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pFB-ERV载体质粒基本信息
pFB-ERV质粒图谱载体图谱和pFB-ERV载体序列质粒序列多克隆位点信息
pFB-ERV质粒载体简介
pFB-ERV载体描述 DNA vector-based systems that allow precise control of gene expression in vivo have become invaluable for the study of gene function in a variety of organisms, particularly when applied to the study of developmental and other biological processes for which the timing or dosage of gene expression is critical to gene function. Such systems have also been successfully used to overexpress toxic or disease-causing genes, to induce gene targeting, and to express antisense RNA. Inducible systems are currently being used by pharmaceutical companies to facilitate screening for inhibitors of clinically relevant biological pathways, and potential applications for gene therapy are being explored. The Agilent Complete Control ecdysone-inducible plasmid vectors are based on the insect molting hormone ecdysone, which can stimulate transcriptional activation in mammalian cells harboring the ecdysone receptor protein from Drosophila melanogaster.2 The system has a number of advantages over alternative systems. Firstly, the lipophilic nature and short in vivo half-life of the ecdysone analog ponasterone A (ponA) allows efficient penetrance into all tissues including brain, resulting in rapid and potent inductions and rapid clearance. Secondly, ecdysteroids are not known, nor are they expected, to affect mammalian physiology in any measurable way. Thirdly, the heterodimeric ponA responsive receptor and receptor DNA recognition element have been genetically altered such that trans-activation of endogenous genes by the ecdysone receptor, or of the ponA-responsive expression cassette by endogenous transcription factors, is extremely unlikely. In addition, it has been found that in the absence of inducer the heterodimer remains bound at the promoter in a complex with corepressors and histone deacetylase, and is thus tightly repressed until ligand binding, at which time high-level transcriptional activation occurs (i.e., the heterodimer is converted from a tight repressor to a transactivator). In transient assays and stable cell lines harboring receptor expression plasmids in combination with a plasmid bearing an inducible luciferase expression cassette, induction ratios of 1,000-fold have been achieved.3 A limitation to the use of plasmid-based vectors for controlled gene expression is the fact that many cell types of academic, industrial or clinical interest are difficult or virtually impossible to transfect using current transfection methods. In particular, primary human cell lines, lymphocytes, neurons and other nondividing cells are best transduced using viral delivery systems. The most popular and user-friendly of these are the retroviral vectors. Infection with retroviruses often yields transduction efficiencies close to 100%, and the proviral copy number can be easily controlled by varying the multiplicity of infection (MOI). This latter feature is particularly important for inducible systems, for which low basal expression and high induction ratios are affected by copy number. Thus infection of the target cell with virus at an optimal MOI should yield a high frequency of clones capable of mediating desirable expression profiles without exhaustive colony screening. With the vectors pFB-ERV and pCFB-EGSH, we have adapted the ecdysone inducible components of the Complete Control System for retroviral delivery. Used together, we have attained induction ratios of >1,000-fold with these vectors in tissue culture cells. OVERVIEW OF ECDYSONE-REGULATABLE GENE EXPRESSION The ecdysone receptor (EcR) is a member of the retinoid-X-receptor (RXR) family of nuclear receptors and is composed of three domains: an N-terminal activation domain (AD), a central DNA-binding domain (DBD), and a C-terminal ligand-binding and dimerization domain (LBD). In insect cells, EcR and the nuclear receptor ultraspiracle (USP) form a promoterbound heterodimer, which regulates transcription (see Figure 1). In the absence of ecdysone, the receptor heterodimer binds to corepressors and tightly represses transcription.4 When ecdysone binds