffftffitt 841 mvmlaalahh lfywdvwfiy nvclakvkgy rslstsqtfy dayisydtkd asvtdwvine 901 lryhleesrd knvllcleer dwdpglaiid nlmqsingsk ktvfvltkky akswnfktaf 961 ylalqrlmde nmdviifill epvlqhsgyl rlrgrickss ilqwpdnpka eglfwqtlrn 1021 vvltendsry nnmyvdsikq y.

[0071] Thus the variants comprise homologous TLR8 amino acid sequences having one or more deletions, additions or substitutions as long as the requisite level of sequence identity across the full length of TLR8 is achieved. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altschul et al. [1990] J. Mol. Biol. 215:403-410; Henikoff et al. [1989] Proc. Natl. Acad. Sci. USA 89:10915; Karin et al. [1993] Proc. Natl. Acad. Sci USA 90:5873; and Higgins et al. [1988] Gene 73:237-244). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Databases may also be searched using FASTA (Pearson et al. [1988] Proc. Natl. Acad. Sci. USA 85:2444-2448).

[0072] In some embodiments, the TLR8 transgenic animal expresses differential levels of human TLR8. In some embodiments, the TLR8 transgenic animal expresses high levels of human TLR8. In some embodiments, the high levels of human TLR8 expression is the result of multiple copy number, the site of integration of the nucleotide sequence encoding human TLR8, and/or the promoter and/or regulatory region operably linked to the nucleotide sequence encoding human TLR8. In some embodiments, the expression level is between a relative CT value of 10 and 10,000, for example about any of between 100-500, 200-400, or 1500-1700. In some embodiments, the expression level is at least about a relative CT value of any one of 10, 100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000. Relative CT values can be evaluated, for example, by obtaining threshold cycle (CT) values for each gene of interest and normalizing to a housekeeping gene using the formula: relative CT=1.8.sup.(Avg CT Housekeeping Gene-CT Gene of Interest)*100,000. In some embodiments, the gene expression is evaluated by the average relative CT. In some embodiments, the Avg CT housekeeping gene is the mean CT of triplicate housekeeping gene runs and/or Avg CT Gene of Interest is the mean CT of duplicate runs of the gene of interest. In some embodiments, the house keeping gene is ubiquitin, myosin, hsp90, and/or actin.

[0073] In some embodiments, the expression level is about the same or greater than the expression level of human TLR8 in TLR8TGCL12. In some embodiments, the expression level is about the same or greater than the expression level of human TLR8 in TLR8TGCL6. In some embodiments, the expression level is about the same or greater than the expression level of human TLR8 in TLR8TGCL23. In some embodiments, the expression level is about the same or greater than the expression of human TLR8 in TLR8TGCL8. In some embodiments, the human TLR8 is expressed in a similar expression pattern in the transgenic animal as human TLR8 is expressed in humans. In some embodiments, the levels of expression of human TLR8 in the transgenic animal is similar to the level of expression of human TLR8 in humans. In some embodiments, the levels of expression of human TLR8 in the transgenic animal is about any of 1, 2, 3, 4, 5-fold the level of expression of human TLR8 in humans. In some embodiments, the levels of expression of human TLR8 in the transgenic animal is similar to the level of expression of mouse TLR8 in mice.

[0074] In some embodiments, the TLR8 transgenic animal has reduced survival (e.g., life span), for example, compared to a wild-type animal. For example, in some embodiments, the transgenic animal survives less than about any of 150, 140, 130, 120, 110, 100, 90, 80, 70, 60 or 50 days. For example, provided herein are transgenic animals comprising a nucleotide sequence encoding human TLR8, wherein the human TLR8 is expressed in the transgenic animal, wherein the TLR8 transgenic animal survives less than about 150 days. In some embodiments, the TLR8 transgenic animal has survival (e.g., life span) similar or substantially the same as compared to a wild-type animal.

[0075] The human TLR8 gene may be of natural or artificial origin. It may be genomic DNA (gDNA), complementary DNA (cDNA), hybrid sequences or synthetic or semi-synthetic sequences. To be expressed, the human TLR8 gene should be operably linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions may be used to increase, decrease, or regulate the expression of a gene or to designate the expression of a gene to certain tissues or to certain stages of development. In some embodiments, the regulatory region increases expression of the gene or increases expression of the gene in neutrophils. The regulatory regions may comprise a promoter region for functional transcription, as well as a region situated 3' of the gene of interest, and which specifies a signal for termination of transcription and a polyadenylation site. All these elements constitute an expression cassette.

[0076] Promoters that may be used include both constitutive promoters and regulated (inducible) promoters. The promoter may be naturally responsible for the expression of the nucleic acid. It may also be from a heterologous source. In particular, it may be promoter sequences of eukaryotic or viral genes. For example, it may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, it may be promoter sequences derived from the genome of a virus, including the adenovirus used. The promoters of the ElA, MLP, HCMV and RSV genes and the like may be used. In addition, the promoter may be modified by addition of activating or regulatory sequences, or sequences allowing a tissue-specific or predominant expression. The promoter need not be a naturally occurring promoter. The promoter may be an inducible promoter. In some embodiments, the human TLR8 gene is operably linked to the human TLR8 promoter. In some embodiments, the human TLR8 gene is operably linked to the human TLR8 promoter and human TLR8 regulatory region.

[0077] Tetracycline-inducible systems can be used, as described in Hickman-Davis et al. (Pediatric Respiratory Reviews 2006 7: 49). Two independent transgenic mouse lines are generated: (1) transactivator mice, in which a tetracycline-controlled transactivator is expressed under the control of a tissue-specific promoter and (2) responder mice, in which expression of the DNA of interest is under the control of a tetracycline-dependent promoter (a minimal RNA polymerase II promoter fused to tet operator sequences). The breeding of these two strains of mice generates a double-transgenic mouse that responds to tetracycline or its derivatives (doxycycline) to control expression of the transgene. There are two possible mirror image tetracycline-inducible systems. In the first system, the absence of doxycycline allows for transcription of the transgene and addition of tetracycline or its derivatives causes transcriptional downregulation (tet-OFF). In the second system transcription of the transgene occurs in the presence of doxycycline and therefore removal of the activator results in transcriptional downregulation (tet-ON).

[0078] Additional useful promoters are the ubiquitous promoters HPRT, vimentin, actin, and tubulin; the intermediate filament promoters desmin, neurofilaments, keratin, and GFAP; the therapeutic gene promoters MDR, CFTR, and factor VIII; promoters which are preferentially activated in dividing cells; cytomegalovirus immediate-early; retroviral LTR, metallothionein; SV-40; Ela and MLP promoters. Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, U.S. Pat. Nos. 5,168,062 and 5,385,839, the contents of which are incorporated herein by reference.

[0079] In some embodiments, a cre/loxP recombinase system is utilized for generation of the transgenic animals. For example, the Cre/loxP recombinase systems described in Hickman-Davis et al. (Pediatric Respiratory Reviews 2006 7: 49) can be used. For this system, the generation of two independent mouse lines requires: (1) mice that contain the target gene or gene segment flanked by two 34 bp, asymmetric nucleotide sequences (loxP) sites in the same orientation ('floxed' sequence) and (2) mice that contain a fusion transgene expressing the Cre recombinase of the P1 bacteriophage. The Cre recombinase promotes recombination by recognition of the loxP sites, and when these two mouse strains are crossed, the floxed gene is deleted and a null mutation is created. Cre/loxP recombinase system is also useful in the targeted mutagenesis of embryonic stem cells in vitro to create `clean` mutations that lack a selection cassette that might interfere with gene regulation, in which pluripotent stem cells containing the gene of interest and only one loxP site with foreign sequence are generated for use in the creation of a transgenic mouse. Several methods have been demonstrated for controlling Cre expression including the creation of fusion proteins containing Cre and having specific ligand-binding domains (i.e., Cre is expressed only in the presence of a specific ligand), as well as a tetracycline-inducible Cre system.

[0080] In some embodiments, the transgenic animal is a mouse or a rat. In some embodiments, the transgenic animal is a mouse. Mouse strains useful for generating transgenic mice include, but are not limited to CD-1.RTM. Nude mice, CD-1 mice, NU/NU mice, BALB/C Nude mice, BALB/C mice, NIH-III mice, SCID.TM. mice, outbred SCID.TM. mice, SCID Beige mice, C3H mice, C57BL/6 mice, DBA/2 mice, FVB mice, CB17 mice, 129 mice, SJL mice, B6C3F1 mice, BDF1 mice, CDF1 mice, CB6F1 mice, CF-1 mice, Swiss Webster mice, SKH1 mice, PGP mice, and B6SJL mice.

[0081] The transgenic animals are produced by introducing one or more trangenes into the germline of the transgenic animal. The methods enabling the introduction of DNA into cells are generally available and well-known in the art and different methods of introducing transgenes could be used. See, for example, Hogan et al. Manipulating the Mouse Embryo: A Laboratory Manual Cold Spring Harbor Laboratory, 2.sup.nd edition, Cold Spring Harbor Laboratory (1994) and U.S. Pat. Nos. 5,602,299; 5,175,384; 6,066,778 and 6,037,521, which are incorporated herein in their entirety. Technology used in developing transgenic animals include pronuclear microinjection (Gordon, J. W., PNAS 77, 7380-7384 (1980) and U.S. Pat. No. 4,873,191), homologous recombination (targeted transgenesis by transferring embryonic stem cells into blastocysts; Thompson et al., Cell 56:313-321 (1989)), RNA interference (RNAi) for silencing of specific gene function; retrovirus gene transfer into germ lines (Van der Putten et al., Proc. Nat. Acad. Sci. 82:6148-6152 (1985)); electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803-1814 (1983)); and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989)).

[0082] Generally, the zygote is the best target for microinjection. In mice, for example, the male pronucleus reaches the size of approximately 20 .mu.m in diameter, which allows reproducible injection of 1-2 pL of DNA solution. The use of zygotes as a target for gene transfer has a major advantage. In most cases, the injected DNA will be incorporated into the host gene before the first cleavage. Consequently, nearly all cells of the transgenic non-human animal will carry the incorporated transgene. Generally, this will also result in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is one method for incorporating transgenes in practicing the invention. The pronuclear microinjection method of producing a transgenic animal results in the introduction of linear DNA sequences into the chromosomes of the fertilized eggs. Bacterial Artificial Chromosome (BAC) containing the gene of interest or an alternative plasmid construct containing the gene of interest is injected into pronuclei (i.e., fertilized eggs at a pronuclear state). The manipulated pronuclei are subsequently injected into the uterus of a pseudopregnant female. Mice generated can have one or multiple copies of the transgene, which can be assayed by southern blot technology.

[0083] The transgenic animals can also be generated by introduction of the targeting vectors into embryonal stem (ES) cells. ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans et al., Nature 292:154-156 (1981); Bradley et al., Nature 309:255-258 (1984); Gossler et al., PNAS 83:9065-9069 (1986); and Robertson et al., Nature 322:445-448 (1986)). Transgenes can be efficiently introduced into the ES cells by DNA transfection using a variety of methods known to the art including electroporation, calcium phosphate co-precipitation, protoplast or spheroplast fusion, lipofection and DEAE-dextran-mediated transfection. Transgenes can also be introduced into ES cells by retrovirus-mediated transduction or by micro-injection. Such transfected ES cells can thereafter colonize an embryo following their introduction into the blastocoel of a blastocyst-stage embryo and contribute to the germ line of the resulting chimeric animal (reviewed in Jaenisch, Science 240:1468-1474 (1988)). Prior to the introduction of transfected ES cells into the blastocoel, the transfected ES cells can be subjected to various selection protocols to enrich for ES cells that have integrated the transgene if the transgene provides a means for such selection. Alternatively, PCR can be used to screen for ES cells that have integrated the transgene. This technique obviates the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel.

[0084] Retroviral infection can also be used to introduce a transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, blastomeres may be targets for retroviral infection. Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida. The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene. Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells. Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoel. Most of the founder animals will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Furthermore, the founder animal may contain retroviral insertions of the transgene at a variety of positions in the genome; these generally segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo.

[0085] Viral vectors may be used to produce a transgenic animal. Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors which are used lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles.

[0086] The retroviruses are integrating viruses which infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). The construction of recombinant retroviral vectors has been described: see, in particular, EP 453242, EP178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, etc. In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus"), MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Defective retroviral vectors are disclosed in WO95/02697.

[0087] In general, in order to construct recombinant retroviruses containing a nucleic acid sequence, a plasmid is constructed which contains the LTRs, the encapsidation sequence and the coding sequence. This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes. Such packaging cell lines have been described in the prior art, in particular the cell line 17 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO90/02806) and the GP+envAm-12 cell line (WO89/07150). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene. Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art. Additional means of using retroviruses or retroviral vectors to create transgenic animals known to the art involve the micro-injection of retroviral particles or mitomycin C-treated cells producing retrovirus into the perivitelline space of fertilized eggs or early embryos (WO 90/08832 (1990); Haskell and Bowen, Mol. Reprod. Dev. 40:386 (1995)).

[0088] Once the founder animals are produced, they can be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic mice to produce mice homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the transgene and the physiological effects of expression.

Methods of Screening for and/or Identifying TLR8 Modulators

[0089] Provided herein is a method of screening for and/or identifying TLR8 modulators using the transgenic animals described herein or cells therefrom. In some embodiments, the cells are primary cells, for example in cell culture, obtained or prepared from the transgenic animals described herein, which comprise a nucleotide sequence encoding human TLR8. In some embodiments, the cells are of a stable cell line derived from primary cells of the transgenic animals. In some embodiments, the cells are an early passage of the primary cells. Provided herein are also methods of screening for, and/or identifying, TLR8 modulators, the methods comprising: administering a candidate agent to the transgenic animal or providing a candidate agent to cell(s) and/or cell culture obtained and/or derived from the transgenic animal described herein; and determining the effect of the candidate agent on the transgenic animal or cell culture (e.g., as compared to an untreated or mock treated control). Further, provided are methods of screening for, or identifying, TLR8 modulators, the method comprising: providing a candidate agent to a cell culture, wherein cells of the cell culture are obtained and/or derived from the transgenic animals described herein which comprise a nucleotide sequence encoding human TLR8; and determining the effect of the candidate agent on the cell culture (e.g., as compared to an untreated or mock treated control). In some embodiments, the cells are from a transgenic animal. In some embodiments, the cells are from a transgenic mouse. In some embodiments, the nucleotide sequence encoding human TLR8 results in inflammation or autoimmune diseases in one or more organs as compared with a control non-transgenic animal and/or elevated levels of cytokine production as compared with a control non-transgenic animal. In some embodiments the effect is a TLR8-mediated response, evidenced by a change in TLR8-mediated cytokine production, cell proliferation, and/or cell surface marker expression. A TLR8-mediated response is one that is stimulable by a TLR7/8 agonist such as R848 and CL075 or a TLR8 agonist such as a TLR8 ligand stabilized immunomodulatory RNA (e.g., SEQ ID NO:3). TLR8-mediated responses are assessed by measuring expression of a cytokine or a cell surface marker. Suitable cytokines are selected from but not limited to TNF-.alpha., IFN-.alpha., IFN-.beta., IFN-.gamma., IL-1.alpha., IL-1.beta., IL-6, IL-8, IL-10, IL-12, IL-23, IP-10, MIP-1, and MCP-1. Suitable cell surface molecules are selected from but not limited CD40, CD80, CD86, ICAM-1, ICAM-2, ICAM-3, and CCR7. In some embodiments, the cytokine comprises one or more of the group consisting of TNF, IL-12, IL-6, MIP-1.alpha., IFN.gamma., IP-10, IL-1.alpha., and IL-1.beta..

[0090] In some embodiments, the candidate agent is selected and/or identified if the candidate agent modulates a TLR8-mediated immune response. In some embodiments, the immune response is evidenced by level or extent of TLR8-mediated inflammation. In some embodiments, the immune response is evidenced by level or extent of TLR8-mediated cytokine production, proliferation, marker gene production, and/or cell surface markers. In some embodiments, the modulation of TLR8-mediated immune response is inhibition of a TLR8-mediated immune response as compared to a control. In some embodiments, the level or extent of TLR8-mediated inflammation in the transgenic animal is reduced upon administration or providing the candidate agent compared to the control. In some embodiments, levels or extent of TLR8-mediated cytokine production, proliferation, marker gene production, and/or cell surface markers in the transgenic animal and/or cell(s) and/or cell culture obtained and/or derived from the transgenic animal described herein is reduced upon administration or providing the candidate agent compared to the control. In some embodiments, the modulation of a TLR8-mediated immune response is stimulation of a TLR8-mediated immune response as compared to a control. In some embodiments, the level or extent of TLR8-mediated cytokine production, proliferation, marker gene production, and/or cell surface markers in the transgenic animal and/or cell(s) and/or cell culture obtained and/or derived from the transgenic animal described herein is increased upon administration or providing the candidate agent compared to the control.

[0091] The control may be cells and/or animals, which do not express human TLR8. In some embodiments, the control may be untreated or mock-treated transgenic cells and/or animals, that express human TLR8. In some embodiments, the effect of the TLR8 modulator on modulating immune responses is compared to its effect in a non-transgenic animal and/or cell(s) and/or a cell culture obtained and/or derived from a non-transgenic animal. In some embodiments, the effect of the TLR8 modulator on modulating immune responses is compared to the effect of the TLR8 modulator in an animal and/or cell(s) and/or a cell culture obtained and/or derived from the transgenic animal described herein that do not express human TLR8 or express a different level of human TLR8. In some embodiments, the effect of the TLR8 modulator on modulating immune responses is compared to the effect of a known TLR8 modulator in a TLR8 transgenic animal and/or cell(s) and/or a cell culture obtained and/or derived from the transgenic animal described herein. In some embodiments, the effect of the TLR8 modulator on modulating immune responses is compared to the effect of an agent which does not affect human TLR8 in a TLR8 transgenic animal and/or cell(s) and/or a cell culture obtained and/or derived from the transgenic animal described herein.

[0092] In some embodiments, the TLR8 transgenic animal is a chimeric TLR8 transgenic animal. In some embodiments, the TLR8 transgenic animal is a TLR8 transgenic animal with germ cells and somatic cells containing a nucleotide sequence encoding human TLR8. In some embodiments, the nucleotide sequence encoding human TLR8 is stably integrated into the genome of the TLR8 transgenic animal. In some embodiments, the nucleotide sequence encoding human TLR8 is extrachromosomal. In some embodiments, the extrachromosomal nucleotide sequence encoding human TLR8 is provided as a minichromosome, yeast artificial chromosome, or bacterial artificial chromosome.

[0093] For example, provided herein are methods of screening for and/or identifying TLR8 modulators, the method comprising administering a candidate agent to a transgenic animal having a genome comprising a stably integrated transgene encoding human TLR8; wherein the transgene results in inflammation in one or more organs (e.g., pancreas, kidney, liver, joints, reproductive tissue, etc.) or an autoimmune disease (e.g., pancreatitis, nephritis, hepatitis, rheumatoid arthritis, diabetes, diabetes-related disorder, reproductive disease, etc.) as compared with a control non-transgenic animal (or elevated levels of cytokine production as compared with a control non-transgenic animal); and determining the effect of the candidate agent on the inflammation or the autoimmune disease of the transgenic animal. Also, for example, provided herein are methods of screening for and/or identifying TLR8 modulators, the method comprising administering a candidate agent to a transgenic animal having an extrachromosomal nucleotide sequence comprising a transgene encoding human TLR8; wherein the transgene results in inflammation in one or more organs (e.g., pancreas, kidney, liver, joints, reproductive tissue, etc.) or an autoimmune disease (e.g., pancreatitis, nephritis, hepatitis, rheumatoid arthritis, diabetes, diabetes-related disorder, reproductive disease, etc.) as compared with a control non-transgenic animal (or elevated levels of cytokine production as compared with a control non-transgenic animal); and determining the effect of the candidate agent on the inflammation or the autoimmune disease of the transgenic animal.

[0094] Further, for example, provided herein are methods of screening for and/or identifying TLR8 modulators, the methods comprising incubating a transgenic animal cell culture with a candidate agent, the transgenic animal cell culture being derived from a parent transgenic animal, cells of the culture comprising a stably integrated transgene encoding human TLR8; and determining the effect of the candidate agent on the cell culture.

[0095] Provided herein are also methods of screening for and/or identifying TLR8 modulators, the methods comprising: incubating a transgenic animal cell culture with a candidate agent, the transgenic animal cell culture being derived from a parent transgenic animal, cells of the culture comprising an extrachromosomal nucleotide sequence comprising a transgene encoding human TLR8; and determining the effect of the candidate agent on the cell culture.

[0096] Further provided herein are methods of screening for and/or identifying TLR8 modulators, the methods comprising incubating a transgenic animal cell culture with a candidate agent, the transgenic animal cell culture being derived from a parent transgenic animal, cells of the culture comprising a stably integrated transgene encoding human TLR8; wherein the transgene results in inflammation in one or more organs (e.g., pancreas, kidney, liver, joints, reproductive tissue, etc.) or an autoimmune disease (e.g., pancreatitis, nephritis, hepatitis, rheumatoid arthritis, diabetes, diabetes-related disorder, reproductive disease, etc.) as compared with a control non-transgenic animal (or elevated levels of cytokine production as compared with a control non-transgenic animal cell culture); and determining the effect of the candidate agent on the cell culture.

[0097] Provided herein is a method of screening for and/or identifying TLR8 modulator, the method comprising incubating a transgenic animal cell culture with a candidate agent, the transgenic animal cell culture being derived from a parent transgenic animal, cells of the culture comprising an extrachromosomal nucleotide sequence comprising a transgene encoding human TLR8; wherein the transgene results in inflammation in one or more organs (e.g., pancreas, kidney, liver, joints, reproductive tissue, etc.) or an autoimmune disease (e.g., pancreatitis, nephritis, hepatitis, rheumatoid arthritis, diabetes, diabetes-related disorder, reproductive disease, etc.) in one or more organs (e.g., pancreas, kidney, liver, joints, reproductive tissue, etc.) as compared with a control non-transgenic animal (or elevated levels of cytokine production as compared with a control non-transgenic animal cell culture); and determining the effect of the candidate agent on the cell culture.

[0098] In some embodiments, the TLR8 modulator is a TLR8 agonist or antagonist. In some embodiments, the TLR8 modulator is an anti-TLR8 antibody. In some embodiments, the TLR8 modulator is a TLR8 agonist or antagonist antibody. In some embodiments, the TLR8 modulator comprises a polynucleotide. In some embodiments, the polynucleotide comprises a TLR8 immunoregulatory sequence (IRS). In some embodiments, the polynucleotide comprising a TLR8 IRS further is not capable of modulating TLR7 and/or TLR9 and/or does not comprise (i.e., lacks) a TLR7 and/or TLR9 IRS sequence. In some embodiments, the polynucleotide comprising a TLR8 IRS further is capable of modulating TLR7 and/or TLR9 and/or comprises a TLR7 and/or TLR9 IRS sequence. In some embodiments, the TLR8 modulator does not comprise (i.e., lacks) an immunostimulatory sequence. For example, in some embodiments, the TLR8 modulator does not comprise (i.e., lacks) a CG sequence or TCG sequence wherein the C is unmethylated. In some embodiments, the TLR8 modulator is not (i.e., excludes) an antisense oligonucleotide and/or does not operate by a RNAi pathway. In some embodiments, the TLR8 modulator is not (i.e., excludes) a microRNA and/or siRNA. In some embodiments, the TLR8 modulator is an antisense oligonucleotide. In some embodiments, the TLR8 modulator is a small molecule.

[0099] In some embodiments, the effect of the TLR8 modulator on modulating immune responses is determined by measuring TLR8-mediated cytokine production, proliferation, marker gene production, and/or cell surface markers. In some embodiments, the effect on modulating immune responses is inhibition of TLR8. Inhibition of a TLR response, e.g., a TLR8 response, includes, but is not limited to, inhibition at the receptor site, e.g., by preventing or blocking effective ligand-receptor binding, and inhibition of the downstream signal pathway, e.g., after effective ligand-receptor binding.

[0100] The concentration of the candidate agent being assayed by the above methods may range, for example, from about 0.001 .mu.M to about 100 .mu.M, although in some embodiments the assay may be performed with a test compound present in concentrations outside this range. In some embodiments, the concentration of the candidate agent is 0.001 .mu.M, 0.01 .mu.M, 0.1 .mu.M or 1.0 .mu.M or greater. In some embodiments, the concentration of the candiate agent is 100 .mu.M, 10 .mu.M, 1.0 .mu.M or 0.1 .mu.M or less. The cell culture may be incubated with the test compound, for example, from about 10 minutes to about 24 hours, although in some cases the incubation period may be outside this range. The density of cells incubated with the compound to be tested may be, for example, from about 1.times.10.sup.4 to about 1.times.10.sup.7 cells/ml, although in some embodiments the assay may be performed using a cell culture having a cell density outside this range.

[0101] In some embodiments, cytokine levels are determined using a commercially available ELISA assay. In other embodiments, cytokine levels are determined using such techniques as, for example, antibody detection and quantitation (e.g., flow cytometry, western blotting, immunohisto/cytochemistry, proteome array assays), and bioassays (e.g., L929 cytotoxicity assay where the amount of cell death is directly proportional to the amount of TNF-.alpha. in the sample). See, e.g., Current Protocols in Immunology, John Wiley and Sons, Inc. (2001).

[0102] Many different cytokines and/or markers can be assayed in the methods described above. Suitable measurable cytokines include, but are not limited to one or more of TNF-.alpha., IFN-.alpha., IFN-.beta., IFN-.gamma., IL-1.alpha., IL-1.beta., IL-6, IL-8, IL-10, IL-12, IL-23, IP-10, MIP-1, and MCP-1. In some preferred embodiments, the cytokine is selected from the group consisting of TNF.alpha., IL-12, IL-6, IFN.gamma., IP-10, IL-1.alpha., and IL-1.beta.. Suitable measurable cell surface markers include co-stimulatory markers (e.g., CD40, CD80, CD86), intercellular adhesion molecules (e.g., ICAM-1, ICAM-2, or ICAM-3), and maturation markers such as, for example, CCR7.

[0103] Also provided by the present disclosure are kits comprising the transgenic mice or cells derived therefrom, and instructions for use of the mice to screen for and/or to identify a TLR8 modulators. In some embodiments, the kits further comprise a TLR8 agonist. In some embodiments, the kits further comprise a TLR8 antagonist.

Candidate Agents

[0104] Suitable candidate agents to screen for TLR8 modulatory activity include, but are not limited to, polynucleotides (e.g., singe or double stranded nucleic acids), polypeptides (e.g., antibodies or antibody fragments), antisense oligonucleotides, and small molecules (e.g., organic compounds having a molecular weight of less than 2000, 1600, 800, or 400 daltons).

[0105] In some embodiments, the TLR8 modulatory activity is TLR8 agonism (e.g., stimulation). In other embodiments, the TLR8 modulatory activity is TLR8 antagonism (e.g., inhibition). Suitable agonist molecules include polynucleotides comprising a TLR8 immunostimulatory sequence (ISS), agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, and small molecules. Suitable antagonist molecules include polynucleotides comprising a TLR8 immunoregulatory sequence (IRS), antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, and small molecules.

[0106] Antibodies

[0107] In some embodiments, the TLR8 modulator is an anti-TLR8 antibody. In some embodiments, the TLR8 modulator is an antibody, which can reduce and/or inhibit a TLR8-induced cellular activity disclosed herein. In some embodiments, the TLR8 modulator comprises an antibody, which can cause and/or enhance a TLR is an agonist antibody. In some embodiments, the anti-TLR8 antibody is an antagonist antibody.

[0108] In some embodiments, the TLR8 antibody directly binds to TLR8. Alternatively, an antibody may combine with TLR8 indirectly by, for example, (a) forming a complex with another molecule that directly binds to TLR8, or (b) otherwise causing the modification of another compound so that the other compound directly binds to TLR8. In some embodiments, the anti-TLR8 antibody specifically binds TLR8, for example, specifically binds TLR8, but not TLR7 and/or TLR9. In some embodiments, the binding of the receptor is measured in a cell-free assay.

[0109] An antibody "which binds" TLR8 is one that binds TLR8 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent, e.g., in targeting a cell expressing TLR8, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody to its particular TLR8 protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). An antibody that "specifically binds to" or is "specific for" TLR8 polypeptide or an epitope on TLR8 is one that binds to that TLR8 polypeptide or epitope on TLR8 without substantially binding to any other polypeptide or polypeptide epitope.

[0110] In some embodiments the anti-TLR8 antibody is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, an intact antibody, a single chain antibody, or an antibody fragment. In some embodiments, the antibody fragment is Fab, Fab', F(ab')2, Fv fragments; diabodies; or linear antibody. In some embodiments, the anti-TLR8 antibody or fragment thereof is isolated (e.g., identified and separated and/or recovered from a component of its natural environment).

[0111] The anti-TLR8 antibody may be from any of the five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated .alpha., .delta., .epsilon., .gamma., and .mu., respectively. The .gamma. and .alpha. classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0112] In some embodiments, the anti-TLR8 antibody suppresses and/or reduces TLR8-mediated cytokine production. In some embodiments, the anti-TLR8 antibody suppresses and/or reduces extent and/or levels of TLR8-mediated cytokine production as compared to, for example, extent and/or levels of cytokine produced during untreated conditions (e.g., media alone or buffer alone). In some embodiments, the anti-TLR8 antibody causes and/or enhances TLR8-mediated cytokine production. In some embodiments, the anti-TLR8 antibody causes and/or enhances extent and/or levels of TLR8-mediated cytokine production as compared to, for example, extent and/or levels of cytokine produced during untreated conditions (e.g., media alone or buffer alone).

[0113] Small Molecules

[0114] In some embodiments, the TLR8 modulator is a TLR8 modulating chemical compound (e.g., small molecule). In some embodiments, the TLR8 modulator is a TLR8 modulating chemical compound (e.g., small molecule), which can reduce and/or inhibit a TLR8-induced cellular activity disclosed herein. In some embodiments, the TLR8 modulator is a TLR8 modulating chemical compound (e.g., small molecule), which can cause and/or enhance a TLR8-induced cellular activity disclosed herein. A TLR8 modulating (e.g., small molecule) may be a ligand that directly binds to TLR8. Alternatively, a compound may combine with TLR8 indirectly by, for example, (a) forming a complex with another molecule that directly binds to TLR8, or (b) otherwise causing the modification of another compound so that the other compound directly binds to TLR8.

[0115] In some embodiments, the compound suppresses and/or reduces TLR8 induced-cytokine production. In some embodiments, the compound suppresses and/or reduces extent and/or levels of cytokine production as compared to, for example, extent and/or levels of cytokine produced during untreated conditions (e.g., media alone or buffer alone). In some embodiments, the compound causes and/or enhancesTLR8 induced-cytokine production. In some embodiments, the compound causes and/or enhances extent and/or levels of cytokine production as compared to, for example, extent and/or levels of cytokine produced during untreated conditions (e.g., media alone or buffer alone).

[0116] Polynucleotides

[0117] In some embodiments, the TLR8 modulator is a TLR8 modulating polynucleotide (e.g., polynucleotide).

[0118] In some embodiments, the TLR8 modulating polynucleotide is a TLR8 polynucleotide agonist. In some embodiments, the TLR8 polynucleotide agonist causes and/or enhances a TLR8-induced cellular activity. In some embodiments, the TLR8 polynucleotide agonist further comprises a motif, which causes and/or enhances a TLR9-induced cellular activity such as an unmethylated CG or unmethylated TCG. In some embodiments, the TLR8 polynucleotide agonist further comprises a motif, which causes and/or enhances a TLR7-induced cellular activity. In some embodiments, the TLR8 polynucleotide agonist further causes and/or enhances a TLR7 and TLR9 induced cellular activity. In some embodiments, the TLR8 polynucleotide agonist does not cause and/or enhance a TLR7 and/or TLR9 induced cellular activity. In some embodiments, the TLR8 polynucleotide agonist causes and/or enhances TLR8 induced-cytokine production. In some embodiments, the TLR8 polynucleotide agonist causes and/or enhances extent and/or levels of cytokine production as compared to, for example, extent and/or levels of cytokine produced during untreated conditions (e.g., media alone or buffer alone). In some embodiments, the TLR8 polynucleotide agonist causes and/or enhances TLR8 induced-immune response. In some embodiments, the TLR8 polynucleotide agonist causes and/or enhances extent and/or levels of immune response as compared to, for example, extent and/or levels of immune response produced during untreated conditions (e.g., media alone or buffer alone).

[0119] In some embodiments, the TLR8 polynucleotide antagonist comprises a TLR8 IRS. In some embodiments, the TLR8 IRS reduces and/or inhibits a TLR8-induced cellular activity. In some embodiments, the TLR8 IRS further comprises a motif which reduces and/or inhibits a TLR7 and/or TLR9-induced cellular function. In some embodiments, the TLR8 IRS does not comprise (i.e., lacks) a motif which reduces and/or inhibits a TLR7 and/or TLR9-induced cellular function. In some embodiments, the TLR8 IRS does not comprise an immunostimulatory sequences such as unmethylated CG or unmethylated TCG. In some embodiments, the TLR8 modulator is not (i.e., excludes) an antisense oligonucleotide and/or does not operate by a RNAi pathway. In some embodiments, the TLR8 modulator is an antisense oligonucleotide. In some embodiments, the TLR8 modulator is not a microRNA or siRNA. In some embodiments, the TLR8 modulator is a microRNA or siRNA.

[0120] In some embodiments, the TLR8 IRS suppresses and/or reduces TLR8 induced-cytokine production. In some embodiments, the TLR8 IRS suppresses and/or reduces extent and/or levels of cytokine production as compared to, for example, extent and/or levels of cytokine produced during untreated conditions (e.g., media alone or buffer alone). In some embodiments, the TLR8 IRS suppresses and/or reduces TLR8 induced-immune response. In some embodiments, the TLR8 IRS suppresses and/or reduces extent and/or levels of TLR8 induced-immune response as compared to, for example, extent and/or levels of immune response produced during untreated conditions (e.g., media alone or buffer alone).

[0121] In some embodiments, a TLR8 modulating polynucleotide comprises an IRS, as described herein, which inhibits and/or suppresses a measurable immune response as measured in vitro, in vivo, and/or ex vivo. In some embodiments, the TLR8 immune response is an innate TLR8 immune response. In some embodiments, the immune response is an adaptive TLR8 immune response.

General Techniques

[0122] The practice of the present application employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, chemistry, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed., 1987); Handbook of Experimental Immunology (Weir & Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (Miller & Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (Coligan et al., eds., 1991); The Immunoassay Handbook (Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (Hermanson, ed., Academic Press, 1996); and Methods of Immunological Analysis (Masseyeff, Albert, and Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993).

EXAMPLES

[0123] Abbreviations: BAC (bacterial artificial chromosome); BM (bone marrow); CT (threshold cycle); CTRL (control); FACS (fluorescence activated cell sorter); hTLR8Tg (human Toll-like receptor 8 transgenic); IRS (immunoregulatory sequence); KO (knock out); MDC (myeloid dendritic cells); PBMC (peripheral blood mononuclear cells); PDC (plasmacytoid dendritic cells); PN (polynucleotides); TLR (Toll-like receptor); and WT (wild type).

Example 1

TLR8 Expression

[0124] TLR8 expression was analyzed in human cellular subsets (FIG. 1). Plasmacytoid dendritic cells (PDC), monocytes, myeloid dendritic cells (MDC), CD4+ T-cells, CD8+ T-cells, and neutrophils were purified by means of magnetic beads (Miltenyi Biotech) according to manufacturer's instructions. RNA was purified with a micro RNA KIT (Qiagen) according to manufacturer's instructions. cDNA from RNA was generated with SuperScript First-Strand Synthesis System (Invitrogen). Threshold cycle (CT) values for each gene were normalized to the housekeeping gene Ubiquitin using the formula: relative CT=1.8.sup.(Avg CT Ubi-CT Gene)*100,000 where Avg CT Ubi is the mean CT of triplicate housekeeping gene runs, Avg CT Gene is the mean CT of duplicate runs of the gene of interest, and 100,000 is arbitrarily chosen as a factor to bring all values above one. Neutrophils showed the highest level of expression. TLR8 was also expressed in monocytes, mDCs and CD4+ T-cells (FIG. 1).

Example 2

Activity of TLR7 and TLR8 RNA Polynucleotides (PN) in Human and Mouse Cells

[0125] PN-based TLR8 ligand stabilized immunomodulatory RNA (5'-M2UGCUGCUUGUG-/glycerol/-GUGUUCGUCGUM2-5' (M2=C6-linker); SEQ ID NO:3) and PN-based TLR7 ligand stabilized immunomodulatory RNA (5'-URCURCUUCUR-/glycerol/-RUCUUCRUCRU-5' (R=7-deazaguanosine); SEQ ID NO:2) were previously identified (Lan et al., PNAS 104:13750-13755, 2007). The effect of these TLR7- and TLR8-stimulating RNA PN was evaluated by measuring production of IL-6 and TNF-.alpha. in human peripheral blood mononuclear cells (PBMCs) and human monocytes (FIG. 2). 5.times.10.sup.5 PBMCs or 2.times.10.sup.5 monocytes from healthy human donors were stimulated with 20 .mu.g/mL, 100 .mu.g/mL, or 200 .mu.g/mL of either TLR8 agonist or TLR7 agonist as indicated in FIG. 2. Twenty-four hours later supernatants were assessed for presence of inflammatory cytokines IL-6 and TNF-.alpha. by standard ELISA procedure. TLR7 and TLR8 RNA PN stimulated production of IL-6 and TNF-.alpha. in PBMCs and, to a lesser extent, in monocytes (FIG. 2).

[0126] The effect of TLR7 and TLR8-stimulating RNA PN were also evaluated in human pDCs by measuring production of IL-6, TNF-.alpha., and IFN-.alpha.. As shown in FIG. 1, human pDCs were negative for the expression of TLR8. 1.times.10.sup.5 pDCs from healthy human donors were stimulated with 100 .mu.g/mL of either TLR8 agonist or TLR7 agonist (Lan et al., PNAS 104:13750-13755, 2007) or medium alone as indicated in FIG. 3. Twenty-four hours later supernatants were assessed for presence of inflammatory cytokines IL-6, TNF-.alpha. and IFN-.alpha. by standard ELISA procedure. The TLR8 agonist did not stimulate the pDCs proving its specificity for TLR8 (FIG. 3)

[0127] The effect of TLR7 and TLR8-stimulating RNA PNs was further evaluated in mouse cells by measuring IL-12 and TNF-.alpha. production (FIG. 4). Mouse splenocytes and PBMCs were prepared from 129 wt mice and TLR7KO mice, and 5.times.10.sup.5 cells were stimulated with 100 .mu.g/ml of either TLR8 or TLR7 agonist as indicated in FIG. 4. IL-12 and TNF-.alpha. were measured by ELISA using standard techniques. The TLR8 agonist did not stimulate mouse splenocytes and PBMCs (FIG. 4).

Example 3

Generation of Human TLR8 (TLR8) Transgenic Mice

[0128] Transgenic TLR8 mice were generated using BAC/ES technologies (Sparwasser et al., Immunology 121:308-313, 2007). Briefly, the human BAC carrying TLR8 gene was obtained from the RPCIB-753 BAC library. The human BAC contains both human TLR7 and TLR8 genes in a cluster. The BAC was modified to knock out TLR7 gene to retain the chromosomal region at the 5'UTR of mouse TLR8 that likely contains the promoter. Human TLR7 gene was knocked out by inserting a neomycin cassette (FRT-PGK-gb2-neo-FRT). The FRT-PGK-gb2-neo-FRT template encoded the neomycin/kanamycin resistance gene, which combined a prokaryotic promoter (gb2) for expression of kanamycin resistance in E. coli with a eukaryotic promoter (murine phosphoglucokinase gene (PGK)) for expression of neomycin resistance in mammalian cells. A synthetic polyadenylation signal inhibited the kanamycin/neomycin expression. The cassette was flanked by FRT sites for later excision by Flp-recombinase. The appropriate modification was introduced into the BAC using bacterial recombination procedures. After purification of the modified BAC, its quality was checked using pulse-field gel electrophoresis and Southern blot analysis. Important regions of the gene of interest (such as exons or proximal promoter), as well as the introduced modification were confirmed by sequencing. The sequence of construct BAC_RP11-1137P1_TLR7-KO: is provided as SEQ ID NO:1.

