质粒名称: | pJRH029 |
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出品公司: | Addgene |
目录编号: | 171060 |
存储实验室: | Jennifer Doudna |
载体骨架基本信息 |
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载体名称: | pCAGGS |
载体出品公司: | Clontech |
空载体大小: | 4718 bp |
完整质粒大小: | 10496 bp |
载体修饰: | -- |
载体类型: | 哺乳动物表达,慢病毒,CRISPR |
筛选标记: | -- |
载体抗性: | 氨苄青霉素,100μg/mL |
生长温度: | 37 ℃ |
宿主菌: | NEB Stable |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | Gag-spyCas9 |
物种来源: | -- |
插入大小: | 5778 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子: | CAG |
标签/融合蛋白: | 3x FLAG标签(插入件上的C端子)2x SV40 NLS(插入件上的C端子) |
克隆方法: | Gibson Cloning |
5’克隆位点: | -- |
3’克隆位点: | -- |
5’测序引物 | AAGGAAATGGGCGGGGA |
3’测序引物 | CAGGAAACAGCTATGAC |
文献引用方法 |
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材料和方法部分: | pJRH029 was a gift from Jennifer Doudna (Addgene plasmid # 171060 ; http://n2t.net/addgene:171060 ; RRID:Addgene_171060) |
参考文献部分: | Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Hamilton JR, Tsuchida CA, Nguyen DN, Shy BR, McGarrigle ER, Sandoval Espinoza CR, Carr D, Blaeschke F, Marson A, Doudna JA. Cell Rep. 2021 Jun 1;35(9):109207. doi: 10.1016/j.celrep.2021.109207. 10.1016/j.celrep.2021.109207 PubMed 34077734 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pJRH029 10504 bp ds-DNA circular SYN 03-FEB-2023 DEFINITION Plasmid for expressing Gag fused to spyCas9-3x FLAG-2x SV40 NLS with the SQNYPIVQ HIV-1 cleavage site in between. ACCESSION . VERSION . KEYWORDS pJRH029 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10504) AUTHORS Hamilton JR, Tsuchida CA, Nguyen DN, Shy BR, McGarrigle ER, Sandoval Espinoza CR, Carr D, Blaeschke F, Marson A, Doudna JA TITLE Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. JOURNAL Cell Rep. 2021 Jun 1;35(9):109207. doi: 10.1016/j.celrep.2021.109207. PUBMED 34077734 REFERENCE 2 (bases 1 to 10504) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 3, 2023 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..10504 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 5..384 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" intron 662..1678 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1602..1624 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1686..1705 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" regulatory 1733..1742 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1739..3238 /codon_start=1 /gene="gag" /product="gag protein from human immunodeficiency virus 1" /label=HIV-1 gag /translation="MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFA VNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKI EEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKA FSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGP IAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTS ILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPG ATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAP PEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ" CDS 3275..7378 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 7382..7447 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 7466..7486 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7493..7513 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(7526..7545) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 7591..7646 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(7645..7664) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(8007..8023) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8007..8023) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(8020..8042) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 8031..8047 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(8055..8085) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8100..8121 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8180..8375 /label=SV40 promoter /note="SV40 early promoter" rep_origin 8226..8361 /label=SV40 ori /note="SV40 origin of replication" primer_bind 8288..8307 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 8381..8515 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8418..8437) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 8472..8491 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(8583..8600) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(8754..9342) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8834..8853) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9513..10373) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 10136..10155 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10374..10478) /gene="bla" /label=AmpR promoter ORIGIN 1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat 61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg 121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc 421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 481 gcgatggggg cggggggggg gggggggcgc gccmggsggg gsggggsggg gsgrggggsg 541 gggsggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct 1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgccaccat 1741 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgatgggaaa aaattcggtt 1801 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct 1861 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact 1921 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac 1981 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt 2041 agacaagata gaggaagagc aaaacaaaag taagaaaaaa gcacagcaag cagcagctga 2101 cacaggacac agcaatcagg tcagccaaaa ttaccctata gtgcagaaca tccaggggca 2161 aatggtacat caggccatat cacctagaac tttaaatgca tgggtaaaag tagtagaaga 2221 gaaggctttc agcccagaag tgatacccat gttttcagca ttatcagaag gagccacccc 2281 acaagattta aacaccatgc taaacacagt ggggggacat caagcagcca tgcaaatgtt 2341 aaaagagacc atcaatgagg aagctgcaga atgggataga gtgcatccag tgcatgcagg 2401 gcctattgca ccaggccaga tgagagaacc aaggggaagt gacatagcag gaactactag 2461 tacccttcag gaacaaatag gatggatgac acataatcca cctatcccag taggagaaat 2521 ctataaaaga tggataatcc tgggattaaa taaaatagta agaatgtata gccctaccag 2581 cattctggac ataagacaag gaccaaagga accctttaga gactatgtag accgattcta 2641 taaaactcta agagccgagc aagcttcaca agaggtaaaa aattggatga cagaaacctt 2701 gttggtccaa aatgcgaacc cagattgtaa gactatttta aaagcattgg gaccaggagc 2761 gacactagaa gaaatgatga cagcatgtca gggagtgggg ggacccggcc ataaagcaag 2821 agttttggct gaagcaatga gccaagtaac aaatccagct accataatga tacagaaagg 2881 caattttagg aaccaaagaa agactgttaa gtgtttcaat tgtggcaaag aagggcacat 2941 agccaaaaat tgcagggccc ctaggaaaaa gggctgttgg aaatgtggaa aggaaggaca 3001 ccaaatgaaa gattgtactg agagacaggc taatttttta gggaagatct ggccttccca 3061 caagggaagg ccagggaatt ttcttcagag cagaccagag ccaacagccc caccagaaga 3121 gagcttcagg tttggggaag agacaacaac tccctctcag aagcaggagc cgatagacaa 3181 ggaactgtat cctttagctt ccctcagatc actctttggc agcgacccct cgtcacaaaa 3241 ctttagccag aactatccga ttgtgcagac cggtatggac aagaagtaca gcatcggcct 3301 ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag 3361 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg 3421 cgccctgctg ttcgacagcg gagaaacagc cgaggccacc cggctgaaga gaaccgccag 3481 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga 3541 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga 3601 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca 3661 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc 3721 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct 3781 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt 3841 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa 3901 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct 3961 gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac 4021 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga 4081 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct 4141 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa 4201 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca 4261 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga 4321 gattttcttc gaccagagca agaacggcta cgccggctac atcgatggcg gagccagcca 4381 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact 4441 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag 4501 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt 4561 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc 4621 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag 4681 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg ccagcgccca 4741 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc 4801 caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca aagtgaaata 4861 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aagccatcgt 4921 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt 4981 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc 5041 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa 5101 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag 5161 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa 5221 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg 5281 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc 5341 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca 5401 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg 5461 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa 5521 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac 5581 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa 5641 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga 5701 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga 5761 catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga 5821 cgactccatc gataacaaag tgctgactcg gagcgacaag aaccggggca agagcgacaa 5881 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgccagc tgctgaatgc 5941 caagctgatt acccagagga agttcgacaa tctgaccaag gccgagagag gcggcctgag 6001 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa 6061 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga acgacaaact 6121 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga 6181 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct 6241 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt 6301 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat 6361 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga 6421 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac 6481 aggcgagatc gtgtgggata agggccggga ctttgccacc gtgcggaaag tgctgtctat 6541 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc 6601 tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact gggaccctaa 6661 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt 6721 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat 6781 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga 6841 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg 6901 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc 6961 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc 7021 cgaggataat gagcagaaac agctgtttgt ggaacagcac aaacactacc tggacgagat 7081 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa 7141 ggtgctgagc gcctacaaca agcacagaga caagcctatc agagagcagg ccgagaatat 7201 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac 7261 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca 7321 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacgc 7381 cgactataag gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga 7441 cgataagagc ctgggcagcg gctcccccaa gaaaaaacgc aaggtggaag atcctaagaa 7501 aaagcggaaa gtgtaagcta gcagatcttt ttccctctgc caaaaattat ggggacatca 7561 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag 7621 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa 7681 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca 7741 tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt 7801 ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt 7861 tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc 7921 tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc 7981 tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 8041 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 8101 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 8161 ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc 8221 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 8281 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 8341 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta 8401 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 8461 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg 8521 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 8581 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 8641 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 8701 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 8761 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 8821 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 8881 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 8941 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 9001 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 9061 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 9121 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 9181 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 9241 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 9301 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 9361 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 9421 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 9481 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 9541 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 9601 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 9661 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 9721 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 9781 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 9841 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 9901 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 9961 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 10021 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 10081 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 10141 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 10201 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 10261 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 10321 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 10381 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 10441 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 10501 cctg //