to the EcR LBD, the corepressors are released, coactivators are recruited to the complex, and transcriptional activation is enabled. In mammalian cells harboring the EcR gene, EcR heterodimerizes with RXR, the mammalian homologue of USP. The EcR–RXR heterodimer binds to multiple copies of the ecdysone-responsive element (EcRE), and in the absence of ponA, represses transcription of an expression cassette. When ponA binds to the receptor, the receptor complex activates transcription of a reporter gene or a gene of interest. To avoid pleiotropic interactions with endogenous pathways in mammalian host cells, both the EcRE recognition sequence and the EcR protein were modified. The EcRE sequence was modified to create a synthetic recognition site that does not bind any endogenous transcription factors. The wild-type EcRE sequence consists of two inverted repeat sequences separated by a single nucleotide: AGTGCA N TGCACT. The EcRE sequence was changed to AGTGCA N1 TGTTCT (and renamed E/GRE). Recognition of the synthetic E/GRE recognition sequence by either a steroid receptor or a wild-type RXR heterodimer receptor is extremely unlikely, as these receptors recognized only the wild-type perfect inverted repeat. The E/GRE recognition sequence has imperfect inverted half sites separated by one nucleotide. A wild-type RXR heterodimer requires single nucleotide separation of the inverted repeats, and the majority bind to direct repeats rather than inverted repeats (EcRE is an exception). The EcR protein was modified to create a synthetic ecdysone-binding receptor that does not transactivate any host genes. Three amino acids in the EcR DBD were mutated to change its DNA-binding specificity to that of the glucocorticoid receptor (GR), which recognizes the half-site AGAACA.2 Like all steroid receptors and unlike RXR receptors, the GR protein homodimerizes and recognizes two inverted repeat sequences separated by three nucleotides. The GR–EcR fusion protein (GEcR) retains the ability to dimerize with RXR and activate, with ponA-dependence, reporter genes that contain the synthetic E/GRE recognition sequence. The GEcR receptor was further modified by replacing the EcR AD with the more potent VP16 AD. The result of all the modifications is the synthetic ecdysone-binding receptor VgEcR. VgEcR is a fusion of the ligand-binding and dimerization domain of the D. melanogaster ecdysone receptor, the DNA-binding domain of the glucocorticoid receptor, and the transcription activation domain of herpes simplex virus (HSV) VP16 OVERVIEW OF REPLICATION-DEFECTIVE RETROVIRAL GENE TRANSFER SYSTEMS Non-replicating retroviral vectors contain all of the cis elements required for transcription of mRNA molecules encoding a gene of interest, and packaging of these transcripts into infectious virus particles (Figure 2). The vectors are typically comprised of an E. coli plasmid backbone containing a pair of 600 base pair viral long terminal repeats (LTRs) between which the gene of interest is inserted. The LTR is divided into 3 regions. The U3 region contains the retroviral promoter/enhancer. The U3 region is flanked in the 3′ direction by the R region, which contains the viral polyadenylation signal (pA), followed by the U5 region which, along with R, contains sequences that are critical for reverse transcription. Expression of the viral RNA is initiated within the U3 region of the 5′ LTR and is terminated in the R region of the 3′ LTR. Between the 5′ LTR and the coding sequence for the gene of interest resides an extended version of the viral packaging signal (ψ+), which is required in cis for the viral RNA to be packaged into virion particles. In order to generate infectious virus particles that carry the gene of interest, specialized packaging cell lines have been generated that contain chromosomally integrated expression cassettes for viral Gag, Pol and Env proteins, all of which are required in trans to make virus. The gag gene encodes internal structural proteins, pol encodes reverse transcriptase (RT) and integrase, and the env gene encodes the viral envelope protein, which resides on the viral surface and facilitates infection of the target cell by