[0129] The modified BAC construct, BAC_RP11-1137P1_TLR7-KO (FIG. 5), was transfected into an ES cell line, C57BL/6NTac ES cell line. The C57BL/6NTac ES cell line was grown on a mitotically inactivated feeder layer comprised of mouse embryonic fibroblasts (MEF) in DMEM High Glucose medium containing 20% FBS (PAN) and 1200 U/mL Leukemia Inhibitory Factor (Millipore ESG 1107). For manipulation 1.5.times.10.sup.5 ES cells were plated on 3.5 cm dishes and transfected one day later with purified BAC_RP11-1137P1_TLR7-KO DNA and Lipofectamine.TM. 2000 Reagent from Invitrogen (Catalog No. 11668-027) according to manufacturer's protocol. From day 2 onwards the medium was replaced daily with medium containing 200 .mu.g/mL G418 (Geneticin; Invitrogen; Catalog No. 10131-019). Seven days later single clones were isolated, expanded and analyzed by Southern Blot.

[0130] The ES clones generated were validated for presence of the TLR8 gene integrated on the genome by using southern blot techniques. Five ES clones were chosen: Clone 8 and clone 6 with about 1-2 copies of TLR8 integrated in the genome, Clone 12 with 2-4 copies of TLR8 integrated in the genome, clone 16 with about 5 copies of TLR8 integrated in the genome and Clone 23 with about 15 copies of TLR8 integrated in the genome, respectively.

[0131] ES cells were injected into the blastocoel of 3.5 day old mouse blastocysts from BALB/c females. Subsequently, the injected embryos were transferred to the uterine horns of appropriately timed pseudopregnant recipient BALB/c females. Embryos gestated for about 18 days and the resulting pups were chimeras, whose tissues have developed from both the ES cells carrying TLR8 gene and the recipient blastocyst cells of BALB/c background. This mix of starter cells was visible in the mouse's coat, which exhibited patches of coat color from the host embryo and patches from the injected ES clone. Chimeras with 50-75% ES contribution (based on fur color) were then put to breed with C57BL/6 animals to pass germline transmission.

Example 4

Analysis of TLR8 Chimeric Mice

[0132] The clones described above were evaluated in more detail. Chimeric ratio was about 30% TLR8 transgene and 70% wild-type. Chimeric mice of four out of five clones (clones 6, 12, 16 and 23) died before germline transmission. Only chimeric mice of clone 8 were able to breed and pass germline transmission. Clones 6, 12, 16, and 23 resulted in chimera lethality.

[0133] To determine TLR8 expression 200 .mu.l of blood was harvested from mice and RNA and cDNA were prepared according to standard procedures. TAQMAN assay was used to evaluate TLR8 gene expression. TLR8 expression correlated with survival time (FIG. 6A-B). The highest expressing clones (6, 12, and 23) caused lethality of the chimeric mice. Only Clone 8 with about 1-2 copies of TLR8 integrated in the genome survived (FIG. 6A-B) and passed germline transmission (TLR8TGCL8 mice). Additionally, increased inflammatory cytokine levels were detected in the serum of TLR8Tg mice (n=15) as compared to control WT C57BL/6 mice (FIG. 6C).

Example 5

Gross and Histopathological Analysis of TLR8 Chimeras with High TLR8 Expression

[0134] To further evaluate the phenotype of the chimeras with high expression of TLR8, biopsy specimens from chimeric mice of clone 12, 23, and 6 were harvested from mice that showed evident sign of distress and needed to be euthanized. Organs were fixed in formalin and embedded in paraffin. Organs from wild type C57BL/6 (CTRL B6) mice were also harvested as controls. Sections were stained with hematoxylin-eosin. Blinded evaluation of the liver, kidney, intestine, lung, brain, heart and pancreas was conducted by a pathologist during the course of the study. Inflammation was scored 1 to 4 as follow: 1=Minimal; 2=Mild; 3=Moderate; 4=Marked. Statistical significance among groups was calculated with a Mann-Whitney U-test with P values comparing chimeric mice to CTRL B6 animals. P values were considered statistically significant at p.ltoreq.0.05.

[0135] As shown in Table 5-1, histopathology of the TLR8 chimera's organs revealed multi-organ inflammation with massive autoimmune pancreatitis. Inflammation was found in liver, kidney, and pancreas of human TLR8 expressing mice. Pancreas histological data revealed abundant lymphocytes and macrophages/neutrophils infiltration, while acinar cells were intact. Kidney histological data revealed interstitial inflammation, glomerular changes with segmental hypercellularity, and pyelitis renal pelvis.

TABLE-US-00002 TABLE 5-1 Histopathology of the TLR8 Chimeric Mice Organs Kidney Liver Pancreas Glomerulo- Kidney Cholangio- ID Name Pancreatitis nephritis Pyelitis hepatitis CL#12 1-1 3 2 0 1 CL#12 2-2 3 3 0 3 CL#23 138800 4 3 3 2 CL#23 3-1 4 -- -- 3 CL#23 4-1 4 3 2 4 CL#23 T4720 4 3 0 2 CL#23 136833 4 3 3 0 CL#23 138782 4 3 2 1 CL#23 138785 4 3 3 2 CL#23 3-2 4 3 3 4 CL#23 5-4 4 3 3 3 CL#23 T4715 4 3 3 1 Average 3.7 2.4 1.8 2.0 SEM 0.2 0.1 0.4 0.3 P value <0.001 <0.001 N.S. <0.001 CTRL B6 #1 0 0 0 0 CTRL B6 #2 0 0 0 0 CTRL B6 #3 0 2 3 0 CTRL B6 #4 0 2 0 0 Average 0 1.0 0.6 0 SEM 0 0.3 0.4 0

Example 6

Pathological Characterization of Chimeric Mice with High TLR8 Expression

[0136] To further analyze the phenotype observed in chimeric mice with high expression of TLR8, pancreatic cytokine production was determined (FIG. 7). Pancreases from TLR8 chimeric mice of Clone 12, 6, or 23 (total number of mice=6) or CTRL WT C57BL/6 mice (total number of mice=6) were harvested and RNA was extracted with fibrous tissue RNA extraction kit (Qiagen) according to manufacturer's instructions. cDNA from RNA was generated with SuperScript First-Strand Synthesis System (Invitrogen). Relative CT values for each gene were normalized to the housekeeping gene Ubiquitin using the formula: Gene expression=1.8.sup.(Avg CT Ubi-CT Gene)*100,000 where Avg CT Ubi is the mean CT of triplicate housekeeping gene runs, Avg CT Gene is the mean CT of duplicate runs of the gene of interest, and 100,000 is arbitrarily chosen as a factor to bring all values above one. Results show a strong up-regulation of a number of inflammatory cytokines such as IFN-gamma, TNF-.alpha., IL12, IP-10, IL-1.alpha., and IL-1.beta. in the chimeric mice compared to the C57BL/6 control mice. (FIG. 7A-F).

[0137] Aberrant recognition of self RNA and DNA has been implicated in the development of pathogenic autoantibodies in human lupus patients and in mouse models of this disease. The presence of nucleic acid-specific autoantibodies in the serum of the TLR8 chimeric mice was assessed using commercially available kits from Alpha Diagnostic. Sera was collected from TLR8 chimeric mice of clone 12 when the mice became moribund. Significantly increased levels of anti-ANA Ig, anti-dsDNA Ig and anti-RNP Ig were detected in the serum from the TLR8 chimeric mice (FIG. 7G-I). Thus, over-expression of hTLR8 is associated with development of anti-self nucleic acid antibodies.

[0138] In addition, myeloid dendritic cells (MDCs) were analyzed in chimeric TLR8TGCL23 and wild type B57BL/WT mice (FIG. 8A-D). Chimeric and wild-type mice were sacrificed and spleens were harvested. Flow cytometric analyses were performed using fluorochrome-conjugated monoclonal antibodies to mouse CD11c, and costimulatory molecules CD80, GITRL, OX40L and CD40 (BD bioscience). These data show that MDCs were highly activated inTLR8TGCL23 chimeric mice (FIG. 8).

Example 7

Analysis of Mice Receiving Bone Marrow from TLR8 Chimeras

[0139] To examine whether epithelial cells or leukocytes are responsible for the phenotype observed in chimeric mice with high levels of TLR8 expression, bone marrow (BM) from TLR8 chimeras of Clone 6, 12 and 23 was transferred to recipient C57/BL6 (SJL) mice and animals were monitored for BM uptake and pathology. Recipient mice (C57/BL6 (SJL)) were irradiated with 900 rad using a cobalt irradiator. Four hours later, 2.times.10.sup.6 BM cells extracted from the femurs of TLR8 chimeras were transferred intravenously. Reconstitution of the BM of the recipient mice with the BM of TLR8 chimeras was verified using flow cytometry on blood samples. 25 mice were obtained of which the BM was reconstituted with 80% of TLR8 BM.

[0140] Mice that were transplanted with TLR8 transgenic mice bone marrow cells (TLR8TG>C57BL/6SJL mice) were monitored daily. Mice died very quickly within 30-60 days after introduction of the TLR8 BM or were euthanized when moribund (FIG. 9). Wild-type mice reconstituted with bone marrow from huTLR8 chimeras developed a spontaneous inflammatory disease remarkably similar to that in the original ES chimeras. In contrast, wild type mice reconstituted with WT BM (C57BL/6>C57BL/6SJL mice) did not show any signs of illness. These data suggest that high level TLR8 expression in the hematopoietic compartment is sufficient to cause the phenotype observe in chimeric TLR8 mice.

TABLE-US-00003 TABLE 7-1 Histopathology of the TLR8TG> C57BL/6SJL Bone Marrow Transplanted Mice* Kidney Kidney Salivary Small Clone BM Pancreas D1 D2 Liver Gland Intestine CL 23 4 1 0 1.5 3 NT CL 23 3.5 0 2 3 NT NT CL23 3.5 0 1 2.5 3.5 NT CL 6 NT 0 0 1.5 2 NT CL 6 4 0 0 2 2.5 NT CL 6 4 0 0 3.5 2 NT CL 12 4 0 0 1.5 3.5 0 CL 12 4 0 0 2 2.5 2.5 CL 12 4 0 0 1.5 3.5 2.5 CL 12 4 0 1 2.5 4 1 CL 12 4 0 0 2.5 3.5 0 CL 12 4 0 1 1.5 3.5 1 CL 12 4 0 0 2 1.5 1 CL 12 4 0 0 2.5 3 0 CL 12 4 0 0 1.5 3 1 CL 12 4 0 0 1.5 2 1 CL 12 4 1 1 2 4 2.5 CL 12 4 0 0 2 1.5 3 CL 12 4 0 0 3 1.5 0 CL 12 4 0 NT 1.5 3.5 1 CL 12 4 0 0 2.5 3.5 3.5 CL 12 4 0 IS 1.5 2 2 CL 12 4 0 NT 2 3.5 2 CL 12 4 0 0 2.5 2.5 2.5 CL 12 3.5 0 0 0 0.5 1 Average 3.9 0.1 0.3 2.1 2.7 1.4 SEM 0.03 0.06 0.11 0.14 0.19 0.25 P value <0.001 <0.001 0.61 <0.001 <0.001 0.23 B6 CTRL 0 0 0 1 0 1 B6 CTRL 0 1 0 1 0 0 B6 CTRL 0 1 0 1 0 1 B6 CTRL 0 1 0.5 1 0 1 Average 0.0 0.8 0.1 1.0 0.0 0.8 SEM 0.00 0.25 0.13 0.00 0.00 0.25 *Gross Pathology: pancreas pancreatitis, kidney D1 glomerulonephritis, kidney D2 pyelitis, liver cholangiohepatitis, salivary gland inflammation, and small intestine inflammation.

[0141] To evaluate the histopathology of TLR8TG>C57BL/6SJL mice, biopsy specimens were harvested from mice that showed evident signs of distress and needed to be euthanized. Organs were fixed in formalin and embedded in paraffin. Organs from C57BL/6SJL (CTRL B6) mice were also harvested as controls. Sections were stained with hematoxylin-eosin. Blinded evaluation of the liver, kidney, intestine, lung, brain, heart and pancreas were conducted by a pathologist. Inflammation was scored 1 to 4 as follow: 1=Minimal; 2=Mild; 3=Moderate; 4=Marked. Statistical significance among groups was calculated with a Mann-Whitney U-test. P values compare chimeric mice to CTRL B6 animals. P values are considered statistically significant at p.ltoreq.0.05. As shown in Table 7-1 histopathology of the TLR8TG>C57BL/6SJL bone marrow transplanted mice revealed multi-organ inflammation with massive autoimmune pancreatitis.

[0142] Flow cytometric analyses of T cells of TLR8TGCL12>C57BL/6SJL and wild-type C57BL/6SJL mice were performed using fluorochrome-conjugated monoclonal antibodies to mouse CD4, CD8, CD44, CD62L (BD bioscience) (FIG. 10A-B). 30-40 days after bone marrow transplantation, TLR8TG>C57BL/6SJL mice (n=19) were sacrificed and blood and spleens were harvested. C57BL/6SJL WT mice (n=9) were used as controls. Dot blot analysis (FIG. 10) and cumulative data (FIG. 11) show that naive cells were CD44 low and CD62L high, effector cells were CD44 high and CD62L high, and effector memory cells were CD44 high and CD62L negative. P values were calculated with a Mann-Whitney U-test and were considered statistically significant at p.ltoreq.0.05. The presence of effector and effector memory cells shows that T cells were highly activated in TLR8TG>C57BL/6SJL mice.

[0143] In addition, flow cytometric analyses of MDC of TLR8TG>C57BL/6SJL and wild-type C57BL/6SJL mice were performed using fluorochrome-conjugated monoclonal antibodies to mouse CD11c, and costimulatory molecules CD80, GITRL, OX40L, PDL-1, and MHC CLASS II (BD bioscience) (FIG. 12). TLR8TG>C57BL/6SJL mice were sacrificed and spleens were harvested. C57BL/6SJL wild-type mice were used as controls.

[0144] These data suggest that high level TLR8 expression in leukocytes and not epithelial cells is responsible for the phenotype observed in chimeric TLR8 mice. In these mice, the bone-marrow originated from the TLR8-transgenic animal while the hosts are wild-type. Therefore, this means that the entire hematopoietic compartment carries the transgene but the tissues (and thus all epithelial cell) are wild-type.

Example 8

Transgenically Expressed TLR8 is Functional

[0145] PN-based TLR8 ligand stabilized immunomodulatory RNA (5'-M2UGCUGCUUGUG-/glycerol/-GUGUUCGUCGUM2-5' (M2=C6-linker); SEQ ID NO:3) and PN-based TLR7 ligand stabilized immunomodulatory RNA (5'-URCURCUUCUR-/glycerol/-RUCUUCRUCRU-5' (R=7-deazaguanosine); SEQ ID NO:2) were previously identified (Lan et al., PNAS 104:13750-13755, 2007). To confirm that human TLR8 expressed from the transgene in TLR8TGCL8 mice is functional and sensitive to stimulation, the effect of TLR8-stimulating RNA PN (SEQ ID NO:3) on cytokine production was assessed. 5.times.10.sup.5 blood cells from TLR8TGCL8 transgenic mice or C57BL/6 WT animals were stimulated with TLR8 agonist at different concentrations as shown in FIG. 13. 24 hours later supernatants were harvested and assayed for mouse IL-12, TNF-.alpha. and IL-6 using standard ELISA procedures. In addition, 5.times.10.sup.5 bone marrow cells from TLR8TGCL8 transgenic mice or C57BL/6 WT animals were stimulated with TLR8 agonist (SEQ ID NO:3) at different concentrations as shown in FIG. 14. Mouse TNF-.alpha. and IL-6 production was assayed in supernatants harvested after 24 hours. Only cells from transgenic animals responded to the TLR8 agonist demonstrating that human TLR8 is functional in these mice (FIGS. 13 and 14).

Example 9

TLR8 Mediated Diseases

[0146] The role of TLR8 in disease and/or disease susceptibility was evaluated by using the TLR8 over-expressing mice and/or bone marrow from the TLR8 over-expressing mice as described below.

[0147] Spontaneous Arthriti.

[0148] hTLR8Tg chimeric mice from clone 12 (n=6) and C57BL/6 CTLR animals (n=6) were euthanized 90 days after the birth. Paws and joints were fixed, sectioned and stained with toluene blue. Representative sections of joints from WT mice (FIG. 15A) and hTLR8Tg mice (FIG. 15B) were examined. All of the hTLR8Tg mice showed signs of swelling in fore limbs and hind limbs. The histological changes, degree of inflammation and cartilage damage were evaluated by a pathologist in a blinded fashion and scored 1 to 5 as followed: 1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe. Two paws and two ankles were evaluated for each animal and the scores were summed to obtain the histological disease score (FIG. 15C).

[0149] Rheumatoid Arthritis

[0150] To investigate whether TLR8 had a role in rheumatoid arthritis, wild-type (C57BL/6) and TLR8 transgenic mice (TLR8TGCL8) were immunized per a published immunization schedule and protocol (Campbell, Eur J Immunol 30:1568-1575, 2000). On day 0 of collagen immunization, collagen (Chicken Type II Collagen from Chondrex; 2 mg/mL) was emulsified with Complete Freund's Adjuvant (CFA from Chondrex; 5 mg/mL concentration of Mycobacterium tuberculosis H37Ra) as follows:

[0151] (i) one volume of CFA was mixed with an equal amount of the collagen solution;

[0152] (ii) mixing was continued until a stable, stiff emulsion resulted;

[0153] (iii) to ascertain the desired stability of the emulsion, 1 drop of emulsion was added into a water-filled beaker (the emulsion was considered stable if it remained in the water as a solid); and

[0154] (iv) 100 .mu.l was injected subcutaneously at the base of the tail.

[0155] A second injection was performed at day 21. Animals were assessed for redness and swelling of the 4 limbs and the cumulative score of each mouse was the sum of the score obtained for each limb. The Clinical Score Guidelines were as follows: 0-Normal; 1-Mild, but definite redness and swelling of the ankle or wrist, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2-Moderate redness and swelling of ankle of wrist; 3-Severe redness and swelling of the entire paw including digits; and 4-Maximally inflamed limb with involvement of multiple joints and a clinical score allocated.

[0156] Animals were sacrificed 80 days after CIA induction. The two front joints of each animal were used to prepare RNA to measure expression of various genes using TAQMAN assays (46 joints per group). The data shown are cumulative of two independent experiments. The TLR8 transgenic mice had a higher cumulative clinical score and a significant increase in the incidence of symptoms with a clinical score 4 or greater over time than wild-type mice (FIG. 16A-B). Based on this data, expression of TLR8 was confirmed to play a role in the development of rheumatoid arthritis. FIG. 16C-E shows significantly elevated expression of hTLR8, F4/80 and TNF in the joints of TLR8 transgenic mice (TLR8TGCL8) as compared to wild-type mice (C57BL/6). In addition, the level of expression of hTLR8 was found to correlate with the clinical score (FIG. 16F). Moreover, expression of F4/80 and TNF correlated with levels of hTLR8 expression (FIG. 16G-H). Likewise, expression of various pro-inflammatory cytokines (e.g., IL-1B, IL1A, IL-6, IL-18, IP-10, MMP-9) was found to correlate with hTLR8 levels (data not shown).

[0157] Diabetes

[0158] To investigate whether TLR8 had a role in diabetes, C57BL/6-SJL WT Mice were transplanted with WT C57BL/6 bone marrow cells or with TLR8TGCL12 bone marrow cells as described Example 7. Mice were monitored daily. Blood was collected from moribund mice, and glucose levels in the blood were assayed. Blood glucose levels in mice reconstituted with TLR8 BM were significantly elevated as shown in FIG. 17. 62% of the mice showed a level equal or exceeding 200 mg/dL and were therefore considered diabetic (See Lee et al., Nutrition Research 26:474-479, 2006; Serrano et al., Proc. Intl. Soc. Mag. Reson. Med. 15 2463, 2007; and Tanquilut et al., J. of Med. Plants Research 3: 1066-1071, 2009, which are incorporated by reference in their entirety).

[0159] Blood Disorders

[0160] Cellular subsets were isolated from spleens of B6.SJL mice transplanted with bone marrow from TLR8TgCL12 mice. Expression of hTLR8 was evaluated by TAQMAN. Cumulative data from at least three independent experiments is shown (n=5-10, mean.+-.SEM). Human TLR8 is expressed in monocytes, neutrophils and dendritic cells of the hTLR8Tg mice (FIG. 18A). The expression pattern huTLR8 in leukocyte subsets from these mice is very similar to its expression in human leukocytes.

[0161] Approximately 5.times.10.sup.5 PBMC from TLR8Tg chimeras or C57BL/6 WT animals were stimulated with a TLR8 agonist and 24 hr later supernatants were harvest and assayed for cytokine levels by ELISA (n=4 mice, mean.+-.SEM). The RNA-based TLR8 agonist ORN-8L (300 .mu.g) induced secretion of IL-12 and TNF by PBMC from hTLR8Tg mice (FIG. 18B-C).

[0162] Mice from TLR8Tg Clone 8 line were injected intravenously with a TLR8 agonist. Serum was collected 6 hr later and assayed for cytokine levels (n=3 mice, mean.+-.SEM). The RNA-based TLR8 agonist ORN8-L (300 .mu.g) or the small molecule-based TLR8 agonist CL075 (5 .mu.g) induced secretion of IL-12 in vivo in hTLR8Tg mice. Thus human TLR8 is functional in these mice

[0163] The number of various white blood cells was assessed in peripheral blood and in spleens of TLR8Tg chimeras or age-matched C57BL/6 controls (FIG. 19A-B). TLR8Tg mice develop lymphopenia (decrease in number of circulating lymphocytes), granulocytosis (increase in number of granulocytes, which are primarily neutrophils) and monocytosis (increase in number of monocytes). As shown in FIG. 19C there is a significant decrease in circulating lymphocytes paralleled by an increase in granulocytes and monocytes in the blood of the TLR8 expressing mice as compared to WT controls.

[0164] Cells from spleens from TLR8Tg clone 12 chimeric mice and from WT mice were stimulated in vitro for 2 hr with PMA (5 ng/ml) and ionomycin (500 ng/ml). The percentage of CD4 and CD8 T cells producing TNF and IFN-gamma was increased in TLR8Tg chimeric mice as compared to CD4 and CD8 T cells from wild type control mice (FIG. 20A-C).

Example 10

Screening for and/or Identifying TLR8 Antagonists and Agonists

[0165] TLR8 antagonists and agonist can be screened for and/or identified using the transgenic mice expressing human TLR8 described above.

[0166] In Vitro Assays

[0167] Human primary monocytes and splenocytes from TLR8TGCL8 mice are used to screen for human TLR8 antagonists and agonists. Cells are stimulated with TLR8-specific ligands and the activation or inhibition of TLR8-related immune responses are measured by standard in vitro assays described herein (e.g., cytokine production, cell proliferation, marker gene expression, and/or cell surface markers).

[0168] In Vivo Assays

[0169] Clone 8 TLR8 transgenic mice either with germline transmission or TLR8 chimeras or without germline transmission are used to screen for TLR8 antagonists and agonists. Furthermore, new chimeras can be generated using TLR8 transgenic mice with high copy number TLR8 ES expressing clones. Mice are injected with either TLR8 agonist or antagonist candidates. TLR8 agonists activate, while TLR8 antagonists inhibit the TLR8-dependent signal transduction pathway and TLR8-associated immune responses. Mice are injected with TLR8 agonist known in the art prior to injection of TLR8 antagonist candidate. Alternatively, TLR8 antagonist candidates can be assessed in spontaneous disease mouse models in the absence of administration of a TLR8 agonist

[0170] Autoimmune mouse models employing the TLR8 transgenic mice of the present disclosure include, for example, models for rheumatoid arthritis, diabetes, pancreatitis, glomerulonephritis, pyelitis, cholangiohepatitis, and reproductive disorders.

Sequence CWU 1

1

71145837DNAArtificial SequenceSequence of construct BAC_RP11-1137P1_TLR7-KO 1gaattcagct gtgaatacgt ctggtcctgt gcttgttttg gttggtaggc tattaattac 60tgcctccatt tcagaacttg ttattgatct attcagggat ttgacttttt cctgatttag 120tcttgggagg gtgtatgtgt ccaggaattt atccatttct tgtagatttt ctagtttatt 180tgagtagagg tgattttagt attctctgat aatagtttgt atttctgtgg gatcagtggt 240aataacccct ttatcatttt gtattgtgtc tatttgattc ttctctcttt tctcctttat 300tagtctggct agtggtctat ctattttgtt aatgttttca aaaaaccagc tcctggattc 360actgattttt tgaagggttt ttcatgcctc tatcttcttt agttctgctc tgattttagt 420tatttcttgt cttctgctag cttttgaatg tttgctctcg cttctctagt tctttttatt 480gtgatgttag ggggtcgatt ttagatctgt ccctgctttc ttttgtgggc atttagtgct 540ataactttcc ctctaaacac tgcgtttgct gtgtcccaga gattctggta tgttgtgtct 600ttgttctcat tggtttcaaa taacttattt atttctgcct taattttgtt atttatccag 660tagtcattca ggagcaggtt gttcagtttc catgtagttg tgcagttttg agtgagtttt 720taatcctgaa ttttaatatg attgcactgt ggtctgatag actgttatgg tttccattgt 780tttgcatttg ctgaggagtg ttttacttcc aattatgtgg tcaattttag aataactgtg 840atgtggtgct gagaagaatg tatattctgt tatttgaggt ggagagttct gtagatgtct 900attagttgtg cttggtccag agctgagttc aagtcctgaa tatccttgtt aattttctgt 960ctcattgatc tgtctaatat tgacagtggg gtgttaaaat ctcccactat tattgtgtgg 1020gagtctaagt ctccttctag gtctctaaga acttgcttta tgaatctagg tgctcctgta 1080ttgggtgcac atatatttag gataattagc tcttcttgtt acattgatcc ctttaccatt 1140atatagtacc cctctttgtc tttttttaat ctttcttggc ttaaaatctg ttttatcaca 1200gactaggatt gcaacccctg ctgggttttt tttctttttt tttttttctt ttttttttag 1260atggcgtttt gctcttgttg cccaggctag agtgcagtgg catgatcttg gctcaccaca 1320acccccacct cccaggagca agcaattctc ctgcctcagc ctcccgagta gctgggatta 1380caggcatgtg ccaccacgcc ttgctaattt tgtattttta gtggagatgt ggtttctcca 1440tgttggtcag gctagtcttg aactcccgac ctcaggtgat cccgctgcct cagcctccca 1500aagtactggg attacaggtg tgagccacct cgcctgacca acacctgttt tttttttttt 1560ttgctttcca tttgcttggt aaatattcct ccatcccttc attttgagcc tatgtgtgtc 1620tttgtacatg agatgcatct cctgaataca gcacactgat gggtcttgac tctttatcca 1680atttgccagt ctgtgtcttt taattggggc atttagccca tttacattta aggtgaatac 1740tgttatgtgt gaatttgatc ctgtcattat gatgcttgct ggttattttg cccattagtt 1800gatgcagttt cttcatagtg tcgacggtct ttacaatttg gtacgttttg cagtggctgg 1860taccagtttt tccttttgat atttagttct tccttcagga gctcttgtaa ggcaggcctg 1920gtggtgacaa aatgtctctc agctttgttt ttctgtaaag gattttattt ctccttcgct 1980tatgaagctt agtttggctg gatatgaaat tctgggttga aaattccttt tttaagaatg 2040ttgaatattg gcctctcttc tggcttgtag ggtttctgcg gagagatctg ctgttagtct 2100gatgagcttc cctttgtggg taacccgacc tttttctttg gctgcctttg acattttttc 2160cttcatttca accttggtga atctgatgat tatgtgtctt ggggttgctc ttctcgagga 2220gtatctttgt ggtattctct gtatttcctg aatttcaatg ttggcccgtc ttgctaggtt 2280ggggaagttc tcctggataa tatcctgaag agtgttttcc aacttggttc cattctcccc 2340atcacttcca gatacaccaa tgaaacatag gtttggtctt ttcacatagt cccatatttc 2400ttggaggctt tatttgttcc tttcattctt ttttctctaa tcttgtcttc acgttttatt 2460tcattaagtt gatcttcaat atctgatatc ctttcttcca cttgatcaat tcggctatta 2520atatttgtgt atgcttcaca aagttctcgt gctgtgtttc tcagctccat cagatcattt 2580atgttcttct ctaaactggt tattctagtt agcaattcct ctaacctttt ttcaaggttc 2640ttagcttcct tgcattgagt tagaacatgc tcctttagct ctgaggagtt tgttattacc 2700caccttttga agcctacttc tgtcaattgg tcaaactcat tctccattca gttttgtttc 2760cttgctggcg agaagttgtg atcctttgga ggagaagagg cattctggtt tttggaattt 2820tcagcctttt tgcactggtt tttcctcatc tttgtggatt tatctacctt tggtctttga 2880tgttggtgac ctgcagatgg ggtttttgtg tgggcatcct ttttgttgat gttgatgtta 2940ttgctttctg tttgttaatt tttcttctaa cagtcaggcc tctctgcttc aggtctgctg 3000gagtttgccg aagtccactc cagaccctgt ttgcctgggt atccccagcg aaggctgtag 3060aatagcaaag attactgcct gttcctgcct ctggaagctt catcccagag gggcactcac 3120tggatgccag ctggagctct cctgtatgag atgtctgtca acccctgctg ggaggtgtct 3180cctagtcagg aggcacgagg gtcagggacc cacttgagtt ggcagtctgt cccttagcag 3240agctcgagca ctgtgctggg agatccactg ctctcttcag agctggcagg caggaacatt 3300aaaatatgct gaagctgtgc ccacagccgc cccttccccc aggtgctctg tcccagggag 3360ataggagttt tatctataag tccctgactg gggctgctgc ctttctttca gatgtgccct 3420gcccagagag gaggaatcca gagaggcatt ctggctacag tggctttgcc aagctgcagt 3480gggctatgcc tagttcgaac ctcctggcag ctttgtttac actgtgaggg gaaaaccacc 3540tactcaagcc tcagtaatgg catacacttc ttcccccacc aagcttgagc atcccaggtc 3600aacttcagac tgctgcgctg gcagcaagaa tttcaagcca gtggatctta gcttgctggg 3660ctctgtaggg gtgggatccg ctgagctaga ccacttggct ccctgactgc agcccccttt 3720tcaggggagt gaacagttct gtctcgctgg tgttccaggc gccactgggg tatgaaaaaa 3780aaaaactcct gcagttagct cagtgtctgc ccaaatggcc aaccagtttt gtgcttgaaa 3840cccagggccc tggtcacata ggcacccaaa ggaatctcct ggtctgtggg ttgcaaagac 3900tgtgggaaaa gtatagtatc tgggctggaa tgcactgttc ttcatggcac agtccctcat 3960ggcttccctt ggctagggga gggagttccc cgaccccttg cacttcccag gtgagatgac 4020accccaccct gcttctgctc accctccgtg ggctgcacct actatctaac cagtcccaaa 4080gagatgagct gggtacctct gttggaaatg cagaaatccc ccaccttctg cattgatctt 4140gctgggagct gcagaccgga gctgttccta tttggccatc ttcccagcca cctggtaatt 4200taactcttat gggccaaaat ggcaaaagtc atctgtggtc aaatcacaga gcccattacg 4260tctgtcaaag gtcatggaat tgataagatt ttggagttag atggaacatt catgactgaa 4320tgattcccat cttattttac agatgaggaa actaaggctc aaagagacta agtaactggc 4380ttagtaaggt tacacagttc cctagaggaa aagctggggg tagaaaatgg ggcttttcgg 4440ccaggcgtgg tggctcccga ctataatccc agcactttgg gaggttgagg tgggtggatc 4500acttgaggtc aggagtttga gaccagcctg gccaacatgg taaaaccccg tctctactaa 4560aaatacaaaa attagccggg catggtggca cacacatgta attccagcta ctccagaggc 4620tgaggcagga gaatcatttg aaccagggag gcagagattg cagtgtgctg agattgagcc 4680agtgcactct agcctgggtg acagagagag actctgtctc aaaataaaca aacaaatgaa 4740caacaacaac aacaacaaaa agaaaatggg ggcttttctg ttctttaagc agagatgttt 4800gcattaaccc aggcaacttc aatgattcta atcactagtg cataacagtg aaggagctta 4860ttaaacctca gggcagtgag gaaaagcaaa aaatacggta cggtaacttc catcagaacc 4920tgaaatactt gcatccactg atttggttgt cctttgcctt cctccaaaaa caaggtcttc 4980atttcactca ctgtgtttgc gtggtccttt ttcaggtcgt tatgctaaca tgagaaccaa 5040aataacaaca ctgggtaatg gagatttttt aaataatggt gatctgtaag gcagtggttc 5100tcaaagtgag gccctggact agcagcatct gcatcaccaa aaacctcagg agaaagttga 5160aaaccaccct gatagttaag tccatgctag ttgctaatgt aatggtccct gtctgttgcg 5220taaggaagga tcatttcctt ctctgataaa aatggctctg gctctggtaa gttatactca 5280aatttaatta tatttcatgg taaatcttgt cagctgaaaa gttatgccaa cttggtggac 5340aatggcagag aatacaagtt tctgacttac ctcaaggttt gaatgtacct agagcagaag 5400tttctgatat ttgtactcac ttaaccaaca actataattg ccgggcactg ttgtggttgc 5460ttgggatatt tcagttgtta ctaatgcttt ggttttatgg aaagacaaag acaatgctgg 5520tcttctagca gcacaaagta taaggtaatg ttcctatatt attcatagtc cattggcaaa 5580aatgcagtta ttatcaaaaa acattaaaat taaaagtatt taagtcattt gtaataaggg 5640attcaacaaa cactcaatat ctaagtaatt tccaatgatt tcagtaatat gagtattggc 5700tttaaaaagt aatgttttag agccaagtgc agtggctcac gcctgtaatt ccagcacttt 5760gggagtcaga ggcaggagga tcgcttgagt cttagagttc aagacctgcc taggcaacat 5820ggcgagaccc tgtctctaca aaaaatttaa aaattagctg ggcatggtgg tgcatgcctg 5880tggtcccagc tactcaggag gctgagatga gaggattgct tgagcctgga atatcaaggc 5940tgcagtgagc catgatcaca ccactgcaat ccagcctggg tgacagagtg agactttgtc 6000tcaaaaaaaa caaaacaaaa aaacaaatag agaataaaag gtttagaaac agatacaact 6060ggctctagga aaacaactca actgttgaga gcaataagtc atatgtactg acagcctatc 6120aaattgagat gttaataaga aatcggccaa cttttttttt tttttttttt ttttaacaga 6180aggtaaggag aatgcaggtt aaccagtgga atttcaggtc agttcagtca agagagtttt 6240gactatgcct gttggtctca aacttggtta tcaattggaa tcacatgagc agccttgaaa 6300attccaaata cccagactac acccaagagt agttaaatca gaagctttgg tgtcaacata 6360gtaatcagga actttgtaat gatcccattc cggtgtgcag ccgaaattga gaagctccag 6420tttaaggtca tgggccatga agctttcaag ttctgccacc tcctctcaac attaatgtgt 6480atcagctttg ggaaactaca tgtagaaggt atgaagaatg gcagaaagta tataaaggga 6540caaatcacca ccattaattc cacaaatcag tacaggtcag tagctacagt ttggagaatt 6600taaaacattt tcctctttag taagaattcc tacaaagaaa tgcccattta aaatcctgat 6660ctatttcaat tgcttgccag aaatgttgta ccaatttgca ctctcaccca aagtccctaa 6720atcctgacaa acactgtgaa ttgatcatct ttttcctgtt ttccaatctg ataatttaac 6780agtactgggg tattgtttta tttgcatttc tgtaagaact ttattttatt gactacaaat 6840attttacttt ccaggatcta taaagcaata tagcaatata aataatatta tataagcata 6900ctcaaacctt gttaacctat caccgattat tgcaatacta tttttagatt ttttcttcaa 6960ttgcaaaaat actttattac tgattaaaaa tagtatatat gcactgataa aattttgata 7020aatgcaaaga caagtaatga aaatcatcag caataacaat ggctaaaagt ggagtataca 7080tacaaacaaa tacttgtttt gaaacttttt ttggtttact cttagcatag tatctttttc 7140catgtcatgt agtattctaa tgtgtgactt ttggaggctg catagtattc taaagtaata 7200tgttttgaat ccaatattct tcaaaaaaat atggaacaac agatcttggc ttaaatatga 7260atttttttac atttctgttg tatatactac aagtggtgga ttgctacttt ggtggttccc 7320gatgaaccat aactcctggt attcacactt ctgtggtgta tccttccata ttaacctcgg 7380gctcggccaa taaataggac attagcaaga atgatgtaag caaatgcttg ataagtgctg 7440gcacactgga gcttatgtct cttgaaatgc tcactctcag gatcaaacca ccatgaaaag 7500aaacccaagc tagccacatg gggaagaaaa gaggaaaaga tcactccctc tcaaccttcc 7560ccactgggga actagacatg tgaacaaagc catctgatta caacctcaca ggagacccca 7620actgaaacca accaaagaac agcccagcag agccccagcc aaccaaagaa tcatgaacaa 7680ataaagtggt tactgtttta agcagatatg ttttgggttg gtttgttaca cagcaagaga 7740taagcaaaat gtgacagcat ctaatatgaa tacctgcttt agatatgaaa aagaaaccct 7800tagatatgaa aaagaaaccc ttaggatcat aaactcaaga agaatttttt gtttttatct 7860agtcagattg ctgaggtcct ggctgtttag atgacttaca gaaagaactt gggtagtaga 7920gtcaagatat gaatagtaac ccagttttat tactagagat ttgacttcgt tgagcatcag 7980ctttcttgtg ggcgagttag gttttaacaa tagctgcctt gcaggtgttt tatttttaag 8040cattatgaat gatggtgcct ccagttccat aacaagatga aggcatatgc agatgtgagt 8100taacattttc attggctggc catgtatgtt gaatcccaaa caagtggtct tcactacacc 8160tcaccactca caaatctact tatgagtctc agatggtgag ctagatggtc agtaataccc 8220acctcaggaa aactgcttct gagtttacaa gacagcaggc ggaacagctc aaagaccaat 8280gcatgcataa atagtctttc ctttttttca ccaggtcaac tactcgatta tttgcactgg 8340gggccttttt tcaaagcatc tgaccttttg gactcgtctg gtgaaatcta gtatgtactt 8400aggaccagcc ttcaacacaa aggaaggttt ttgttacatt cgtttgcata tcctgtcaag 8460gatatcttca gaacactttc atttcacagt aaaatgaaac ttacggaaga actaagcagt 8520gaagtgcttc atactttcat ttaaaagggg aatgagaaac aaaagacaag gttaattatg 8580acaccggggc tttacaatgc taaaaatatc ctatatacaa agggatatgt aggctgtgtt 8640ctttttccat gtcattacaa agaacaggct caaggtatct gcaaatttct aataaaaata 8700ttattacttg aaaaatgtcc atggttgtat ctaactgaag ggattaaaac ttttagactt 8760gttatacaaa taccatgata cttgtcatca aagtcctcct tttcatcaag cagaccctga 8820cttacagcat ctgagagtgg aaaaaactag catgaaccgt aaggcagtgg gattcatacc 8880tacctctcac tcacacgttc atccaaacca agcaagttgc ataaagcaac ctgagacttt 8940atacttccac atgctggcaa cacagcatta actagactcg ggcaacttgt ttaaggggtt 9000tatttaggtt cctatcagta aagaggtgtt tttccagctt tgggagaata tgtatgatgc 9060caagtttttt tttttttaag tttaaatact tttataagtt taatgttggc attggatgtt 9120tacatattaa ctggtacaat ttacatcggt tttatactta cctatttttg caaagatgtc 9180atgtacacaa agatacatat tcaataactg agaaacaagt gaaacttctg atctatactt 9240tcaggtttgt ttcataaaga tatttctgat aaggattatc agcatacaag tttcacctct 9300tccagaagga cactgtcatt ttatctaaga gtttactctg ggttttattg cagaacacaa 9360attccctcaa ttgctttttt cttgcaggtg tctttttcca gaattttttc ttataaccag 9420cctttctcct cacccaaagg ccagaatgaa gtcgaaaaaa cctatagata acagctttca 9480cagttgttct cttgcctttt tgtgtactga cgtatattac agttctgact ggcagcttca 9540ggacacttgg aagcaagggg gccactctgt tggggatcac tgcagtatgc ccacatgtca 9600ggtttctgac agatgtgatc agcctgggag tggaggaaac aaatggtgtc tgaatatgac 9660taaaacgtcc agtagacaat gcagaaataa gagaggcatt cctggcacag tttcggtggg 9720ctggagatgc aaaaatattc aggagccata gattcctgaa gctgctctca tcgcaccagc 9780aaaggcagag gcagccatct tgccagcctg ctatgatgcc aagtttcttt cttttttttt 9840tttttttttt tgagatgaag tctcgctctt gtcccccagg ctggagtgca atggtgtgat 9900ctcggctcat ggcaacatcc acctcccggg ttcaagcaat tctcctgcct cagcctcccg 9960agtagctgtg attacaggtg cctgccaaca cacccagcta attttcgtat ttttagtaga 10020gacggggttt caccatgttg tacaggctgg tctcgaactc ctgaccttag gtgatccgcc 10080cgccttggcc tcccaaagtg ctgggattac aggcgtgagc caccacgccc agccaatgcc 10140aagtttctat tgaaccactt tgatgtctat gtctgagtca aatggcagag ccgggactca 10200aatctaggtc ttctgtccac gaccatatct ttgttcaatc accactctgg atgctgatca 10260agtctgacct tccttttcat ttcagagcat tcaccccagg aatccttcaa tgtttaaatc 10320tctagattaa aatatggtac tgctgggcat ggtggctcac tcctataatc taaaaaaatt 10380gggaggctaa ggtgggagga ttgcttgaga ccaggagatc aaggccagtg tggaaaacat 10440agtgagaccc tgtttctaaa aataaataag taaaatagaa taaaatatgg cactaaacta 10500gggtggccag ataaaataca ggacacccaa ttaaatttga atttcagata ataaataatt 10560ttttagtaga agcatgtccc gatttgctaa atttggcaac cctacactaa accttaccta 10620cccaccaagg gctgttccca gtcctcctca tttccaccta aaaagaaagg agaaatacaa 10680caagacctag aaaacaaagt gtgagttgcc ccagccagtg agaggccagt tctcaaaaga 10740taaagagaag aactgtatcc tctccatcca gtgatctctc ttgagctaat ttacagatgg 10800gggactagac caataacgta ggatcttctg tgccatcact cagactgggg tagttcactg 10860gagccacagc taactcttca gaagaagatc tttcggaggc ccaaggctat ttatcgcact 10920agtgcttgca ataccaaaga tcagaaacca cccaaacgtc cgttagagaa atggttaagt 10980aaattgtgaa acttctagaa aatgggatgt tttccagcta tgagaaaaaa aaaatgcaga 11040tgctcctgag ataaaggtat agaaggaact gcaaatgaaa aaaacaagag agtaagagag 11100tatatgctaa cgaagtatga caaaggcagg gccagaagaa tatgttttta tttgctagta 11160tgcctaaaga aactctggaa agataagcaa ggaactaata agaatggttt cttgtgggga 11220tgggtgtggg attgggtgga tggtctacaa gaagagcaga aaagttttta ctatgtatct 11280acctatgtat ttatgtatgt atgtatatat gtatgtatat gcatggatgt atgtatagat 11340acatttatct gtacagaaaa gagttaacat ggtagccagt tgccatggct caagcctata 11400attccagcac tttgagaggc caagggtgga tcgcctgagg tcaggagttc gagaccagcc 11460tggacaacat gatgaaaccc catctctact aaaaatacaa aaaattaacc aggcatggtg 11520gggggcgcct gtaatcccag ctactcggga ggctgaggta ggagaatcac ttgaacccag 11580gaggcggagg ttgcaatgag ccgagattgc gccattgcac tccagcctgg gcaacaacag 11640tgaaactcga tctcaaaaac aaaaaaccaa gaaacatggc aggcctgaga ctgctctcct 11700tagaaaggcc tgcttgtaag gttggccttt ggctcatgtc tgggaacttg gatttcaggg 11760tggttcccac cattcccaga actaagaaga gtggctcact gctcttacac tgtttgtaca 11820aataatatgg tttttgctgc acacctgctt tcctattggg aggctggaat tttggtatgt 11880gctgggcaga gtgtgcctac atgaccagcc ccgagtaaac accctgggca ctgaatctct 11940aacgagcctc cctggtagac agcagttggc atgtgttttc tcaatttgtt gcagaaggaa 12000tcaagtacat ccatgtgact aaagtcggag gggatgcttg gaaggttggg cctggtttct 12060tctgggcatc acctcatgca ccattgtcct ttgctgcttt tggttcgtat cttttcactg 12120caataaatca tagctgtgac catactttat tgagttctgc gactcctagt ggatcacgaa 12180gcctggggtg gcggagtctt ggggacctcc gacacagtac acagtctctg tctctctctc 12240tctctctctg tcccacacac cccatatctt tctattcagc tctatgtaag aattctgtac 12300cacacaaata tgtattaaaa tgaaataaaa aataaaaaag gcctttgctt tatatttgcc 12360taaccaaaag ctccattcaa tctgcttttc tttccttctt tgtttccttc cttcttttct 12420tctttctttt ctcctgtcct tcattctttc ctttccttcc tttctgccgg cctgccttgt 12480ctccctccct ccccaccctt tctttccttc tctctttctt tccttcactc tctccctttc 12540ttcctttgtt tctctgtctc tagttttcca tttactacgt agaagttctt accatatgac 12600tatgtatttt ccagcaaaat tgaagaaaaa gttaaaaagc tattgcccta tatttgccaa 12660ctaaaagccc cattcaaaat gtctcaaggg gagagagaga agaaaagcaa aagaaacaaa 12720gaatacaact aacttcactg cctcactctc tattaaggag acagaaaatt ctcatagctt 12780ttaaacaaca gtgtcaggtt tggtgtgttt ttctgttgtt cagctttaac acagcactgt 12840ttcaaaacct taaggctttg tcctagaaga aaatgttcct gtttgaacat ataaaacaat 12900ccagcatata actagaaata caggtctgcc aatcatggta acacttttgt gacaaaactt 12960aaagttatct ttggcggtga tatacatttt taagaagtta ttacatatgc aaagattgta 13020gagaaattac agagacacaa tcaaactcac tatgtaaaca ctgagtagtt cttgaatcag 13080aaagaaaata aagctataaa agatattttg gggacaaatt gaaaaaatgt gaatacagaa 13140tctatatgag atattacaaa ttattagttt ttaaatgtta ctttaagttc tgggatacat 13200gtgcagaatg tgcaggtttg ttacatgggt atacatgtgc catcgtggtt taccgcacct 13260atcaacccga catctaggtt ttaagcccca catgtattag gtatttgtcc taatgttgtc 13320tctccccttg ctccccaccc cctgataggc ccgagtgtgt gatgttcccc tccctgtgtc 13380catgtgttct tattgttcaa ctcgcactta tgagtgagaa catgcggtgt ttggttttct 13440gttcctgtgt tagtttgctg agaatgatgg tttccggctt catccatgtc cctacaaagg 13500acatgaactc attctttttt aaggctgcat agtattccat ggtgtatatg tgccacattt 13560tctttatcca gtctattatt gatgggcttt tggattggtt ccaagtcttt gcatttgtga 13620atagtgctgc aataaacata cgtgtgcatg tgtctttata gtagaatgat ttataatcct 13680ttgggtatat accagtaatg ggatggctgg gtcaaatggt atttctggtt ctagatcctt 13740gaggaattgc cacgctgtct tcctcaatgg ttgaactagc ttacacgccc gccaatagtg 13800taaaagtatt cctatttctc cacatccttt ccagcatcta ttgtttcctg actttttaat 13860gattgccatt ctaaatggcg tgagatggta tctcattgtg gttttgattt gcaattctct 13920aatgaccagt gatgatgagc ttttttttca tgtttctttg ttgcataaat gtcttctttt 13980gagaagtgtc tgttcatatc tttcactcac tttctgatgg ggttgtttgt ttttttctta 14040taagtttctt tgagttcttt gtagattctg gatattagcc gtttgtcaga tgagtagatt 14100gcaaaaattt tctcccattc tgtaggttgc ctgttcactc tgatgatagt ttcttttgtt 14160gtgcagaagc tctttagttt aattagatcc catttgtcaa ttttggcttt tgttgccatt 14220gcttttggtg ttttagtcat gaagtctttg cccacgccta tgtccagaat ggtattgcct 14280aagttttctt ctagggtttt tatggtttta ggttttatat ttaagtcttt aatccatctt 14340tagttaattt ttgaataagg tgtaaggaaa gggtccagtt tccattttct gcatacagct 14400agccagtttt cccagcacca tttattaaat agggaatcct ttccccattg cttgtttttg 14460tcaggtttgt caaagatcag atggttgtag atgtgtcgtg ttatctctga gggctctgtt 14520ctgttccatt ggtctatatc tctgttttgg taccagtatc atgctgtttt ggttactgca 14580gccttgtagt atcgtttgaa gtcaggtagt gtgtaatgcc tccagctttg ctctttttgc 14640ttaggattgt cttggctata tgggctcatt ttttattcca tatgaaattt aaagtagctt 14700tttctaattc tgagaagaaa gtcaatggta tcttgatggg gatggcattg aatctataaa 14760ttacttttgg cagtatgtcc attttcacga cattggttgt tcctatccat gagcatggaa 14820tgttcttcca tttgtttgtg tcctctctta tttccttgag cagttgtttg tagttctcct 14880tgaagaggtc cttcacgtcc cttgtaagtt ggattcctag gtattttgtt ctctttgtag 14940caattgtgaa cgggagttca ctagatttgg ctctctgttt

