质粒名称: | pX459-SpCas9-NG |
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出品公司: | Addgene |
目录编号: | 171370 |
存储实验室: | Masahito Ikawa |
载体骨架基本信息 |
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载体名称: | pX459 |
载体出品公司: | Clontech |
空载体大小: | 9175bp |
完整质粒大小: | 9173 bp |
载体类型: | 哺乳细胞表达、CRISPR |
筛选标记: | Puromycin |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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基因突变: | pX459-SpCas9-NG was constructed by replacing the Cas9 coding sequence of pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988) with that of pX330-SpCas9-NG (Addgene plasmid #117919) using AgeI (R0552, NEB, MA, USA) and FseI (R0588, NEB) restriction enzymes. |
启动子: | U6 for sgRNA; CBh for Cas9-2A-puroR |
克隆方法: | 限制性内切酶 |
文献引用方法 |
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材料和方法部分: |
pX459-SpCas9-NG was a gift from Masahito Ikawa (Addgene plasmid # 171370 ; http://n2t.net/addgene:171370 ; RRID:Addgene_171370)
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参考文献部分: | Precise CAG repeat contraction in a Huntington's Disease mouse model is enabled by gene editing with SpCas9-NG. Oura S, Noda T, Morimura N, Hitoshi S, Nishimasu H, Nagai Y, Nureki O, Ikawa M. Commun Biol. 2021 Jun 23;4(1):771. doi: 10.1038/s42003-021-02304-w. 10.1038/s42003-021-02304-w PubMed 34163001 |
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载体基本信息
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载体简介
载体序列
LOCUS pX459-SpCas9-NG 9173 bp ds-DNA circular SYN 22-JUL-2021 DEFINITION SpCas9-NG-mediated genome editing.. ACCESSION . VERSION . KEYWORDS pX459-SpCas9-NG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9173) AUTHORS Oura S, Noda T, Morimura N, Hitoshi S, Nishimasu H, Nagai Y, Nureki O, Ikawa M TITLE Precise CAG repeat contraction in a Huntington's Disease mouse model is enabled by gene editing with SpCas9-NG. JOURNAL Commun Biol. 2021 Jun 23;4(1):771. doi: 10.1038/s42003-021-02304-w. PUBMED 34163001 REFERENCE 2 (bases 1 to 9173) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 22, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..9173 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 268..343 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 440..725 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 727..1003 /label=chicken beta-actin promoter intron 1004..1231 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1243..1252 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1252..1317 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1324..1344 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1369..3084 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 5470..5517 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5533..5586 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 5587..6186 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(5587..5606) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6083..6103 /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind complement(6214..6231) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6220..6427 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6436..6576 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6436..6565 /label=AAV2 ITR rep_origin 6651..7106 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6738..6757) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6948..6969 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7123..7142) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7242..7264 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7302..7320) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7388..7492 /gene="bla" /label=AmpR promoter CDS 7493..8353 /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 complement(7711..7730) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8524..9112 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9013..9032 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 841 gggggggcgc gcgccrggsg gggcggggsg gggsgrgggg sggggsgggg sgaggcggag 901 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 961 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg 1021 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac 1081 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct 1141 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct 1201 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag 1261 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg 1321 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac 1381 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac 1441 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag 1501 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag 1561 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc 1621 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc 1681 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag 1741 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc 1801 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg 1861 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc 1921 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc 1981 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg 2041 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc 2101 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag 2161 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag 2221 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc 2281 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac 2341 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag 2401 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc 2461 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc 2521 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc 2581 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg 2641 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc 2701 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg 2761 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc 2821 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag 2881 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc 2941 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa 3001 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa 3061 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat 3121 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac 3181 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg 3241 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac 3301 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag 3361 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc 3421 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa 3481 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc 3541 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac 3601 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga 3661 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa 3721 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag 3781 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac 3841 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc 3901 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc 3961 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag 4021 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga 4081 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg 4141 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag 4201 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat 4261 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac 4321 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa 4381 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc 4441 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt 4501 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca 4561 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa 4621 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc 4681 agcaaagagt ctatccggcc caagaggaac agcgataagc tgatcgccag aaagaaggac 4741 tgggacccta agaagtacgg cggcttcgtt agccccaccg tggcctattc tgtgctggtg 4801 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg 4861 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag 4921 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag 4981 ctggaaaacg gccggaagag aatgctggcc tctgcccggt tcctgcagaa gggaaacgaa 5041 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg 5101 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac 5161 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct 5221 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag 5281 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctcg ggccttcaag 5341 tactttgaca ccaccatcga ccggaaggtg taccggagca ccaaagaggt gctggacgcc 5401 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg 5461 ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggaa 5521 ttcggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct 5581 ggcccaatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg 5641 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat 5701 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg 5761 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg 5821 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg 5881 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc 5941 aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt 6001 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc 6061 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc 6121 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt 6181 gcctgagaat tctaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc 6241 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 6301 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 6361 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg 6421 ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 6481 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 6541 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc 6601 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg 6661 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 6721 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 6781 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 6841 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg 6901 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 6961 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt 7021 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 7081 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc 7141 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 7201 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 7261 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 7321 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 7381 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 7441 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 7501 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 7561 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 7621 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 7681 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 7741 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 7801 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 7861 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 7921 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 7981 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 8041 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 8101 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 8161 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat 8221 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 8281 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 8341 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 8401 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 8461 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 8521 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 8581 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 8641 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 8701 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 8761 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 8821 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 8881 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 8941 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 9001 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 9061 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 9121 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt //
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