direct interaction with cell type-specific receptors; thus the host range of the virus is dictated not by the DNA vector but by the choice of the env gene used to construct the packaging cell. The packaging cell line is transfected with the vector DNA, and at this point either stable viral producer cell lines may be selected (providing the vector has an appropriate selectable marker), or mRNAs that are transiently transcribed from the vector are encapsidated and bud off into the cell supernatant. These supernatants are collected, and used to infect target cells. Upon infection of the target cell, the viral RNA molecule is reverse transcribed by RT (which is present in the virion particle), and the cDNA of the gene of interest, flanked by the LTRs, is integrated into the host DNA. Because the vector itself carries none of the viral proteins, once a target cell is infected the LTR expression cassette is incapable of proceeding through another round of virus production. Recent advances in transfection technology have allowed the production of high titer viral supernatants following transient cotransfection of the viral vector together with expression vectors encoding the gag, pol and env genes (Figure 2),5, 6 obviating the need for the production and maintenance of stable packaging cell lines. For example, Agilent pVPack gag-pol and env-expressing packaging vectors consistently give rise to titers of >107 infectious units (IU)/ml when cotransfected with the pFB-hrGFP control vector (Agilent Catalog #240027), using a 293-derived cell line for virus production. Description of the Vectors The pFB-ERV vector was derived from the high-titer MoMLV vector pFBNeo5 for efficient delivery of the ecdysone receptor proteins VgEcR and RXR (Figure 3). In the vector pFB-ERV the ecdysone receptor and the neomycin-resistance open reading frame (ORF) are expressed from a tricistronic message with the neomycin resistance ORF expressed at the end of the message. Thus, maintenance of infected cell lines in G418 ensures expression of the transcript encoding the receptor genes. The tricistronic transcript is expressed from the CMV promoter, which is flanked by unique EcoR I and Fse I sites so that a cell type-specific promoter of interest may be substituted. The viral promoter within the 3′ LTR has been deleted to make this a self-inactivating (SIN) vector. Upon infection and chromosomal integration into the target cell genome, the SIN deletion is transferred to the 5′ LTR, resulting in an integrated expression cassette in which only the CMV promoter is active. Cells containing an estimated single integrated viral expression cassette can be selected in as high as 1 mg/ml G418, although 600 μg/ml is routinely used. The vector pCFB-EGSH contains an ecdysone-inducible expression cassette inserted between the viral LTRs in the antisense orientation relative to that for the viral promoter (see Figure 4). The U3 promoter within the 5′ LTR of the vector has been replaced with the CMV promoter to increase production of viral RNA in packaging cells, thereby increasing the titer of the viral supernatants. Potential interference from the proviral 5′ LTR is obviated due to the SIN deletion. The inducible expression cassette contains a multiple cloning site that contains three contiguous copies of the HA epitope(3× HA) positioned for fusion at the C-terminus of the protein of interest. A second expression cassette in which the hygromycin-resistance gene is expressed from the TK promoter is located downstream (relative to transcription from the LTRs) of the inducible cassette. A pBR322 origin and ampicillin-resistance gene allow pCFB-EGSH to be propagated in prokaryotes. The pCFB-EGSH-Luc vector contains the luciferase reporter gene and is intended for use as a positive control vector to test the expression of the VgEcR and RXR receptors in pFB-ERV-containing cell lines. The pCFB-EGSH-Luc vector is derived from the pCFB-EGSH vector and has the luciferase gene inserted in the MCS. The pCFB-EGSH-Luc vector does not contain the HA epitope sequence. pFB-ERV载体限制性酶切位点 pFB-ERV, 11067 bp version 011006 Enzymes with 1-10 cleavage sites: #sites -- Bp position of recognition