gtctgttatt ggtgtatagg 15000aatgcttgtg atttttgcac attgattttg tattctgaga atttgctgaa gttggttatc 15060agcttaagga gtttttgggc tgagatgatg gggttttcta agtatacaat catgtcatct 15120gcaaacagag acaatttgac ttcctctctt cctatttgaa tatgctttat ttctttctct 15180tgcctgatta ccctggccgg aacttccaat actatgttga accttgtctt gtgccggttt 15240ccaaagggaa tgcttccagc tttttcccat tccgtatgat attggctatg ggtctgccat 15300aaatagctct tattattttg agatatgttc catcaatacc tagtttattg agagttttta 15360gcatgaaggg atgttgaatt ttattgaagg ccttttctgc atctattgag ataatcatgt 15420ggtttttgtc attggttttg tttatgtgat ggattacgtt tattgatttg catttgttga 15480acaagccttg catcccaagg atgaagctga cttgattatg gtggataagc tttttgatgt 15540gctgctggat tcagtttgcc agtattttac tgaggatttt tgcattgatg ttcatcaggg 15600atattggcct gaaattttct ttttttgttg tgtcactgcc aggttttggt atcaggacaa 15660tggtggcctc ataaaatgag ttagggagga gtctctcttt ttctattgtt tggaatagtt 15720tcagaaggaa tggtaccagc tcctctttgt acctctggta gaattcggct gtgaatacat 15780ctggtcctgt gctttttttg gttggtaggc tattaattac tgcctcaact tcagaacttg 15840ttattgatct attcagggat ttgacttctt cctggtttag tcttgggaga gtttgtgtgt 15900ccattaattt ttccatttct tctagatttt ccagtttatt tctgtagagg tattgattgt 15960agtctctgat ggtagtttgt atttctgtgg gatcagtggt gatatccctt ttatcatttt 16020ttattgtgtc tatttgattc ttctctcttt tcttctttat tagtctagct agtggtctat 16080ctattttgtt aatcttttca aaaatccagc tcctggattc attgattttt tgaagggatt 16140tttgtgtctc tatctccttc agttctgctc tgatcttatt tcttgtcttc tgctagcttt 16200tgaatttgtt tgctcttgct tctctagcta attgtgatgt tagggtgtcg atatcagatc 16260tttttaactt tctgatatgg gaatttagtg ctataaattt ccctcttaat actgctttag 16320ctgtgtccca gggattctgg tacattgtat ctttgttctc cttggtttca aagaacttat 16380ttatttctgc tttaatttca ttatttaccc agtagtcatt caggagcagg ttgttcagtt 16440tccatgtagt tgtgcggtgt tcagtgagtt tcttaatcct gagttctaat ttgattgcac 16500tgtggtctga gagactgttt gttgtgattt ccattatttt gcatttgctg aggagtgttt 16560tacgtccaat tatgtggtca tttttagaat aactgcgatg tgctcctgag aagaatgtat 16620attctgttga tttggggtgg agagttctgt agatgtctat taggttcgct tggtcgagag 16680ctgaattcaa gtcctggata ttcttgttaa ttttctgtct cgttgatctg tctaatattg 16740acagtggggt gttaaagtct ctcactatta ttgtgtggga gtctaagtct ctttgtaggt 16800ctctaagaac ttgttttatg aatctgtgtg ctcctgtatt gggtgcatat atatttagga 16860cagttagctc ttcttgtttt attgatccct ttaccattat gtaataccct tcttcgtcta 16920ttttcatctt tgttggttta aagtctgttt tatcagagac taggattgca accccccccc 16980cttttttttc tttccatttg cttggtaaat attcctccat cactttcttt tgagcctatg 17040tgtgtttttg cacgtgagat gggtctcctg aatacagcat accaataatg ggtcttgact 17100ctatccaatt tgcctgtcca tgtcttttaa ttggggcatt tagcctattt acatttaagg 17160ttaatattat gtgtgaatct gatcctgtca ttatgatgat agctggttat tttgcacatt 17220aattgattca gtttcttcat agtgtcattg gtcttcatat tttggtgtgt ttttgcagtg 17280gctggtactg gtttttcttt acctatttag tgcttccttt gggagctcct gtaaggcagg 17340tctggtggtg acaaaacacc tcagcatttg cttttctgga aaggatttta tttctttttt 17400gcttatgaag cttcatttgg ctggatatga aattctgggt tggaaattct tttctttatg 17460aatgttgaat attagccccc actctcttct ggcttgtagg gtttctgcag agagatccac 17520tgttagtctg atgggcttcc ctttgtaggt aacctgacct ttctctctgg ctgcccttaa 17580tattttttct ttcttttcaa ccttggagaa tctgatgatc atgtgtcttg gggttgctct 17640tctcgaggag tatcttagtg atgttctctg tttttcctta atttgaatgt tggcctgtct 17700tgctaggttg gggaagttct gctggataat atcctgaagt gtgttttcca acttgtttcc 17760attatccctg tcacttttag ctacaccaat caatcgtagg tttggtcttt tcacatagtc 17820ccatatttct tggaggcttt gatccttcct tttcattctt tttctctaat cttgtcttca 17880cgccctatat ttgtaagttg atcttcaatc tctgatatcc tttcttctgc ttgattgatt 17940cacctattga tacttgcgta tgcttcacga cattctcgtg ctgtgttttt cagctccatc 18000gggtcattta tgttcttctc taaactggtt attctagtta gcagttcctg taacctttta 18060tcaaggttct tatcttcctt gcattgggta gaacatgttc ctttagctca gaggagttta 18120ttattaccca ccttctgaag cctacttctg tcaatttgtc aatctcattc tccgtccagt 18180tttgtgtcct tgctggagag gagttgtgat catttggaag agaaaaggca ctctgctttt 18240tggaattttc agcatttttg cacttgtttt ttctcatctt catggattta tctgcctttg 18300atctttgatg ctgatcacct ttggatgggg tttttgtgtg gaggtccttt atgttgatgt 18360tgatgttatt gctttctgtt agtttttcta ataggtccct cttctgcagg tctgctgcag 18420tttgctggag gtctactcca gaccctgttt gcctgggtat caccagtgga ggcctgctcc 18480ttctcctgga agctttgtcc cagaggggca ctggcccgat gccagctgga gctctcctgt 18540atgaggtgtc tgttgacctc tgttgggagg tctctcccag tcaggaggca caggagtcag 18600ggacccactt gaggaggcag tctgtccctt agcagagttc aagtgctgtg ctggggggaa 18660tcctccacgt caggatcagc tgctcttttc agagctggca ggcaggaaag tttaagtctg 18720ctgaactaca cccacagctg ccttttcccc caggtgccct gtcccaggga gatggaagtt 18780ttatctgtaa ttccctgact ggggctgctg cctttctttc agagatgccc tgcccagtga 18840ggaggaatct agagaagcag tcaggccaca gccgctttgc catgctgtgg tgaattctgc 18900ccagtctgaa cctcccagcc tccttagcac tgtcagggga aaactgccta ctaaagcctt 18960agtaatggtg gatgcccctc cccccaccaa gctcgattgt cccaggtcca tttcagactg 19020ctgtgccggc agtgagaatt tcaagccagt ggctctttgc ctgctgggct ctgtaggagt 19080gggacccact gagcccgacc acttggctcc ctggcttcag ccccctccag gagagtgaaa 19140ggttctgtct tgctggggtt ccaggtgcca ctggggtaca aaaaataact cctgcagcta 19200gctctgtgtc tgcccaaaca gccacccagt tttgtgcttg aaacccaggg ccctggtggt 19260gtaggcacac aagggaatct cctgatccgc gactgcaaaa acgatgggaa aaacgcagta 19320gctgggctgg gtagcacagt ccctcatggc ttcccttggc tgggggaggg agatcccttg 19380gctccttgta ctttccaggt gaggtgatga cccaccttgc ttctgctcac cctctgtggg 19440ctgcacccac tgcctaacca gtcccagtga gatgaacttg gtacctcagt tggaaatgca 19500gaaatcacct gccttctccg ttggtctccc tgggagctgg agaccggagc tgttcctatt 19560cagccacctt gccagattgg ccaatattac aaattatgaa ttctcttagg tgtcatcctt 19620atttcaggag atacaggatt aagtgtttag ggttaaaatg tcgtgttgtc tgcaacattt 19680taatgattag ggaaataaat acattatata cacatacaaa aaagtaaaac caatgtggct 19740cactatcaac aattgttgaa ttcagacagc tatatgggct gcattgtgca attgtttcaa 19800tttttcaata tatctgaaaa ttttttaagt aaaaagttgg gaagaaaaaa gattttcttt 19860taaaaactag tatattaata taaatgaatt caagtagaat gatataatca gttattagaa 19920aatcaattac cactcagcta ttgaatccac catactgagt taagaagaac ttagtatctt 19980ttgaataaga aaacataggc tttattgatg cttgactact agacaagttg caactcctag 20040taaattcaac cagctagtga ctgtaattag atgactgatc acgtcaatca attaatctgc 20100ctaatgatcg acctctgaaa attggcctaa ttaattgttc ttattttcat gatcactttc 20160attttcatca tcataaagag tgatctctta agtacctaga atggatcctg aggacaacac 20220attcttaact caaggggtct gcaatatcag tgtgggaact aggagatatg aatgaaacat 20280aaataaccag gctgagtgtg gtggcttgtg tctataatcc cagcactttg ggaggctgag 20340gtgggtggat cacctggggt caggagtttg agaacagcct agccaacata gtgaaacccc 20400atctctagta aaaatacaaa aataagctag gtgtggtggt gtgcacctgt agtcccagat 20460actcaggagg ctgaggcacg agaattgctt gaatctggga ggtggaggtt gcagcaagcc 20520aagatgacac cactacactc cagtctgggg acagagcgag actctggccc caaacaaaca 20580aacaaacaaa caaccataaa taaccacgca agaaagcata atatatatgc taaattcata 20640atttcataat ctcttgggat cttagcatat aaaacctggg tgtgtcttta ttgatcatac 20700agcctcatcc ctcactttaa agataaatgg tttattttta tgtagtcaaa tttttcagtg 20760ttttctttaa gactctggga ttgttgctgt tggttccttt tccttgtttg tttggtgtta 20820tcgtcatact tagcactact tttctacttc aagattagaa aaaatattga cctatagttt 20880ctttcatata tatatatata tatatatata attatacttt aagttctagg gtacatgtgc 20940acaacatgca ggtttgttac atatgtatag atgtgccatg ttggtgtgct gcaactccaa 21000aagagtcatg cccatttgaa ttaattttgg aaatgtctgt ttttcttttt gtacttggtt 21060catagcaact gccctagggc accaaccatc cagggaaact ttgatgcatc ttacactttg 21120gtcctgatga gagggaggca taaatgagaa aagctcttat tctgcagtta atacagagca 21180agcggctgag tttgaagcag ccatggagaa cattctagaa cagggttgtc tagtcgtttg 21240gctcccttgg gtcacattgg aagaagaatt gtcttggggc cacacataaa atacactaac 21300aatcgctgat gagcttaaaa aaaaatctca taatatttaa agaatgttta caaatttgtg 21360ttgggccgca ttcaaagctg tcctgggctg catgtggccc atgggtggtg ggttggacaa 21420gtttcctcta gaatcaacaa aattctgcaa ttccgtgtgc taacagaaga gccttaccag 21480ttgtggtgac aggctagaca gccaggtaga gaattcaata ggagcggaaa taactgtcca 21540cgaacagcgt agggcctgac aatctgaaag aagcaatatt ctacctagtt cgaagatctt 21600aagcttacac agaaagggcg cttacactga tacagaaaca aaactatgca gaatccaatc 21660aaatgattaa cttaggtggg gtttgcagtc tttttgtaga agaagaaagg attggccggg 21720cgcggtggct cacgcctgta atcccagcac tttgggaggc cgaggcgggc ggatcacgag 21780gtcaggagat cgagaccaac cccgctaaaa cggtgaaacc ccgtctctac taaaaataca 21840aaaaattagc cgggcgtagt ggcgggcgcc tgtagtccca gctacttggg aggctgaggc 21900aggagaatgg cgtgaacccg ggaggcggag cttgcagtga gccgagatcc cgccactgca 21960ctccagcctg ggcgacagag caagactccg tctcaaaaaa aaaaaaaaaa aaaaaaaaaa 22020aaaagaagaa gaaaggattg ggtattgtat ataattcacc tccaaagacc tgggcagcat 22080gattgcaccc ctgtaaagct atgggtttac cagtagcagc taggtacata ggtttagagt 22140aacccattca ggttcctcag aaggacctga tgagctcttg gctgaaatca ctgattgttc 22200aaaacctgca ggatactgag aaggcaaacc ccaaatttca tgggataaaa tgacagtttg 22260tacgagaaaa atcaccttag atgtctacct ttccaagtct cagcacccag ttaaaatctc 22320taattaaaag tcatctcagt aactgaggtt gcctttcact tcccgaatgc ccaaacattt 22380ttcaactctg ggaaccttta aaaatgagaa cagaaataac caagacactt aaaaacattg 22440tatcgatgtt tcctggttaa ggcatctgca caaacttggg gttgtatatg cttgaatagt 22500taagagaaat atagcctatt aagacaatag cttgtgactc ccagataaag tatataattg 22560agtgattgat cttttcaagt tgaaatctaa ataatttatg tggacactta gaaatttaat 22620aaaacaaaac tagttttgtt tcaccatact tctttttcct ctctcaaaaa aaaatttttt 22680tatactttaa cttctagggt acatgtgcac aacgtgcagg tttgttacat atgtatacat 22740gtgccatgtt ggtgtgctgt acccattaac tcgtcattta acattaggta tatctcctaa 22800tgctatccct cccccctccc tccaccccac aacaggcccc ggtgtgtgat gttccccttc 22860ctgtgtccaa gtgttctcat tgttcaattc ccacctatga gtgagaacat gtgatgtttg 22920gttttttggc cctgcgatag tttgctgaga atgatggttt ccagcttcat ccatgtcccc 22980ataaaggaca tcaactcatc cttttttatg gctgcatagt attccatggt gtatatgtgc 23040cacattttct taatccagtc tatcattgat ggacatttgg gttggttcca agtctttgct 23100attgtgaata gtgctgcaat aaacatacgt gtgcatatgt ctttatagca gcatgattta 23160taatcctttg ggaatatacc cagtaatggg attgctgggt caaatggtat ttctagttct 23220agatccttga ggactcgcca cactgtcttc cacaatggtt gaactagttt acagtcccac 23280caacagtgta aaagtgtgcc tatttctcca catcctctcc agcacctgtt gtttcctgac 23340tttttaatga ttgccattct aactggtgtg agatggtatc tcattgtggt tttgatttgc 23400atttctctga tggccagtga tgatgagcat ttttttcatg tgtctgttgg ctgcataaat 23460gtcttctttt gagaagtgtc tgttcatatc ctttgcccac tttctgatgg ggttgtttgt 23520ttttttcttg taagtttctt tgagttcttt gtagattctg gatattagcc atttgtcaga 23580tgagtagatt gcaaaaatgt tctcccattc tttaggttgc ctgttcactc tgatggtagt 23640ttcttttgct gtgcagaagc tctttagttt aattagatcc catttgtcaa ttttggcttt 23700tgttgccatt gcttttggtg ttttagacat gaagtccttg cccatgccta tgtcctgaat 23760ggtattgcct aggttttctt ctagggtttt tatggtttta ggtcttacgt ttaagtcttt 23820aatccatctt gaattaattt ttgtataagg tgtaaggaag ggatccagtt tcagctttct 23880acatatgtct agccagcttt cccagcacca tttattaaat agggaatgct ttccccattt 23940cttgtttttg tcaggtttgt caaagatcag atggttgtag atgtgtggta ttatttctga 24000gggctctgtt ctgttccatt ggtttatatc tctgtgttga taccagtacc atgctgtttt 24060ggttagtgta gccttgtagt atagtttgaa gtcaggtagc gtgatgcctc cagctttgtt 24120cttttggctt aggattgact tggtgatgcg ggctcttttt tggttccata tgaactttaa 24180agtagctttt ttccaattct gtgaagaaag tcattggtag cttgatgggg atggcactga 24240atctataaat taccttgggc actatggcca ttttcacgat attgattctt cctgtccatg 24300agcatggaat gttcttccat ttgtttgtgt cctcttttat ttcgttgagc agtggtttgt 24360agttctcctt gaagaggtcc ttcacatccc ttgtaagttg gattcctagg tattttattc 24420tctttgaagc aattgtcaat gggagttcac tcatgatttg gctctttgtc tgttattggt 24480gtgtaacaat gcttgggatt tttgcacatt gattttgtat cctgagactt tgctgaagtt 24540gcttatcagc ttaaggagat tttcggctga gacaatgggg ttttctagat ataaaatcat 24600gtcatctgca aacagggaca atttgacttc ctcttttcct aattgaatac cctttatttc 24660tttctgctgc ctgattgccc tggccagaac ttccaacact atgttgaata ggagtggtga 24720gagagggcat ccctgtcttg tgccggtttt caaagggaat gcttccagtt tttgcccatt 24780cagtatgata ctggctgtgg gtttgtcata gtagctctta ttattttgag atacgtccca 24840tcaataccta atttattgag agtttttagc atgaagggct gttgaatttt gtcaaaggcc 24900ttttctgcat ctattgagat aatcatgtgg tttttgtctt tggttctgtt tatatgttgg 24960attacgttta ctgatttgcg tacgttgaac cagtcttgca tcccagggat ggagcccact 25020tgatcatggt ggataagctt tttgatgtgc tgctggattc ggtttgccag tattttattg 25080aggatttttg catcgacgtt catcagggat attggtctac aattctcttt ttttcttgtg 25140tctctgccag gctttggtat caggatgatg ctggcctcat aaaatgagtt agggaggatt 25200ccctcttttt ctattgattg gaatagtttc agaaggaatg gtgccagctc ctccttgtac 25260ctctggtaga attcggctgt gaatccgtct cgtcctgggc tttttttggt tggtaagcta 25320ttattgcctc aatttcagag cctgttattg gtctattcag agattcaact tcttcctggt 25380ttagtcttgg gagggtgtat gtgtcgagga atttatccat ttcttctaga ttttctagtt 25440tatttgcgta gaggtgttga tagtattctc tgatggtagt ttgtatttct gtgggatcgg 25500tggtgatatc ccctttatca tttcttattg cgtctatttg attcttctct cttttcttct 25560ttattagtct tgctagtggt ctatcaattt tgttgatctt tccaaaaaac cagctcctag 25620attcactgat tttttgaagg gttttttgcg tctctatctc cttcagttct gctctgatct 25680tagttatttc ttgccttctg ctagcttttg aatgtgtttg ctcttgcttc tctagttcat 25740ttaattgtga tgttagggtg tcaattttag atctttcctg ctttcgcttg tgggcattta 25800gtgctataaa tttccctcta cacactgctt taaatgtgtc ccggagattc tggtacgttg 25860tgtctttgtt ctcgttggtt tcaaagaaca tctttatttc tgccttcatt tcgttatgta 25920cccagtagtc attcaggagc aggttgttca gtttccatgt agttgagcag ttttgagtga 25980gtttcttaat cctgagttct agtttgattg cactgtggtc tgagagacag tttgttataa 26040tttctgttct tttacatttg ccgaggagtg ctttacttcc aactatgtgg tcaattttgg 26100aataggtgct gtgtggtgct gagaagaatg tatattctgt tgatttgggg tggagagttc 26160tgtagatgtc tatcaggtcc gcttggtgca gagctgagtt caattcctgg atatccttgt 26220taactttctg tcctgttgat ctgtctaatg ttgacagtgg ggtgttaaag tctcccatta 26280ttattgtgtg ggagtctaag tctgtttgta ggtctctaag gacttgcttt atgaatctgt 26340gtgctcctgt attgggtgca tatatattta ggatagttag ctcttcttgt tgaattgatc 26400cctttatcat tatgtaatgg ccttctttgt ctcttttgat ctttgttggt ttaaagtctg 26460ttttatccga gactaggatt gcaacccctg cctttttttg ctttccattt tcttggtaga 26520tcttcctcca ctcttttatt ttgagcctat gcatgtctct gcatgtgaga tgggtttcct 26580gaatacaaca cactgatggg tcttgactct ttatccaatt tgccagtctg tgtcttttaa 26640ttggggcatt tagcccattt acatttaagg ttaatattgt tatgtgtgaa tttgatcctg 26700tcattaggat gttagctggt tattttgctc gttagttgat gcagtttctt cctggcgtca 26760atggtcttta caatttggta tgtttttgca gtggcttgta ccggctgttc ctttccatgt 26820ttagtgcttc cttcaggagc tcttgtaggg caggcctggt ggtgacaaaa tctctcagca 26880tttgcttgtc tgtaaaggat tttatttctt cttcacttat gaatgttagt ttggctggat 26940atgaaattct gggttgaaaa ttcttttctt taagaatgtt gaatattggc ccccactctc 27000ttctggctta tagagtttct gccgagtgat cagctgtcag tctgatgggc ttcccttgtg 27060ggtaacccaa cctttctctc tggctgccct taacattttt tccttcattt caactttggt 27120gaatctgaca attatgtgtc ttggagttgc tcttctcgag gagtatcttt gtggcgttct 27180ctgtatttcc tgaatatgaa tgttggcctg ccttgctagg ttggggaagt tgtcctggat 27240aatatccaga agagtgtttt ccaacttggt tccattctcc ccgtcacttt caggtacacc 27300aatcagatgc agatttggtc ttttcacata gtcccatatt tcttggaggc tttgtttctt 27360tctttttatt cttttttctc taaacttctc ttctcgcttc atttcattca tttgatcgtc 27420aatcactgat actctttctt ccagttgatc gaatcagcta ctgaagcttg tgcattcatc 27480acatagttct cgtgccatgg ttttcagctc catcgggtcc tttaaggact tctctgcatt 27540ggttattcta gttagccatt cgtctaattt tttttcaagg tttttaactt ctttgtgatg 27600agtttgaact tcctccttta gctcggagaa gtttgatcgt ctgaagcctt cttctctcaa 27660ctcgtcaaag tcattctcca tccagctttg ttccgttgct ggtgaggggc tgcattcctt 27720tggaggagga gaagcactct gatttttaga attttcagtt tttctgttct gttttttccc 27780catctttgtg gttttatcta cctttggtct ttgatgatgg tgacatatag atggggtttt 27840ggtgtggatg tcctttctgt ttgttagttt tccttttaac actccggacc ctcagctgca 27900ggtctgttgc agtttgctgg aggtacactc cagatcctgt ttgcctgggt atcagcagcg 27960gaggctgcag aacagcgaat attgctgaac agcaaatgtt gctgtctgat tgttcctctg 28020gaggtttctt ctcagagggg tacccggcca tgtgaggtgg cagtctgccc ctactggggg 28080atacctccca gataggctac tcgggggtca gggacccact tgaggaggca atctgtccat 28140tctcagatct ccagctgtgt gctgggagaa ccactactct cttcaaagct gtcagacagg 28200gacatttaag tctgcagagg tttctgctgc cttttgttca gctatgccct gcccccagag 28260gtggagtcta cagaggcagg cagccctcct tgagctgtgg tgggctgcac ccagttcaag 28320cttcctggcc actttgttta cctactcaag cctcagcaat gacaggcgcc cattccccag 28380cctcgctgct gccttgcagt tcaatctcag attgctgttc tagcaataag tgaggctcca 28440tgggcgtggg accctccgag ccaggcgcgg gatataatct cctggtgtgc catttgctaa 28500gaccattgga aaagcacagt attagggtgg gagtgacccg attttccagg tgctgtgtgt 28560cacagctttg cttggctatg aaagggaatt ccctgacccc ttgtgcttcc caggtgaggc 28620aatgcctcgc cctgctttgg ctcatgctcg gtgtgctgca cccactgtcc tgcgcccact 28680gtctgacaag ccccagtgag atgaacccag tacctcagtt ggaaatgcag aaatcaccca 28740tcttctgcat tgctcacgct gggagctgta gactggagct gttcctattc agccatcttg 28800gaaccaccct acagtttctt tcaatacttt tcatgttcaa tgtcttacac ttaaattttt 28860caactacatg aaatttattt tgatataaga aatgaggttg ggccaggcgc agtggctcac 28920acctgttatc ccaaaacttt gggaggctga agtgggcgaa tcatgagatc aggagttcaa 28980aaccagcctg gccaatatag tgaaacccca cctctacaga aaatacaaaa aattagccga 29040gcgtggtggc gggtgcctgt aatcccagct actcgggagg ctgaggcagg agaatcgctt 29100gaacctggga ggtggaggtt gcagtgagca gagattgcgc cactgcactc cagcctgggt 29160gacagagtga gactacatct caaaaaataa ataaaaagac aaatgaggtt ggacaatagc 29220ttgatttttt aaaaactcca agccagttgc ccaataatcc attttgccat tgatgtgact 29280tgaaactaaa cacccatatg tgcttttact taattctgct cacttctcag tttcactgat 29340gcactggtac tatgccattg gaggcaatac agcattctaa tgcatttaca gatatggaag 29400agaaatcctc cccttttatt ttcattcaga aattgcctgg ctattctcac atgtggtctt 29460accccagtca gacccaacac ctcactttta taacaaatat ttggtaatgc accctttgtt 29520atatgaaagg agatatttgt ggataatgta accccagtct tcatgataaa caaaaaggcc 29580cagctgattt ccaaaatgca ccccagttta gaatcagtct ggcaagtatc acatgaaatc 29640ctattggtat ttgattggga tcacacagca tttgcaaatc aatttaagca acatttctat 29700ttttacaata ttgtggcctc tagccccaat aacaattatt tctcttcatt tacttatatc 29760tttgtgtctg ggcagggtcc tgccaggaaa cagacggcat gttaaagtga gaaactgatg 29820agttcagcaa agtgactatt tatatatttg gacaggattt aaggaagtaa gaaaggatga 29880tgcaacactt cagaggggag tctttccgac ctcaggctga aggagaagga acgattactg 29940gaattcaggg aggagagcat caccaaacaa gagcttcatt agaggactgc agccaacgca 30000gggccaggcg gagggagcca gagggaggca ggctctgctc