site -- AarI 3 5297, 6078, 7229 AatII 7 978, 2200, 2253, 2336, 2522 3692, 10993 Acc65I 8 647, 1847, 2966, 3609, 4792 5409, 7341, 8593 AccI 6 1968, 2058, 3212, 3762, 6234 8953 AccIII 1 8514 AclI 3 4990, 10300, 10673 AflII 3 202, 1265, 8466 AflIII 9 164, 2820, 3249, 5283, 5458 7215, 7390, 8545, 9182 AgeI 2 1841, 6125 AhdI 6 687, 733, 1274, 8633, 8679 10070 AleI 2 5607, 6402 Alw44I 6 1054, 5445, 7377, 8998, 9496 10742 AlwNI 3 323, 398, 9593 ApaI 5 1238, 5081, 5754, 5787, 7013 ApoI 4 87, 1128, 2064, 2986 AscI 2 3231, 8537 AseI 2 2085, 10246 AvaI 10 577, 610, 643, 1241, 2019 4259, 4340, 5700, 8556, 8589 BamHI 4 3167, 6272, 6425, 6539 BbeI 7 615, 1656, 4376, 6598, 6939 7704, 8561 BbsI 10 2714, 3947, 4148, 5101, 5201 5567, 7033, 7133, 7499, 11060 BbvCI 6 453, 4448, 4454, 4526, 4538 6744 BciVI 7 656, 1987, 3038, 7913, 8602 9391, 10918 BclI 1 8508 BfrBI 1 8519 BglI 10 2163, 2285, 2356, 3926, 4122 4859, 5264, 5906, 7196, 10189 BglII 2 1678, 4266 BlnI 3 5119, 7051, 7542 BlpI 4 4349, 5850, 6283, 6817 BmgBI 4 2774, 5508, 6562, 7440 BmrI 6 2373, 5318, 7250, 7644, 8928 10120 BmtI 6 6, 16, 26, 197, 4772 8461 BpmI 7 1799, 4049, 4592, 6277, 6934 8414, 10160 Bpu10I 10 337, 412, 453, 1548, 4448 4454, 4526, 4538, 5730, 6744 BpuEI 8 5370, 5920, 7302, 8096, 9288 9550, 9827, 10695 BsaAI 8 2001, 2417, 5282, 6316, 7214 8007, 8524, 8934 BsaBI 1 2807 BsaI 10 694, 715, 782, 1414, 1802 6310, 8640, 8661, 8728, 10142 BsgI 3 5668, 6184, 6559 BsiWI 1 4187 BsmBI 8 976, 1093, 1337, 1396, 1582 4337, 4630, 8831 BsmI 7 3317, 4894, 5114, 5147, 5712 7046, 7079 BspHI 3 9902, 10910, 11015 BspMI 7 3085, 3951, 5298, 6079, 7230 7601, 7964 BsrDI 6 3420, 5072, 7004, 7931, 10129 10311 BsrGI 3 1541, 3252, 6017 BssHII 6 563, 3232, 3551, 6681, 8102 8538 BssSI 4 8297, 9355, 10739, 11046 Bst1107I 1 8953 BstAPI 4 322, 397, 4392, 9000 BstEII 2 1346, 4961 Bsu36I 3 1276, 5939, 5996 BtsI 4 4015, 4124, 10468, 10496 ClaI 2 5534, 7466 DraI 3 9939, 9958, 10650 DraIII 3 1874, 5326, 7258 DrdI 4 6454, 7726, 8871, 9284 EagI 7 961, 2679, 3311, 4040, 6728 6962, 7611 EcoICRI 3 580, 3403, 5733 EcoNI 2 1647, 4048 EcoRI 1 2064 EcoRV 2 308, 383 FseI 1 2676 FspI 3 6059, 7805, 10295 HincII 5 2058, 3663, 4551, 6234, 10614 HindIII 3 2957, 5192, 7124 KasI 7 615, 1656, 4376, 6598, 6939 7704, 8561 KpnI 8 647, 1847, 2966, 3609, 4792 5409, 7341, 8593 MluI 1 8545 MmeI 10 684, 1383, 3823, 4811, 4936 5336, 7268, 8630, 9372, 9556 MscI 7 827, 1368, 1668, 3537, 6158 6548, 7785 MunI 2 11, 21 NaeI 5 2677, 4388, 4735, 6837, 8205 NarI 7 615, 1656, 4376, 6598, 6939 7704, 8561 NcoI 7 2439, 2688, 5651, 5783, 6161 7560, 8137 NdeI 4 1664, 1672, 2312, 9004 NgoMIV 5 2677, 4388, 4735, 6837, 8205 NheI 6 6, 16, 26, 197, 4772 8461 NotI 1 6961 NruI 2 3004, 3885 NsiI 1 8519 PciI 6 164, 2820, 3249, 5458, 7390 9182 PfoI 6 771, 2848, 3327, 6275, 8717 8826 PmlI 3 2001, 5282, 7214 PpuMI 8 499, 1477, 1925, 3082, 4723 5924, 6065, 8530 PshAI 2 1015, 2731 PspOMI 5 1238, 5081, 5754, 5787, 7013 PstI 4 1178, 1360, 7754, 10316 PvuI 4 1034, 5532, 7464, 10442 PvuII 6 286, 361, 4541, 5707, 6021 7809 RsrII 2 4035, 8220 SacI 3 580, 3403, 5733 SacII 3 151, 4431, 4677 SalI 2 2058, 6234 SanDI 1 8530 SapI 5 3070, 3202, 8054, 8264, 9059 ScaI 2 6134, 10553 SexAI 3 1474, 4663, 6458 SfcI 10 182, 1178, 1360, 5216, 7148 7754, 8446, 9447, 9638, 10316 SfoI 7 615, 1656, 4376, 6598, 6939 7704, 8561 SmaI 5 643, 1241, 2019, 5700, 8589 SnaBI 2 2417, 8524 SpeI 1 897 SphI 3 2788, 3502, 8106 SrfI 1 1240 SspI 3 3532, 3877, 10877 StuI 2 3813, 6773 TatI 10 1541, 2296, 2376, 2409, 2460 3252, 6017, 6134, 8988, 10553 TfiI 8 2837, 3469, 3911, 4493, 4603 8190, 8324, 9157 Tsp45I 9 1282, 1491, 6364, 7826, 8132 8837, 8932, 10332, 10543 Tth111I 8 633, 1473, 3992, 6358, 6457 7820, 8579, 8926 Van91I 2 5415, 7347 XbaI 3 464, 2037, 2972 XcmI 2 5741, 5813 XhoI 2 4259, 4340 XmaI 5 643, 1241, 2019, 5700, 8589 XmnI 3 5176, 7108, 10670 ZraI 7 978, 2200, 2253, 2336, 2522 3692, 10993 Enzymes that do NOT cut molecule: AsiSI BstBI BstXI Eco47III FspAI HpaI PacI PmeI PsiI SbfI SfiI SgrAI SwaI
pFB-ERV质粒序列载体序列
pFB-ERV, 11067 bp version 011006
NOTE: The following sequence has been verified for accuracy
at the junctions. The remainder of the sequence has been
obtained from existing data.