tccctcttcc tgcccttcag 30060tttccaacca cggcctccta ttggccaaac ccaaccagaa gccagcgagc aaggggctac 30120tgatgaagca catatagctc agcctccaga gacacagaac aggataaaag gctgagacag 30180tgggtctggt ggggcaaaga gaaagcttgc acctgccagg taaagcatta tagtccccat 30240cctcccccca ccaccttagt tcttgtgcat ttcccatcag ttttcttcca aagcatttca 30300gatctcactg atttgaatgg gacctcttct tctattacca atttggatca gtaattattt 30360atgtatgaga aaactattga tttttacata tcgttgcata gcagagtggt ttaataagga 30420gacatttggt ttttactgcc tgtgtgggta tcacttgcta tgtgacttga ggcaaatcca 30480atatttcttc tgttataaat tccagtattt gtaaaaaatg ggtaataaga tctctatttt 30540atatagtttt agtatttaat gagataatac atataaagtc attaaaacag tgtctggctc 30600ataaaaaacc ctcaataaat gtcacttatt actgtatctg gtttttgagc tgctctattg 30660caccattgag ttttcagccc agtatatgtt aaccctgatc attatctgca gaagtccccg 30720tgccacactc tacatcatcc aaattctctc caggtggact aagtagatta aagaacttta 30780aacataacta ccatattttg gctctatcta caaaatgtcc aataatcagt taagaaagga 30840acaattctct tggggcccac actttgagaa gcaaatgcag ctgaactttt ttagaggaaa 30900gtgagtgaac caactggtag ctttgccact gcttaaaaac cagcatcctt tccagctggg 30960tctaagacag aataaggtaa atttagatat gtctctaata tatctataga acagtggttc 31020tcaacccggg gtgtttttgc cccttagggg ataatttgca atgtctggag acatctgtga 31080ttgtcataac tggaaggggg cagtgctatt ggcatctagt gggtatagag caagggtgct 31140accaaatatc ctatggtgca acagagaatt atctggtcaa aaatgtaaat agtgctgagg 31200gtgagaaacc ctgctataaa aacgaaagaa atttggtcta cagagttgtt tggatttaga 31260caagacgttg ccccaatagt ggtgatagaa ataagaggaa ccccgtgctt ttgcaaagcc 31320catatctggg gtggcttaaa taatcatgct cctccccatc ccccgacctg atctttgtag 31380ttggaaactc cagggctggc tgcctgtagt ctttgtgact acacttcctg cctcccatca 31440cttcatctca gaagactcca gatataggat cactccatgc catcaagaaa ggtattttaa 31500acattggaac acatatagat aatttaagta ggtagatgta tgtgctgtta taaggaagtg 31560gggaggagag aagagggaac cgaaatcata tgcacaaaaa ttttttttag aatataaata 31620aaaaatgtgg tagtctaaaa tgtcaattct tcaaagataa agttaggctt tcagtaacgt 31680tagaaatggt tttctggaat atgtctccag tctacctaac tttgaggaag taaatactgt 31740aaatagatgt ttcaaacgca ttttaaagca atgatcctag catgtcttta agctacagta 31800ttgtgctgtc tttgaaatgt aaactttgat gtcttctctt tctcttagtt gatgctattg 31860ggcccatctc aagctgatct tggcacctct catgctctgc tctcttcaac cagacctcta 31920cattccattt tggaagaaga ctaaaaatgg taagaacagc tcagagaacc ttaaaaagtg 31980ttatctgtaa tctttgtgga aacaactgaa accagctggc aagagcaata ttgaagaatc 32040tgtacttagg ttatttgctg ggggaaagtg cttcctgata tttcacaatt ggcattaatg 32100aagggggcat gtcacaattt cagattaatc aacgcttgct ctgttcaact tcctacaaga 32160attaaatatg tgctgtgggg aggaggagca gatgtttgaa ttggggacat agcttctatg 32220tatctcattt cttcagccta caattttggc tttaaagcca taacaaatca ctgaattact 32280gaagttactt tgtgcttttt ccagcatatg gtgttgtctt aatgactgtg tggatgaaag 32340tgtgtgggca ggctcatagc aataaaatac gggaaatccc cgggcttgag tgctgtcaaa 32400gaaaactaaa tttggacagt agataaagat actatcagga ctattgcaat cggcagaaag 32460agacctcagt atagaaaggg gctcaattcc aaatacagcc aaagaccagt aaagatttct 32520ggccaaggag tagagtgggg gtcagtggat ggaaaattac taagaggaaa catcaagggt 32580aaaaggattc tggctaaacc gacctgacag gattcttgct gaagacaggc cagggtgatc 32640agacctcacc tgtggatggt gggagatgag gaatttgatc agatattgag ggtgatcaca 32700taccaagagg agtggattat caataaaatg acttagcagg attcctgctt gaactgggca 32760atgcaaagat ggacatgaag ccaaaggccg aagcctaggg gtgtagtaga gcctgattaa 32820gttgaattaa ggagagtctt tgtcagcgct ggctctccca gtcactagtt gggggggcct 32880tgtgcctgtc atcaaagtcc tctgaaactc aatttctctg actatgaaat aggcattaga 32940atccctcccc tgttgccttc cagggccact gtgaggctca aataatagac tatttttcaa 33000gtcctttgca agtggtatga tgcaagtgtg agttattagg tatgccaaaa cttagtcgga 33060aaaagacgtc aagggccttt ttctgaaatt attttgtcac ttaaatcaga cacattctag 33120atccgaatgt tagctcctag gctcattttg tgtcaaagtt ctaatgaagc attaaccatg 33180gggctattgt tacaaaggaa acaactgctt acggtttcat ttcctagaaa cccagatgtc 33240tattttaatg caaacctatg cccacatctg tctttgcccc ttgatgggtg gcataatggg 33300aatgatagta atacagagag ctcacatttc ttgaccactc aactatcatg ctgagggcta 33360gatagacatg attctatttt ggcctcaaag tagccctata aggtagagat aacgaaactg 33420gggctttgag aggttaagga gcttgggtgg ctctgaaagc tgtgctgaag actcttctgt 33480tcttcctaga ccaagcccag cacacacgca ataaagatga ggttggatat gatggcttcc 33540tactcaagta caaaggggaa atagtatatc ttttctaaga aaagacgtga aaataatttt 33600caatataaga aattcaaaag gcaaaaaagc acagggaaaa tattcaactg tattgagtca 33660tatggcagat cctttgatct agagattaca cttttagaaa ctcttcttaa agaagtgacc 33720atgagactgg ataaaaaaat gtggcacata tacaccatgg aatactatgc agccataaaa 33780aggaatgaga tcatgtcctt tgcagggaca ttgatgaagc tggaagccat tatcctcagc 33840aaactaacac aggaacaaaa aaccaaacac cgcatgttct cacttataag tgggagctga 33900acagtgagaa cacatggaca cagggagggg aacaacactc actgaggcct gtaggaggag 33960ggtggggcag gagagagcat tagggtaaaa agctaatgca tgctgggctt aatacctagg 34020tgatgggttg atctgtgcag caaaccacca tggcacgttt aactatgtaa caaacctgca 34080catcctgcac atgtacccca gaacttaaaa aaacaagcaa taaaataatt ttaaaaaaac 34140aaaagaagtg atcgtggaca tggaaaacta tttaccaaga tggtcagtgc agccaggcaa 34200aaaaaaaaaa aaaaaaaaaa tcatgtccca tgttgggaag gggtgaatta attgtagtag 34260actcattaaa tggaatatta tgtaatcatc aaatcatgtt ttttaaaata atactgaatg 34320acctaagaaa gcactcatgg tataatgtta aatgaaaaaa gcaagctaga aatggataag 34380taccgtgtat tcctcatgtt tttactgcac ctgctaggca aatactagat gctcactaaa 34440tgttggataa tctgtgatga tggtttacat aaacacatgt gttgcatatt ctaatttcat 34500tcaacatccc tactttataa ccattttaca gttggcaaat cagaggctca tgaggtcaag 34560tgatttatga aagtcagaga gctcttacat gacagaacaa ggacttaaaa ccaaattttt 34620gtactgacaa agccttggct gttactagaa tgcttctcac catgtgaaat agatgcaggg 34680atgggaaatt actattagaa gggaccatct cccaaaatgt caatagtggt tcagcaaatt 34740taaaagtaaa aatattattc tgctcttaac ctataggaaa tttctttatg gctaaaaaaa 34800ggttattaag taatcaattt attaaattaa tacaatctga ttatttaaaa atttggaacg 34860ctgtactaaa attaaaaatc atcattacag attaaccagc cagtacctct gcaccccaag 34920aataaataat gtatatcccc gaaactcacc gaagtttagg gctggggttg gcaaactatg 34980gcccatgggc tatatcccac ctgctgtaca gctcatgagc taaggggttt ttttttaatt 35040gttgttttta aaagactgaa aaatatcaga gcaaaattac tattttgtga catataaaag 35100ttacattcaa gtttcagtgt ttacaaatgg ttttattgtt tgagtatttg tttacttatt 35160gttgataagt gcttttgcac tacgatggca aactattcaa ggagttgggt agtgtgacag 35220agaacctgat ggcctgcaaa gattaaacca tttactaact ggccctttac agaaaaagta 35280cgtcaggccg gggcttatag aaaacaaagg gataaggtat aaggtcaaat aggtttgaga 35340gccctatggt ctttggtgac tgttgtgatg cataatagct gttgagttcc taatttatgt 35400aagacaactt tatatccttt tattctttta gtttgaaaac taagtctgtt gggctaaaat 35460gataggaagt aaatgataac tctctccttt ttttaaaaaa aagcaagtgg tttacaacct 35520tgtacttaaa cgttttggtg acataatgaa actgatattc atggtatttg tactttacag 35580agattaaact aaaattaaaa atatttcaaa attcacaaat aggggatatt tgttaataaa 35640tctatttggg aaattcctag cagaggctca gtctataaaa tgaatagcat ttcagcaact 35700tcccttattc acagtgcttg gttattctct agggagacat acacaacaca tctctagtta 35760ccaaacaatt cagtgtgata taaacatggc aaaaagtcaa tgaatttgag ggcaaggttt 35820ccagcaatcg ccccggccat tgcttacttc ttccatgccc tttctaagtt ttcttcagcc 35880aggcagccat cccctctggt ttctcccaga cccccgctgc aggctccccg ccatcacaga 35940aagcccctcg ctcacacgtc ttggctcaag caactctttg tcttagaaat gcagatccca 36000acatttcctt ttaaactcag gcaacttggc ttttttctgc tctgtgatct tgaaagtcgc 36060ttggaggaac agctgagtgc atggggctgt tgtcctctca gggctaacat gttgtagccc 36120agggggtgcc caggggcctt tctgactggt tggttagttg ggtaaaagag tagagtcagg 36180agagcaggaa atcctttctt aactcactat aaaaataaaa gcgttcccca ggcctcaaat 36240agtctcatct caagataaat ttccttttgc caagattgct gctgaaaata atccattgta 36300gccagataat agctatgcaa agaatatata atagactggc aggggcatgc ctaccgattc 36360aatacagaaa ggtgagggtt tcatttgctg gggtgtagtg ggtgggagaa ttccttattg 36420caatcacact ctacttctcc atccagaaaa ctctccaacc ctcctggagg actctccatt 36480ttctcctctt tctcctcctt gtgtacctac ctagaccatc tgctcccata tgtcctgtct 36540gacttcctgt tccagttacc tatcactgcg taagagatca cctcaaaatg caatggcttc 36600aaacaacaac aatcatatac tgctttctat catgggtcca ggagttgact ggactcatta 36660ggcagctctc ccacagggtc tctcttgggg tggcagtcag gcggtgactg cgactggaat 36720cacctgaaga ctcactctcc aggtctgatg cctgggctag gagactcaac agctaggtgc 36780cgaagcagct gcagctcctc aagtgtctct gtctccatgt ggtctctcta atatggtggt 36840tgtcgtatag ccaggcttct tacaagggtg atgactcagg actccaaagc aagtgggtga 36900gagaaaggga gagagggaga aacagggaga gagagagaga aagtgtgtgt gtgccagtac 36960gcgcgaggtg aaagctgtat tgcctgtgaa ctacccacca tgtctttcgt cctcttgaca 37020ggaaacctcc tagaaatgtt tgctgtctcc aaatccctct ccttacgttc ttccaagaac 37080tttgaagtca tattttatgt agctactcct tcaaaacata tctggtgttc ggccagttct 37140tacgccctcc agcactgcta cctgggactt ctgcttgaat gactgtaata gcctctcaac 37200tagtctccct gctttcaccc ttgcccctca ctgtctattc tcaacacagc agccagcagc 37260atccttctca aatgtaagtc agaccaactg attgtcagct caaaaatttg caatgcatct 37320gcattccacc cagagcagag accgccatcc atggaatggt agagaaagcc caacatgctc 37380agggacactc cctctctgac ttcatctcct attgttctcc tacaccccct gcttcagcaa 37440tattggcccc gttgccattt ttgtgaatat tctagcatgt tttcaccttg gggcctttgc 37500tccaggctaa tccatctgtc tggaatgcat ttcccctgga tgtctgttat ggatgacttt 37560gtcctttcct tgaggtcttt gtttagatat caacttctta atgatgccta tccaagctgc 37620cctatttatc gtcacaatcc taccccacat tcctgatcct tttcactctg ccctgttttc 37680tttttcagta acacttatca cttgacatgc aatatcattt ctgacagtta tatatttttg 37740tgattattta gagaacataa gctatagttg agtggaaatc ttttctattt tgtccactga 37800tgtcccaaac acctagagaa gtacctggca tgttgcaggc atcaataaat acttgttgaa 37860tttttccttt ttcacaattt ccttctacgt tgttatgatg agatcttatt tcctctgtaa 37920tttgatttta aaagttttaa taaaaaacaa tacatattat ttatgataaa aagtcaaaga 37980gtagagaagg gtataacata aaaatagaag tccccctctt cccagggaag gcccctttat 38040accactgccc agaagaaatt gctattaaag gtttcttgtg tattctttcc tacttttctc 38100tgcaaataca aatatatgca tatatattta tcataaatgc attatatgtt atatgttatt 38160ttaatgctgc tttaaaaatc ccctttattt tttgtaactt agtagtagat catgcatagc 38220tttttatgtc gatacccaca gctctaccac attcttttta agggacattt gatattttac 38280tattggtagt ttcccatttt taaccattct ctcaaatcaa tggattgtca tgtaattctt 38340cctattctta ctatttcaga aagctgaatc aaactagcaa aatagtttta tctaaagaca 38400tataaggccg ggcgtagtgg ctcttgcctg taatcccagc actttgggag gctgaggcag 38460gcagaccacc tgaagtcagg agtttgagac cagcctggcc aacatggtga aaccccgtct 38520ctgctaaaaa tacaaaaatt agctgggagt ggtggcggct gtctgtaatc ccagatactc 38580aggaggctga ggcaggagaa tcacttgaac cgggtaggca gaggttgcgg tgatccaaga 38640tcgggccagt gtactccagc ctgggcgaca gagtgagact ctgtctcaaa ataaataaat 38700aaataaataa taaagacata taatgcttac tttaaagaaa aacaaaacaa aacatgtact 38760agttattttt ttcctccctc tgtggaattc ttagaaggtt tatggtagtt tgaagctttg 38820catggaccat tttgaaacag cagcagcctg aggttccagg gggttatgaa gactcccagc 38880tgaggacaga ccctggcaga taagtttcag ggggctctac accaaccatt agagtcatag 38940aataagcaca atagaaaagg accattaagg tcagttagcc aaactccaga gtttgttgat 39000gagaaagtca aggttcagga taattcagtt ggtagccctg tagcagacag agagactgaa 39060aacaaatctg actttcagtt cacgtggtgc taacccctag aataaataaa cacgaggaga 39120aatcagacta atcccagtct tcttctaact tgtcacaaga cacaaaccac ttaccttcac 39180ttcctcattt tttccatcta atagttccca gttatataca tgtccttctc actcctctga 39240ttgcaaccag acatctctta caagtttaca aagttttgaa gataaaaacg ctatttggaa 39300agcgtaaagt taaaaacagc ttggtaaatg tttttttttt tttctattag taattcgatc 39360tctacaactg taaatattgt ggtaggaatc taatacagat ctaaaatcag taaaattcaa 39420tcttgaatat gggcttcagt cctgccatca aaatagtgca tccaggtgga taggttttgc 39480caccttgaag agttgtttat tcaaactttt gtttgaagag taggaaagca gtgttacctt 39540taggcctgac ttagcccttg ccccacaatc tattgttttt tctcaccata gatttccctg 39600acagcagaga gagagttctg tgctcaagag atacacacag cttctgacaa tagagcagca 39660gagtatttgg ttcctaattg agcaggaatg gtgtttgact catcatcatt tccctacttt 39720gtctagcaca gtaccttgca cagagtagat tctcaataat gtttgttgaa tgactgtggg 39780agcatataat tcataatgga gacaaagctc aatgaggctt taaatttcta aatccacaaa 39840atgccctcat gtaacattgc tggatgatat ggtttagctg tgtccccacc taaatctcac 39900cttgaattgt agctcccata atccccacgt gttgtgggag ggacccagtg ggaggtaatt 39960gaatcatggg ggcgggtttt tcccatgctg ttctcatgat agtggataag tctcacaaga 40020tctgatggtt tcataaacgg cagttcccct gcacatgctc tcttgcctga cgccatgtaa 40080gacgtaattt tgctcctcct tcaccttcca ccatgattgt gaggcctcct cagtcatgtg 40140gaactgtgag tccattaaat ctctttttct ttataaatta cccaaactcg gatatgtttt 40200tattagcagc atgagaacag actaatacaa tggacattgg atgcaattca tttaaaaaat 40260catcttaaaa atatctttct tttttctccc tcaagttggt cccactcaaa acataaacac 40320accatttttt tttttttttg tcttgagaca gagtcttgct ctgtcaccca ggctggagtg 40380cagtggtatg atcgtggctt actgcaacct ctgcctcccg agttcaagca attctcctgc 40440gtcagcctcc tgagtagctg ggattacagg tgcatgccac catgcccggc taattttgta 40500tttttagtag aaatagggtt tcaccatgtt ggccatgctg gtctcaaact cctcacctca 40560ggtgatcctc ccgccttgga ctcccaaagt gctgggattt catgtgtgag ccagtgtgcc 40620cagccaccat tttttaatac ttgtaaattt ttcctataaa aacaaaccaa tttctctatg 40680ccccaaaacc gctaagtagc acaaaataga aacattagag taccaagaat acttgaactg 40740aaaaggaaat taatcaaaat gcagacacac attataccaa gtgcatttgc tgtagctgtg 40800taaggcaact tgaatagaat tggtcaacaa tgagtctgaa tcttggtttg aaattgcctg 40860tctgatctct gcttcctcat cagtaaaatg agaatattta tatggccttt caacttcagt 40920gtgagggatc aatgatgtaa tataaacaac aagtctgcct tagaacctgg cacaccataa 40980gtaataaaag gcagccaata ttttaaaaaa tacacaaatc atggtctgat ggctgtccaa 41040tataaattct ctattttcca ttttaactaa agagacgata tattgagaaa atagaaacac 41100ctgtgtgtat gaaatcaccc attcccattt ttacaataat tagtttgcta attgagcatc 41160caaatttacc cagtgtattt gcatgtgtaa ttagctgtga ttcaatacca aagccaggcc 41220tatcatggta tactatgcta ttttacaagt caaattactg aaagatgcat gtctttaggc 41280aatcattaca aataaaaaaa aaaaaaccga agcaaaacaa aataacatag attatttgta 41340tcagatggac aaaacagacc tggcttgatg ccgaaccctt aaatctcaaa ataacgatag 41400ttgaagctaa ggttccagct taagtctgaa gcaggtagtt tccaatggct tgaaaggaga 41460aatttctaca ctgaaggaaa tttccattgg aataaaggaa tatttcacac ttttaagtca 41520tcttctctag atggtctttt gggtatactt tctctttaaa taacagattt agaagcactt 41580tgttcatttg tttagaatta attccattca caagtttaac acagcctaag gtttggtcta 41640gaccaggggt ctgccagcta tgacctctgg gctaaatctg tcccttcacc tgcttttttt 41700tttttttttt ttccaacctg tgagctaaga atgggtttta ctattctaat aaatagtgag 41760ttcatttttc tccctcacct gcttgatcag agcccaactt tctcattgca gttaatcttc 41820cttctggcat ggatcttgga atgcaaactt gctgggatct ccgagttcca ggcttcccgt 41880gcagccggtg tggagagcca agagatgttt tgtttggcat aaagcattcc aagggtcagt 41940gggcttgggc tcaactattg agcataggac aagggcagcc ccatcctgac tgtgactctt 42000cccacaagag acaaacgagc tctgtgcttt cactggggtt tcaggttcaa agggacagag 42060cgtctgagaa aaaggattat gaaagagtcc gtctgcagct ccacttcccg tgcccttcca 42120atgataccat cctcgtttct tctgtggcat gctccccact tcaatccttc cttcagaggc 42180cccaaaccct cctggtctct ccttgtcacc ttgtgaaaat ctgatcttca gggaaaaatt 42240ccttactatt tatactagta taatgtgaat cttctatggg attttaagaa agttcaaagc 42300cttggtttac tcagcaaata tttagcttgc actcactatg tggcgggcat cctaatgatg 42360gagtatatgt aaagacaaaa aaagtttccg gacctcaaag tgttctccat ctataggggc 42420agatgactga gttgacatct cgagaagtag aatagcagag tggctaagag tgccagctct 42480gtctcaatca cctaggtctc acctcagcat taatttcact ttcctcattg taaatgagca 42540tatctcttag aattgggata agcattaaat aatatagact tggaatgaat ttgcttagaa 42600ctaattccat gcacaagttt atcacagcct aaggtttggt ctagaccaga ggtctgccaa 42660gtatgacctg tgggctcaat ctgtcccact acctattgtt gttgttgctg ttgtttttta 42720atgacctgtg agctaagaat gggttttact attctaatta gttacattct caatggttat 42780ttaagtacct ccataatatc ctcaattttg cctaaaatat ttaccatctg gccctttaca 42840gaataagttt gctgacttat tggtctggac caatgctatc taataaaact ttctgcaatg 42900atgaaaatgg tctctatctg tacccttgaa tacagcagcc actagcctaa tgtggctttt 42960tgagctcttg aaatatagtt agtgtgacta agagattgaa ttttaattaa tttaaattta 43020tggagccaca tgtgactatg acattagagc agctctagac agcctgaagt ctaaagactc 43080tatgctttgt cggtgctccc ctctctcaat tgaatcaact accctgaggc tgcatgagtc 43140aaggggaagg ccacactctt caatcagatt ttttgccctg gactggcttt cattgtctac 43200tagaaaatgc ttaatgggaa gtgcttagaa aatgtacatg ggcatacact taattaatct 43260aagttgctgc tttgtctgta tccattaaat ctgctttatt ttggggtaaa ctacagtaga 43320agttggcttt ttcaaccctg caaagcctta aaattcagga tgtcttactc aacttaaagt 43380gtagagttgc agccagagca caactgtatt tccttctagc cctgcttgca gaatggctaa 43440cttcagtcct atttcatttc tcttgtaaga ctgctaaaaa cagtaagaag ccaccaacat 43500cattatgaat attgccaaat catttcgcct aagagtaaag tcacagttgg catgtgttct 43560gccctccaag acaagatagc ataggtgaca gttttatcag atatcttgtg atggcataat 43620ataggccacc cagctttcca gcctctgata tctgagtctt cccaatagcc tgatgacatc 43680cgcatcacat attttaggtt cgctcatgga cagtaactta tttccaaatt ctatactggt 43740taaaattagg tttgcatttg tgcaatagaa aatccaattg acattggctt agcataacaa 43800ttttttgatt tctcataaac tcttggcagt cagcaggtcc aagccattat ttctgctctg 43860ctctctgagg tcatataagg aaggatctgg atgctctggg tcatctacgt catctaactg 43920gttgctgtgc catccctagc tcatttttct catgtgcatt gcccaagatg gctggctaca 43980acatccacat tacaagaagc caggtggaag cagacaggag aaagaggaga aaggggaact 44040gccccaccgt ttaaggacat gtcccagaaa ctgtacacct cacttcctcc caaatttcac 44100tggctatcac ttagtcatat agccacactt agctgcaagt gtgtctggga gatataatta 44160tttttcacag tggtatatgc ccaactacaa atggaggttc tgtcattatg agatgagaga 44220aaggcagaaa acatgttgag agatgtgtag caatctctgg actccacggg gataaaaaag 44280aattgagagt atcaaaattc aggatcaaaa tcaaaattaa agataaaaaa tatcaataac 44340tatcacctgg aataagaaca acgtacagtt cagctacaca tatacaagtg gcagcatctt 44400gtctggaagg aactaatggt ctttctacat tgtattttag atatgtattt ttttttctcc 44460cttccacagg attttgagct ccttaagggc agagactttg tgtctcctgc tcctagtagg 44520catccaacac gtatctgtca actgaaagaa tgaatatgag tcagtagata catattagaa 44580ttctaatatc cactggctgg gtccttggtg tgtcccatat tgttgtttct gtgtccatca 44640ttcttttgca gggtatcttc tactgggcac agaacctgcc tcagaggggc atatgggtaa 44700tgaactacca agaaaggagt agaacccagt tcttccaacc ctccacccag agtgcttttc 44760acaacctcat gtgtaataag tgcagtagga gatgagagga gggagtgatt acttctgtct 44820ggtttgatcc cagaaggttt tttgaagaaa gtgtttttga atgagacatt atgaaaacag 44880agcttcttaa accttttccc ccaaggaatc cctgggcaga tagaagagac agaaatctga 44940cctctgctta gtctgggggt atagactgaa ggaacctact caaaggagaa atttttctca 45000tttttcttta cttcacgatt catatatgca ggcattcatt ctttcattca tgtatctcac 45060agacataacg aggtcctaat taagtgccag gcattgtttt

acatgagacc acaagaggcc 45120ctaccctctt gcagcttaca ttcttgtaca gaatagacat catacgaata agcaacataa 45180atcatcaaga taatttctga ccgtggtaag ggctatgacc gaaatcaaac agggtagtca 45240gttacagagt gcatatacct ctctgtgcct cagttgactc atctgtaaaa tggagataat 45300aatagaggtc taggctaggc atggtggctc atgcctgtaa tcccagcact ttgggaggcc 45360gaggtgggtg gttcacttgg ggtcaggcat tccagaccag cctaaccaac atggtgaaac 45420cccgtctcta ctaaaaatac aaaaattagc caggcctggt ggtgcatacc tgtaatccca 45480gctacttggg aggctgaagc aggagaatcg cttgaacccg ggaggtggag gttgcagtga 45540accgagatta tgccattgca ctccagcctg ggcaataaga gcgaaactca gtctcaaata 45600ataataataa taataataat aataataata gtctataatt ccaaaaccca aaactgaaag 45660ctttgtccta actcagttga ttgcaaacat aatatgatct gaatgcattt ggaggtagat 45720cttgacctga actgaagtta tttattcttt ttaataaata aatgagttat ttattctttt 45780taataaataa atgagtcatt tattcttttt aataaatgag ttattctttt taataaataa 45840taaactgagt tatttattct ttttaataaa taataaataa ctgagttatt tattcttttt 45900aataaataat aaatgagtta tttattcttt ttaataaata ataaataact gagttattta 45960ttcttttttt tttaataatt ccacttagag tggacaatcc tatatgtcac tgcagaaatt 46020ttgtgtgttt gattatggaa tgctgcccca ggcctcaata gttattacat aatttagggt 46080acatgtagcg tattaccttc taaaatttga aaaattccga attccaaaac acatgtagca 46140ccaaaggttt cggataaggg attgaagacc tgtagtatcc attattgtga ggattaaatg 46200aatgaatata tggaaaacac ttaaaatgat gcctggcatg tggtaagtgc tacgtaagtt 46260aactactatt actattatta tcactattct tacatgagaa gatatttaga taagttggtc 46320agggaaagcc tctctgagga tgtgtcactt gaataggcaa ctaaggggtg gtaatgaccg 46380ggctgtggga agaggaggag aaagatgatt tcaggaatag gaaacagcaa gtgccaagac 46440tgtggtggtt acaaggctgg cttgaatgca gaacagaaaa cagaccagat ggctgatatg 46500tggtaaagga ggggaaagat ggctcaaggt cagagaggta ggctgaagtc agaacaccct 46560tgatataagc aatggtagag actttggatt tcatttaaag tgtaatagga agacattata 46620gttgatctga ttcaggttta taaagaacgc tctgatgctg ttggatgaat gaattataga 46680ggagaagggg gagcagggag agcaatttgg agtctagcat agtggtccag atgagaccta 46740atgactaatt ggagttggga ggtggtaata gtcaaagaga aaagtggaca ggtgcgagaa 46800aaaagtttag aaataagtgg ggggcggggg aggttttctg attaatttgc attctaattt 46860ataatatgtc actgtgtaga ggctaaaaat ttcacagtca ttgtctcagg tgtgttaagg 46920ccagtggcgt gctggacccc acttgaaatt ggccatggag ggaatattta cactatagaa 46980attgacaaat gctacaaatc aagacaacaa atcaggcaaa gcttcttgtt aaacatttac 47040catcacacca ctggtgaagg tgacttgatt tttccacaac taaacttcct tcatttcaca 47100gcctccattt tccctgatca cgaaaacact taaactaggc acatcctcgg aaacgcagta 47160tgaggactgc tgtgtcaatc acttcatgtt tttaactcaa ttcagcgatc ctcccacttc 47220ttcccaggct ctcatttagg tacatgggaa tgggatggga agagggacct ggttcatgat 47280tgtcatttac ccaccttggc cccctctgaa gtacaactcc actctctgct ttacaatatc 47340actctgggca gcattaccaa ttgcctcctg atagtgggat ctatgaaccc attatgtctt 47400tggacaaaag catagccagg ggttgggtcc agggcctggg atcctataac cgtacaaatc 47460ctattatcag ggactataaa atcctattat cagggaccat agccatccct ctatcttgac 47520tcaactcctc ctccctgagt agtgaacatt tttcctaaat ctctgagaaa gactggtgct 47580ctagaaagat gtaccatatt tatttaaggg cttcctgtac ccactggcat attgccatat 47640attctgaggt atctgagtgc tccttttgag aaacatagcc ttaaaggata agtagaaatc 47700tggtgggtga aaatggtagg gaagaggact tctaacggag ggacttgcaa gtcagggaac 47760ttgggtttat cgactagtga ggctagtaga ggaattcaat caggtaagcc ggacaagtag 47820acagggtaca aattatggaa gactttggat gccatgataa aaagcttcag ctcatactgt 47880aaaaaataaa ataaaataag aaggttgggt gcagtggctc atgactgtaa tttcagcact 47940ttgggaggct gaggtgggac gatcgcttga gcctgggaaa caatttcaag gagttcacag 48000caagaaactg actgattaag gtttgggaag cttgatagat agggtagact gggaaagtga 48060gagaggaggc tttggagtgg accaaggata gagggatctc agctgatatt atgtcagcta 48120aaacctcaaa gcaaggagga tgttaagaac aatgaaggag gtcagctgga ctctcaatgt 48180ttttaacgat agggaggaaa agataggggg gtgacaagaa gaagagacaa ttttgtacct 48240ctaactccaa caaactttag acctgaaaaa tcccttctga gccatcttgc attggagaaa 48300aaaaattgct tatttacctc caattagagg aattaaggga agtaggattt ttttgttttt 48360cttttgagac agggtcttgc tctgtcaccc tggctggggt gcagtggtgt gatcacggct 48420cactgcaacc tcaaactctt gggcttaaga ggtcctccca actcaacctc ccgagtagct 48480gaactacagt tgtgtgccac catgcccagc taatttttta ttttctgtag agagaggggt 48540ctcacgctat gttgcccagg ctagtcttga actctggcct caagcgatcc gcctgccttg 48600tcctcccaaa gcgttggtat tagaggcatg agccaccaca tctggtggaa gtaggcattt 48660ggtttcttag ataacaacat gattggttga ttcagtcact tgggaagata aaagcattaa 48720ctgagctaga tccctatggt agagacacag gctggaccac tccatgcgta agtactaaac 48780taaaaccagt gttctggagt agacattgct agaaatcctg aaacttgaga gccagtccac 48840ggttaaagca ttctgtaagg cagagccagt ggaaggtaat aaggtgattt ttaaagctct 48900tctgcacttc ccatattccc ttttagggcc tttctcccta gggtcccagt gtctgtcatg 48960ctaaacctag atgcacaaca atcatcttta tgggtagttt cccatatgtc ccagtttgcc 49020tgacagactc ttggtttatg cctatagtct tggtgtaatt attaccagcc ccacttcatt 49080cttgtaagta tactaatgga tcagttatac ggttcctctg attatgtatc acctaggcag 49140tgccctgact ctactactat ctcctctcca aatttatgta atgtaaaccc aatgtgtagg 49200gaaaatgctc atcctaaaat ctccttggag gggataattt gcaagattct ttgcaaaaac 49260aatccaagac aagagccaga ttatggaatg tcagtgccag aatggcagga atgtatgttt 49320tctaatcaaa tgccacttac tactgggtaa ccttgggcta atcagttaat attgctgagc 49380gatgtcttca tttgtaaaac gggaatctta gaatattctg agactcaaat actatgaaag 49440actcatgtaa tgtgtaccag ggcaggttta gcaggccgac ataaattgca ctaaagtctt 49500catgtgttat ttttcatggg tgtatccata ttctaacatt tcttcaccct ccaaatttca 49560gactttggca gtgaatctat ggctctgcaa ttttagtgtt ccatgtaaca acgaatagga 49620aaatgctgct tctaccctct cgaaagctat tttgctaaag agctaagatg ctaaaagcta 49680aatatgtaac taaatagttg caaatctcag taactgacaa atacagtcat gggtcactat 49740agggctcgag gaagttccta tactttctag agaataggaa cttcggaata ggaacttccg 49800cgccgcacac aaaaaccaac acacagatca tgaaaataaa gctcttttat tggtaccgaa 49860ttcgccaggg agctctcaga cgtcgcttgg tcggtcttta ttcgaacccc agagtcccgc 49920tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat 49980accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg 50040ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa 50100tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac 50160gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc 50220gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt 50280acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag 50340cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg 50400agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc 50460agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg 50520cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac 50580cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga tcgtctgttg 50640tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc 50700atcttgttca atggccgatc ccatggttta gttcctcacc ttgtcgtatt atactatgcc 50760gatatactat gccgatgatt aattgtcaac acgtgctgct gcaggtcgaa aggcccggag 50820atgaggaaga ggagaacagc gcggcagacg tgcgcttttg aagcgtgcag aatgccgggc 50880ctccggagga ccttcgggcg cccgccccgc ccctgagccc gcccctgagc ccgcccccgg 50940acccacccct tcccagcctc tgagcccaga aagcgaagga gcaaagctgc tattggccgc 51000tgccccaaag gcctacccgc ttccattgct cagcggtgct gtccatctgc acgagactag 51060tgagacgtgc tacttccatt tgtcacgtcc tgcacgacgc gagctgcggg gcggggggga 51120acttcctgac taggggagga gtggaaggtg gcgcgaaggg gccaccaaag aacggagccg 51180gttggcgcct accggtggat gtggaatgtg tgcgaggcca gaggccactt gtgtagcgcc 51240aagtgcccag cggggctgct aaagcgcatg ctccagactg ccttgggaaa agcgcctccc 51300ctacccggta gaatgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact 51360tcaagcttat aacttcgtat gcattagccc ttctttgcaa aacacaactg cctagtttac 51420caaggagagg cctggctgtt taaattgttt tcatatatat cacaccaaaa gcgtgttttg 51480aaattcttca agaaatgaga ttgcccatat ttcaggggag ccaccaacgt ctgtcacagg 51540agttggaaag atggggttta tataatgcat caagtcttct ttcttatctc tctgtgtctc 51600tatttgcact tgagtctctc acctcagctc ctgtaaaaga gtggcaagta aaaaacatgg 51660ggctctgatt ctcctgtaat tgtgataatt aaatatacac acaatcatga cattgagaag 51720aactgcattt ctacccttaa aaagtactgg tatatacaga aatagggtta aaaaaaactc 51780aagctctctc tatatgagac caaaatgtac tagagttagt ttagtgaaat aaaaaaccag 51840tcagctggcc gggcatggtg gctcatgctt gtaatcccag cactttggga ggccgaggca 51900ggtggatcac gaggtcagga gtttgagacc agtctggcca acatggtgaa accccgtctg 51960tactaaaaat acaaaaatta gctgggcgtg gtggtgggtg cctgtaatcc cagctacttg 52020ggaggctgag gcaggagaat cgcttgaacc cgggaggtgg aggtggcagt gagccgagat 52080cacgccactg caatgcagcc cgggcaacag agctagactg tctcaaaaga acaaaaaaaa 52140aaaaacacaa aaaaactcag tcagcttctt aaccaattgc ttccgtgtca tccagggccc 52200cattctgtgc agattgagtg tgggcaccac acaggtggtt gctgcttcag tgcttcctgc 52260tctttttcct tgggcctgct tctgggttcc atagggaaac agtaagaaag aaagacacat 52320ccttaccata aatgcatatg gtccacctac aaatagaaaa atatttaaat gatctgcctt 52380tatacaaagt gatattctct acctttgata atttacctgc ttaaatgttt ttatctgcac 52440tgcaaagtac tgtatccaaa gtaaaatttc ctcatccaat atctttcaaa ctgttttgtt 52500aactaatgcc atatatttgt aagtatctgc acacttgata cagcaacgtt agatggtttt 52560gatggtaaac cctaaaggag gactccaaga gtgtgtattt atttatagtt ttatcagaga 52620tgacaattat ttgaatgcca attatatgga ttcctttcat tttttgctgg aggatgggag 52680aagaaaccaa agtttataga ccttcacatt gagaaagctt cagttttgaa cttcagctat 52740cagattcaaa aacaacagaa agaaccaaga cattcttaag atgcctgtac tttcagctgg 52800gtataaattc atgagttcaa agattgaaac ctgaccaatt tgctttattt catggaagaa 52860gtgatctaca aaggtgtttg tgccatttgg aaaacagcgt gcatgtgttc aagccttaga 52920ttggcgatgt cgtattttcc tcacgtgtgg caatgccaaa ggctttactt tacctgtgag 52980tacacactat atgaattatt tccaacgtac atttaatcaa taagggtcac aaattcccaa 53040atcaatctct ggaataaata gagaggtaat taaattgctg gagccaacta tttcacaact 53100tctgtaagct ttattgtgtt tcatagtttc cgttcttctt ctgtgagaac aaggataatg 53160gcattaaaaa atcagctttt ggtcattata aattgtcttc tattaaaaca catatacaca 53220taaaatcact tgaagacaat ttaaacatct tctgaaatgg atcaagagga agggaaactg 53280aaaataatgc aactcagaaa ccacagagta ttttgacatg aggttaagca ccgtggtttg 53340ttgtaggaaa ataacagcac accaacagat ggtttttatc tgaattcttt ggtaatcttg 53400acatgtcatt cttctaactt tctgagggcc ctcagtgcag ttttgtagga ctggagctgt 53460tcacagacgg tccccacaaa gctctgaacg tggggcttct ctgctgactg gcctctggtt 53520ggctccaccc cggaaggaac tcccagattc tccatgaatt ccgcttccac catcaagcct 53580tggtccaagc ccctttcaac cttgacttgg ccaggaagtg tcctttctct tcagatagat 53640actacacctt agcaagactt ggcattttta gaatccaagc caagggaggc acttggcaag 53700gcaaatgtta tggatgagaa aaaggcaaaa caagtgtctg cagtttgtag aggagagaga 53760ggatgagtct gattgtagcc ctgaccctga gtcaggatct ctcggcccca tttgcaggtc 53820tacttccagc tccatctgtc tggacactct tttaggtcca gatcatctct tacatgtggc 53880caaggaatat agagtatgca aggggatgta gcgacctgag agtgtgagta acttgtgccc 53940atctccaagg aagctgtgat ggggatagca aggacacaca ctcttcttat ttataatgcc 54000tttccccctc ccatgagata tgctttttat ttacttcctc cttctcatcc taagtcgggt 54060gaacaagagg accaggttgc acatcctact acttatttat ggcccaattt taacatgggg 54120gtggagttga ggttggaatt gttcctccgc ctctgctgca catgctcagt aagcaagaac 54180actgttgatg ggaaaggctt agtcacagac agtgggagca catccctcct tggagctttg 54240ggtcgctgtg ctccagaaac agttagttat agcacaccct gctcctggca tctactggaa 54300ggtgaagccc ttgaccctaa gaaacattgg gaatgatttg taccctccaa agtccaatag 54360ctatgtcgga gggaaacgat caaagaacat gattgaggag actcaaacag agatgtgctt 54420cagacaacac caagacagaa aattaatcca ttttaccaag ttaacaatgt actgaaggcg 54480aacaagagac caacccacct gccaaccaac gctatgaaga aggagggttg atcagtctgg 54540ctaacatggt gaaaccccat ctctactaaa tatacaaaat tagctgggcg tggtggcaca 54600ctcctgtaat cccagctact cgggaggctg aggcaggaga atcgcttgaa cctgggaggc 54660agaggttgca gtgagctagg atcaagccac tgcactccag cctgggtgag agagtgagac 54720ttggtctcca aaaaaaaaaa aaaaagaagg agggttaaaa agagaataag tcccaaactc 54780ataagatggt gtggaaaggg ccctggtgac ataggggcca cccatgccag tgagaatgaa 54840atcacaacag ggcagtttca cactgtttca ggtttttatt ttttcttctt cttctttccc 54900tcctttcttc ttttgcctcc ccctccctct cggtttcctt tttggctcta gacacccaca 54960gcaagtgtca agcaatgtac aagaatgaaa agaagacagc cgttgttgca ggtggatgct 55020tctgtttgga aggtgtggtt ttgtgtgcac ttttggttgg aaacctatgt ctctctcaca 55080cacatgtccc ccacctgctt cagtgagcat gatcttctaa aattggctcc atggaatctt 55140ccctaggtga cactggctgg actctgacac attcaaattc attctttcta cttatctcaa 55200ctttcaagca ctaattcagt ctttaagatt cccctaatct caggttgtaa ttcatttcta 55260acatgtttgt catgacatca gtggttgcat gtcttcatat gtttatcact gccccctctt 55320gggaatttat attaattaaa cattttggaa aagctcagac ttaaaatgac tgagaaggtt 55380catgttacag tgtctcacac tgaacactca ctaaaaatag tctggtaaaa tccagaagga 55440aatgggaagt cctgggggcc tcatggcagt tttcagactt ccctagagca aagatgagcc 55500agacagcggc tcccacaaac agaaaatggc tctcaatatt cttctggcca gtggcaacta 55560ctgagggtaa aactagacag agacggctaa ctaggaagcc atattgctct tgtatgacac 55620atggctgctt tacttctcac tggccaaggc aaaaccatga ggcattgatt ccaggggatt 55680tcataacagt gaattgcatg agttgacttg agcaaaagaa gtttgtagta acatctccat 55740gaaccatatc tgaggcccat tatccagggg gaccacactg ctgagaagat tgcttgggct 55800gttttgggag ccagaactag gttgttgtcc tgctatcacc ctgggatggt ggttacatcc 55860ctgcaccagg tccagatgga ggcccaccaa acagaactta gtaacaggtg acatgcatta 55920aataaaacag tttatagctc ccatctctct tctgggccta cttttctcat gattgatttc 55980tttgcctttg gattgaggca ttgtaactaa aattcccagt taatttacaa cgttccaagg 56040gttgtttcaa cacatcactc ctgtccatta acacattgtg atggattaat ttgtgagatg 56100atcaactctc cagaacaggc cacctaagaa aacagaacac aggaagcatg ttctgaatgg 56160agaagatgca ttttaaagta atcccaactt tggctcctgc tcatgagggc aggcccacaa 56220agggaatgac aggcaaggag ggagtgagga ggatcttagg aagaagggct caagatgaag 56280tcactggtat cccgacaatg tcattcagta aggggtggag gggaaggagg aagggaagtc 56340acatctatta agcagcaaat gcaaattaaa tgcttccttt attcccttga atcttcacat 56400cacctctatg aggctagtat tagcagctcc atcttaaaga tgatgggtta actcagagat 56460aaaggtgaga tcagagaggc aaagtcaact gctcaatctc tgggacccag gagtccgcat 56520ccctggaata cttttgaagc aactggcttg cagagcagtt tgggaagcac cacatcttcc 56580tttgtgacac cagggactgt tattacaggg atcactttag taaggaaagc ccagaccccc 56640aagctgtgct caatttcctt ccttttgtgt ataaactttg gtgctctcaa cctacttccc 56700gggagtggag ctgcacttaa actcaaattg cttcccgctg ggcactgttt ttggacaggt 56760ataatttaac tatataggac tggcatttct gtttagggcc caaaggtgaa gacaatttga 56820acaagaacac tagggcagaa ttagatagac aggggcaggt ggttaatgga attgaagtct 56880cgtaagtcct gatagagttg ctgattacat ttgtgggtaa aaggatgtct ttaccgtaac 56940ttccataaca tcttactgat agacgctgtg gttctactta tgtggcgcca agcgccacct 57000agtggggtgg gaaatccctc ctggatgctg atgttctgct tctgagaccc ttagacgggc 57060ttccaaaggg ggatgtggga agaaaatgta agaacagata tagtcatgtt tttaaaagta 57120tgtctgtggc tatttttatc atatacccag tgtattaata cagaaatatg tagggctgtg 57180tgttgaatgt ttactgatgg gagggcagtt tacaaagaag ttgaaggcca cagactattg 57240aatatttgtt tctatcattt atttccattg tttttgcttt aagtctcaaa atggggccag 57300tcatgaattt ccttttgccc atgcagatgt agatgtatag ccttgagtta actgggtgac 57360cgatcctctt tttcctacat ttccattttt tatagactgt ggcccccatt ttaaaactgt 57420atttgagtat cagtaatctt tcttgcacat tctagctgaa cattctctct ctctcatatt 57480tccgagcaat ccaaccacta accattccct tctgtcacca ccccccgccc tataaatagc 57540attttgaagc atgcagtaac ttgccagttt gaggatttag gtctttttca tcctgcttgc 57600ctacttgact gtcagttcat gaagacagtt ggtgaacagg catgatgctt gggtgtttca 57660cgcccgttaa ttaagtcaaa gatgatgggg cgcttacttg ctaagatata tatacatgta 57720cacacctacc tatgtattct gtatgcacat atatatagct gtccatatat gtttgtgtat 57780atgcacacat atacatgcaa gcatgtatat atttgtgtgt attagaatta ctgataatta 57840gaattactga attactgatg gccatgtttt gcatagatta attgtaaacc taagtaagat 57900aaaccataaa aagtactcag cacagggtct gacatctatc aaacactcca taaataactt 57960tttaaattct ttttattact ttttaaattc tttttattac tttttaaatt ctttttattg 58020ctttggatat ctaatcaagt atataatctt tttttttttt ttttttttga gacagagtct 58080cactctgtca cccaggctgg agtgcaatgg tgcgatctca gctcactgca acctccacct 58140ccgagttcaa gcgattctcc tgcctcagcc tctccagtag ctgggactac aggcacacac 58200caccacacct ggctagtttt tgtattttta gtagagacgg ggttttgcca tgttggccag 58260gctgttctcg aactcctgac ctcaggtgat ccacctgcct cagcccccca aaatgctggg 58320attataggca tgagtcaccg cgcctggcta atcaattaca taatctcaag aggctaattc 58380tatattcttc atacagtgaa aatttttaac atttttcttt tagtatacag tatctgggaa 58440attttttcta ctcatcactt actctgtaca taaagtttat gttcaacatt tcatgtacta 58500tattcataca ttatttaaca atttttttaa tttaaagggt gacactcaac gtattgcttt 58560tccatttgtt acatattctc aatcaaaatt cgttttctct agatttaaat ccatcaaaat 58620ttccagaaat gtcccagata cccagacgtc ctgtaggctt taaatataat ttttcatctt 58680caataatctt ctatttacca taagaatgct gaccacttcc tgcttgtagc ttatctgagg 58740aaagtccctt ttagttgatc caagttccca gatttcccca attctgcttt ttcctgtgat 58800cccacaagaa gttccaaaag tatgaagtag cgccaaatgt taacttgcat aaaaccctgt 58860gatgatactt gtaagcatca gcattgagtt cttcctgttc ctacccaccc ctactcctga 58920tttcctcctg actctgggta taattggtgt tctccatggt cacatgtgag tttaaattct 58980tccagatgtt ttgataaaac ttcattttaa aagccatgtt gaaggagacc atagcagagt 59040ttgtatgcaa aggtatggag tggtttcttc tgagaatggg cccagcaggg cttacctgcc 59100tataagaggc actggtctcc aaagttgtgc caagtctagc cttcgtgaaa ttcacctggg 59160aatttagaat cccagttgga atgcttgatg tttaggtgtt tcatgcctgc taattaagtc 59220aaagacgttg gggtgcttac ttgctgagat atatatatat atatatatat atatatatat 59280atatatatat atatatatgt acacatccac atatatattc tgtataccca catatatacc 59340tgtccatgta agtttgtgtg tatatgcata catatacatg caagtgtgta tatattttgt 59400gtgtgtgtgt gtgtgagaat taccgataag ctcatagcca tgctttgtat agattagtta 59460taaaactaag tgttcagaat tgttggggct tgagggagct ggttactcac accctctcca 59520gctgagcaat tcacactgca gacagtgcag ctgagagtga gcaggtgcat gaattccata 59580ggccatagct tggacttgca ctcttgcaac tctccagtgc ctcccggtag caaagaagaa 59640acgcaagcca tcaatccaga acaaaggatc cctgtcacag agagctcacc ttagggcagc 59700agaaacaaac agagggtgca ctccacctat aggaaggctg tcccaagaga acaagagcca 59760ggagacttga ggcagcagca gcagggacag tttccccact gtcaccaatc ccacaagtca 59820ctctgtctct tgtgagaagc ataggaagga ggaaagggga agagggacaa gtccccaccc 59880ttccgtattt caggggttga tttcgaaatg tagggagaag agaaaagttc ctctaaagta 59940ccatcctgaa gctaataaat gaataaattc tcaaccagat tgtctacact gaggagacgg 60000gtctgtaaaa catggtgccg tgcttggact catgacatga aaaagactct tccctgcaga 60060gcagagacca aagcttgcag gtttcccctc attatgaatc tactgtacag aagatagaca 60120gagtccctac cttcatgggg ctaaagatat aatttccaag