1 GAATTGCTAG CAATTGCTAG CAATTGCTAG CAATTCATAC CAGATCACCG
51 AAAACTGTCC TCCAAATGTG TCCCCCTCAC ACTCCCAAAT TCGCGGGCTT
101 CTGCCTCTTA GACCACTCTA CCCTATTCCC CACACTCACC GGAGCCAAAG
151 CCGCGGGACA TATACATGTG AAAGACCCCA CCTGTAGGTT TGGCAAGCTA
201 GCTTAAGTAA CGCCATTTTG CAAGGCATGG AAAAATACAT AACTGAGAAT
251 AGAAAAGTTC AGATCAAGGT CAGGAACAGA TGGAACAGCT GAATATGGGC
301 CAAAGCGGAT ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC AGGGCCAAGA
351 ACAGATGGAA CAGCTGAATA TGGGCCAAAC AGGATATCTG TGGTAAGCAG
401 TTCCTGCCCC GGCTCAGGGC CAAGAACAGA TGGTCCCCAG ATGCGGTCCA
451 GCCCTCAGCA GTTTCTAGAG AACCATCAGA TGTTTCCAGG GTGCCCCAAG
501 GACCTGAAAT GACCCTGTGC CTTATTTGAA CTAACCAATC AGTTCGCTTC
551 TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG AGCTCAATAA AAGAGCCCAC
601 AACCCCTCAC TCGGGGCGCC AGTCCTCCGA TTGACTGAGT CGCCCGGGTA
651 CCCGTGTATC CAATAAACCC TCTTGCAGTT GCATCCGACT TGTGGTCTCG
701 CTGTTCCTTG GGAGGGTCTC CTCTGAGTGA TTGACTACCC GTCAGCGGGG
751 GTCTTTCATT TGGGGGCTCG TCCGGGATCG GGAGACCCCT GCCCAGGGAC
801 CACCGACCCA CCACCGGGAG GTAAGCTGGC CAGCAACTTA TCTGTGTCTG
851 TCCGATTGTC TAGTGTCTAT GACTGATTTT ATGCGCCTGC GTCGGTACTA
901 GTTAGCTAAC TAGCTCTGTA TCTGGCGGAC CCGTGGTGGA ACTGACGAGT
951 TCGGAACACC CGGCCGCAAC CCTGGGAGAC GTCCCAGGGA CTTCGGGGGC
1001 CGTTTTTGTG GCCCGACCTG AGTCCAAAAA TCCCGATCGT TTTGGACTCT
1051 TTGGTGCACC CCCCTTAGAG GAGGGATATG TGGTTCTGGT AGGAGACGAG
1101 AACCTAAAAC AGTTCCCGCC TCCGTCTGAA TTTTTGCTTT CGGTTTGGGA
1151 CCGAAGCCGC GCCGCGCGTC TTGTCTGCTG CAGCATCGTT CTGTGTTGTC
1201 TCTGTCTGAC TGTGTTTCTG TATTTGTCTG AAAATATGGG CCCGGGCCAG
1251 ACTGTTACCA CTCCCTTAAG TTTGACCTTA GGTCACTGGA AAGATGTCGA
1301 GCAGATCGCT CACAACCAGT CGGTAGATGT CAAGAAGAGA CGTTGGGTTA
1351 CCTTCTGCTC TGCAGAATGG CCAACCTTTA ACGTCGGATG GCCGCGAGAC
1401 GGCACCTTTA ACCGAGACCT CATCACCCAG GTTAAGATCA AGGTCTTTTC
1451 ACCTGGCCCG CATGGACACC CAGACCAGGT CCCCTACATC GTGACCTGGG
1501 AAGCCTTGGC TTTTGACCCC CCTCCCTGGG TCAAGCCCTT TGTACACCCT
1551 AAGCCTCCGC CTCCTCTTCC TCCATCCGCC CCGTCTCTCC CCCTTGAACC
1601 TCCTCGTTCG ACCCCGCCTC GATCCTCCCT TTATCCAGCC CTCACTCCTT
1651 CTCTAGGCGC CCCCATATGG CCATATGAGA TCTTATATGG GGCACCCCCG
1701 CCCCTTGTAA ACTTCCCTGA CCCTGACATG ACAAGAGTTA CTAACAGCCC
1751 CTCTCTCCAA GCTCACTTAC AGGCTCTCTA CTTAGTCCAG CACGAAGTCT
1801 GGAGACCTCT GGCGGCAGCC TACCAAGAAC AACTGGACCG ACCGGTGGTA
1851 CCTCACCCTT ACCGAGTCGG CGACACAGTG TGGGTCCGCC GACACCAGAC
1901 TAAGAACCTA GAACCTCGCT GGAAAGGACC TTACACAGTC CTGCTGACCA
1951 CCCCCACCGC CCTCAAAGTA GACGGCATCG CAGCTTGGAT ACACGCCGCC
2001 CACGTGAAGG CTGCCGACCC CGGGGGTGGA CCATCCTCTA GACTGCCGGA
2051 TCGAATTGTC GACGAATTCG CCGTTGCATT AGTTATTAAT AGTAATCAAT
2101 TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC
2151 TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG
2201 ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA
2251 TTGACGTCAA TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC
2301 ATCAAGTGTA TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT
2351 AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC
2401 TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC
2451 GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA
2501 TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA
2551 AAATCAACGG GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC
2601 AAATGGGCGG TAGGCGTGTA CGGTGGGAGG TCTATATAAG CAGAGCTGGT
2651 TTAGTGAACC GTCAGATCCG CTAGTGGCCG GCCGCCACCA TGGAACAAAA
2701 ACTTATTTCT GAAGAAGACT TGGCCCCCCC GACCGATGTC AGCCTGGGGG