acttggccgg gtgtggtggc 60180tcacacctgc aatcccagca ctttgggagg ccaaggcaga tggatcatct aaggtcagga 60240gttcgagacc agcctggcct cggtgaaatc cctaaacata caaaaaatta gccggacgtg 60300gtggctcatg cctgtagtcc cagctactca gaaggctgag gcacaagaat tgcttgaacc 60360caggagatgg aggttgcagt gagccgaggt tgtgccactg cactccagcc tgggtgacag 60420agcaagactt catctcaaaa aaaaaaaaaa agaaagaatt tccaagactc attattattg 60480attgggttct acaatttccc ctaggcattt ctgaaatggt atagaatata gctatgaaat 60540ttgtattctg cattacatag atgtaatatt cagaaactgt tgattgcatt catccatcca 60600tctatccatc catccatcca tccatccatc catccatcca ttcagtcagt cagtcaataa 60660atatttatgg tgtggccacc atgggcctgg catgatgctt agggtgagga gacactggcg 60720agccaaaata gaaatagtcc ctgttctcct ggagcttatt cagaaatcca ttaatcagat 60780tattgcactg aaggagattc aagtgtgatg aaggaaagga agaggtttct ataagagtga 60840tgacttggaa aagggagcga aaccagggag caggagaggc gggctgccct gaggaaggcc 60900acccaaggtg agatgtgaaa aatgagttaa ttgaaccagg caacactgaa agatgaccat 60960atttgggggc gggagacaaa gagtgtgtct tagtcatatg gcatttggga gccttcgaat 61020caccaagtgg atggagatgc tgagtcagta ggtgcccata gtctacagag ctcaaagaat 61080agagctgagc tggaaagatg cagttgaaca ctcctgtggc aatgggagta atggacacct 61140tgggcggggt cttgctactc aaaccatggt ccctggacca gcaacatggg catcccctgg 61200gagttcatta gacatgctga ctctcaggct ccacctcaga cctgctaaat cagacacaca 61260ggcagaagat gaccctgtcc catcaatgac aaggtttaca accagagatt ttcagggtga 61320ggataatacc acaatatccc agcagtgagt atatatttat tgtgagacag tctcagtgat 61380ttttatttta tttttttaag acagcgtctc actctgtcgc ctaggttgaa ctgcagttgt 61440gaggtctcag ctcactgcag cctttgcctc ctgagcacaa gcaaacctcc acctcagcct 61500ctcgcgtgtg cccccacact tggcgagttt ttttagtttt ttttgtagag acaaggtcca 61560ggctggcctc gaactcctgg gctcaagcaa tcctttttca ccttggcttc tcaaagtgct 61620aggattacag gtgtgagcca ctacacctgg ccagtcctag tgatttaggt atcaactccc 61680caacctgtta cagtggctct taactttgcc tgcaccttgc aattacctgg gcagctttaa 61740aaaaagacta tgtctgggtt ctaccataaa gattgtgatt taattggttt gggctacaga 61800ctgagctttg ggattttaaa gctggagggc gattccaaca tgcaggcaaa gttgagaacc 61860actgcttcag agtcattgct tgattgcaac agattctcaa gcatgagcag gcatcaaaat 61920cacctgaggg gctagttaaa acagattgct gggccctact tctcagcttc tgattcagta 61980ggtctggagt tggaacccga gaatttgcat ctcttacaag ataggtccaa gtgatgctgg 62040tcttgctcta ttgagcggtt gtcaaacttg actggctaat agagtcacct atgggtgcct 62100aggccttgtg ctagagcccc tggattaata gatatgaagg caaagtcctg acatctatgt 62160tttaaataaa aacccaccca gatgatcctg atgtagatga cctaaagatg aacttttgca 62220aaaccctgct ctagacacaa tttatattta taaccttaag cactgagatg acactgggag 62280gtggagtaat cttcgagaat gccatctctt ctttctcttt tccttcatcc ccattcaagc 62340tgatcctaaa ctgtatgcat tgcctcaggg ggcacgtgca ggggaagcaa cctagtcggc 62400ttttgtcttc tacaggaagg cccaaaggcc ttaggggatc atagaagagc ccgtggaaca 62460aagcgactct agggcatcaa ccaggtgtcc aggaagagaa gggatagggc tgcttttctg 62520ccccttctgg agaaagacgt gaagagggaa ggggaggaag gtaatattaa cagatcaggg 62580aggtctgtca gacctgagaa aagtgggacc ctgccaactc cacgtgagaa atgatcaatc 62640ttccacagag ttgagatgaa ggagttgaat taaaaaatca attgtgtacc ccgcttccac 62700acaccccact ctggtttgcc actgcatcag actcagaagc tgtacccaat ccctggcagg 62760caaggactcc tgtgaaagga aaataccttg ggcccccaac atcgctaagc taaaaggaaa 62820agtcaagctg gaaactgctt agggcaaacc tgcctcccat tctattcaaa gtcatccctc 62880tgctcactga gatagatgca tattctgatt gcctcctttg gaaacgctaa tcagaaactc 62940aaagaatgca accgtttgtt tctcacctac ctgtgacctg gaagccccct ccctgcttcg 63000agttgtcccc cctttctgga agaatcaatg tacttcttac agagattgac tgatgtctca 63060tgtctcccaa aaatgtgtaa aaccaagctg tgccccaacc accttgggca catgtcgtca 63120ggacatcctg aggctgtgtc acaagcacgc gtccttaact ttgtcaaatt aaactttcta 63180aattaactga gacctctttc aaattttcgg ggttcacact ccccactcct ccgagggtct 63240gagttgatct tctggagagt gaggtaggat gaaagctcag agacaaacac agaatggagt 63300cataaaaaac aaacgtctct gcacatttca gttggtggac taagtcacag cagatacatt 63360agacatatgt ttaactattt gaacattttt cacctgggta tatttcttgc ataattgtat 63420ttgttttgtt ttcaaaattt aactcagtaa gtagatttag aatatacatt gggaattctg 63480agctcgattt tgtatcattc atcaattata aaagtgaccc ctccctagca tagaacgtag 63540ttaaaatcct ctgtctgttc caaaatggga cgagcaaaag atcgtttcat gtttcctttt 63600cttacatctt aagcactttg gtgcctatag ggcaaactat gaatcaaagt tgttttttat 63660ttagaaactt ggatttcctt aactgaagat gctcaaaagc ctgccgggcg cggtggctca 63720cgcctgtaat cccagcactt tgggaggctg aggcgggcgg atcacgaggt caggagatcg 63780agaccacggt gaaaccccgt ctctactaaa aatacaaaaa attagctggg cgtggtggcg 63840ggcgcctgta gtctcagcta ctcgggaggc tggggcaggc gaatggcgtg aacccgggag 63900gcggagcttg cggtgagccg agatcgcgcc acagcactcc agcctgggcg acagagagag 63960attctgtcta aaaaaaaaaa aaaaaaaaaa agcctgaagc aaaactttta gacatgtgta 64020catttcataa acttaatccc agcactttgg gaggctgagg caggagaatc gcttgagccc 64080aggagtttga gaccagcctg ggcaacatag caagaccccg tctctacaaa aaataaaaaa 64140ttagctgggc atggtgtgtg cctgtagtct cagctatttg ggaagctgag gcaggaaggt 64200cacttgagct gagaatttga ggctgcagtg agccgcgatt gcaccactgc actccagcct 64260gggtgaaaga gcgacacact gtttaaaata taaaataaaa taaaatgaaa aaataaaatt 64320ttatactgta cgttgccaca caaaaattat tacctacgag tctgatcagg aagcccgaga 64380tattgggtgg tgggcgccat cttgtggcag catggttatt aacatcttgg ccattggctg 64440gatcttcaag ccaatgctga cacgtgaagg cgtttctgca gtccctgaaa aaaggtgcag 64500gtgcaggaag gaagaaacaa aaatgccttt ctcgaatgct aaaagcactg attctggaga 64560taggaagact tgcaaaagag aactagaaaa aaagaggaaa taaaacatat tggggggaag 64620ggcagaggag tcctgagact gtgaggaatc aaatgaagat tccatggaga agtagcctgt 64680ataacaggaa tggcccttct caaatcttct ccatgccaag ccccgggcct caaatctagc 64740aggttgttct tgtgcttttc caacagccct catatatttg aatcaattat ccccaacatc 64800ttcccacata ttttcagcat ttggtactga tccctctacc agatgccctg tccccaacaa 64860gcagcctcgc ctcctatatc tgcccaccaa tggatgcgtc ctcaaattct gtcctctcta 64920catcaaacat gttttatgtc accgacttca tcatcatcat ctcacaaaac ccagattctt 64980tcctcacaga gcctatctgc attcgttctc attccaagga gaggtggccc ttcccttcct 65040aaattgcaag cccacccacc actgcccctg gttcattcac tttccctact tgggaaagcg 65100gctccatgag gttgactctg tccgttgcac atgtgaccac tcttctccat cctgccggct 65160tttcctcctc acttgaacac acttctttta aaattatctt ccctccaaag atgtttgtcg 65220cagcattgtt tgtaatttca aaaaattagg acaagcacaa atgccaatca atagaggatt 65280cattaagcaa aataccacat gcataaccgg ctacagaaca cagccactga aaaaaaagaa 65340caagacagat ctgcatatcc aaaccagaat gctagctgga aaaaaaaaat gatgcagaat 65400agcacgtatc atgtgaactc agttttcaaa aagaattctg tacgtaatta tatgtcagta 65460tatacatagg aaaaatcttg aagaacatat accaaaccat taacaatggt caactctgag 65520gagtggaatt tggagagctt tcatctgatg tgttctacat tcctttattg ggatatttag 65580gaccatgtaa taacattata ctcagaaaaa ttttatatac aaaagaaact tcccttaatt 65640ctatcaatat catcctctgt ttccctttaa ttttcaccaa catccatcca tccatccatc 65700cattcgtccc catccatttt caccaacatc catgtgtaag accaggctgg atggatacaa 65760cagcccttgg gctcaaaact tgtacagtct ggtgaatgta acacggagcc accaaaatgt 65820catccagcag agaaagtggc aagtacgaca gagctttccg ttcagatcca agctttgctg 65880ctgaatttgg gtaaatgact gagcctttat gagcctgttt cctcctctga aaatggggat 65940tatagtaaca agcagagaga acaggagtgg tgggtgggta gcgccttaag taggtgttgg 66000tgcagaggac tccatcccat ctgggccaca gaccatcagg aggcacgtgg ggacttgggg 66060acaaagaaaa caagcagccc atatctgccg gcaggacctg gcaaagtggg cgtgacacca 66120acaagttgca tcccagaggt gaccacaaag cgcccaggat ttgcaaagct aaggtcaagt 66180ggaggcagga ggagacagtt tcaggaaaca cctgtggcct atctcacccg tgtcttccct 66240ggccagggaa gggcagccgc ttaaaggggc caagggaagc tgcagcgtgg ctcctaaaca 66300tggacgcaca gtagctgtcc tctcaggagc tctgcacccc cacttaaaac caggatcaca 66360agagatttgt gtaaaggaag cctcttgtgc atttcttcat ctgaatcgtt gacttaaaag 66420tttcttccac gtaagtcctt tcactttagc tttgtatata tttagcattg atttcacaac 66480aaacaactga ttccaggcac ttggtgtttt cccagacctt gagctttttc cagggttgca 66540cgttatttcc atatgtggta cttcgcggca gccccagtat ggggaagcca tttgctgcct 66600tatgaattgt ctggtcatcc actcgagagg aaagggctgt gtccagggct ggggaaggcc 66660ccaagagtgg ttcttcaggc ctactgttgg caacagccca ggaatgtgtg aagtctgtaa 66720caacaagcgt agtaataata attccagctc tgctagttga acgctcactt tgtgccagct 66780acttttctaa gagttccacg atatgaacaa tttaatcctc aaagccaaca tctgagaaag 66840atactactat tcttatccca atttgacaga tgagaaaact aaggctcaga gaggcttgtc 66900tgctggtgat ttgtgtgtct gggaagtgca cgttgtctgg caggcttgcc tggcagaagc 66960ggttcatgac aggtcttctc cattttggaa tcaagatctg tgtcagcatg gatggcaagg 67020ggaagagtaa ggaaagattg gtctaggaga tgcccaggag cttttggcct cacagtgcag 67080ggtggggaga gcagacctgg ccaccaagga ccagtttctg ggcttgggca gaagccaatc 67140atctggctac tttacaagca gatggagctt cttcctctca gtaaccttcc tgccctggtg 67200tcaattccat tttcattttc atcttttatg gtactggttg tgtggagaaa actgaaggct 67260catttggtat aagtaagtaa ctcattttta tgagtttgct tgacatagat actagactgt 67320atgtgtgtat atatacatat aaacatgcat atgtacataa aaaatatata tatatcatat 67380atacatgatg atacacacac acacacacac acacacacac acacatatat atatatacgt 67440atacaagcat gctttacaag gccaattgac tggtctacaa ttggctgaca cttggtggcc 67500tagaagccag ggtatgtgag tctcgctttt ctagaaagct gacaaactct ccagttccaa 67560ggatccttgc tcagtcaacg gctggaagtc atttttactt cgctgttttt tgtttgtttg 67620tttgtttgtt tttttagaca aagtctcatt ctgtcaccca ggctagagtg cagtggcact 67680atcatggctc actgcaatct ccacctcctg ggctcaagcg atcctcccac ctcagccacc 67740cgagtaactg ggactacagg tgcacaccac catgcctggc taatttttgt atttttttag 67800agacaaggtt ttgccatgtt gcccgggttg gtctcaaacc cctgagcaca agtgatcctc 67860ctgcctcggc ctctacaaag tgctggaatt acaggtgtga gccactgcac tcgatccatt 67920cttacttact ttctttactt tatttccaag caaatgtttg gagggaaacc aagagacttg 67980gatgcggcca gccgaggcct ttgggtttac aatcacaaat gtttttggtt tgcccatgaa 68040ggcccaggct gcactctctg atgtcacagg aatcacctct caaaccatgc accaggtctt 68100gaattccctt agggtgtgat ctttagaggt ccatctaggt atacccaccc aagccattct 68160ttgactgctg acaggccttc cttcataaca aggtgttcca cagtccattt atatatggat 68220gtcatctctg cccaccctgc tgccaatttg gttttctccc actcctgggg tgtaaggcaa 68280gatgaaacat atcacatccc gttctaaact ttattcttgt ggccaggggt cagcaaactt 68340tttctgtaaa gggccagatg gcaaatatct taggttttac aggccaagaa gcaaatttgg 68400catattatgt agctacttat atagtaaaat aaaaatttcc acaattatgt aattgatgaa 68460actcaaaatg taataataat aatcgaaggc agtttttttg tagtataggt ttaataatga 68520gaagaatgga atcatttttg gaggtgctaa cattctgctt ggttggaatt taaagttagt 68580gttctgtatc agcaaatcca ttgccaatgt tcatctaaaa atgttttcac ttctgggccg 68640gatttcgttc aaaggctgca gtttgctgac ctctgctctt ggttacacct tttgaggccc 68700ttgctctccg agcataaaat ggaatccatt tatcagacta aatcgggaag attaaatttt 68760ccagcctcac gaatgctcag ccattgactc actcgttcat acaatgaaca ctcattgagc 68820ttatactaca tgccaggtgc tggaggaggc atggggcgcc caggagaaag atgctcgctt 68880tgcggccaca gcccagtggg agggagaccc atacctaccg gtgctgtctc agaaacttgt 68940ggaacaaaga tgaagcaatg ttcatgttat tcgcctacat ctgtgaatta cacaaggaag 69000acgagtttga gaaatccgaa gttcagtaca aatttatggt aactttttta aaaaagaata 69060cactgaagtt ttcttagtga atggaataat gttccctttt tctcccctgt acacacaaat 69120acacaaaaac taacaaaaat acgtcgtgtg tgtctgattt gggttgtatt taaatcattt 69180cataaatgac tttttcccat aacttcagtt tcaaagtttt aaagcacagt caattaatga 69240tttggcaaca gctaagaaat cacaagttcc cttcttttca tgtaaacttc tgtaaaacac 69300acgctacgtt ctgctgatgg taaatagagc catttcagga agttagccag tttctcttct 69360cggccacctc ctgcatagag ggtaccattc tgcgctgctg caagttacgg aatgaaaaat 69420tagaacaaca gaaacatggt aagccacttc tatttcttta gcaaagcttt ccaacagaat 69480atggggtttc tgacccagaa atctgggttg gtggcaaatg gtgtgagcct agaaagtaat 69540aaatgggcaa ataaggataa aaattaaaga tcgaaacaac tgtaaatgca ggtaaagcgg 69600cttgctatga tctttaattt gtgcacacgt tagtataaag gaattagaga gtaaattttg 69660aaaatcaaat gcagtgatga tcttactaat ttggacagga aaataagaaa atttcaagtt 69720agaaattgaa ctggaaatat tacttactgg ccctaccaga gacaatatcc tcttccagaa 69780caacagggtt ggaagagaag gtgagggaaa tattcttcct ttgctatttc tgtagaaaag 69840gacaaactct cttccttcac atacataggt caattgctag atcctagtga agcctgagct 69900taacctactg ttggaggctt aaagttcgac attaattgct acttttcttg gtcagagttt 69960taaataatta ggttggtaca aaaaactgtg attacttttc caccaaccta ataacatgct 70020acaatttctg taattattat tttacactgt caagacatag caggtggtcc gtttttgtta 70080ttgtcaagaa ctgtcagact aaaaatgaac tttacacttc tttttaaatg atacattttc 70140tagaaaattc aatgaggttt aagagcaatt gaaaagtctg atttcaagag agtctcatcc 70200aaaatgtact atatattttt ccccaaagtc cttggagtta attttgacaa caatttaaag 70260tacacttaag tcttttgaag ttaatgggtc tgccacccag gttggagtgc agtggcgtga 70320tctcagctca ctgcaacctc cgcctcccgg gttcaagcga ttctcctgcc tcaacctccc 70380aagtagctgg gactacaggt gtgtgccacc acgcctggct aatttttgta tttttagtag 70440agacggggtt tctccatgtt ggccaggctg gtctcgaact cctgacctca ggtgatccgc 70500ctgtctcagc ctcccaaagt gctgggatta caggcatgag ccaccgcgcc cggcctgaag 70560ttaattttta tacccaccta atgttcatta tggatcttga aggtaaatta attctgcact 70620aaaattttac aatgctttac aaaatgactg taggtggccc atatggaatt cggtcaactg 70680ggccaatgac acatatggga ttgcagttga aattatccaa ttcctacttg atatttgtaa 70740gctgctgtga tagccagtat aattgtactg taagaatgtg gtaaatagcc ggggcccggt 70800ggctcacgcc tataatccca gcactttggg aagccgacgt gggcggatca cttgaggtca 70860gtaggtagag accagcccgg tcaacacggc aaaacctcgt ctctactaaa aatacaaaaa 70920ttagccaggt gtggtggtac gcacctgtag tcccagctac tcaggaggct gaggcaggag 70980aatcgcttga gcccatgagg tggatgttgc agtgagcaaa gatcgcacca ttgtactcca 71040gcctgggcaa cggagtaaga ctctgtttca aaacaacaac aacaacaaca acaacagatt 71100ggtaaataga gtaataataa aatcaaatta aacttgcaaa aaatggccac tttgctccca 71160ctggtggcca atggaggtca aggacctggc tgacctcctg cctaaaggca gaggttgtta 71220gccttcgcaa tggactcaaa tcagaggggg agctttcaaa actcctgctg cccagactga 71280accccagatc aatgaaacca aaatctctgg atacagggct tggcatttgt agcttttaga 71340gttcctaagt atctctactg tgcagccaaa gttaagaatc agtgccttag aacatcaaca 71400gttttttggt ccttttgtta aaaagcacag tccgtttttt taggtggcta gaaatgctcc 71460aggaagagct gaaatgtatt taccagccac cttggtttga ttttagaaag caaaatagaa 71520gttctaagta tgctttctct gaaaagctga gactgcagat aagagtgagg gcagttgatg 71580gagttcattc tcctctttca atcactgctt ctcatccttt cattataata atctaagaat 71640ctcagagatt atgaaagaga aagcagtctt atggaagacc ccagactcac agaatattag 71700ggtgtgtttc acagggaagg atgtcattac ccacagttag tctttgaaac gcagttggac 71760attatttgta agtgcatcat agtgtcgcct ccaggttcca ttgaggggaa cgtcattcca 71820atgcaacatc tctgagttca tctgggttat taaatggggt tgagggattt gttattttta 71880aattagtagc cccaatttag gactactcaa gaccatagga caagcctgtc caaccctcgg 71940cctgcgggct gcatatggcc gaggacagct ttgaatgcag cccaagacaa attcataaac 72000tttctgaaaa tattatgcat ttgtttttta gcccatcagc tactgttagt gttagtgtat 72060tttatgtgtg gcccaagaca attcttcttc ttccagtgtg gcccagagaa gctgaaagat 72120tggacacccc tgctataaga cacagtaata taaatacata acctgtggtt ctggattggc 72180attagcagat acaggctgtg ttgattttgc agaaagttac aaagagctgc tagttggtgt 72240gtatgtctaa aatcagtaga tttcctgtgg ttctaaggaa tgacaaagaa tctggaagtt 72300ctctgtggta gcctgctcag tgcagaaagg gaacgtggaa aatccgccac cagcatttga 72360gtcttggagg ttccacatag ggctatcagg tctctgctga tcactgaaac cagatcatgg 72420ccaactagcc ccttggcttc agccctccca attcattaac tactcaggta aatctagggt 72480cactttcaac tctaccacct accatctgag tgaccttgaa aacattcatc tctctgagcc 72540tcaggtccca tgtctgtaaa gcaggggcct catggacttc tttgggtttt tttgtttttg 72600tttttgtttc tgaggattaa acaaatgctc cctaccctat ttcccagcat ccagtaacac 72660agtttttcat atttttgtgt atgttaagtc aggacccatc tctttaatga taagtgcact 72720taatgtggtc atgttttctt ttgtcttcca aagctgttag tgaatccatt gaatttggga 72780tgggtaaaat aaagtatcta ttattaattg taaatttcat ctaaagtgac aaatcctacc 72840tgcataacca tttcttaatt tcctttcatc atgtatcagt ggtcaacatt gttaactgcg 72900aatgaatcag aatccatcaa aaattagaac tatttccagt ctggcaaaaa ttcagctctg 72960gttgaatcca aacattgtgc tgaagcagct aagtaattca actgaggaga ttaattacat 73020gttataatca ataggttctc ttgacacttc agtgttaggg aacatcagca agacccatcc 73080caggagacct tgaaggaagc ctttgaaagg gagaatgaag gagtcatctt tgcaaaatag 73140ctcctgcagc ctgggaaagg agactaaaaa ggtaaaaagc tgttaattcc aggaagacag 73200ctttacgccc ctcccagacc acctgcactg cacactacgt ggaatttatt ttagtctcac 73260atggcagcgt ccctaccttt gtgcccacac atctggtctc cgccctggct gcagccctcc 73320ccttcaggcg aattctgggt gtgtcctatc tgctcattgc aactcccagc gaatgagttt 73380tcagcgaagg cagactttct gacctgttct tcaaactgca ctggtctttt aaaaacgtgt 73440ttggtggcca tcagcatcca atttcagaag aaagatttgg gtgaggactg agagaggctg 73500ttgttgttgt gctgtctgtt tccttcagaa tctgcagaag aaaattggca ggtcatgtac 73560tgtggaccta accaaaggac aaatgatgta tggaaaatag aaaaactgtt gtgaaattgc 73620ttcctcatta gcaataactg tatttggcag ggagaggaga agttgggcac attttttttt 73680cttttttttt tcatgattca tacgttttct ttaaagaagt gggttttgct tttcactggg 73740tgctctaaga caaccccagt gaaagatctg gaccacgaag acccagtcat cctcataagg 73800gtgttcattg cagcaagctc aagggcatgc caggcaaagg ccttttttct ggcagcttga 73860acttgtctca gcagagggtt tcacagaaca actgtcattt acctgttctc tgctcttact 73920tgattcgttt cccaggactg ctgaaacaaa gtaccacaaa cttggtggat caaaacagca 73980gaaatatatc ctctcacagt tctggaaacc acaagtcaga aaccaatgtg ttgttggcag 74040ggttggttcc ttcttaaggg gctagaggga aaatctgttt catgctcctc tcccagcttc 74100tggtggtagc tagcaattct tgatgctctc tggcttgccg ctgcatctct ctagccttca 74160cctctcctca tgtgggtggc cttctttcct gtgtgtctat ttccaaattc cccttttctt 74220ataaggggac cagttattgg atcagggccc accttaattc agtagatccc attttaactt 74280gatgacatca gcaaagtcca aataaggttg tattcacagg taccaggggt tagaacttca 74340agttatctat taggggacac aattcaacct aaaaactccc cttttttgat tctctattct 74400gccacttcta ctcaatccag gttcttcact tcatcagctc ccaatctaat acttatctta 74460tttctagtaa gcatctcttc cttatcttaa ctggtccctg gggcctggcc cgagccccat 74520tataccatca gctgttgaca tcaagggtgg acttctcttt cggcacagaa ggcacagggc 74580tgtaggcttc agccttctct gctttgctct gccccatcta ctgttcatcc acctgctttc 74640cattttgcta aactttgtag aaaattcttg tcagctgttg tctcctccta cactttcttt 74700gatcttagag gattctattc ttttactatg gctttaatcg gagcacccga ctgttaggtt 74760caaccaacag aagttggttg tgctctctca ctctttcttt ctctctctct ctctttctct 74820ctatttgcat agtggtattt tttttttcct ctattttatt ggcagaattg ccatttctct 74880aagttattgt agagttgctg tttctctatt ttatttgcat atttctcttc tgccaggctg 74940gattgtttct attgattggt tctgctgtaa tgagggtgac ttctcattag tatccttctc 75000acttcatctg ggaccagatg ccctttgata tccttttgga gccacaactt ttggtagtca 75060gaggcatggg tgtggctcaa aggaagaact tggctcagaa ggtgcagctc ttgctgggcc 75120tttggtctct gctctgtctt ctgagatcag tggctgctgg gacctggggt tcccccatgc 75180cgggcatggt cacacagcac tcctatggac ttgagcagag

caccctgcaa agtgagcatt 75240agcaatccat tccaactctg tgcagtcctg cacggaatat agaaggtgga gcaatgacag 75300tctccccaac ttctctgcaa gcaacctgct caccatttct tgcccttccc atttatgtac 75360ttttcaaaat caggttattt ggaatttgtc gactcatgtt tcttacttca gtactttttt 75420gggagggcag cattagaaac ctcaaactct taactaaaaa atgtctttgg gaatgttctg 75480gccattttca tggcccacaa tttgctttaa gctgctttag actctcccag aggctatttt 75540catcccgaaa gaacagagca gagctcaaaa gactccagtt ttggtctcta gcagccccta 75600gaggatttcc ccctcaattc ctctctgcct tgtatgaaat agaattggat ttgaaatcgg 75660atgttgaggc cttacctcca ggctagtgag gccacacaag atggatcctc tggacccgcc 75720caagtgtcca cctaaacatg agttaccaac taacaatgtt ttgtttagca tgcaaaggga 75780gtggtctgga atctggcctt gccctgacat attctccttg ggccttttta aaaaaataat 75840ttgtgttaat ctgtagttaa aaattataat aaggacctga caaacactac ctcagtcaga 75900tgatcaaggt acacataaat agtgaaagtc atgttgatag catgcaccct tcatatgata 75960tggctagaat ggccctgcac ttctgtgatc ttcctcccct agactcatca gctcgatcta 76020atcataacaa aagcatcaga taagtccccg cccagggaca ttctacataa ccatttccct 76080tcccagttat atttttctcc acaatacttt ccaccatcta acattctatc tttcaaaatg 76140ggcaagtatt ttagcctggt ttgttcattg ttttatctgc aactcaaata cagttcctga 76200aataaaatat ctgcctaata aatatttaat gaatgaatga atatagcatt gccttatccg 76260tttaattgcc acatggtatt tcattgtgtg aacataatat cgtttattta cccagactac 76320tactcatagg catttagatt atttccggtc ttttgctatt gctaacagcc tttgcaatga 76380acatccttgt atacagacat ttgcatatat gagggtgtgt ctttaggatc tacttctaga 76440attgaaattg ccaactccaa gtatatgttt ccaattgtga tagatattac acattaccct 76500ccatcttaga ggtggtgtta atttagattc ctgccagcaa aatttaagag tgtttgtttc 76560cccatatcct caactgccta acagaatcag tgaaaaatgg tatgacagtg taatttttga 76620gtgaggttga gtatcttttc ctatgcttta agagcaattt atgtttcctt tttatgtgaa 76680ctgtctgtta atatattttt tcaatttttc tattgggtta tttgtctttt cattaatgca 76740tatacctgtt acatatttat accaagtatg tattaaatac taacatattg atgaaacaga 76800gcaaaaagcc tagaaataga tccaaataac agaagagtta gtatgtgata caggaagcct 76860ataaaatcag tgagcaaaag accatccaat taataacgtt agggtaaatg ggtctccatt 76920tagaaaaaaa taatgtgggt ctacacctca cattttatac ctaaacaatt ccagtgggat 76980aagaaaatga aatcataaaa aattactagg aaaaagatga gaaaattgtt cataaaactg 77040aagtgtggaa gatcctttat gccttacact gccctgagtg atctcattca tacccatggc 77100ttcaattgtc atgaatccca aattcattcc tctgtcagaa ctctcttctg agcttcagac 77160ccacatactc agctgcctac tggacacctc tacttgaata tcacaaactc aactcaaaag 77220caaacctgtc aaatttaatt actagtagcc ctaccccaaa caatcttcct gctcagtgaa 77280tgacacccat ccctccaggt gcacagacca ggaacctaga agtcactctg attgcatccc 77340tctccctcac aacctctacc tccctttatt catccattgc tatgtctctc aaatgtacct 77400cccaaatatc tcttgaacgc gttcttttct atctctattg ccaccaccct agttcaaact 77460cccatcatct catgactgaa gttctgtgcc ctcttgccag tgaacactgt agaatcaatc 77520taaacatggt gccaccctgc ttaaaaacct tcaaaggctc acatcacttc tcagatgaag 77580agattgggga gacgttggta ataggacaca aaatttcagt taggcaggag gaaaaagttc 77640tattgaagaa ctctattgta caatatggtg actatagtta ataacaacat attatacact 77700tgaaaatcac taagagagtc cattttaagt gttctcatga ccaaaaaatg ataagtatat 77760gaggtaatgc atatgtgaat tagcttgact gaggcattct acatgtatac atatttcgaa 77820acatcatgtt gtacatcata aatgcataca ctttttagtt gtcaatttaa ttaatatttt 77880ttaaacctac tctggccttt ttttcctttt ttgagacggg tggtctctgt cccccatgct 77940agagtgcagt gcgcaatcat ggctcactgc agcctccacc tcccagtctc aggcgattct 78000ccagtctcag cctcccaagt agctgggacc acaagcatga gccaccatgc cccgctattt 78060gtttttgtat tttttgtaga gatgggatct cgccacatgg cccagtctgg tgtccaactc 78120ctgagctcca gtgatccacc tgcctcagct tcccaaactg ctgggattac aggcgtgagc 78180cactgtgcct ggtccactct ggtctttact caagtccctg gctttctctc agtctcttaa 78240acttatgtgc ttagtaagat gaggactgaa aaatgtccac agaacatagt gacatggaga 78300tactgagaac ctcaacgaca tctccattag ccacttcctc tgtgccattc cagtcctctg 78360ggccccactg tggcaagcag tcctaccatg gcaaacatga aagctgatgt gccttgtctt 78420agacccacac catatctctc tgaattcctg tcccagggct tctctggagg tacagcctgg 78480gaaactcacg ggaatagaca cagggccttt gcacatgctg ctcccttttc ctgaaaaatt 78540cctttgacat cttggttgtg ccttacacat gcctactcaa ccttaggatt gcagttcagg 78600tttcactcct tttttttttt tctttttgag acggagtttc actcttgttg cccaggctgg 78660agtgcaatgg tgtgatcctg gctcaccaca acctctgcct cctgggttca agtgattctc 78720ctgcctcaac ctcctgagta gctgggatta tagtcatgca ccaccacgcc cagctaattt 78780tgtattttta gtagagacag tgtttctcta tgttggccag gctggtctcg aactcccgac 78840ctcaggtgat cggcccgcct cggcctaggt tccacttctt tatggaaatc ttccccagtt 78900gccttgacta ggccaaagtc ccctcttctt aggctcttac agtgtcatgc acttcttttt 78960tatcacagtg taaaccttgt aatgttgtgt ttaagtcata tctgttgtac ccatgagact 79020gggagccaat tcatatattg tgagtgtaat cgaacagact tcccaggcca cccactagct 79080aatcaaggca gggatgagtc cggaaagtga ctttgaaatc tagcaatgtt ggaacttgga 79140aatcacacag gctgagatct gctcaggtgc ctgaacaaat atagcattgc ctgtggcgtc 79200tccctcaaag tgccttgcat gtctgagccc cgttgcccct tcctttggtg tgcctgtgtc 79260tcccggtaca gatgtgaagc ctggagacct gtggctgcct ctgcaggagc tccatgtttt 79320caagccataa atcatcttag aattcatagc atctagatat attagttttc tattactgca 79380gaacaaatcg ctcccaaatg tagaggcttc aaagaatgcc cattgattgg ccttaatttc 79440tgtaagttag aatctgggca ggtttgcctg agttctccac tccaagtctc ataaagccaa 79500gctgggctgt catctggagg ctctgagtaa aaatttgttt ccaggttcat ccagattgtc 79560aggtgatttc agttccttgc agttgttgtt cgactcacta ccccaccacc accccgaaaa 79620cctcatttcc ttgctagctg cctgcagaga gccactctca gcttccacag gctgcttgca 79680ttccttgttg tggggccgct acctcctcaa gccagaaata gggcatccag ttcttctcat 79740gcatcctacc cctctgactt ttccttctgc cgataaccag aaaaaacgtt ccgccttcaa 79800acgctcgtat gattagacta agcccatcca gataaattcc catatgccat atactataat 79860gtcatcacag cagtaatacc cgggacaaaa ttcatggggg tcatcttaaa attctgccta 79920tcacaccagg tatagtagag gcttgtttta gtgcaagtta aacattaagc agcaacatca 79980cgatagtgct gcatttgaaa ataactacta gcaactgaac atgtctggga gttctgctcc 80040actttaattt ccatctcaaa aggagctggg ttttccttgg ctgttacaaa tgggcaataa 80100tgattgagct taagaataat caatgtccac ataaaaatct tttataacat agtgagagtg 80160tgacatataa aggtgttagt tcaccggccc taaattttag gagaattttt aaaaaggcac 80220ttatctggtt taatccataa taaagacatg agttgggctt tagtgaaaaa tctaggctgg 80280tttctgtgtt cagtgaaaga agatttgaga gttctcttaa ttacaaccct tgatcaaacc 80340taccacatta atctgtttat tgcattgtat ggttaccaaa agtgatatat tcagccctct 80400atttattaag aaacagttac agaaagtgag gcactctcct gtgttactga gggtgcataa 80460aaatataaag caccatgtgt cttccctaga gaagtttcaa aactagcaag caaatagcta 80520ttaatgctaa tgtttgtgtg atagggaaca tatgagtagt aattattcca caaacaattt 80580tttgagtgct gtttacattt gaggcacagt tcaggcacga ggatttcaaa aggagattgt 80640gtagcatgat ggcttgttaa aaatatgatt ttggaatcag atttgctcaa gtcccagtgc 80700tacagcatac catccttcaa aaaggtactt aagtctctga gtttgttttc tcatctgcaa 80760aatataaata ataagaggac ctactgcgtc atgttcttgt gagcattaat gtgggtgatg 80820aaatgtttat gaagcactta gcacaatacc tgacattttg tttgttatta ttatcaacat 80880aaagtgccca ctttccagtc atgcaagaag aaaacataat atatgtcacc atagaagtat 80940agaacaattg tgggaaatac cagtaagaga gatatagctg tataaataag gtaaagatga 81000ctgcctagaa gatctaggat gataccatat tagaagttgc atctgaactc tccttgggga 81060ctggccaaag tttcatcaag tgtcatgtca gtaggttggt gctataaata tatagcttgc 81120aaagctatag acttactata aaccatagct gtggtccagc ttagactcat tatggtggtg 81180gagtatcttg attaatggcc tctgcagaag cttcccaggt cttctcatca tcataatctc 81240agatagcttc atcttcaact tccttttttt tgttgttttt gagacagggt ctcactctgt 81300catccaggat ggagtgcagt ggcacaatca tggctcactg cagcctcgac ctcaggagct 81360caagccatcc tcccacttca gcctcccgag tagttgggac tacaggcatg caccactacg 81420cccggctaat tttttcattt ttttgtagag tcagggtctc cctatgctgc ccagtctggt 81480ctcaaactcc tgggctcaaa ccatctttcc acctcggcct cccaaaatgt tgggattaca 81540ggtgtgagcc accacacaca gcccatcttc aacttctttt agcaccatga agctgaacat 81600agtaaaaaag taaaatcatt ctggacctaa tctgatgcaa tttatttaat tgttaagtga 81660atgcacacat caaaattcat acaagtatgg ggcagcgctg ctaatttatt tacaaaacac 81720ctggcaaata ctgctactct aatactgtgc ttccactttt gattttcctt aggaaaacat 81780gttccttcag tcgtcaatgc tgacctgcat tttcctgcta atatctggtt cctgtgagtt 81840atgcgccgaa gaaaattttt ctagaagcta tccttgtgat gagaaaaagc aaaatgactc 81900agttattgca gagtgcagca atcgtcgact acaggaagtt ccccaaacgg tgggcaaata 81960tgtgacagaa ctagacctgt ctgataattt catcacacac ataacgaatg aatcatttca 82020agggctgcaa aatctcacta aaataaatct aaaccacaac cccaatgtac agcaccagaa 82080cggaaatccc ggtatacaat caaatggctt gaatatcaca gacggggcat tcctcaacct 82140aaaaaaccta agggagttac tgcttgaaga caaccagtta ccccaaatac cctctggttt 82200gccagagtct ttgacagaac ttagtctaat tcaaaacaat atatacaaca taactaaaga 82260gggcatttca agacttataa acttgaaaaa tctctatttg gcctggaact gctattttaa 82320caaagtttgc gagaaaacta acatagaaga tggagtattt gaaacgctga caaatttgga 82380gttgctatca ctatctttca attctctttc acacgtgcca cccaaactgc caagctccct 82440acgcaaactt tttctgagca acacccagat caaatacatt agtgaagaag atttcaaggg 82500attgataaat ttaacattac tagatttaag cgggaactgt ccgaggtgct tcaatgcccc 82560atttccatgc gtgccttgtg atggtggtgc ttcaattaat atagatcgtt ttgcttttca 82620aaacttgacc caacttcgat acctaaacct ctctagcact tccctcagga agattaatgc 82680tgcctggttt aaaaatatgc ctcatctgaa ggtgctggat cttgaattca actatttagt 82740gggagaaata gcctctgggg catttttaac gatgctgccc cgcttagaaa tacttgactt 82800gtcttttaac tatataaagg ggagttatcc acagcatatt aatatttcca gaaacttctc 82860taaacttttg tctctacggg cattgcattt aagaggttat gtgttccagg aactcagaga 82920agatgatttc cagcccctga tgcagcttcc aaacttatcg actatcaact tgggtattaa 82980ttttattaag caaatcgatt tcaaactttt ccaaaatttc tccaatctgg aaattattta 83040cttgtcagaa aacagaatat caccgttggt aaaagatacc cggcagagtt atgcaaatag 83100ttcctctttt caacgtcata tccggaaacg acgctcaaca gattttgagt ttgacccaca 83160ttcgaacttt tatcatttca cccgtccttt aataaagcca caatgtgctg cttatggaaa 83220agccttagat ttaagcctca acagtatttt cttcattggg ccaaaccaat ttgaaaatct 83280tcctgacatt gcctgtttaa atctgtctgc aaatagcaat gctcaagtgt taagtggaac 83340tgaattttca gccattcctc atgtcaaata tttggatttg acaaacaata gactagactt 83400tgataatgct agtgctctta ctgaattgtc cgacttggaa gttctagatc tcagctataa 83460ttcacactat ttcagaatag caggcgtaac acatcatcta gaatttattc aaaatttcac 83520aaatctaaaa gttttaaact tgagccacaa caacatttat actttaacag ataagtataa 83580cctggaaagc aagtccctgg tagaattagt tttcagtggc aatcgccttg acattttgtg 83640gaatgatgat gacaacaggt atatctccat tttcaaaggt ctcaagaatc tgacacgtct 83700ggatttatcc cttaataggc tgaagcacat cccaaatgaa gcattcctta atttgccagc 83760gagtctcact gaactacata taaatgataa tatgttaaag ttttttaact ggacattact 83820ccagcagttt cctcgtctcg agttgcttga cttacgtgga aacaaactac tctttttaac 83880tgatagccta tctgacttta catcttccct tcggacactg ctgctgagtc ataacaggat 83940ttcccaccta ccctctggct ttctttctga agtcagtagt ctgaagcacc tcgatttaag 84000ttccaatctg ctaaaaacaa tcaacaaatc cgcacttgaa actaagacca ccaccaaatt 84060atctatgttg gaactacacg gaaacccctt tgaatgcacc tgtgacattg gagatttccg 84120aagatggatg gatgaacatc tgaatgtcaa aattcccaga ctggtagatg tcatttgtgc 84180cagtcctggg gatcaaagag ggaagagtat tgtgagtctg gagctaacaa cttgtgtttc 84240agatgtcact gcagtgatat tatttttctt cacgttcttt atcaccacca tggttatgtt 84300ggctgccctg gctcaccatt tgttttactg ggatgtttgg tttatatata atgtgtgttt 84360agctaaggta aaaggctaca ggtctctttc cacatcccaa actttctatg atgcttacat 84420ttcttatgac accaaagatg cctctgttac tgactgggtg ataaatgagc tgcgctacca 84480ccttgaagag agccgagaca aaaacgttct cctttgtcta gaggagaggg attgggaccc 84540gggattggcc atcatcgaca acctcatgca gagcatcaac caaagcaaga aaacagtatt 84600tgttttaacc aaaaaatatg caaaaagctg gaactttaaa acagcttttt acttggcttt 84660gcagaggcta atggatgaga acatggatgt gattatattt atcctgctgg agccagtgtt 84720acagcattct cagtatttga ggctacggca gcggatctgt aagagctcca tcctccagtg 84780gcctgacaac ccgaaggcag aaggcttgtt ttggcaaact ctgagaaatg tggtcttgac 84840tgaaaatgat tcacggtata acaatatgta tgtcgattcc attaagcaat actaactgac 84900gttaagtcat gatttcgcgc cataataaag atgcaaagga atgacatttc tgtattagtt 84960atctattgct atgtaacaaa ttatcccaaa acttagtggt ttaaaacaac acatttgctg 85020gcccacagtt tttgagggtc aggagtccag gcccagcata actgggtcct ctgctcaggg 85080tgtctcagag gctgcaatgt aggtgttcac cagagacata ggcatcactg gggtcacact 85140catgtggttg ttttctggat tcaattcctc ctgggctatt ggccaaaggc tatactcatg 85200taagccatgc gagcctctcc cacaaggcag cttgcttcat cagagctagc aaaaaagaga 85260ggttgctagc aagatgaagt cacaatcttt tgtaatcgaa tcaaaaaagt gatatctcat 85320cactttggcc atattctatt tgttagaagt aaaccacagg tcccaccagc tccatgggag 85380tgaccacctc agtccaggga aaacagctga agaccaagat ggtgagctct gattgcttca 85440gttggtcatc aactattttc ccttgactgc tgtcctggga tggcctgcta tcttgatgat 85500agattgtgaa tatcaggagg cagggatcac tgtggaccat cttagcagtt gacctaacac 85560atcttctttt caatatctaa gaacttttgc cactgtgact aatggtccta atattaagct 85620gttgtttata tttatcatat atctatggct acatggttat attatgctgt ggttgcgttc 85680ggttttattt acagttgctt ttacaaatat ttgctgtaac atttgacttc taaggtttag 85740atgccattta agaactgaga tggatagctt ttaaagcatc ttttacttct taccattttt 85800taaaagtatg cagctaaatt cgaagctttt ggtctatatt gttaattgcc attgctgtaa 85860atcttaaaat gaatgaataa aaatgtttca ttttacaaga ggagtgtatg ataaatatat 85920catagagaaa ttggtcttta atataaaaga aattgccata tacactgaat tttttcagaa 85980ctctttttaa aaaactattt ggtagaaatc aaaggggaag cagttttcat gacactttta 86040ctttaagata cttattaata gataaattct atcttgattc cctactcaga agacataaag 86100tcagaatgcc tggctgttgg tagcctttgt gcaattcccc caaatgaaac aactttggca 86160accctttcca cttctactgt ccccttggtt cctctgcatc agtccatagc atcctctatc 86220cagtatgaat cttgagatat ctaatgaaat ttacctgaga ataactagaa attatccaag 86280cataagaaaa ggaagttgct tcagaatgaa aagaagataa acctccaata taccatcttt 86340cctttttagt taaatcttac agcatgagtt accttttaat atgtgcttct aagaaactga 86400ccaaaataat gtgtcatagt gttatttaat acgcacaaag tggaaagcag tgcaagtttg 86460ccaaggacaa tttaattttg tcacattgca tgctgttttg tgaccatgaa gagtttatac 86520aaagatgttt atgcttgtgc ttgttgaggt atagggacaa atatctaaaa gcaagatcag 86580atgggtgtgg tatctcacac ctataatcct tggattaaaa tctacctcaa ttgtaggact 86640accagttgaa ccacatgctt cccactgccc tcagcaaagg gcaccttagt tagaggaaag 86700gtagagcctt tctatggagg aggaatttgt gaggtttgag ttttatcagc tacctgggag 86760tcagaccctg atagattctc cttcacactc cctggacctt ttcctgccaa gtggaggctc 86820tcactcagag gaaatctcca ttcttttgat gcaggtcatt catactcaga tattctgcac 86880tgttcaagca ataaaaattg aatgagcacc tattatgtac accagttggc actgtgtcaa 86940aatgtacttg tgcagagacc ttggatcatt ggtgacaggt cttcttctcc tctgcatttt 87000tctcaagacc aggcctcagt gtagcatgtt tccatggagt gaaagagggg aaggaagagt 87060gggctttgga aagtggcagc tgtgtcatag cagtcagcct ctgtgtatgt gaaggacttt 87120ccagagcccc cccactaaag cctccatgct cctcctggga ctgccacagt tcttgaaact 87180atccatacag tcttcatgag ttatttttaa tttttttttc ttcttttctc tttcctcctt 87240ttcccctttt ccccactccc tagttagatc tttaaaaatg caattgtaac ctttatcttc 87300ccttcaccag acactcccta cagggcaagc ttatgtatac gcttacctaa aagctccaga 87360gccagaaatc tctcccactc ggggactgcc tcaagagaca gcagtcaatt tacaacctaa 87420agcatgccca caacaaaact ctctcccacc tggaggatat cttgaggcaa tggtcacttt 87480acaacctagt tctgcctgca atggcaccag ctcaaccacc tggtacataa gacacaaaag 87540caagttgcat agacctcacc ttctcactcc cttccctgca tgccattaat gccaactccc 87600cctttaaaag cccctgcttt ctgccccaaa agcaaagtga tacccttaaa gtcaggagcc 87660tatacttctt ccccctaagc taatttttgg aataaaagtc attttattga gaacctccat 87720aaactgttgg tgggaatata aattagtaaa ccatgatgga gaacagtttg gagtttcctc 87780aaagaactaa aaatcgaatt accatatgac ccagcaatcc cactgctggg tatacaccca 87840aaagaaagga agtaattata ttgaagagat atctgcactc ccatgtttgc tgcagcactg 87900tttacaatag ctgagatttg cagcaaccta agtgtctatc aacagatgaa tggataaaga 87960aaatgtggta cacatacaaa atggagtact agtcagccat aaaaagaatg agatcctgtc 88020atttgcaaca acatggatgg aactggaggt cattatgtta agtgaaataa gccagacaca 88080gaaagacaaa tatcaaatgt tctcacttat ttgtgggatc taaaaattaa aacaattcaa 88140ctcatggaca tagagagtag aaggatggtt accagaggct gggaggggaa gtggaagcta 88200ggggaggtgg ggatggttaa tgggtacaaa aaaatagaaa gaatgaatta gatctactat 88260ttgatagcac aacagagtga ctatagtcaa taataattta actgtacttt ttaaaaataa 88320caaaaatcgt gtaattggac tgtttataac tcaaagaata aatgcttgag gggatggata 88380tcccattctc catgatgtga ttacccattg catgtatcaa aacatctcgt gtacccccta 88440aatatataca cctactatgt atccacaaaa actaaaaata aaattttgtt taaaaagtca 88500ctttctttat accacatctc acccttgtta attggactct gcgaggggtg aacaactgga 88560cctgtgattc agttaaaatt agatcctcag gcaccttctg ttgagaaaga ataggtctca 88620aatgttgcaa atctctttta cctttctcaa ggttctagcc tctcctatca ccaatttagg 88680taagaatata aaatcatcag gcctgtgtta cctcaactat cttcctcttt gatccaaaac 88740gtatacttag tggacaaagg ctttttggac aactaatatg tgctaagtct taaggtgggt 88800tcagaaatgg ccaggatcca tcaactaatc aatggataaa taaaatgtga tctatccata 88860caatggaata ttattcagcc ataaaatgta atgaagcact aatactatga tgcaacatgg 88920atgaactttg aaaacatcat gctaatagac caataacagg ctctgaaatt gtggcaataa 88980tcaatagctt gctaaccaaa aagagtccag gaccagatgg attcacagcc gaattctacc 89040agaggtacaa ggaggaactg gtaccattcc ttctgaaact attccaatca atagaaaaag 89100aggggatcct ccctaactca ttttatgagg ccagagtcat cctgatacca aagccgggca 89160gagacacaac caagaaagag aattttagac caatatcctt gatgaacatt gatgcaaaaa 89220tcctcaataa aatactggca aaccgaatcc agcagcacat caaaaagctt atgcaccatg 89280atcaagtggg cttcatccct gggatgcaag gctggttcaa tatatgcaaa tcaataaacg 89340taatccagca tataaacaga accaaagaca aaaaccacat gattatctca atagatgcag 89400aaaaggcctt tgacaaaatt caacagcctt tcatgctaaa aactctcaat aaattaggta 89460ttgatgggat gtatctcaaa ataataagag ctatctatga ccaacccaca gccagtatca 89520tactgaatgg gcaaaaactg gaagcattcc ctttgaaaac tggcacaaga cagggatgcc 89580ctctctcacc actcctattc aacatagtgt tggaagttct ggccaaggca attaggcagg 89640agaaggaaat aaagggtatt caattaggaa aagaggaagt caaattgtcc ctgtttgcag 89700atgacatgat tgtatatcta gaaaacccca tcgtctcagc cccaaatctc cttaagctga 89760taagcaacat cagcaaagtc tcaggataca aaatcaatgt acaaaaatca caagcattcc 89820tatacaccaa taacagacaa acagagagcc aaatcatgag tgaactccca ttcacaattg 89880cttcaaagag aataaaatac ctaggaatcc aacttacaag ggatgtgaag gacctcttca 89940aggagaacta caaaccactg ctcaaggaaa taaaagagga cacaaacaaa tggaagaaca 90000ttccatgctc atgggtagga agaatcaata tcgtgaaaat ggccatactg cccaaggtaa 90060tttatagatt caatgccatc cccatcaagc taccaatgac tttcttcaaa gaattggaaa 90120aaactacttt aaagttcata tggcaccaaa gaagagcccg catcgccaag tcaatcctaa 90180gccaaaagaa caaagctgga ggcatcacgc tacctgactt caaactatac tacaaggcta 90240cagtaaacaa aacagcatgg tactggtacc aaaacagaga

tatagatcaa tggaacagaa 90300cagagccctc agaaataacg ccgcacatct acaactatct gatctttgac aaacctgaga 90360aaaacaagca atggggaaaa gattccctat ttaataaatg gtgctgggaa aactggctag 90420ccatatgtag aaagctgaaa ctggatccct tccttacacc ttatacaaaa attaattcga 90480gatggattaa agacttaaac gttagaccta aaaccataaa aaccctagaa gaaaacctag 90540gcattaccat tcaggacata ggcatggaca aggacttcat gtctaaaaca tcaaaagcaa 90600tggcaacaaa agccaaaatt gacaaatggg atctaattaa actaaagagc ttctgcacag 90660caaaagaaac taccatcaga gtgaacaggc aacctacaga atgggagaaa attttcgcaa 90720cctactcatc tgacaaaggg ctaatattca gaatctacaa ggaactcaaa caaatttaca 90780agaaaaaaaa aaaacaaccc catcaaaaag tgggcaaagg acatgaacag acacttctca 90840aaagaagaca tttatgcagc caaaaaacac atgaaaaaat gctcaccatc actggctatc 90900agagaaatgc aaatcaaaac cacaatgaga taccatctca caccagttag aatggcaatc 90960attaaaaagt caggaaacaa caggtgctgg agaggatgtg gagaaatagg aacactttta 91020cactgttggt gggactgtaa actagttcaa ccgttgtgga agtcagtgtg gcaattcctc 91080agggatctag aactagaaat accatttgac ccagccatcc cattactggg tatataccca 91140aaggactata aatcatgctg ctataaagac acatgcacac gtatgtttat tgcggcacta 91200ttcacaatag caaagacttg gaaccaaccc aaatgtccaa caatgataga ctggattaag 91260aaaatgtggc acatatacac catggaagac tatgcagcca taaaaaatga tgagttcatg 91320tcctttgtag ggacatggat gaaattggaa atcatcattc tcagtaaact atcgcaagaa 91380caaaaaacca aacaccgcat attctcactc ataggtggga attgaacaat gagaacacat 91440ggacacagga aggggaacat cacattctgg ggactgttgt ggggtggggg gaagtgggag 91500ggatagcttt aggagatata cctaatgcta aatgacgagt taatgggtgc agcacaccag 91560catggcacat gtatacatat gtaactaacc ggcacattgt gcacatgtac cctaaaactt 91620gaagtataat aataaaataa aataacagaa acaaagcaga catagacagt atgcaaatta 91680aaaaaaaaaa agaaaacatc atgctaagtg aaagaagcca gacacaaaag accctatatt 91740gtatgattcc actgatatga aatgttcaga ataggcaaat gcacagagac agagagtagg 91800taagtagttg cctgaggcag tagagggttg agggattggg agaagatggg catggctatt 91860caggggtctg gggtttcttg ttgaagttat gcaaatgttc taaaattgat tggagtgatg 91920attacacaac tctgtgaata ttctgacagc aatatttctc ttcctggccc attttttggg 91980tggtttttct ggcattgctg caccatgtct gaagctcctg gcacttacac tcttgaagtc 92040ttctctatca gctttcccgg accctctgct tttttcttgg aattgatcaa gatttgccag 92100atcatagttt ggcactgacc tctgattgtt ggcttctgta aagaacccca aactacagga 92160attggaatgc ctggtctggt caaaggagag gatctggttg attctggaca aatcactcca 92220cctcgctgag tatgattctt catctataga gtggactgaa tgatctccaa gtactttcaa 92280atatgaaaac tactctagtt tcctcaagct gccacaaact gcacggctta aaaaacagaa 92340gtttattgtc tcatagttct ggaagttaaa agtctgaaat caaggtgtct gcagggccac 92400gctccctctg aaacctatag agaaagatcc ttcttgcctc ttttggcgtc tggtgtttgc 92460cagcaatcca cagtattcct taccttgaag acgcaggact ccagtctctg cctctgttgt 92520cacatggcca tcttccctct atgtctctct cctcttctta agaacagaag tcatagtgga 92580ttaaggaccc accctactcc tactatgacc tcatcttgac taatcacatc tgcaacaacc 92640tcatttccaa atgaggtcag cttctgaggt cctgggggtt aagacttcaa catgtaattt 92700tgtggggatg cagttcaacc cataaccaca accgtggctt ttcttcctta gaagttactg 92760agaactccat gaaagttgaa aatgcccttg tgaatgggag agtcattcac gactattgga 92820agaaaaaggc aattttcctg tcggctatta ataaagctat tatagcaata tggcaaggtt 92880tctactctga ttatccttga acttaaagtc ataatagtat ccattatttc cagtgagtac 92940aagggaaggt cacattactg tgttctcttc attgctgccc ttaaatgctt tcagggaaag 93000ggggaataga taaagcccca ggccttactc agcaatcggg gcaagacgta gcagtgtttt 93060gaacctgaag gagacaaccc agggcttcct gtgccaatga ctcagaatgg aaaatggtct 93120ttataaatgt ccctccagtg tgcaaagggt agagactaga atttaaaatt catcagatat 93180taataagaaa aaccaaagca aacaaaaaat ctacttatga tcatagaaca ttgccattgg 93240ggagctgttt attatttgag cctttttaaa atttcttttt ttgtgtgtga gaaacatatt 93300tctaggagta gagagaaaag gctcccctgt tttatgtact cagtctttcc ctcctttgag 93360attaccccta aactatttta gggatagctc cctggttatc tcagtaatga tgaccccaga 93420agttcagaga taattaagag ccatgtctgg gtttgtgaga gaagcccaaa tgcagttatt 93480caaaccagaa accagagcct cgttgttgac tcttccctct caccgtgaga tccaaccact 93540catcaaggac tctccatttg acctcctaaa ttcttctccc atccatcccc catctttgtg 93600tccatcatca acaccccttg tccaagatac ctgcattgct cactagcttc ctggtatcca 93660ctgtgttccc tccaattcag ttccagaagc tagagtgatt cttatcaaaa gtgtggcttc 93720ttctgaatta ggctgacaat ctcaactcct ggagaagacc cacaatccct ttgtgacctt 93780ccatttgtgc acccctccag cctccactct aagcaccccc accagttgtc ttcggtttgt 93840gctaagaaca caccaaactc ccatctcagc acctacacac atacgactgc cccaaggcac 93900gtctgactcc agacttcact tagctaattc ctgcttaccc tacagggaga ggttaaatgc 93960cactgagagc agctttattc taacctccaa atcccactca gacgtccccc ttatactatc 94020ttctcagcat ttaccacaag ttttgttgga taaatgatag agaaatgaat agtgtggcta 94080caacacgtgt cttccattag actgtaggcc ccaaagatca gggaaggtga cttttagtcc 94140atcactctgt ccccagtatt catcaccacg cctgatacac aacaaagact ttcaataaat 94200acttttcaag tgaatgaatg aatgagcaaa ggttcaggtt ggcttctcta gctacaccct 94260aggacagata cagtaaggtc aggccacatt gtcaaatgtc ccactgtagc agtcatgcat 94320aagggtaggc cttctgcacc ttccatgttc ccattgccca ctcatggtgg caagcatgct 94380gattgtctct tgagtactac acagcaccaa caatctcagg taccatgatg ctgattccat 94440gaggctctga gcctgggcaa gaatcattta cattcttatt gagtgttggg ctatggttca 94500gagcaagcac agcataagcc aagaagaagg caaaggatgt tatcatgaaa tggaagaagt 94560gtgtaaattg agtgcaacta tgtagaaatc atgcgatggt ccataaacaa atatgggcat 94620aagagcaagt ctgaattcag gctcatggaa ggctggtgtg gctctgccag catgtgctag 94680ttcactagtt ctatgccacc attcaaagca gaatttcccc aagtcaattc catacgtaag 94740tactccatta aaaacgctcc tgagttcaaa caggtttgga aacatcttag ttactatagt 94800atttctcata tgccttgtga tctccaaaag agaactatac tacactgtgt cccaaactta 94860cttgatcacg attccttttt ttcttttttt gcagggcatc tcctagagct aatgttctct 94920ctctctcttt ggaaatgcag atttgaaggc ctgagattta cccagcctaa tgaacccctg 94980aaaatgttcc caggtatatg atgatagtca ccatattgac atgaattgtt ctgttttaaa 95040aacagaaaaa aaaaccacaa taatctaggc catgtccctt gaggccttta tatcaagatg 95100attagctcct tgaactggtc ctattaggtc tctagaggag agattattgc ttctgaagag 95160agatgtgaca acaaggcaga atatgattca gaagcggtaa ataataaaac taaaaatgat 95220agcttctcct ttggtacagg gtggagaggt taatattcat ttctaaagca tcattagcca 95280tataaagtat aatttcagca cttgacataa actgaaaaca cacataatcc tcccattgag 95340tgggctaacc ttggctgtgg ctggatgagc tctctaggga cttattaggg aatatggtac 95400tttcaaagat gttaaatcca gtggaagtga ctgaagcaat tagtctctga tcttgagaga 95460atttccacct ttgaatcagc aaaccctatc caaccgaagt tgagtactct agagatgata 95520ttgtttatac tggaaaccaa tatgaaatat gtgaatgtat gtataatgta catatataga 95580cacatgtaga ggagtattta tatatagata gagatgttca ttattgactg cttagtagat 95640ataaagaaga gaataaaatt taaaaataag gaaataagga aaatgagagc atgatgtagt 95700aatttttcta tttctattta aatgttcttt cctaacatcc agtttgttgt ggagtcacca 95760aaaagcatca cctcatcaaa acacacacac acacacacac acacaccaac aaaagacata 95820acctaccagc caaaaaaaag cccaggacca gacggattta cagctgattt ctatcagagg 95880tgcaaagaag aaaagagctg ggactctttc tactgaaact attccaaaaa attgaaaagg 95940agggactcct ccctaactca ttctatgagg ccagcatcat cctgatacca aaacctggca 96000gagacacaac aaaaaaagaa aacttcaggc caatatcctt gatgaacatt gatgcaaaaa 96060tcctcaacag gctgggtgtg gtggctcatg cctgtgatcc cagcactttg ggaggccaag 96120gtgggcagat cacttgagag cagcagtttg ggaccagcat ggcaaaaccc cctctctact 96180aaaaatacag aaattagccg ggtgtggtgg cttgtaatct cagctacttg ggtggctgag 96240gcatgagaat cacttgaacc caggagacgg agcctgcagt gagccatgat tataccactg 96300cgctccagcc caggggacag agcaagactt tgtctcaata aaaaaagtcc tcaacaaaat 96360actggcaaac tgaatccagc agtacatcaa aaagctaatc aaccacaatc aagtaggcct 96420catccatggg atgcaaggtt ggttcaacat actcaaatca atagatgtaa ttaatcacat 96480aaacagatct aaagacaaaa accacatgat tgtctcaata cgtgcagaaa aagcctttga 96540taaaattcaa cattccttca tgttaaaaac tctcaataaa ctaggtattg aaggaacata 96600cctcaaaata ataagagcca tgtatgacaa acccatagcc aataccatat tgaatgggaa 96660aaatctggaa gtattcccct tgaaaacaag aaagacaaaa tctgcatcat tccctgaggg 96720gcatttcttt tcctggctca gcacataggc taaaaagagg ccaagagtga gcagatctgt 96780gagctgtggt tcaacgtctg gtggtgggga agagaaaaga agaaagaatt gggaaggaga 96840gagaagaaac aaggccttgt gagtggggaa agatgcaggt aggagtcagt caagtattta 96900tcttgaggtg tctatctcac atcagtttct ctttgaggca gtggttttca aattcggcct 96960atatcaccta aagggcttgt taaaacacag tctggggcat gcctgacaag tgtgcagctc 97020cagtgagtta ccaggtgctg ctgatgctgc tgattgggga acacactttg agaaccactg 97080gtctaagcat cccccaagtg cacaactcaa aatcaacaat ttctacctcc accttagaca 97140gcactttgct cctgtatcat ggcacttctt tgttttatct tggtcagagt tagtagcttg 97200atctccctcc tctgtaaatt ctgtagggga ctgaagattg taaattcctg gaaggcagag 97260gccacagttt actcatgcct gtatcctcca gagcacaaag tacggtacgt attagttagc 97320agttgtttaa tcaatgtttg tggaataaaa ttctagaaag tgatctgata aatatgtgta 97380ttatgtaact gatgcataca ttaacctttc cagagattta catggtttgt ttactgatgg 97440caatatctta aaaccaaaca aatgcatttc taatgatgtt catttaagca atgcaccaga 97500tctgttgagg ggatatagtt ttggcaaaga cgtgctgact ggttcttaaa ccacaacggc 97560tggatttttc tggctttcct gctcctctga cttatcagga gagctttgcg tacctcccga 97620gcctccaaca gccagaagtt tcaagttagc cactaatctg aacacacgat tatcatgttc 97680tgatcacaaa atatgaaaca ttacaaaagg aaggctcatt taattttcac atgtacacag 97740tccaaagata aggtgtggct ccttattcgc attactctgc gaacccaaat gcacataggg 97800agccacacca gatgggagtg gctccctatc tgcattactc tgtgaaccca aaatatctga 97860gacaagcctc agtcaattta gaaagtttat tttgccaagg ttaaggatac acctatgaca 97920cagcctcaag agggcctgac aacatgtgcc caaggtggtt tgggtgcagc ttagttctat 97980acattttagg gagatatgag acatcagtat gtgtaagatg tacattggtt cggtccagaa 98040aggctggaca actcgaagca ggggagggga ctttcaggtc ataggtagat aagatacaaa 98100aggttgcatt attttgggtc tctgatcagc ctttcactga atatacaatt cacatggaag 98160aggagttcga gatcagcctg gcgaacatgg caaaactcca tctctactga aataccaaaa 98220attagctggg tgtagtggca ggtgcctgta atcccagcta ctctggaggc tgaggcagga 98280gaattgcttc aacctgggag gcagagggtg cagtgagctg ggatcgtgcc actacactcc 98340agcctaggcg acagagcaag actccatctc aaaaaaaaaa aaaaaattgt aatcattttg 98400actgtaaaat ataatttcca taaacccttt tataaccttt tattaaggag tgggttaaca 98460ttccaagaaa acttgttaat ctgacacagg ggcccatatg ctggtcttgc atcagtgtac 98520cttcaatatt aatggttaat ttatagagaa actgaactaa ttttatctct caaaattggc 98580ctttacaatc tcttacacca tcctcttcca tggtagtccc tgggccttga ggagttgaat 98640agttttaatt tctggattgg tgcctcacaa acacagctta ttttgattgg catcttctac 98700caggtctgaa gatgaggctt taactggtgt cagtgtttaa gatttagcag gacttggtgt 98760cttttttaga cccaggagtc aaagccttga aacttaatgg cacaaagact ttaagagcaa 98820aatacagaaa gttacatgga tgtgataatc ttaatttaaa acaattttta aagtctcagt 98880ttttttctaa gcaaatcaaa agttagtaac aatgacatag gaattatttt gataaaatgt 98940aaaatttgtt tgttagacca gtcaccaaaa ggcaaaagaa aagaccttct gcagtgcaat 99000tgctgttcct atgggtgtcc atttagataa cctgcaagtc aaactaatga aaagggtaca 99060tcaattaatc agacatagga agagtgtttc ttgggtaata agtgaaaatt tttggattca 99120tagaacaatt taaagccaag agcacagaat gttatgttgg aataaaatat ttcctttaga 99180catttataaa acatttttag catcagactt caacaaacag ttagaaccta agggaaaaaa 99240aatcttacag gaactgaaaa tgagttgaag gatagagtta ttatttcagg ccttttaaaa 99300agggagagaa agctgaaaat agcaagacac aaaaaagttg aactttgggt tcaaaaaaat 99360taaaatctct ggtaatttta ttaagaataa accaatacct taagaaaatt ccaggccagg 99420cgtggtggct cacacctgta atcctagcac tttgggaggc tgaggtgggt ggatcacctg 99480aggtcaggat ttcgagacta gcctggccaa tatggtgaaa ccctgtctct actaaaaatg 99540caaaattaac caggcatggt ggcatatgcc tctaatccca actacttggg agactgaggc 99600aggagaatca tttgaacctg ggaggcggag gttgcagtaa gccactgcac tccagcctgg 99660gcaacaagag caaaactccg tctcaaaaaa aaaaaaaaag aaaattccat tgttctaccc 99720aattctttag tgtataagtg ctctttaaat attaaagccc aatctctaga aagaccatca 99780taaataattt ccctttaatt ttaggcaact tgatcataaa gacccttttt ttccataaat 99840tctcttttta caaaccttat tacagcttac acaggccatt catgatattc atgacatgct 99900tggactttgt ggattgtcct gaacatatct ctttcttaaa caaccagtca ttttattcta 99960ggacaaaaat ttaccataca atattttttc tcatataaaa ttattttgtt tttaaccttt 100020cttgccaaaa atacctcttt acatttataa ctttttttac attgctttta tttactggtt 100080accattactt tgttttataa ataactttta aataaacttt gaattagaca aaaattattt 100140tcttttaaat aagaacacat ttcttttttt tagaaaaaat gttttcttat cacttaaaaa 100200aattggaaat gacccagaca ttcaatgaat atctattatt taactttaga ttttaaattt 100260tataagttta tttataagca tttatctcat tacatttacc taattaattt ttaacatagt 100320ttaaccagat tacttataaa aactgtgata gttatcattt aaagttattt ccctgttaac 100380cattttataa cctgtgaatt tcaggttttc ctaagtgaga accttaaggt tagataaata 100440gtttttttgg tgtgtttttt tttttttgcc aataactcag gatttagctg ttttcattaa 100500ttgaacaata ttaaatgatt tatcaaagtt tacataaaga taattttcct ttgggctgca 100560ttcatagctt tacaaccctc atgccaaatt ttaacatgta gcagcgataa aatataaaac 100620cactttacca ataaatctaa acaataatgt atgttgacaa ttctgaagcc atttctaatt 100680ctgtttcatg aatactttca aaaccagctt atttactaaa aatttacttg agtcatatga 100740acttgaaaaa gcatttggct taaagtattt ttttctgata atttgatttt ggctgggtgc 100800agtggctcac atctgtaatc ccagcacttt gggaggccaa ggtaggcaga tcacttgagg 100860tcaggagttc gagaccagcc tggccaacat ggtgaaaccc cgtctctacc aagaatacaa 100920aaattagccg tggtggcaca cgcctgtact gccagctact tgggaggctg aggcacaaga 100980atcacttgaa cctaggaggt agaggttgta gtcagctgag attatgccac tgcactccag 101040cctgggcaac agagcaagac tctgtctcaa agaaaagaaa atttgattta agcgcttttt 101100tttcctttaa gccaattaat tagagcttgt tggtgtattt ttagtagtga aacattatat 101160acatgacaca taaatacata gatatattag acatgcagat agaccttata gattcataag 101220acctcttctt tttcctccta ttttagactt ccaatttctt gatgacccat tttattaccc 101280taggtagctg tcagctagat agccccaaat ttgcatatca aaggaatagc tctcaggtga 101340aaaatcagat attgaaattt atatctcaag gtagagagag agagtctggt ggtgctagga 101400gattaaaaac aaatgccaaa tcaaacataa atttatagaa atctatcagg attgtataag 101460gaaataaatt ttatttagat aggtagttct attaaataca tttagtctct atcttttaac 101520tggatctctg agctctgagc agagcaccca ttgaatcctg ggtctccaaa aagcgagaat 101580tattatggga ctagaacaca tgatgctttt acagtatact tttttttaaa aaaaaagata 101640tttctttaag tgtctaaact atactcttcc ttactttaaa cacccaagag aaacctctgt 101700tataataact attttagtta aagaaaaaaa gaaagaaaga aatcaggtaa cacaataaaa 101760aaacaagcag tttaagatct gagatgaact tgtctcttta cgctctttgt gttccataag 101820gaaaaacaga ggtttctccc caaaagggag tctggcactg tctgttttct ttaaggaatc 101880ccatgctatt agaaactgta ttggttccct catgcagcag agggtggcaa aagaaagtag 101940agacagcaga agtaaatggc gaaaacagaa ttcagtctac tgagaagaga aaaaaaactt 102000tttctcaaaa aaaaaacaag gtcctaggag agaaaaaaaa accataacaa cctttttaaa 102060tacacacaca cacacacaca cacacacaca cacacacaca catcatcatc atcatcatca 102120tcttggatat tagcttttaa ttaaactgac ttttaaccat tgagctccat ttaaaaaaaa 102180acctttttaa tctcattacc atattttaac taggataaat tgctgatatt tcaaaagtaa 102240cacaaatatc aaaccagaaa gggctttaga aaccaaaccc agactgtctt gatgaaaaaa 102300aagtgggggc agaagcttag ctatggaact gctgcatggg gcgacagcca ttactctttc 102360agtttggctt ggctaacaaa aactggcctt attatgtaaa taaagccctc agggtagtca 102420aaatcaagaa cctttctctt ttatcccctt ttgctggact gtttttcctt ctccctgctt 102480tttttttttt tttttccagc tgtggaattt agccaattca gaggtcttgt tccccgtaat 102540gtggaacttt ccttcagatt tgatcaagtt ggatagagtt ggtcaaaccc aattgtcagg 102600cctctgagct caagctaagc catcatatcc cctgtgatct gcacgtacac atccagatgg 102660ctggttcctg ccttaactga tgacattcca ccacaaaaga agtgaaaatg gcctgttcgt 102720gccttaactg atgacattgt cttgtgaaat tccttctcct ggctcatcct ggctcaaaag 102780ctcccccact gagtaccttg tgacccccca ctcctgcccg ccagagaaca attccctttg 102840actgtaattt tcctttatct acccaaatcc tataaaacgg ccccaccctt atctcccttc 102900gctgactctc tttttggact cagcccacct gcacccaggt gaaataaaca gccttgttgc 102960tcacacaaag cctgtttggt ggtctcttca cagggacacg catgaaattt ggtgccgtga 103020cttggatcgg gggacctccc ttgggagatc aatcccctgt cctcctgctc tctgctctgt 103080gagaaagatg cacctacgac ctcaggtcct cagaccgacc agcccaagaa acatctcact 103140aatttcaaat ctggtaagcg gcctcttttt actctcttct ccaacctccc tcactatccc 103200tcaacctctt tctcctttca atcttggcgc cacacttcaa tctctccctt ctcttaattt 103260caattccttt cattttctgg tagagacaaa ggagacatgc tttatccatg gacccaaaac 103320tctggtgccg gtcacggact gggaaggcag ccttcccttg gtgtttaatc attgcaggga 103380tgcctctctg attattcacc cacgtttcag cggtgtcaga ccacccaggg atgcctgcct 103440tggtcattca tgcttagcgg caagttccgc ttttctgtgg gaggggcaag taccccaacc 103500ccttctctcc gtgtctctac cccttctcca cttttctggg ggaggggcca gaacccctca 103560accccttctc cttcaccctt agtggcaagt cccgcttttc tagggggcaa gaatccccaa 103620tcccttattt ccgtgccccg acctctcatc tctgcacctc aaccccttat ttccgtgccc 103680cgaccccttt cccgcttttc tggaggttaa gaaccctcga accccttccc tccatgtctc 103740tactctctct tttctctggg cttgcctcct tcactatagg caaccttcca ccctccattc 103800ctccgtctcc cttagcctgt gctctcaaga acttaaaacc tcttcaactc acacctgacc 103860taaaacctaa atgccttatt ttcttctgca atgccgcttg accccaatac aaacttgaca 103920gtggttccaa atagccagaa aatggcactt tcaatttttc cattctacaa gatctaaata 103980attcttgtca taaaatgggc aaatggtctg aggtgcctga cttccaagca ttgttttaca 104040cattggtctc tccctagtct ctgtgcccag tgcaacttgt cccaaatctt ccttctttcc 104100ctcccgcctg tcccctcagt cccaacccca agtgttgctg agtctttcta atcttccttt 104160tctacagacc catctgacct ctcccctcct ccccaggctg ctccttgcca ggccgagcta 104220ggtcccaatt cttcctcagc ctccgctcct ccaccctata atctttttat cacctcccct 104280cctcacaccc ggtctggttt acagtttcat tccgtgagtg gccctccccg acctgcccag 104340caatttcctc ttaaaaaggt ggctgaagct aaaggcatag tcaaggttaa tgctcctttt 104400tctttatcag atgtctccca aatcagtgag catttaggct ctttcatcaa atatgaaaaa 104460cccagcccag ttcatggctc gttcggcaac aaccctgaga cgctttacag ccctagaccc 104520taaaaggtca aaaggccgtc ttattctcaa tatacatgtt attacccaat ccgctcctga 104580cattaaataa aactccaaaa attaaattcc ggccctcaaa ccccacaaca ggacttaatt 104640aacctcgcct tcaaggtgta caataataga gacagccaag tagcaacata tttctgagtt 104700gcaattcctt gcctccactg tgagacaaac cccagccaca tctccagcac acaagaactt 104760ccaaacgact aaaccgcagt agccaggcat tcctccagaa ccgcctcccc caggagcttg 104820ctacaagtgc cagaaatctg gccaccaggc caaggaatgc ccgcaaccca ggattcctcc 104880taagccgtgt cccatctgtg cgggacccca ctggaaattg gatggaaatc ggactgttca 104940actcacctgg cagccactcc cagagaccct ggaactctgg cccaaggctc tctgactgac 105000tccttcccag atcttctcgg cttagcggct gaagactgac gctgcctgat tgcctcagaa 105060gccctgtaga ccatcacaga tgccaagctt taggtaactc tcacagtgga gggtaagtcc 105120gtccccttct taatcaatac ggaggctacc cactccacat taccttattt tcaagggcct 105180gtttcccttg cctccataac tgttgtgggt attgacggcc aggcttctaa acctcttaaa 105240actccccaac tctggtgcca acttagacaa tactctttta agcactcctt tttagttatc 105300cccacctgcc cagttccctt attaggccga gacactttaa