2751 ACGAACTCCA CTTAGACGGC GAGGACGTGG CGATGGCGCA TGCCGACGCG
2801 CTAGACGATT TCGATCTGGA CATGTTGGGG GACGGGGATT CCCCAGGTCC
2851 GGGATTTACC CCCCACGACT CCGCCCCCTA CGGCGCTCTG GATATGGCCG
2901 ACTTCGAGTT TGAGCAGATG TTTACCGATG CCCTTGGAAT TGACGAGTAC
2951 GGTGGGAAGC TTCTAGGTAC CTCTAGAAGA ATATCAAATT CTATATCTTC
3001 AGGTCGCGAT GATCTCTCGC CTTCGAGCAG CTTGAACGGA TACTCGGCGA
3051 ACGAAAGCTG CGATGTGAAG AAGAGCAAGA AGGGACCTGC GCCACGGGTG
3101 CAAGAGGAGC TGTGCCTGGT TTGCGGCGAC AGGGCCTCCG GCTACCACTA
3151 CAACGCCCTC ACCTGTGGAT CCTGCAAGGT GTTCTTTCGA CGCAGCGTTA
3201 CGAAGAGCGC CGTCTACTGC TGCAAGTTCG GGCGCGCCTG CGAAATGGAC
3251 ATGTACATGA GGCGAAAGTG TCAGGAGTGC CGCCTGAAAA AGTGCCTGGC
3301 CGTGGGTATG CGGCCGGAAT GCGTCGTCCC GGAGAACCAA TGTGCGATGA
3351 AGCGGCGCGA AGAGAAGGCC CAGAAGGAGA AGGACAAAAT GACCACTTCG
3401 CCGAGCTCTC AGCATGGCGG CAATGGCAGC TTGGCCTCTG GTGGCGGCCA
3451 AGACTTTGTT AAGAAGGAGA TTCTTGACCT TATGACATGC GAGCCGCCCC
3501 AGCATGCCAC TATTCCGCTA CTACCTGATG AAATATTGGC CAAGTGTCAA
3551 GCGCGCAATA TACCTTCCTT AACGTACAAT CAGTTGGCCG TTATATACAA
3601 GTTAATTTGG TACCAGGATG GCTATGAGCA GCCATCTGAA GAGGATCTCA
3651 GGCGTATAAT GAGTCAACCC GATGAGAACG AGAGCCAAAC GGACGTCAGC
3701 TTTCGGCATA TAACCGAGAT AACCATACTC ACGGTCCAGT TGATTGTTGA
3751 GTTTGCTAAA GGTCTACCAG CGTTTACAAA GATACCCCAG GAGGACCAGA
3801 TCACGTTACT AAAGGCCTGC TCGTCGGAGG TGATGATGCT GCGTATGGCA
3851 CGACGCTATG ACCACAGCTC GGACTCAATA TTCTTCGCGA ATAATAGATC
3901 ATATACGCGG GATTCTTACA AAATGGCCGG AATGGCTGAT AACATTGAAG
3951 ACCTGCTGCA TTTCTGCCGC CAAATGTTCT CGATGAAGGT GGACAACGTC
4001 GAATACGCGC TTCTCACTGC CATTGTGATC TTCTCGGACC GGCCGGGCCT
4051 GGAGAAGGCC CAGCTAGTCG AAGCGATCCA GAGCTACTAC ATCGACACGC
4101 TACGCATTTA TATACTCAAC CGCCACTGCG GCGACTCAAT GAGCCTCGTC
4151 TTCTACGCAA AGCTGCTCTC GATCCTCACC GAGCTGCGTA CGCTGGGCAA
4201 CCAGAACGCC GAGATGTGTT TCTCACTAAA GCTCAAAAAC CGCAAACTGC
4251 CCAAGTTCCT CGAGGAGATC TGGGACGTTC ATGCCATCCC GCCATCGGTC
4301 CAGTCGCACC TTCAGATTAC CCAGGAGGAG AACGAGCGTC TCGAGCGGGC
4351 TGAGCGTATG CGGGCATCGG TTGGGGGCGC CATTACCGCC GGCATTGATT
4401 GCGACTCTGC CTCCACTTCG GCGGCGGCAG CCGCGGCCCA GCATCAGCCT
4451 CAGCCTCAGC CCCAGCCCCA ACCCTCCTCC CTGACCCAGA ACGATTCCCA
4501 GCACCAGACA CAGCCGCAGC TACAACCTCA GCTACCACCT CAGCTGCAAG
4551 GTCAACTGCA ACCCCAGCTC CACCCACAGC TTCAGACGCA ACTCCAGCCA
4601 CAGATTCAAC CACAGCCACA GCTCCTTCCC GTCTCCGCTC CCGTGCCCGC
4651 CTCCGTAACC GCACCTGGTT CCTTGTCCGC GGTCAGTACG AGCAGCGAAT
4701 ACATGGGCGG AAGTGCGGCC ATAGGACCCA TCACGCCGGC AACCACCAGC
4751 AGTATCACGG CTGCCGTTAC CGCTAGCTCC ACCACATCAG CGGTACCGAT
4801 GGGCAACGGA GTTGGAGTCG GTGTTGGGGT GGGCGGCAAC GTCAGCATGT
4851 ATGCGAACGC CCAGACGGCG ATGGCCTTGA TGGGTGTAGC CCTGCATTCG
4901 CACCAAGAGC AGCTTATCGG GGGAGTGGCG GTTAAGTCGG AGCACTCGAC
4951 GACTGCATAG GGTTACCCCC CTCTCCCTCC CCCCCCCCTA ACGTTACTGG
5001 CCGAAGCCGC TTGGAATAAG GCCGGTGTGC GTTTGTCTAT ATGTTATTTT
5051 CCACCATATT GCCGTCTTTT GGCAATGTGA GGGCCCGGAA ACCTGGCCCT
5101 GTCTTCTTGA CGAGCATTCC TAGGGGTCTT TCCCCTCTCG CCAAAGGAAT
5151 GCAAGGTCTG TTGAATGTCG TGAAGGAAGC AGTTCCTCTG GAAGCTTCTT
5201 GAAGACAAAC AACGTCTGTA GCGACCCTTT GCAGGCAGCG GAACCCCCCA
5251 CCTGGCGACA GGTGCCTCTG CGGCCAAAAG CCACGTGTAT AAGATACACC
5301 TGCAAAGGCG GCACAACCCC AGTGCCACGT TGTGAGTTGG ATAGTTGTGG
5351 AAAGAGTCAA ATGGCTCTCC TCAAGCGTAT TCAACAAGGG GCTGAAGGAT
5401 GCCCAGAAGG TACCCCATTG TATGGGATCT GATCTGGGGC CTCGGTGCAC
5451 ATGCTTTACA TGTGTTTAGT CGAGGTTAAA AAACGTCTAG GCCCCCCGAA
5501 CCACGGGGAC GTGGTTTTCC GTTGAAAAAC ACGATCGATA ATATGGAACA