ctaaattatc tgcttccctg 105360actattcctg ggctacagcc acacctcatt gccacctttt cccctagctc aaagcctcct 105420tcacatcctc cccttgtatc tccccacctt aacccacaag tataagacac ctctactccc 105480tccttagcga ccgatcatgc accccttacc atcccattaa aacctcatca ctcttacccc 105540gctcaatgcc aatatcccat cccacagcat gctttgaaag gactaaagcc tgttatcact 105600cgcctgctac agcatggcct tttaaagcct ataaacgctc cttaccattc ccccatttta 105660cctgtcctaa aaccagacaa ggcttacagg ttagttcaga atctgcgcct tatcaaccaa 105720attgttttgc ctatccaccc cgtggtgcca aacctatata ctctcctcaa tgcctccctc 105780tacaacccat tattctgttc tggatctcaa acgtgctttc tttactattc ctttgtaccc 105840ttcatcccag cctctcttcg ctttcgctta gactgaccct gacacccatt aggctcagga 105900aattacctgg gctgtactgc cgcaaggctt cacaggcagc ccccattact tcagtcaagc 105960ccaaatttca tcctcgtctg ttacctatct tggcataatt ttcataaaaa cacacgtgct 106020ctccctgctg atcgtgtccg attaatctcc caaacctcaa tcccttacaa aacaacaact 106080cctttccttc ctaggcatgg ttagtgtggt cagaattctt acacaagagc caggaccgca 106140ccctgtagcc tttctgtcca gacaacttga ccttactgtt ttagcctagc cctcatgtct 106200gtgggcagcg gctgccgctg ctttaatact tttagaggtc ctaaaaatca aactatgctc 106260aactcactct ctacatttct cataacttcc aaaatctatt ttcttcctca tacctgatgc 106320atatactttc tgctccccgg ctccttcagc tgtactcact ctttgttaag tcccacaatt 106380accattgttc ctggcccaga cttcaatccg gcctcccaca ttattcctga taccacacct 106440gacccccatg actgtatctc tctgatccac ctgacattca ccccatttcc ccatatttcc 106500ttctttcctg ttcctccccc gatcacgctt gatttattga tggcagttcc accaggccta 106560atcgccacac accagcaaag gcaggctatg ctatagtaca agccactagc ctgcctctta 106620gaatctctcc tcattttctt tccattgtgg aaatctatca tcaaggaaat aacttctcag 106680tgttccgtct gctattctac tactcctcag ggattattca ggccccctcc cttccctaca 106740cattaagctc gaggatttgc ccccacccag gactggcaaa ttagctttac tcaacatgcc 106800ccgagtcaga taactaaaat acctcttagt ctaggtagac actttcactg gataggtaca 106860ggcctttcct acagggtctg agaaggccac cgcagtcatt tcttcccttc tgtcagacat 106920aattcctcag tttagccttc ccacctctat acagtctgat aacagaccag cctttattag 106980tcaaatcagc caagcagttt ttcaggctct tagtattcaa ggaaaccttt atgtccctta 107040cggtcctccg tcttcaggaa aagtagaacg gactaaaggt cttttaaaaa cacacctcac 107100caagctcagc caccagctta aaaaggactg gacaatactt ttaccacttt tccttctcag 107160aagtcagacc tgtgctcaga atgctacagg gtacagccca tttgagctcc tgtatagacg 107220ctccttttta ttaggcccca gtctcattcc agacaccaga ccaacttaga ctgtgcccca 107280aaaaacttgt catccctact atcttctgtc tagtcatact cctattcacc gttctcaact 107340actcatacat gccctgctct tgtttacact gccggtttac actgtttctc caagacatca 107400cagctgatat ctcctggtgc tatccccaaa ctgccactct aaactcttga agtaaataaa 107460taatctttac tggcaaggct atgctgaacc tccttaggca ctctctaatt agatgtccta 107520ggtcctccca attcttagtc ctttaatacc tgttttctcc ttctcttatt ccgtttagtt 107580tttcaattca tacaaaactg tatccaggcc atcaccaata attctaaatg acaaatgttt 107640cttctaacaa ccccacaata tcacccctta ccacaaaaac ttccttcagc ttaatctctc 107700ccactctagg ttcccacgcc gcccctaatc ccgctcgaag cagccctgag aaacatcgtc 107760cattatctct ccacaccacc ccccaaaatt ttcaccgtcc caacacttta ccactatttc 107820gttttatttt tcttattaat ataagaagac aggaatgtca ggcctctgag ctcaagctaa 107880gccatcatat cccctgtgac ctgcacgtac acatccagat ggccggttcc tgccttaact 107940gatgacattc caccacgaaa gaagtgaaaa tgacctgttc gtgccttaac tgatgacatt 108000gtcttgtgaa attccttctc ctggctcatc ctggctcaaa agctccccca ctgagtacat 108060tgtgaccccc actcctgccc gccagagaac agcccccttt gactgtaatt ttcctttatc 108120tacccaaatc ctataaaaca gccccacctt tatctccctt ggctgactct cttttcggac 108180tcagcccgcc tgcacccagg tgaaataaac agccttgttg ctaacacaaa gcctgtttgg 108240tggtctcttc acacggatgt gcatgaaacc aatgaaaaaa agaccaaaac aacaaccaaa 108300acagaaacaa agaacaacaa aaaacagcta agcaaagcaa acaatgccac aatttatagg 108360ttggtgcaaa agtaattgtg ctttttgcca ttaaaaagaa tgcaaaaccg caattacttt 108420tgcaccaacc taatatatga ttaccaagcg ctctaatggt aaggataaat taaaaccagc 108480tgattgttaa ttttaacttt agccaagaca aaagcccaat tcagctactt acctaggaat 108540ggggctcagg ctgaacactg ctctctacca tctttgaagc aggaaaaaac tcaaactcgc 108600cttccctgtt ggaagtgagc tgaaattcca gaaaggagtt acctgcactc ttcatcatgg 108660aagcaggaaa acttgccttc ctttttggga gcaagtgaaa ctccagaaaa ggagttatac 108720agcaaaataa actttagatc tcaaccaaat ttggagagat cagggattct cttggtcggg 108780ggtgggggtg tctcccaggc ctcagccaat tgtcctactg gcttgagcca taaagatagc 108840tcaagctggt accaagcacc aacagtagat ttgtcaaagg tcaggggtac ctccactcag 108900aatcccttca tggttaccaa tttgtgaatc caaaatatct gagacaggcc tcagtcaatt 108960tagaaagttt attttgccaa ggttaaggat gcacccatga cacagcctca agaggtcctg 109020gctacatggg cccaaggtgg tcggagcaca gcttggttct acacatttta gggagacgtg 109080agacatcaat caacatgtgt aagatgtaca ttggttccgt ccagacaggc aggacaactc 109140gaatcagggg agggggcttc cagatcacag gtagataaga gacatttgtt gcattctatt 109200gggtctctga ttagcctttc actgaatata caattcacat gggagaggag ggtggaggaa 109260tagtcactta tgcctggtct gccttggtga aacgacaggg cagaggaagc aatcagatat 109320gcatttgtct cacatgagcc tcagagggat gactttgagc tctgtctgtc ctttgtccac 109380gaggaatttc cttgtgggca aattgtgagg gaggtatgtc tttttttttt ttttatcttt 109440gtagctgtct tatttaggaa taaaatggga ggcaggtttg cttgacgcag ttcccagctt 109500gacttccctt tagcttagtg attttggggt cccaagattt attttccttt cacaactccc 109560atctgatata tttaaggggg aattcttttg tgcctacagc attgttagtg gaatgtggag 109620cgacagcaag ggatgggcct agctggaatt taccaccagg gggcttggac tctttctcat 109680gcgtcaacac caatcactgc caataggggg cagcaacaat ttgcctacag cctttaggct 109740ttggttttag actcaaggag ctcagggtgt ggaatctcca ttgtttttcc tctgaaactt 109800ggcttcttat ctgcctgcgg actcaaatcc tcctcctgcc aatactacaa cagccacaga 109860acaaatgcat tgaaactgag agaactggta agaggctaga tctagtgtaa atcagagaat 109920gggcctcata aaaggagcag tttggaggaa cctaatcctt cacagtttgt ctagcaattt 109980ctaggttcaa tcaattgagg gcttaactgg taaactcaag tggaaatttt ttattggctc 110040ctggtgatgt cactcctggt gagtgtggga gaactggttt tctaggtgtg gggattcttt 110100attctctgaa acacccacta ggaaacgccc tgaggccttt tcaccttctg tggtgtcagg 110160acctctcatg taactggata cagtgctttt cccaaacaga ctatacacaa cagaaactca 110220aaaaatagaa acgctaagtg ggaataaggg aaataaacta caagagtagt ggttctcgac 110280cttggctgca catgggaatc acctgggagc ttcaatccgc aaggaccaat gccagggacc 110340cccctacccc ctgcccccga gatttggatt aactggtctg catgcaacca gggcagctgg 110400cgttttgaaa ggtccccagg tggttcccat gtacagccaa actgagaacc attgtcagat 110460gtgcagtctt tgctcctggg tcaattaatt cctcacgccc actaactact ttagatgtct 110520tatgctttga gcacatttaa aataaaaccc aaaacttaat aaaacaaagt tagtacacag 110580agaaatagga acctgattaa aatagaacat gaagtcaagt cagtttggtt ttgttttaaa 110640tctccaattg ccaccattaa ttctgctccc aaaggtcaat aatgctagcc tgctatttgc 110700tttgttcact tctgaatgaa gcacttgtgt ttgctgacct tcaaacactt ccaggttgtc 110760taagaggaag ccatgtggca ctttgacttt tgtgtgacct ggcaatttgt ttacatcaaa 110820tcccttttaa ccctctgcag ttgacagtgg agaattcagt attttcatat gctgcctaac 110880ctctagggtg gaaggcttta aacttgattt aggtcaagtt ggcagtgacc caggtgagcc 110940cccaggaaac tgcacttcca cagccaaaac gtggccatcc accaacttag gcatgacagt 111000tacagttttg aacctgctat taagtcccac aattctcttg gttagttaga gtaatcaaac 111060ctgtcaaggg taggcagaac tggaggagtg aagaatggtt ttgagggtac agtggaaaaa 111120tcaactttgg agtatgacaa gccagcctca acactcagtc actcttacca gctgtgagat 111180cttaggtggg tttatcactg gctgtgagct tgacttgcct atctatacta taaaggaata 111240aaagcccctt caggtggttt ttcttaaata aatggaatag acctgtcatg tgctggtaaa 111300tgtttaacaa ctggttctca aggtggtggg ggcagccctg atttggagta tttccatggt 111360gtaaatactg tcaccatggc agatctcaag acaccagtgt gacagctctg aatgtggaat 111420tgggaagagg tatgcgccat ctgggataaa ccatctccta gtacctacct agaatgcagc 111480agacactcag gatgttatcc cctgcccttc cccctcatag gaacatctat gctgacagag 111540acctttgcca ttattaaagc atttttacaa acactatctc attacattct caggtgagat 111600gggcagggcg ggtcttattt ctacttttta cagacaagaa gcctcaggct cagagagact 111660ggacaaaatt gctcaggggc acacagcaag taatctacaa aatgagcata gcaggcagag 111720tagcagcccc aaaacatgtc tgcatcctaa tccctggaac ctatgaatat ttggttacat 111780ggcaaaaggg aattttgact gcagacggat ttaatattgc tagtcaggtg acctccaagt 111840agggagatca tcctagatta tccgggtggg cctagtgtag tgacaagggc ccttaaaagt 111900gaaggagggc agctcaagag gagagtcagg gggaaatgtg actatgcagg actggtcaga 111960gagatgtgac attgccagct ttgaagatgg aggaagcaag gaatctagaa ggcctctaga 112020agctggaaag gcagggaagc agcttctccc ccagaacctc catgaaaaga tgtagcctac 112080caggtacgtt gattttggcc cagtgagact tctgacctcc agaactttaa gatgatcaat 112140ttgtgttgtt taagctgctg gatctgtggt cctttgctac tacagggata ggaaatgaat 112200tcaacgggac tggcaatcag atctccttat ccctcaaatc tcagctcaaa tttcacctct 112260tgagggagcc atgtttttcc tgccttttct aggcctctgg ttttacccga gttctgataa 112320tatctggctt taggcactca ccagatggtt gtgaatcttt gcatacttgc tggttttctc 112380cctaatcccc aagtggcaag aatattcttt tcactttgca tcactttcca ctagcacagc 112440tccttcccca agaccacagc ataataactg cattcagaat gtgtgagtgt gaaaatagag 112500gaatgaatga atgggagctg acattaacaa tagtactgca ccccagcacc accaggaatc 112560acgatgctga gctgtcactg ttgcagaaca attaaaagca tctcctgcta gtatcctgag 112620ctacctaccc ccttcctgca ggaaagactt cccgtcttcc cactggctca cagaagtggc 112680cttttcctcc ggaaaccagc gtgcatctgt tagcatcttt tgtcttttgt gggaaaggga 112740ggattctggc tccagactga agagggacag gcatggtgtg tgggttcaca gagccccaga 112800ctccagactc tgatgccagc atctttgtct cagcaaaggt cataattgga tttggatgtt 112860taaggcagtc actgatttac tgttctcatt tttcaagtgt gagaaatact aactctgtgc 112920tttgcttact tctaaaggaa caaaggcagt atgagcagcc tttaaatact gccacataat 112980acactgagag cagacatgac cacgtgctac aaagagtcag ggctacgtag gaaagttcac 113040atcggcgtgg gaagtgaaag taagccacgt caccgtttct gtcacgtgac agctgtgggg 113100tgtggagcaa ctcactgagc ctctttcctg gtgtgtaaga tggggataat gatagctccc 113160attcactgac cgcttccaaa gtgcctgacc ctgttctaag ccttcactgt catcctgatt 113220ttatagttag acagtttgtc agtgtcagaa gcagaattgg ctacatagtt tgcaagtcca 113280gtgccctggt tcaaaaatga ttaagaattt caagatggtg gtagcagagc aaaaaaccaa 113340gcccggagcc ctccttagtg tggctcttat gtgactgcac aggctgcaca ccctgggcag 113400aattagtata caaatcctgc agcctgactc cagaaccttc catcttaaca tctatgccac 113460aagagctgtc tcctaaggtg gccgtgaggt tggaaatgag actaaggcat tcctggcaga 113520gggcacagtg tgaatgatct ggaaatacag gcgtggacag gaagatgagg aaggaatgat 113580aaactctcat ggctaaagct ttggtgagct gaaggaaaca gaagtgtgct ggtaggcagt 113640actggctgca aactgtgccc tccctgaagg ccaactataa agtgcaaggc ccttgtactt 113700tataggcgac tggtgttgag gatcgctgga aggtttagaa gcagggggta gcatggacgc 113760aggcagagaa ctgcccctag ggggaagtgt ggagcagagc agactggggc ctgggcaccc 113820gctagaggag gccgttacaa ccacactcta gggatctcat agtgagaggc tcagggagta 113880gaggaaggaa acagggaaaa ctgaaggtct tcaaggtaag tagaaattat agctttggaa 113940tacgatctgg aaacaggaaa ctgggggaaa ggaaaagctt ctgatgcctg ctggcctggc 114000cttctgtttg agcgccatca ggcccgtatg gagtatcatg atgtacacag actgcctttg 114060cacccataga aaacagggta catctcttca ggaagtagaa actctttgag caaaagaaat 114120cgatttaact tcctaaaaac aacagctgtt actggctata aactcagtca tttctcaatt 114180tgggtttttc ccccaaaata ttcccttcca attttcagag gcataatcag ttctgtgatt 114240atgttgcttg cacataatta gatcacacag taataataat aactgtaaac tctggaggaa 114300acagagtgca attaaaacca gaaagaaaaa aatctcccaa acctctccca gattcttctc 114360tcaaacttgg gagtggggca cctcaagccc agtgaaggaa tcaacatatt ttaccccaaa 114420atatatttct ttgacatatt ttgagatggc tgttcacagg gcccgcaaac agaagcagcc 114480ctacaaaact gtctcatgtg gcgaagattt gcatctgcag agaaaaacta cgttgataca 114540gccaggcttt ctctgaggct ctctcttgtc catatctagg aaaaactgag agtctgatac 114600ctttaaaggt ctgaaagaaa catttactat ctattctgtc tgagagcggc tacctctgag 114660atttctccta tgtaacaaga acgcctttgc cagccaggcc tcctcttctc cccatgccat 114720aatctctttg ccaagatcca agcctccagt ctttctgtaa cctcaaggct tctgtacccc 114780actgcgggga ttggtaatca ctctatggtt ctcctccatg tgcacaataa atttgtatgc 114840tttttctcca attaatctgc cttttaattt attattatta taaatttttt ttgagacgga 114900gtcttgctct gttgcccagg ctggagtgca gtggtgtgat ctcagctcac tgcaacctcc 114960gcctcccagg ttcaagcaat tctcctgcct cagcctcccg agtagctggg attacaggtg 115020cacgccacca agcctggcta atttttgtat ttttagtaga gacagggttt caccatgttg 115080gccaggctgg tctcaaactc ctgaccttgt gatccgcctg ccttggcctc ccaaagtgct 115140gggattacag gcgtgagcca ccatgcccgg cctattatat atattttaat gctctctctc 115200agcagagtca agaggattag ttatcggtgt gttggacata ctgagtatag ggggtctgtg 115260ggatatgtaa gaagaaatga ctactgggct gttgcatatg tggttcttca tctcattttg 115320ttttagagcc agagtctcac tctgtcactt agactggagt tcagtggcga ggcttttttt 115380tttttagaca gggtcttaac tctgttgttt aggctggagt gcagtggcac aatcatagct 115440cactgtaacc ttgaactcct gggctcaagc aaccctccca cttcagcctc ctgagtagct 115500gagatgacag gcacgtacca ccactcctga attttttaaa aaatgtttta ggcatgtacc 115560accacaacta aaaattaggc gtgtgccacc acacctagta tttaaaaaaa tgttttcgta 115620gagacggtga tctcgttatg ttgcccaggc tagtctcaaa ctcctggacc catgtgatcc 115680tctggcctca agcgatcctc cagcctcagc ctcagcctcc caaagtgctc agattatacc 115740cagtgtgccc agtctattaa tctgcctttt ttgagttgat ttttcagtaa accttcagag 115800ggcgaagggg aggttttccc ttggccccta ccctggattc aaatcctagc tcccacttcc 115860caacaatgtg actttgggca agtcaggtaa cctggttctc ttctataatg tgagggaacc 115920attctagcac ttaacctcaa tcatactgca ttaccttgtt ctgaagttta aatgagaaat 115980tcttagacta gccttcagca tgtagtaaag caggccataa atgtcagata ttatcataat 116040ctccagattt tctacagtga ttcttgaatg acatttaaga tcagaaacat ccatgtaatt 116100ctcatattac tatcattgtt atgtattctt tctctccgaa tgaagaatga aggtaccatc 116160cactgacacc acagtcacga ggttgggtca agccttatga ccaccctaga agcctagaca 116220gaagggttca aaggctcttc ctctcatatg ggctagtaat aaacagggtc tctaacagaa 116280tagcggaaac ctgagttgag catgttcatg agggagggga agctgacgcc cacatcagca 116340cttgttttcg aagaggagct gtcttggccc agagccgtgg ctcacgcctg taatcccagt 116400attttgggag gccaaagtgg gagaattgct tgagcccaag agttcaagtc tagcctgggc 116460aacatagtga gaccgtgtct ctagaaaaaa tttaaaaaat tagacaggca tggtggtgtg 116520tgcctgtgat cccagctact cgggaggctg aggtgggaag attgcttgag cctaggaggt 116580cgaggctaca gtgagctatg atcacaccac tgcactccag cctgagcaac agagcaagac 116640cccgtcttaa aaaaaaaaaa aaaaaaaaaa agaggagctc tccagattaa taacaatctc 116700cccagttttc atgattcagg cgtcaaatcc tgctgcccca tatggttctc accactcaac 116760gagtattatt tacagagggc cgtcgttgct atgtcctgga gatacagaga caaataggac 116820ccaggccgac cactgaggag atccaacctg atggggaaag aagacagaga acacacaagt 116880ggggctagac acggtggctc acacctgtaa tcccagcact ttgggaggct gaggcgagtg 116940gatcaccaga gatcaggagt tcaagactgg catggccaac atggtgaaac cctgtcgcta 117000ctacaaatac aaaaattagc cgagtatggt cctgtagtcc cagctactgg ggaggctgag 117060gcaagagaat cgcttgaacc caggagggag gggttgtagt gagccgggat tgcgccactg 117120tactccagcc tgggcgacag agagagactc catctctaaa taaataaaaa taaaaagaga 117180acacacaggt ggcagcaatt gggacaaatg tcactgcaga agaccttcca agggggtatt 117240ggggggtgag gaccacattt tgccttaggg tgacaaagaa ggttcacaca gtgggccact 117300ttccggggtg ttggtgatct gaatcttgat ggggagggtg gttacatgcg cgtacatggt 117360tgtctagcac tcatcgaggt acattcaaga tgaattttat gtcaattaaa cctcagtcag 117420gttgatagta aagagtctga aacatcgtgt tagcacttag tgggctcttg ggaattcttg 117480cagaatgaga acgtgagggg tatggagggt gggagtactg gcactttctg catccccagg 117540agctggctcc acattgctcc tggctctgca tcctgctttt gggtggccac ggggagaaga 117600cagggctgaa gccagcctcc acagcggagg gtcctctagc aaggcccagc agggcctcag 117660ggagttcaga gggaggcttg cttttgcttg cttgtctcaa cacaggccca ccttgactaa 117720acacaggctt cttgctgggc gggccagctc tgagccttgc aggccacttt atcacgttgg 117780tggctcccat ctgttcttag agtccacatt tccactgggt tgagctctca gccacctctg 117840cctaggctgc tctcttctga aactttcttc ttcctctgcc tcccctactt cccctcaccc 117900cttcctcacc cctcccattc tctctcccct agggcagagg acccagctgg agcagggctg 117960acaggagccc ctgagaggag aacttagagg cctgagaccc gttgagggtg tgtgtgtgtg 118020tgtgtgtgtg tgtgtgtgtc tatgtttggg agaatttgga taaagagaca ggctggtagc 118080ggtggctccc acctgtaatc ccagcacttt gggaagccga ggtgggctga tcacttgagg 118140tcaggagttc aaaatcaacc atgccaaatg gtgaaacccc gtctctacta aaaatacaaa 118200aattagctgg gcgtggtggc acgcacctgt aatcccagct acttgggagg ctgaggcacg 118260agaatcactt gaacccggga ggcagtagct gcagcgagcc gagatcatgc cactgtactc 118320cagcctgggc aacagagcga gactccgtct caaaaataaa aaataaaaaa taaaaaaaaa 118380acagagacag ccaaagtagc aatgtgcctg tgagagggcc taagttttcc ccctcagggt 118440agtcttgtca gatgaaatac aggatgccca gttaaattgg aatttcacag aaataaaata 118500atttctatgg tctcaaatat tgcatgggac atattcatac taaaaagcta tttgttattt 118560acctgaaatt cccatttaaa tttgagggcg tcttccattt ttattggcta aatctggcca 118620ccctccctca gggtgtgacg gcaggaagcg ggagagaaca cagatgtccc ctgttatctc 118680cctgggccct gcgtggccct ctcagggaac cacaagccag ggccaagccc atccaatcct 118740cctgcaccag gagcagcaga agccatgccg ctaatggcag tcacctcggt ggaagtagag 118800cgggcctgtg gcggcgtgag gcttttctgc tctgcttctg tggaagtgat tatccaataa 118860ctagtaggaa tcgggcagtt ggcagacgcc tcccatgcta taaagccaca caaggcaggc 118920aagtgtgggc acagatgaga ggctgagagc ggaggtagga tgtcacccct gccctcgcca 118980gcctcatctg ttttcccagg cgtcccccaa ccactaccca cgtggattct ccctcgaaga 119040gccccctttc agggccccat tggcacttca ctcttgaggg aggcctagga gaagctagtg 119100gaaagttact tccacataaa cggtcagaca gacggatgat gaaaccacag gaaaccacag 119160ctcctcccct gccggggcac caggccagaa gccgacctgc taggcttggg gaccccttct 119220ggtcttgcta ggggctcaga gaccctgggg aagtccctta ctgtgtgtct catgtcttca 119280cttatggatg ggatgccagc tgccctctca tgccctcaca gggctgttgg gaggcaaggg 119340tgagctagca gatgcaaaat aatggcaaaa ttaagaaacc aagtacctgt acaaatgtac 119400acctttatag tggttcaatt tggccacacg tgcttctact tagagaaaca ccatacatga 119460atattcaggc acatatacat atcactatat gatcctacga gcaacttccc tacctcactg 119520aaaatatgat tcacttcacc aaaatgtaat gctatggtca cattctacaa ggaaaacatc 119580caaagcacag ccactgtaag tgaggatagt tctcaactgg gcctatggtg ggctggctga 119640ctacacttgc agtctagaac ggggcctact gcagactggt ctgcagttag tttgatctag 119700ggttatgaca gcagttgact tgaaaataac tttcctcctg cccctttaag aacacgggtt 119760ccttcccttg cagataagca ggaagtcccc ctccctctgt cttggtgctt ggtggtgtgt 119820ttcacttcct ttcaaaggtg ttttcttact aaagaaaccc taagcccagg gtcacattct 119880gtttctttca caggcatact caactgcgtg ttgttgattc tgtgacaagt ttcttgagca 119940agaaaataca cagaaaaaat gttaaatgta taaaatatga aagtaaaaca tcaataatgt 120000tttaaatatt taaaatataa aagtaatgca tgcttctggt aaaaatgtaa atggtagagg 120060tttctctatt tcctttcccc agaggtaagc tcttaaaatt cccttccgga aatggtctct 120120ctacacctca cttttttatt gcacagaagg gatcagacac tatttatgag gctctttatt 120180ttccacttaa gagtattctg gctgggcgcg gtggctcaca cctgtaatcc tagcactttg 120240ggaggccgag gtgggtggat cacgaagtca ggagttggag accagcctgg ccaacatggt 120300gaaaccccat ttctactaaa aatacaaaaa gtagctggtg tggtggtggg tgcctctaat 120360cccagctact cgggaggctg aggcaggaga attgtttgaa

cccaggaggc agaggttgca 120420gtgagccaag atcgcgccac tgcactccag cctgggtgac agagcgagac tctgtctcgg 120480gggaaaaaaa aaaagagtat tctttggaca tccttccaca gcatatcatg aataaatctg 120540cctcaatctt tttttaacag ctgcatagtt ttccattgtt tgggtactct atcatgtatt 120600taattaatct cccaattaat atacacttag gttgattgta gcttttactc ttatctacaa 120660tgctatgggg gaaagatcta gatgtttgtg catgtgtatg tatatgtata tgtaagtgtg 120720ttagtatggc tatagaatga gagtcctgtc actggactta aactggaaat gctgggttca 120780actggaaatg ctgttttaat tgtgacagcc accggtgcct ctgaaaagtc cctacgaatt 120840gacccttcca acaacagcat ataagagctt tcacttttct ttccctgcat tctggccaac 120900tggagccact actcaacctt ttcatttttg tcagtctgaa atgagcagag aggtcctcat 120960tgtttgattt gcatttgaaa gcctaccata cacgggcatc ttctcatgtt gttcttagct 121020cagtgaggag ggccttagag accagacttt ggaaccagat agagctggat accgtcccag 121080ctcagccact tgccaactgt gatctctgga gcctcggttt atccatctgt aaaatgagca 121140tccttagact aaattcatgg gatttactga gaaggtgcaa caaagcaaca aaaaatgttt 121200atcactacaa atgttagtgt gtttctcatc caaacaaaca aaaaaacata tatatatata 121260tatatatata tatacatatt tacatatagt catgcatcgc ttaacaatgg ggatacgttc 121320taggcgattt cctcattgtg cgaagatcac agagtgtact tacacaaacc tagatggtac 121380agcctactac atacctaggt tatatggtac agcccattgt tcctagcttg caaacctgta 121440cagcatgtca ctgcactaaa tactgtaggc aactgtaaca caatgctaag tatttgtgta 121500tctaaaatta gaaaaggtac agtaaaaaat acagtattat tttatgggat cactgaaatg 121560tcatcacaca gtacatgact gtgtgtgtgt gtatatatat atatatatat acacatacat 121620atatgcacat acacacagtc ttatactgta tatgtataca gatgtgtgta tatatatagt 121680cttataccat atatgtatat agcgtatgtg tgtgtgtgtg tgtgtgtata tatatatata 121740tatatacagt gtgtgtatat atatatatat atatacacac acacacacac acacacacac 121800acacacatac acagggatat atatatactt ttttgaccta gtcaatggcc aatttaaccg 121860tctctacagc atctgagatc tcaccttctc ctcactgctg cccctgcttg agggtagaaa 121920atgtaaataa aatgctgaaa aataaaactt aaaactgtca cattcaatgg ttggtcttac 121980tagctgagca accttggcca tggtacttct cggagctcca gtttctttct ctgtaaaatg 122040gggtgttcac tctacctacc ccacaatgtc actgggagaa tgaagaggcg cttaaggcag 122100tgcctggctc tggcaagcac tcgataaatg atccttgtgg ttgttcttct tggccaggga 122160tccccttaag tgctcaggcc actttcccca gtgggatagg aggaaaatag tcacctcaac 122220taacagaggg taatctatca aacacctact gggctagttg aagacccttt gggcctataa 122280acactgcagt agggtgtggt ggaagggcat ctccaactct ctggtccagt ctttgagaca 122340caattagatc ctgccatgct cagcaagcag gttgagccat cagaccctgt tttttttgtt 122400tgttgttttg ttttgttttg ttttttcttt ctttctctct ctctttctct ctttccttcc 122460tttctttctt tcgacggggt cttgctctgt gacccaggct ggagtgcagt ggtgccaagg 122520ctacagctca ctgcagcctt gacctcacgt gctcaagcca cccttccatc tcagcctgcc 122580aagtagctgg gactacaggc atgtactaca atgcctagct aatttttcta ttttttgtag 122640agacagggtt ttgccgtgtt gcccaggctg gtctcgaact cctgggctca agcgatcctc 122700ccacctcagc ctcccaaagt gctgggatta caggcatgaa ccacctcgcc cggcctcact 122760cctccattgt cttttaatct cctttgtaga ggtcatcaac aaccccattc gttagcgttt 122820aacaagaaaa gaacttcttg gggtggggcg gggagtggga agttattgtg aatctccttt 122880gtgagaaaca aatgccattt tctgaatgat ttttgccacg ttttattttt taaaaatttt 122940ctcctttgga agcagggtgc actgtgtata ctgtcatgtt gctcagatga caggtaagtt 123000acccgtgagt aggcataagt gctgatgtcg tttctgggtc cttcaatacc acacagaatc 123060tccttgggtt tatgcatggt gaggagagta attcgccctc atcaaggcta tgatttatcc 123120cgccataact gagctaaggt ttttgtgaag acagctgaaa tatccacacc atgcagggaa 123180cagagacaca ttaaacaaca acacatctca aaaaatacta cttttgcagt ggagaaacct 123240gagtggtaac acattaatca attgatccaa gttaacatcc caataacgga acataccaac 123300ttcatgggcc cccggctgtg agacaccgag aaggtcacag tgtccctcgc tcgagtagca 123360ttcctgccaa aaaggcaagc ccaaatctaa tcccgaggaa acatcagtca aacttaaatc 123420aagggaaatt ccacaaaata agcggccagt cttcttcaaa aatagtgtca tggaatacag 123480acaggctgag aaagttctaa attaaaagat actagtgggg catatcaatt acatgccatg 123540catgatcctg gattggaaca tggaccggaa aaagctgtta taagaatgtt attgggctaa 123600tagacaaaat ttgaataagg tctgcacatc agataatagt cttgtagcaa tgtgacattt 123660cctggtactg acaattgtac tgtggctatg ttggagaatg accttattag aaaatgcata 123720cctagtattt gtgggtaaaa aggtgtcata ttcacaactt attctcacat ggtctagaaa 123780aaataagtaa gtaggtagac agaaagaacg ataggtagat agaaaaatag atatagatag 123840atagatagat agtaaaatat tgataaagca agtatggcca attattaaca actggtgaat 123900ctgggttaag ggtataccac agttctttac tgtctttttg cagcttttct gtaagtttga 123960aattattcta aagtgaaaaa ttttaaacat tctagtaact gcaaccatgg tgcatgttac 124020aaactaaata ataaataagt cctaattaat aaaggaagcg aaacattaac gttgcagaaa 124080tgtaaaacca aaaacaaatg acaaagcact ctccctctgc tggcagtttt agcattcttg 124140tacacagaat gatcattgat tttgttttgt tttgtttttc aagttcctct tagtacaaaa 124200tttaaatctc tttcatttaa catgcctgca caaagtactc agtaggtgcc aagcaatgac 124260tcacaacaaa gccagttccc aatggctctt gttctcattg gaagtgaagg ccatattcta 124320tatgtatgta tcctctctgc cttcagtaga gaaggcagga acactgggag gaaggaagaa 124380ggtaaaaaaa tctttattaa aatgcctcct gacatgtcca cttggactct actaggcaaa 124440tcaaatgtcc tacattccat ccgagctcct gatcatctat cctaaaacct gagcacactg 124500cgcctcccca tcccctgccc agttaagttt ctccacttca gtggatggcc actccatcct 124560tccagttgct cagaccaaaa tctttgcatc agcctggact cctcactttc tctccccctc 124620cataccctgt agatcagcaa atcctattgg ctctaccttc aaaatacatc cagcatctgt 124680ccactcatca ccactccact ttcccatcct ggtctgtcct ccacctgaat tgttgcaatg 124740gcctgtaaag cagcctccct gctcctagtc tagtctcaac ccaggagcca gagtggctgt 124800ggtccctttc aaacctgaat caggtcatgg gacttctctg atggcaaact gccagtggct 124860tcctaactca tgcaaaaaaa ggaaggccaa agttcttaaa atggcctggg aagctcctca 124920tgtttggcac catccatccc acactcccat aagggtgatt tgacctattt ggcttcacgt 124980tgtacgactc cagggaacac tgctgctctc ctgcttcagc catgctggct ttcgtgtgtg 125040gtcatggtcc ctgctcaggg ccttggagtt ggctgtgacc tctccctgga gcacctcccc 125100caggcatctg cgtggctcac ttcctctttc ccttgaagtc ttgcctctga tggcttacct 125160ggaccatccc tcctacacac tccctgttca tttcccttac cctactctca ctttccatgg 125220tcatcttcta gtatactatg cacactactt atttttaaaa atatgtattc tcgattccac 125280acaaaagtcc ctctccctct ccctctccct ttccctctcc cctctttcca cggtctccct 125340ctccctctct ttccacggtc tccctctccc tctctttgtc tccctctccc tctctttcca 125400cggtctccct ctgatgccaa gccgaagctg gactctactg ctgccatctc ggctcactgc 125460aacctccctg cctgattctc ctgcctcagc ctgccgagtg cctgcgatta caggcgcgcg 125520cccccacgcc tgactggttt tcctactttt ttggtggaga cggggtttcg ctgtgttggc 125580caggctggtc tccagctcct aaccgcgagt gatccgccag cctcggcctc ccgaggtgcc 125640gggtttgcag acggagtctg gttcactcag tgctcaatgg tgcccaggct ggagtgcagt 125700ggcgtgatcg cggctcgcta caacctccac ctcccagccg cctgccttgg cctcccaaag 125760tgccgagatt gcagcctctg cccggccgcc accccatctg ggaagtgaag tgaggagcgt 125820ctctgcccgg ccgcccatcg tctgggacgt gaggagcccc tctgcctggc tgcccagtct 125880ggaaagtgag gagcgtctct gcccggccgc catcccatct aggaagtgag gagcgcctct 125940tcccggccgc catcccatct aggaagtgag gagcgtctct gccgggccgc ccatcgtctg 126000agatgtgggg agcacctctg ccccgccgcc ccgtctggga tgtgaggagc gcctctgccc 126060ggccgcgacc ccgtctggga gatgaggagc gtctctgccc ggctgcccca tctgggaagc 126120gaggagaccc tccgcctggc aaccgccccg tctgggaagt gaggagcatc tccgcccggc 126180agccacccca tccgggaggg aggtgggggt cagcccccac caggccagcc gccccgtccg 126240ggagggaggt ggggggggtc agccccccgc ccggccagcc gccccgtccg ggagggaggt 126300gggggggtca gccccccgcc cggccagccg ccccgtccgg gaggtgaggg gtgcctcttc 126360ccagccgccc ctactgggaa gggaggagcc cctctgcctg gccagccgcc ccatccggga 126420gggaggtggg ggggtcagcc ccctgcccgg ccagccgccc cgtccgggag ggaggtgggg 126480gggtcagccc cccacccggc cagccttccc gtccgggagg gaggtggggg ttcagccccc 126540cgcccggcca gccgccccgt ccgggaggtg aggggcgcct ctgcccggcc gcccctactg 126600ggatgtgagg agcccctctg cccggccacc accccgtctg ggaggtgtac ccaacagctc 126660attgagaacg ggccgggatg acaatggcgg ttttgtggaa tagaaacggg gcaaaggtgg 126720ggaaaagatt gagaaatcgg atggttgccg tgtctgtgta gaaagaagta gacatgggag 126780acttttcatt ttgttctgta ctaagaaaaa ttcttctgcc ttgggatcct gctaatcggt 126840gaccttaccc ccaaccctgt actctctgaa acatgtgctg tgtccactca gagttgaatg 126900gattaagggc ggtgcaagat gtgctttgtt aaacagatgc ttgaaggcag catgctcgtt 126960aagaatcatc accactccct aatctcaagt acccagggac acaaacactg cggaaggccg 127020cagggtcctc tgcctaggaa aaccagagac ctttgttcac ttgtttatct gctgaccttc 127080cctccactat tgtcctatga ccctgccaaa tccccctctg cgagaaacac ccaagaatga 127140tcaataaaaa aaaataataa aaaataaaaa ataaaaaaaa aaaatatgta ttctctcttt 127200ctggcccgct agaaagtaaa ctccttaaag gcagaagttt tgtttattga gatatccttg 127260gctcctacaa caatgcttgc cacacagtag taactcaata aaaattggtt gaacaaataa 127320agactatttc aaaaattaag catgggagat caatgtagtt gcatattaaa atgatttcta 127380taaaaataaa catgatcagt cgagatcttt tttttttttt tttttttgag acggagtttc 127440gctcctgtgt cccaggctag agtgcaatgg tacaatctcg gctcactgca acctccgcct 127500cccgggttca agcgattctc tcctccctca gcctcccagt agctgggatt gcaggcgcct 127560gctaccacac ccggctaatt tttgtatttt tagtagagat gcgggtttcg ccatgttggc 127620caggctggtc tcgaactcct gacctgaggt gatccaccgc ctcggcctcc caaagtgctg 127680ggattacagg catgagccac cgcgcccggc ccagtcaaga ttttttaaca aaatttttaa 127740attatgtctt tcaacaaagg tgtttattct gggttttgga gtttgcattc aaaattttgc 127800attcaaatct gaatttagct ttgtgattcc tgagtttcag tttccttcca gcaaaaccaa 127860aaatagaagg acttggcttg aagggtttta actgaggatt aaattagatg atacacctgg 127920cactggttta ataaatgaga gtacaagctt tttctcattt tgtcattctg taaatctacc 127980cagagttaag ccaaacttca caagttaaat tctctacaag acagccctca cttcagacac 128040cagtccccag ggtcactcac tcttccaacc aactggctaa aattgaggga ttcccgtgac 128100caccctctgg ctcaataatt tgctagaacg attcacagaa ctcagggaag tgctatactt 128160acagttttat tacagtaaaa ggatacagcc caaagaagaa atgcgcaggg caacatctgt 128220cagggttcca agtgcaatgc ttctgtcatc cttaaggacg tgttatcctc ccagcctgaa 128280agtgtgacaa tactcaagga gtaatgtcag tcaaggaggc tcacttgagc tttggtgtac 128340taaaatgttt actgaggcct actgaggcct catacaggca tgattgatag aaccactggt 128400cacatggttc attctccagc tccccttttc ccctccagag gttgggctta tatctcatgg 128460cttaaagccc caaccctcta atcacatatt tgatctttcc ggcctggcca gcccctattc 128520tgaatcgttt cattagcata aactgtccag tgtgagccac ctcattatca taaacaatca 128580agcctggtcc aaggggccca ccatgaataa caaagacact ccaatcactc aggaaattct 128640aagggtttag aagctacatc tcaggaacta gagataatgg ccagccaaat tcttcattac 128700acaaaaccaa acacagtgtt ctagctaaaa agtaaggcat ttatgcaaac ataaccacag 128760agtacaaaat ataaaattca gacattaaaa tacctcctcc tggcccaaac taaaattgtg 128820ttcctgtctg tgtatacata gaaaaataga aaaatttaag tgcaaatgta tgctcactct 128880ttgtgccaag gaccttaagt agtaaaaaaa aaaaaaaaaa aaaaaagact aggaagccat 128940tttcatgaat gagtaaattt aggtctttag aagccacgtg tgtaatttca aatctgagaa 129000atggatgctt gtatgacaaa aactaacaat atacaccacc atcaccacca ctgccactac 129060caaaacaaaa tcaggtctat tattgtttgg ttagcactgg gaacaattcc agtattggtg 129120aaccatccta tgtttaggta aaatacacat tctgagttga ctgagtttca tatatgactg 129180aatctgaatt tggctccttc tcatcttaga tatacaatct tcctcctaaa gtatggattc 129240tatgagatct agcacctaaa attatgccta aaggactatg ggcccacagt tgaatacaag 129300gagttttggt ttatggtctt ggctctgcaa accaactgaa tacccttgaa tgggtggctt 129360aacctctctg tgctgaattc catcctctgc aaaatattga tacgaatatc tagtcccttt 129420atctgcctac aagtacttta aggataaaat gacacaaaag gtgagagaat attttgatga 129480agttcactaa aactagataa atatgagttg cattacataa ggaattacta aacaccaaaa 129540tgtaaggtga cacctataag ctggtcttcc ccccgccccc accagctgtt gtccaatttg 129600ttgctttctc tgagctggtg cccaaaccct ttcaggtaag ctgctggtat agatggtgaa 129660atttagatca agcagagaat attcagaggt aaatggtaaa ttaaatttca aaagcagggt 129720tattggttta tcacattcat cggtagccac gccatcactt ccattttttt ctcaaaaaac 129780gctgttggtg gccgggcgca gtggctcaca cctgtaatcc cagcactttg ggaggccaag 129840gcaggcagat cacgaggtca ggagattgag aacatcctgg ctaacacagg tgaaaccccg 129900tctctactaa aaatacaaaa aaaaattagc cgggcgtggt ggcgggcgcc tgtggtccca 129960gctactccgg aggctgaggc aggaggatgg tgtgaacccg ggaggcggag cttgcagtga 130020gccgagatcg cgccactgca ctccagcctg ggcgacagag caagactctg tctcaaaaaa 130080caaaacgaaa caaaaaaaac aaaaacgctg ttggtgactt tctctagttt aagaaaccgt 130140tcctttagtt tggggtccac ggacacccaa gagatttttg aagagaatct agggaactta 130200gaaggaaaaa aacccatctt tattttcatt aacctctgaa tgttagcggt ttcttcaaat 130260acgcatgtag tcaagaaacc tctgaaatct tagcagttcc tgtgcttgta tcaacaaaag 130320aaattacaga tatttttata tctgttaacg ttgtgtagat atcaaacact gatcactatt 130380ttgaatttat agtggatatg agactgactg ctagactgtg ttctttaata tataaagaag 130440cacatatatt acatcacaat tattaaaaac ttttggtaac tgtatgtaaa tataattttc 130500atttgtgatc ccaggttttt gttttaagca ttgacataat tctgagaaag gatccatgag 130560ggggtggggt ggtccccaac cccctgctgt gcagttagga accgggccac acagcaggaa 130620gtgagacgcc agtgatcgag cattaccaca tgaccgcctg agctatgcct cctgtgagat 130680cagggcggca ttagattttc ataggatcgc caaccctatt tcaaactgca catacgaggg 130740atccaggttg tgcactcctt atgagaagct aatgcctaat gatctcaggt ggaacagttt 130800catcctgaaa ccagcaccct cccgaccctg tccatggaaa gattgtcttc cacgaaaccg 130860gtctctggtg ccaaaaaggt taggaaccgc tgatctatgg gactattggg tcagtttcaa 130920ttctccttca ttggccttgg tacatgcaag tcaattctga ggcacaagta agctgaaagt 130980tgcagccatc cctcagacag gaaaaaataa cccggcagaa aggaagagaa cagaactggt 131040gtgtgcacct cacagaggcc tagaaaacgg tggcggctgc tccaaaacac cagctccaag 131100ggcacctagg ctcagatcga agcattgcag gtggcaaaga ggctggagca tcctgtgtac 131160acactcagac tgatccacca gtgaaaggaa ggggttaaag aggaggagca tggaaaactt 131220ccaggagagc tgacaaactt agctgcaaag tcaggataaa acagtccaag aaccccaaca 131280aggaactact ggaggtgatg tcggagagtt cctctctctc ataaagatca aagatttctt 131340gggattgtct gctgtgcaca agcgtggccc ggccctgggt caacaagaat gtccctcaac 131400tggaaagaaa atagagggat gaagaattcc aattctggca ctggtgccac cagctctact 131460ggtgccagca gtctgagaac cttggtctgg tatccgcacc tcctcccagg tgctctggga 131520atgggtaagg gaactaagat gctgtggccc ttaagctctg ccggtcaaat caacctatga 131580tgaagctcta tagcattgtt aagaagccca ttgttttgta ctttaaaatt tgttttcctc 131640tagtttccta ataaaaagtt tttttttttt tcctaaattg gcaccacttt ctcagctcaa 131700gaagaacagg gagtttccac acctgagaag tgttaatggg catgacctga gagggcatta 131760taggcagagg aagaggcacg tgtgtaaggg cccattcctg ggtccaggaa aagacagtag 131820cactggaggc actcaaagaa tgagaatcta aaaagggcag gactggcttc agaatttggg 131880ggacacagta caaaatagaa atgtggggta agaatttcaa aatggcggca gcagtacatt 131940aagccaagca ccagggactt ctgagcgtaa ggcccagtgc agctgcacag ctgtgcacct 132000gtgaagccag tcctggagag gccaaatcgt cagggtcagc caaggaagtt gatcttggcc 132060tatgtgggtt ttcccctcag atttgagcca gtgctccatt acctacactc cattttcctc 132120accataaagc tattaaccat cttctgctga tgatctggga actatatttt atataaagct 132180ttggcaccca cagaaaaaac acattaaaaa atacaaacac ttgttacttg ctaagcataa 132240ttctgcttca caatggaatc aaaggtttgt catttttaac acagaaagcc attgggagtg 132300tgtaatgcct cccctttcaa tatgctattg aacctactaa tttttttagc cattgtaata 132360tgaatgactt cttagaaaaa tattgtagta aggccaggct cacgcctgta atcccagcac 132420tttgggaggc cgaggcgggt agatcacgag gtcaggagat caagaccatt ctggccaaca 132480cggtgaaacc ctgtctctac taaaatacaa aaaattagct gggagtggtg gtgtgcacct 132540gtaaccccag ctactcagga ggctgaggca ggggaatcgc ttgaacctgg gaggcggggg 132600ttgcagtaag ccgagattgc accactgcac tccagcctgg tgacagagca agactccgac 132660tcaaaaaaaa aaaaaaagaa aagaaaaaga aaaagaaaaa tatttaagta gtgaaaagct 132720gaggaaaact actaacgttc aggatacaaa ttctctttat tgccagtgcc ttggccatgc 132780aggaggagga aaaagagcag attgaatttc tgtaatgaaa taatttgaaa attggggcag 132840ggaaaatttt aatgtatatg acattattac gatatctatg tttaaatgtc ttcatgatgt 132900aggctgggtg cggtggctca cgcctgtaat cccagcactt tgggaggccg aggtggaagg 132960attgcttgag cccaggaact tgagaccagc ctgggcaaca tagagagaac ctgtttctta 133020aaaaaaaaaa agaaaaaaag agaaaaaaca tctacatgat gaaaaagcta gacagctgat 133080tgagacagaa tataacttta cccttatctg gcgtatggaa caaagccttt aaaaggcctt 133140aggattctag agaagagtag tgtttaaatg aggcggcttc tgagccagcc agtctgagtt 133200caaatcctgc ctttggcttt cgttgtgttt tcttggggaa gttatttaat ccctctagaa 133260ttctctcatc tgcaaaatga ggagaataat ggtgccactg tcataggatt gtgtgaggat 133320tcgggaaata ctacatggaa agcttcccag tatatggaaa gcactcagaa agcacttgct 133380gttattactg ctatgggtac agccatgatt gttactatca ttattctaca acctggcccc 133440tgcctggttc ccagcgttat ttcctgtcac tttctctcat tttacgcaag tgcctgccaa 133500gctaagctac tcattgcccc ctgcgttaac tcgaagattc tctgcttccg tattgttccc 133560tggctgtgtg gaggctgagt aaccacttgg aggggatgtt gtagaggaaa attcaagcca 133620cagaagggaa actgggttga atgagatccc cttttacatt tcttttggct ctaaagtttc 133680catcggatca ataaagcaaa caggacaatt agcagcacgg cactcagcac tgagcacagg 133740cgagggtgtt ctttcactcc tctttgcttt cctgccaccc cctgctcgcc ttcctctttg 133800caccatgtgg gtccgctctg ggtgggagtc ctggagaaag atacttcaac taaagataag 133860gcagagatag gcctcggtgg aaagacttgg tagacagacc tgggcatttg attaagagca 133920aattgcttaa attctcggag cctcaatttc tttagtagta aaatggggat attagtactc 133980actccatagg gtaagaatgg aatcagatat tgtttaaaag ggcttatagc tgaggttaca 134040agatatagct agtctactaa atatcctttc tcttttcatt ttcctgttat tcacaggggg 134100gacttaggct gtgtctttaa ggggttttag attccaatca gtggaaggag ggctctgctg 134160tgagggagac cagatggaga ctgcaggggc cacaggggaa cgtggggtgc aggcaggtgg 134220ggcagatggt aggaggccct gaaactcaga caaagggatc tgggcttgag aaagcaagag 134280ctgggagctc agctttgttc ctgttcttcc gttcctccct cctcttcctt cttctcttct 134340tctggtaagt ttttggcaca aaattaaata ggttcaaact aatagaactc attttcatga 134400attcagactt ctagccaact tctccatggt tagaaatagc cacgactcta tcccaggccc 134460gccctggaaa gcaaaataaa actgcgtccc ctgccacctt ttccgggttc caccagacac 134520agggttggag gggaaccgcc cttcaacgca ggcaatgttg gcacattttt cattgggcgc 134580tgtggcccgt gagatgagga aggcaggaca ggcgcaaagt ggactctact tatctttcac 134640cagagagccc caaggccaga gtgcaccata caacccggag tcacccaagg cctttttttt 134700tggggggggg tctcactggg aacaatgtct gcacccattg taagcagttc ctttgcccct 134760gtgtgcctct gtctttttaa ttgggatcga aaactgctac ctcaatggta gttgtgcccg 134820gcacatacta acagacttat aaatgtagct gcctggggca gggatgatcc tggaacattc 134880tgacatctag aacattaaga ctaaatggat ttaattatct ggggagaaga aagcaagaca 134940attagagtca caggttcaac acctgagttc aagtacctcc ctcatttaag aaaatcaatt 135000catggaaggc tgaggcagag aatggcttga atccgggagg tggaggttgc agtgagccga 135060gatcacatca ctgcactcca gcctgggtga cagagcgaga ctgtctcaaa aaaagaaaag 135120aaaagaaaat caattcaatg cagtgggaga actgttattg agcaattact atccaccaga 135180cgctgtggaa agcacaactg cctagctcct cacactcttc ctggtgattc tcgggtacag 135240actaagagga tgtggggagg acttttacat gctcttggca tatataacag agtggctacc 135300agctgaggta acccaatgat ggctctctaa gcctcagttt ccccatctgg aaatttctta 135360tggaaaggcg gtgggaaaga aaggaaaaaa caaatgtaaa acttacgtta caatgcctga 135420ctcttagaaa atatcaaccg acaggagttg tcatgtttta

tttgctgtaa aactaccaaa 135480agatcttcta tcaacatttt gtaagtgaaa ataataatat atattcatat gtattaatta 135540tatagaaata atatataata atatataaat gtctctatag gagaaataat atcaagacac 135600ctaaaatttt taattaattc taaacactaa gacaattatt ttattatttt tcctcaacct 135660tccatcctaa atcatacgaa taataacaat agaaagagct tactaattgt catgcactgt 135720ccgaagcatt tactcctccc aacaaccctg tgaggtcggt gttgcaatga acccatttta 135780cagatgagaa aactccagca cagaaagatt cattcacatg gccagagtca tgcaactttt 135840aagaatattc ctttggtttg catttccttg tatctgcacc acccttccct ttcctgtctc 135900tctaggtctc ctgaccggtt ggcaggtcag tcccaatcat gggaactctt ttcctttaag 135960cagtcgtctc tctttctctt cctctctgtt tccacctgcc ctccatcagt gtctcagcac 136020agtggaaggc tgcttgctac cgcactggaa aacttctcca gacatttcca agtttgcatt 136080cgtccatctc ctttggagaa ttgcttcagt gaggcctaac cctttctgag gaaaggccct 136140tgggagaaga agccaggaga aggtagtggc actcctttgt catcaggaga cattgggagg 136200aaagaaatga ttttcgacag cagctgggtg aggggaaaca gaatcacaga acctgcggaa 136260acggtgtcgg tacaacggca gctgccagct aaacgggcga aaaggcaccc tgcaagatct 136320tttctgtgtt tcaacgatga agtagactcc caacatggaa caggggcggg ggggctgtta 136380ccagacaggt gacatttggt gcatgcacga acaggtccag tgccccattc ggaggccagg 136440aaggcagcca cccagcagag cattaactag gggaccagac ggtcacctgc ccctgctcac 136500ccccacctcc ttcctcccca ggaaaaagga agtcaagtag atggagaaac ggggaagtcc 136560aagctgctgg cagtggcatt caagggtcta tatataaggt tgactctaat ccccagcgag 136620acaaaagcct gtgccataaa aatgattagc aaggggtttc taaaaacatt tccccaggcc 136680acggaaaagg agaaggaata ttttgcacac tgccttgtct cttagaaata atcacagcta 136740ggcctcttta taaccggcga gtctttttcc tttcacacgc catttgcagg cgcgtggcat 136800ggtatttgta actaattttc agtagcaagt gggtcactgg tattcacaga gtcaggtgcc 136860attttgaaaa ctgcttccct ctacccgaat ctcttatatt tccagatgca gatgcacttc 136920agcgatgtcc tagtgctgga aacgctccct tgaaaatata atttgatttt tgaatttcat 136980cttaagacat tagagataat gtaactgtag tctttggaat tccgcggatc cttctatagt 137040gtcacctaaa tgtcgacggc caggcggccg ccaggcctac ccactagtca attcgggagg 137100atcgaaacgg cagatcgcaa aaaacagtac atacagaagg agacatgaac atgaacatca 137160aaaaaattgt aaaacaagcc acagttctga cttttacgac tgcacttctg gcaggaggag 137220cgactcaagc cttcgcgaaa gaaaataacc aaaaagcata caaagaaacg tacggcgtct 137280ctcatattac acgccatgat atgctgcaga tccctaaaca gcagcaaaac gaaaaatacc 137340aagtgcctca attcgatcaa tcaacgatta aaaatattga gtctgcaaaa ggacttgatg 137400tgtgggacag ctggccgctg caaaacgctg acggaacagt agctgaatac aacggctatc 137460acgttgtgtt tgctcttgcg ggaagcccga aagacgctga tgacacatca atctacatgt 137520tttatcaaaa ggtcggcgac aactcaatcg acagctggaa aaacgcgggc cgtgtcttta 137580aagacagcga taagttcgac gccaacgatc cgatcctgaa agatcagacg caagaatggt 137640ccggttctgc aacctttaca tctgacggaa aaatccgttt attctacact gactattccg 137700gtaaacatta cggcaaacaa agcctgacaa cagcgcaggt aaatgtgtca aaatctgatg 137760acacactcaa aatcaacgga gtggaagatc acaaaacgat ttttgacgga gacggaaaaa 137820catatcagaa cgttcagcag tttatcgatg aaggcaatta tacatccggc gacaaccata 137880cgctgagaga ccctcactac gttgaagaca aaggccataa ataccttgta ttcgaagcca 137940acacgggaac agaaaacgga taccaaggcg aagaatcttt atttaacaaa gcgtactacg 138000gcggcggcac gaacttcttc cgtaaagaaa gccagaagct tcagcagagc gctaaaaaac 138060gcgatgctga gttagcgaac ggcgccctcg gtatcataga gttaaataat gattacacat 138120tgaaaaaagt aatgaagccg ctgatcactt caaacacggt aactgatgaa atcgagcgcg 138180cgaatgtttt caaaatgaac ggcaaatggt acttgttcac tgattcacgc ggttcaaaaa 138240tgacgatcga tggtattaac tcaaacgata tttacatgct tggttatgta tcaaactctt 138300taaccggccc ttacaagccg ctgaacaaaa cagggcttgt gctgcaaatg ggtcttgatc 138360caaacgatgt gacattcact tactctcact tcgcagtgcc gcaagccaaa ggcaacaatg 138420tggttatcac aagctacatg acaaacagag gcttcttcga ggataaaaag gcaacatttg 138480cgccaagctt cttaatgaac atcaaaggca ataaaacatc cgttgtcaaa aacagcatcc 138540tggagcaagg acagctgaca gtcaactaat aacagcaaaa agaaaatgcc gatacttcat 138600tggcattttc ttttatttct caacaagatg gtgaattgac tagtgggtag atccacagga 138660cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga 138720ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat agaaattgca 138780tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg gaatggacga 138840tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg 138900tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc 138960gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa 139020catgagaatt gatccggaac ccttaatata acttcgtata atgtatgcta tacgaagtta 139080ttaggtccct cgactatagg gtcaccgtcg acagcgacac acttgcatcg gatgcagccc 139140ggttaacgtg ccggcacggc ctgggtaacc aggtattttg tccacataac cgtgcgcaaa 139200atgttgtgga taagcaggac acagcagcaa tccacagcag gcatacaacc gcacaccgag 139260gttactccgt tctacaggtt acgacgacat gtcaatactt gcccttgaca ggcattgatg 139320gaatcgtagt ctcacgctga tagtctgatc gacaatacaa gtgggaccgt ggtcccagac 139380cgataatcag accgacaaca cgagtgggat cgtggtccca gactaataat cagaccgacg 139440atacgagtgg gaccgtggtc ccagactaat aatcagaccg acgatacgag tgggaccgtg 139500gttccagact aataatcaga ccgacgatac gagtgggacc gtggtcccag actaataatc 139560agaccgacga tacgagtggg accatggtcc cagactaata atcagaccga cgatacgagt 139620gggaccgtgg tcccagtctg attatcagac cgacgatacg agtgggaccg tggtcccaga 139680ctaataatca gaccgacgat acgagtggga ccgtggtccc agactaataa tcagaccgac 139740gatacgagtg ggaccgtggt cccagtctga ttatcagacc gacgatacaa gtggaacagt 139800gggcccagag agaatattca ggccagttat gctttctggc ctgtaacaaa ggacattaag 139860taaagacaga taaacgtaga ctaaaacgtg gtcgcatcag ggtgctggct tttcaagttc 139920cttaagaatg gcctcaattt tctctataca ctcagttgga acacgagacc tgtccaggtt 139980aagcaccatt ttatcgccct tatacaatac tgtcgctcca ggagcaaact gatgtcgtga 140040gcttaaacta gttcttgatg cagatgacgt tttaagcaca gaagttaaaa gagtgataac 140100ttcttcagct tcaaatatca ccccagcttt tttctgctca tgaaggttag atgcctgctg 140160cttaagtaat tcctctttat ctgtaaaggc tttttgaagt gcatcacctg accgggcaga 140220tagttcaccg gggtgagaaa aaagagcaac aactgattta ggcaatttgg cggtgttgat 140280acagcgggta ataatcttac gtgaaatatt ttccgcatca gccagcgcag aaatatttcc 140340agcaaattca ttctgcaatc ggcttgcata acgctgacca cgttcataag cacttgttgg 140400gcgataatcg ttacccaatc tggataatgc agccatctgc tcatcatcca gctcgccaac 140460cagaacacga taatcacttt cggtaagtgc agcagcttta cgacggcgac tcccatcggc 140520aatttctatg acaccagata ctcttcgacc gaacgccggt gtctgttgac cagtcagtag 140580aaaagaaggg atgagatcat ccagtgcgtc ctcagtaagc agctcctggt cacgttcatt 140640acctgaccat acccgagagg tcttctcaac actatcaccc cggagcactt caagagtaaa 140700cttcacatcc cgaccacata caggcaaagt aatggcatta ccgcgagcca ttactcctac 140760gcgcgcaatt aacgaatcca ccatcggggc agctggtgtc gataacgaag tatcttcaac 140820cggttgagta ttgagcgtat gttttggaat aacaggcgca cgcttcatta tctaatctcc 140880cagcgtggtt taatcagacg atcgaaaatt tcattgcaga caggttccca aatagaaaga 140940gcatttctcc aggcaccagt tgaagagcgt tgatcaatgg cctgttcaaa aacagttctc 141000atccggatct gacctttacc aacttcatcc gtttcacgta caacattttt tagaaccatg 141060cttccccagg catcccgaat ttgctcctcc atccacgggg actgagagcc attactattg 141120ctgtatttgg taagcaaaat acgtacatca ggctcgaacc ctttaagatc aacgttcttg 141180agcagatcac gaagcatatc gaaaaactgc agtgcggagg tgtagtcaaa caactcagca 141240ggcgtgggaa caatcagcac atcagcagca catacgacat taatcgtgcc gatacccagg 141300ttaggcgcgc tgtcaataac tatgacatca tagtcatgag caacagtttc aatggccagt 141360cggagcatca ggtgtggatc ggtgggcagt ttaccttcat caaatttgcc cattaactca 141420gtttcaatac ggtgcagagc cagacaggaa ggaataatgt caagccccgg ccagcaagtg 141480ggctttattg cataagtgac atcgtccttt tccccaagat agaaaggcag gagagtgtct 141540tctgcatgaa tatgaagatc tggtacccat ccgtgataca ttgaggctgt tccctggggg 141600tcgttacctt ccacgagcaa aacacgtagc cccttcagag ccagatcctg agcaagatga 141660acagaaactg aggttttgta aacgccacct ttatgggcag caaccccgat caccggtgga 141720aatacgtctt cagcacgtcg caatcgcgta ccaaacacat cacgcatatg attaatttgt 141780tcaattgtat aaccaacacg ttgctcaacc cgtcctcgaa tttccatatc cgggtgcggt 141840agtcgccctg ctttctcggc atctctgata gcctgagaag aaaccccaac taaatccgct 141900gcttcaccta ttctccagcg ccgggttatt ttcctcgctt ccgggctgtc atcattaaac 141960tgtgcaatgg cgatagcctt cgtcatttca tgaccagcgt ttatgcactg gttaagtgtt 142020tccatgagtt tcattctgaa catcctttaa tcattgcttt gcgttttttt attaaatctt 142080gcaatttact gcaaagcaac aacaaaatcg caaagtcatc aaaaaaccgc aaagttgttt 142140aaaataagag caacactaca aaaggagata agaagagcac atacctcagt cacttattat 142200cactagcgct cgccgcagcc gtgtaaccga gcatagcgag cgaactggcg aggaagcaaa 142260gaagaactgt tctgtcagat agctcttacg ctcagcgcaa gaagaaatat ccaccgtggg 142320aaaaactcca ggtagaggta cacacgcgga tagccaattc agagtaataa actgtgataa 142380tcaaccctca tcaatgatga cgaactaacc cccgatatca ggtcacatga cgaagggaaa 142440gagaaggaaa tcaactgtga caaactgccc tcaaatttgg cttccttaaa aattacagtt 142500caaaaagtat gagaaaatcc atgcaggctg aaggaaacag caaaactgtg acaaattacc 142560ctcagtaggt cagaacaaat gtgacgaacc accctcaaat ctgtgacaga taaccctcag 142620actatcctgt cgtcatggaa gtgatatcgc ggaaggaaaa tacgatatga gtcgtctggc 142680ggcctttctt tttctcaatg tatgagaggc gcattggagt tctgctgttg atctcattaa 142740cacagacctg caggaagcgg cggcggaagt caggcatacg ctggtaactt tgaggcagct 142800ggtaacgctc tatgatccag tcgattttca gagagacgat gcctgagcca tccggcttac 142860gatactgaca cagggattcg tataaacgca tggcatacgg attggtgatt tcttttgttt 142920cactaagccg aaactgcgta aaccggttct gtaacccgat aaagaaggga atgagatatg 142980ggttgatatg tacactgtaa agccctctgg atggactgtg cgcacgtttg ataaaccaag 143040gaaaagattc atagcctttt tcatcgccgg catcctcttc agggcgataa aaaaccactt 143100ccttccccgc gaaactcttc aatgcctgcc gtatatcctt actggcttcc gcagaggtca 143160atccgaatat ttcagcatat ttagcaacat ggatctcgca gataccgtca tgttcctgta 143220gggtgccatc agattttctg atctggtcaa cgaacagata cagcatacgt ttttgatccc 143280gggagagact atatgccgcc tcagtgaggt cgtttgactg gacgattcgc gggctatttt 143340tacgtttctt gtgattgata accgctgttt ccgccatgac agatccatgt gaagtgtgac 143400aagtttttag attgtcacac taaataaaaa agagtcaata agcagggata actttgtgaa 143460aaaacagctt cttctgaggg caatttgtca cagggttaag ggcaatttgt cacagacagg 143520actgtcattt gagggtgatt tgtcacactg aaagggcaat ttgtcacaac accttctcta 143580gaaccagcat ggataaaggc ctacaaggcg ctctaaaaaa gaagatctaa aaactataaa 143640aaaaataatt ataaaaatat ccccgtggat aagtggataa ccccaaggga agttttttca 143700ggcatcgtgt gtaagcagaa tatataagtg ctgttccctg gtgcttcctc gctcactcga 143760gggcttcgcc ctgtcgctca actgcggcga gcactactgg ctgtaaaagg acagaccaca 143820tcatggttct gtgttcatta ggttgttctg tccattgctg acataatccg ctccacttca 143880acgtaacacc gcacgaagat ttctattgtt cctgaaggca tattcaaatc gttttcgtta 143940ccgcttgcag gcatcatgac agaacactac ttcctataaa cgctacacag gctcctgaga 144000ttaataatgc ggatctctac gataatggga gattttcccg actgtttcgt tcgcttctca 144060gtggataaca gccagcttct ctgtttaaca gacaaaaaca gcatatccac tcagttccac 144120atttccatat aaaggccaag gcatttattc tcaggataat tgtttcagca tcgcaaccgc 144180atcagactcc ggcatcgcaa actgcacccg gtgccgggca gccacatcca gcgcaaaaac 144240cttcgtgtag acttccgttg aactgatgga cttatgtccc atcaggcttt gcagaacttt 144300cagcggtata ccggcataca gcatgtgcat cgcataggaa tggcggaacg tatgtggtgt 144360gaccggaaca gagaacgtca caccgtcagc agcagcggcg gcaaccgcct ccccaatcca 144420ggtcctgacc gttctgtccg tcacttccca gatccgcgct ttctctgtcc ttcctgtgcg 144480acggttacgc cgctccatga gcttatcgcg aataaatacc tgtgacggaa gatcacttcg 144540cagaataaat aaatcctggt gtccctgttg ataccgggaa gccctgggcc aacttttggc 144600gaaaatgaga cgttgatcgg cacgtaagag gttccaactt tcaccataat gaaataagat 144660cactaccggg cgtatttttt gagttatcga gattttcagg agctaaggaa gctaaaatgg 144720agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt 144780ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta 144840cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca 144900ttcttgcccg cctgatgaat gctcatccgg agttccgtat ggcaatgaaa gacggtgagc 144960tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt 145020tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc 145080aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata 145140tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca 145200atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca 145260aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg 145320gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaatttt 145380tttaaggcag ttattggtgc ccttaaacgc ctggttgcta cgcctgaata agtgataata 145440agcggatgaa tggcagaaat tcgatgataa gctgtcaaac atgagaattg gtcgacggcg 145500cgccaaagct tgcatgcctg cagccgcgta acctggcaaa atcggttacg gttgagtaat 145560aaatggatgc cctgcgtaag cggggcacat ttcattacct ctttctccgc acccgacata 145620gataataact tcgtatagta tacattatac gaagttatct agtagactta attaaggatc 145680gatccggcgc gccaatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg 145740ctctcaaggg catcggtcga gcttgacatt gtaggactat attgctctaa taaatttgcg 145800gccgctaata cgactcacta tagggagagg atccgcg 145837211RNAArtificial SequenceStabilized Immunomodulatory RNA-based TLR7 Ligand 2uncuncuucu n 11311RNAArtificial SequenceStabilized Immunomodulatory RNA-based TLR8 Ligand 3ugcugcuugu g 1141041PRTHomo sapiensTLR8 Sequence of GENBANK Accession No. NP_619542.1 4Met Glu Asn Met Phe Leu Gln Ser Ser Met Leu Thr Cys Ile Phe Leu1 5 10 15 Leu Ile Ser Gly Ser Cys Glu Leu Cys Ala Glu Glu Asn Phe Ser Arg 20 25 30 Ser Tyr Pro Cys Asp Glu Lys Lys Gln Asn Asp Ser Val Ile Ala Glu 35 40 45 Cys Ser Asn Arg Arg Leu Gln Glu Val Pro Gln Thr Val Gly Lys Tyr 50 55 60 Val Thr Glu Leu Asp Leu Ser Asp Asn Phe Ile Thr His Ile Thr Asn65 70 75 80 Glu Ser Phe Gln Gly Leu Gln Asn Leu Thr Lys Ile Asn Leu Asn His 85 90 95 Asn Pro Asn Val Gln His Gln Asn Gly Asn Pro Gly Ile Gln Ser Asn 100 105 110 Gly Leu Asn Ile Thr Asp Gly Ala Phe Leu Asn Leu Lys Asn Leu Arg 115 120 125 Glu Leu Leu Leu Glu Asp Asn Gln Leu Pro Gln Ile Pro Ser Gly Leu 130 135 140 Pro Glu Ser Leu Thr Glu Leu Ser Leu Ile Gln Asn Asn Ile Tyr Asn145 150 155 160 Ile Thr Lys Glu Gly Ile Ser Arg Leu Ile Asn Leu Lys Asn Leu Tyr 165 170 175 Leu Ala Trp Asn Cys Tyr Phe Asn Lys Val Cys Glu Lys Thr Asn Ile 180 185 190 Glu Asp Gly Val Phe Glu Thr Leu Thr Asn Leu Glu Leu Leu Ser Leu 195 200 205 Ser Phe Asn Ser Leu Ser His Val Pro Pro Lys Leu Pro Ser Ser Leu 210 215 220 Arg Lys Leu Phe Leu Ser Asn Thr Gln Ile Lys Tyr Ile Ser Glu Glu225 230 235 240 Asp Phe Lys Gly Leu Ile Asn Leu Thr Leu Leu Asp Leu Ser Gly Asn 245 250 255 Cys Pro Arg Cys Phe Asn Ala Pro Phe Pro Cys Val Pro Cys Asp Gly 260 265 270 Gly Ala Ser Ile Asn Ile Asp Arg Phe Ala Phe Gln Asn Leu Thr Gln 275 280 285 Leu Arg Tyr Leu Asn Leu Ser Ser Thr Ser Leu Arg Lys Ile Asn Ala 290 295 300 Ala Trp Phe Lys Asn Met Pro His Leu Lys Val Leu Asp Leu Glu Phe305 310 315 320 Asn Tyr Leu Val Gly Glu Ile Ala Ser Gly Ala Phe Leu Thr Met Leu 325 330 335 Pro Arg Leu Glu Ile Leu Asp Leu Ser Phe Asn Tyr Ile Lys Gly Ser 340 345 350 Tyr Pro Gln His Ile Asn Ile Ser Arg Asn Phe Ser Lys Leu Leu Ser 355 360 365 Leu Arg Ala Leu His Leu Arg Gly Tyr Val Phe Gln Glu Leu Arg Glu 370 375 380 Asp Asp Phe Gln Pro Leu Met Gln Leu Pro Asn Leu Ser Thr Ile Asn385 390 395 400 Leu Gly Ile Asn Phe Ile Lys Gln Ile Asp Phe Lys Leu Phe Gln Asn 405 410 415 Phe Ser Asn Leu Glu Ile Ile Tyr Leu Ser Glu Asn Arg Ile Ser Pro 420 425 430 Leu Val Lys Asp Thr Arg Gln Ser Tyr Ala Asn Ser Ser Ser Phe Gln 435 440 445 Arg His Ile Arg Lys Arg Arg Ser Thr Asp Phe Glu Phe Asp Pro His 450 455 460 Ser Asn Phe Tyr His Phe Thr Arg Pro Leu Ile Lys Pro Gln Cys Ala465 470 475 480 Ala Tyr Gly Lys Ala Leu Asp Leu Ser Leu Asn Ser Ile Phe Phe Ile 485 490 495 Gly Pro Asn Gln Phe Glu Asn Leu Pro Asp Ile Ala Cys Leu Asn Leu 500 505 510 Ser Ala Asn Ser Asn Ala Gln Val Leu Ser Gly Thr Glu Phe Ser Ala 515 520 525 Ile Pro His Val Lys Tyr Leu Asp Leu Thr Asn Asn Arg Leu Asp Phe 530 535 540 Asp Asn Ala Ser Ala Leu Thr Glu Leu Ser Asp Leu Glu Val Leu Asp545 550 555 560 Leu Ser Tyr Asn Ser His Tyr Phe Arg Ile Ala Gly Val Thr His His 565 570 575 Leu Glu Phe Ile Gln Asn Phe Thr Asn Leu Lys Val Leu Asn Leu Ser 580 585 590 His Asn Asn Ile Tyr Thr Leu Thr Asp Lys Tyr Asn Leu Glu Ser Lys 595 600 605 Ser Leu Val Glu Leu Val Phe Ser Gly Asn Arg Leu Asp Ile Leu Trp 610 615 620 Asn Asp Asp Asp Asn Arg Tyr Ile Ser Ile Phe Lys Gly Leu Lys Asn625 630 635 640 Leu Thr Arg Leu Asp Leu Ser Leu Asn Arg Leu Lys His Ile Pro Asn 645 650 655 Glu Ala Phe Leu Asn Leu Pro Ala Ser Leu

Thr Glu Leu His Ile Asn 660 665 670 Asp Asn Met Leu Lys Phe Phe Asn Trp Thr Leu Leu Gln Gln Phe Pro 675 680 685 Arg Leu Glu Leu Leu Asp Leu Arg Gly Asn Lys Leu Leu Phe Leu Thr 690 695 700 Asp Ser Leu Ser Asp Phe Thr Ser Ser Leu Arg Thr Leu Leu Leu Ser705 710 715 720 His Asn Arg Ile Ser His Leu Pro Ser Gly Phe Leu Ser Glu Val Ser 725 730 735 Ser Leu Lys His Leu Asp Leu Ser Ser Asn Leu Leu Lys Thr Ile Asn 740 745 750 Lys Ser Ala Leu Glu Thr Lys Thr Thr Thr Lys Leu Ser Met Leu Glu 755 760 765 Leu His Gly Asn Pro Phe Glu Cys Thr Cys Asp Ile Gly Asp Phe Arg 770 775 780 Arg Trp Met Asp Glu His Leu Asn Val Lys Ile Pro Arg Leu Val Asp785 790 795 800 Val Ile Cys Ala Ser Pro Gly Asp Gln Arg Gly Lys Ser Ile Val Ser 805 810 815 Leu Glu Leu Thr Thr Cys Val Ser Asp Val Thr Ala Val Ile Leu Phe 820 825 830 Phe Phe Thr Phe Phe Ile Thr Thr Met Val Met Leu Ala Ala Leu Ala 835 840 845 His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu 850 855 860 Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr865 870 875 880 Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp 885 890 895 Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn 900 905 910 Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile 915 920 925 Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe 930 935 940 Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe945 950 955 960 Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile 965 970 975 Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu 980 985 990 Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro 995 1000 1005 Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr 1010 1015 1020 Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln1025 1030 1035 1040Tyr55PRTHomo sapiens 5Arg Gln Ser Tyr Ala1 5 611RNAArtificial SequenceStabilized Immunomodulatory RNA-based TLR7 Ligand 6nucuucnucn u 11711RNAArtificial SequenceStabilized Immunomodulatory RNA-based TLR8 Ligand 7guguucgucg u 11

Claims

1-6. (canceled)

7. A hematopoietic cell obtained from a transgenic mouse whose genome comprises a nucleotide sequence encoding human Toll-like receptor 8 (TLR8), wherein said human TLR8 is expressed in the cell, and wherein at least one inflammatory cytokine selected from the group consisting of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-12 p40 (IL-12B), chemokine cc motif ligand 2 (CCL-2) and interferon-gamma-inducible protein 10 (IP10) is present in sera from the transgenic mouse at an elevated level as compared to a control, non-transgenic mouse.

8-12. (canceled)

13. A method of screening candidate agents, the method comprising: contacting the cell of claim 7 with a candidate agent; and determining the effect of said candidate agent on a TLR8-mediated response of said cell.

14. The method of claim 13, wherein the effect comprises inhibition of said TLR8-mediated response.

15. The method of claim 13, wherein the effect comprises stimulation of said TLR8-mediated response.

16. The method of claim 13, wherein the effect is evidenced by a change in TLR8-mediated cytokine production, cell proliferation, and/or cell surface marker expression.

17. The method of claim 16, wherein the effect is evidenced by a change in TLR8-mediated production of one or more cytokines of the group consisting of tumor necrosis factor-alpha (TNF-alpha), interferon-alpha (IFN-alpha), interferon-beta (IFN-beta), interferon-gamma (IFN-gamma), interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-12 (IL-12), interferon-gamma-inducible protein 10 (IP10), and macrophage inflammatory protein-1alpha (MIP-1alpha).

18. The method of claim 16, wherein the effect is evidenced by a change in TLR8-mediated production of one or more cytokines of the group consisting of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-12 (IL-12).

19. The method of claim 16, wherein the effect is evidenced by a change in TLR8-mediated cell proliferation.

20. The method of claim 16, wherein the effect is evidenced by a change in TLR8-mediated expression of one or more cell surface markers selected from the group consisting of CD40, CD80, CD86, glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL), OX40 ligand (OX40L), and programmed death ligand-1 (PDL-1).

21. The method of claim 13, wherein the candidate agent is an antibody.

22. The method of claim 13, wherein the candidate agent is a small molecule.

23. The method of claim 13, wherein the candidate agent is a polynucleotide.

24. The cell of claim 7, wherein said transgenic mouse is a chimeric transgenic mouse in which both human TLR8 and mouse TLR8 are expressed.

25. The cell of claim 7, wherein said nucleotide sequence encoding human TLR8 is present at a copy number of from 1 to 5.

26. The cell of claim 7, wherein said nucleotide sequence encoding human TLR8 is present at a copy number of from 1 or 2.

27. The cell of claim 7, wherein the hematopoietic cell is a population of cells comprising bone marrow.

28. The cell of claim 7, wherein the hematopoietic cell is a population of cells comprising splenocytes.

29. The cell of claim 7, wherein the hematopoietic cell is a population of cells comprising one or more of monocytes, myeloid dendritic cells and neutrophils.