5551 AAAACTTATT TCTGAAGAAG ACTTGGACAC CAAACTTTCC TGCCGCTCGA
5601 TTTCTCCACC CAGGTGAACT CCTCCCTCAC CTCCCCGACG GGGCGAGGCT
5651 CCATGGCTGC CCCCTCGCTG CACCCGTCCC TGGGGCCTGG CATCGGCTCC
5701 CCGGGACAGC TGCATTCTCC CATCAGCACC CTGAGCTCCC CCATCAACGG
5751 CATGGGCCCG CCTTTCTCGG TCATCAGCTC CCCCATGGGC CCCCACTCCA
5801 TGTCGGTGCC CACCACACCC ACCCTGGGCT TCAGCACTGG CAGCCCCCAG
5851 CTCAGCTCAC CTATGAACCC CGTCAGCAGC AGCGAGGACA TCAAGCCCCC
5901 CCTGGGCCTC AATGGCGTCC TCAAGGTCCC CGCCCACCCC TCAGGAAACA
5951 TGGCTTCCTT CACCAAGCAC ATCTGCGCCA TCTGCGGGGA CCGCTCCTCA
6001 GGCAAGCACT ATGGAGTGTA CAGCTGCGAG GGGTGCAAGG GCTTCTTCAA
6051 GCGGACGGTG CGCAAGGACC TGACCTACAC CTGCCGCGAC AACAAGGACT
6101 GCCTGATTGA CAAGCGGCAG CGGAACCGGT GCCAGTACTG CCGCTACCAG
6151 AAGTGCCTGG CCATGGGCAT GAAGCGGGAA GCCGTGCAGG AGGAGCGGCA
6201 GCGTGGCAAG GACCGGAACG AGAATGAGGT GGAGTCGACC AGCAGCGCCA
6251 ACGAGGACAT GCCGGTGGAG AGGATCCTGG AGGCTGAGCT GGCCGTGGAG
6301 CCCAAGACCG AGACCTACGT GGAGGCAAAC ATGGGGCTGA ACCCCAGCTC
6351 GCCGAACGAC CCTGTCACCA ACATTTGCCA AGCAGCCGAC AAACAGCTTT
6401 TCACCCTGGT GGAGTGGGCC AAGCGGATCC CACGCTTCTC AGAGCTGCCC
6451 CTGGACGACC AGGTCATCCT GCTGCGGGCA GGCTGGAATG AGCTGCTCAT
6501 CGCCTCCTTC TCCCACCGCT CCATCGCCGT GAAGGACGGG ATCCTCCTGG
6551 CCACCGGGCT GCACGTCCAC CGGAACAGCG CCCACAGCGC AGGGGTGGGC
6601 GCCATCTTTG ACAGGGTGCT GACGGAGCTT GTGTCCAAGA TGCGGGACAT
6651 GCAGATGGAC AAGACGGAGC TGGGCTGCCT GCGCGCCATC GTCCTCTTTA
6701 ACCCTGACTC CAAGGGGCTC TCGAACCCGG CCGAGGTGGA GGCGCTGAGG
6751 GAGAAGGTCT ATGCGTCCTT GGAGGCCTAC TGCAAGCACA AGTACCCAGA
6801 GCAGCCGGGA AGGTTCGCTA AGCTCTTGCT CCGCCTGCCG GCTCTGCGCT
6851 CCATCGGGCT CAAATGCCTG GAACATCTCT TCTTCTTCAA GCTCATCGGG
6901 GACACACCCA TTGACACCTT CCTTATGGAG ATGCTGGAGG CGCCGCACCA
6951 AATGACTTAG GCGGCCGCGA TCCGGTTATT TTCCACCATA TTGCCGTCTT
7001 TTGGCAATGT GAGGGCCCGG AAACCTGGCC CTGTCTTCTT GACGAGCATT
7051 CCTAGGGGTC TTTCCCCTCT CGCCAAAGGA ATGCAAGGTC TGTTGAATGT
7101 CGTGAAGGAA GCAGTTCCTC TGGAAGCTTC TTGAAGACAA ACAACGTCTG
7151 TAGCGACCCT TTGCAGGCAG CGGAACCCCC CACCTGGCGA CAGGTGCCTC
7201 TGCGGCCAAA AGCCACGTGT ATAAGATACA CCTGCAAAGG CGGCACAACC
7251 CCAGTGCCAC GTTGTGAGTT GGATAGTTGT GGAAAGAGTC AAATGGCTCT
7301 CCTCAAGCGT ATTCAACAAG GGGCTGAAGG ATGCCCAGAA GGTACCCCAT
7351 TGTATGGGAT CTGATCTGGG GCCTCGGTGC ACATGCTTTA CATGTGTTTA
7401 GTCGAGGTTA AAAAACGTCT AGGCCCCCCG AACCACGGGG ACGTGGTTTT
7451 CCGTTGAAAA ACACGATCGA TAATATGGAA CAAAAACTTA TTTCTGAAGA
7501 AGACTTGGAC ACCAAACTTT CCTGCCGCTC GATTTCTCCA CCCTAGGTGC
7551 CACGCGGTTC CATGGGATCG TTTCGCATGA TTGAACAAGA TGGATTGCAC
7601 GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC
7651 ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC
7701 AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT
7751 GAACTGCAGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT
7801 TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC
7851 TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT
7901 CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC
7951 GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG
8001 AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG
8051 GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA
8101 GGCGCGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT
8151 GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG ATTCATCGAC
8201 TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC
8251 CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG
8301 TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC
8351 CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA TAAAATAAAA
8401 GATTTTATTT AGTCTCCAGA AAAAGGGGGG AATGAAAGAC CCCACCTGTA
8451 GGTTTGGCAA GCTAGCTTAA GTAACGCCAT TTTGCAAGGC ATGGAAAAAT
8501 ACATAACTGA TCATCCGGAT GCATACGTAG GGACCCGGCG CGCCACGCGT
8551 CCTCACTCGG GGCGCCAGTC CTCCGATTGA CTGAGTCGCC CGGGTACCCG
8601 TGTATCCAAT AAACCCTCTT GCAGTTGCAT CCGACTTGTG GTCTCGCTGT
8651 TCCTTGGGAG GGTCTCCTCT GAGTGATTGA CTACCCGTCA GCGGGGGTCT
8701 TTCATTTGGG GGCTCGTCCG GGATCGGGAG ACCCCTGCCC AGGGACCACC
8751 GACCCACCAC CGGGAGGTAA GCTGGCTGCC TCGCGCGTTT CGGTGATGAC
8801 GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA CAGCTTGTCT
8851 GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
8901 TTGGCGGGTG TCGGGGCGCA GCCATGACCC AGTCACGTAG CGATAGCGGA
8951 GTGTATACTG GCTTAACTAT GCGGCATCAG AGCAGATTGT ACTGAGAGTG
9001 CACCATATGC GGTGTGAAAT ACCGCACAGA TGCGTAAGGA GAAAATACCG
9051 CATCAGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG
9101 TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA
9151 TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA
9201 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA
9251 GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG
9301 TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG
9351 CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT
9401 CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT
9451 AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA
9501 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC
9551 TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT
9601 GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT
9651 GAAGTGGTGG CCTAACTACG GCTACACTAG AAGGACAGTA TTTGGTATCT
9701 GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA
9751 TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA
9801 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT
9851 CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG
9901 GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA
9951 ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA
10001 GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT
10051 CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG
10101 GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC
10151 GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC
10201 GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA
10251 TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA
10301 ACGTTGTTGC CATTGCTGCA GGCATCGTGG TGTCACGCTC GTCGTTTGGT
10351 ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC
10401 CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG
10451 TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG
10501 CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG
10551 TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT
10601 GCTCTTGCCC GGCGTCAACA CGGGATAATA CCGCGCCACA TAGCAGAACT
10651 TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG
10701 GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA
10751 ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA
10801 ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG
10851 TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG
10901 GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA
10951 CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTCTA
11001 AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA
11051 GGCCCTTTCG TCTTCAA
