质粒名称: | pNICKclos1.0 |
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出品公司: | 爱迪基因 |
目录编号: | 73639 |
存储实验室: | Sheng Yang |
载体骨架基本信息 |
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载体名称: | pIMP1-ptb |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 4806 bp |
完整质粒大小: | -- |
载体修饰: | -- |
载体类型: | E、 大肠杆菌梭状芽孢杆菌穿梭载体 |
筛选标记: | 梭状芽孢杆菌红霉素 |
高拷贝/低拷贝: | 低拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Cas9 nickase |
物种来源: | S. pygogenes |
插入大小: | -- |
基因突变: | D10A |
GenBank编号 | -- |
启动子 | ptb |
标签/融合蛋白: | -- |
克隆方法: | -- |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | SpCas9N-R TACGAGCTGTCCGTTTGAGA |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pNICKclos1.0 was a gift from Sheng Yang (Addgene plasmid # 73639 ; http://n2t.net/addgene:73639 ; RRID:Addgene_73639) |
参考文献部分: | CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Li Q, Chen J, Minton NP, Zhang Y, Wen Z, Liu J, Yang H, Zeng Z, Ren X, Yang J, Gu Y, Jiang W, Jiang Y, Yang S. Biotechnol J. 2016 May 23. doi: 10.1002/biot.201600053. 10.1002/biot.201600053 PubMed 27213844 |
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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 pNICKclos1.0 11082 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Genome editing for gene pyrE (CAC-002) in Clostridium acetobutylicum ATCC 824. ACCESSION . VERSION . KEYWORDS pNICKclos1.0 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11082) AUTHORS Li Q, Chen J, Minton NP, Zhang Y, Wen Z, Liu J, Yang H, Zeng Z, Ren X, Yang J, Gu Y, Jiang W, Jiang Y, Yang S TITLE CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. JOURNAL Biotechnol J. 2016 May 23. doi: 10.1002/biot.201600053. PUBMED 27213844 REFERENCE 2 (bases 1 to 11082) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11082 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 14..48 /label=J23119(SpeI) promoter /note="bacterial promoter (Registry of Standard Biological Parts BBa_J23119) modified to end with an SpeI site" misc_RNA 69..144 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 2304..6410 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" primer_bind complement(6422..6439) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6422..6438) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6431..6453) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(6647..6666) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6766..6788 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6826..6844) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6912..7016 /gene="bla" /label=AmpR promoter CDS 7017..7877 /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(7235..7254) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8048..8636 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8537..8556 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8790..8807 /label=L4440 /note="L4440 vector, forward primer" protein_bind 8924..8945 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8960..8990 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8998..9014 /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)." primer_bind 9003..9025 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 9022..9038 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 9022..9038 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 agcttggctg cagttgacag ctagctcagt cctaggtata atactagtta tacttaaatc 61 acaggctggt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt 121 gaaaaagtgg caccgagtcg gtgctttttt gcggccgcat tcttatgaaa attcaagtgc 181 tctaactgta atttttccag gaataaatta tggtcatgaa aagcctcttc tgtattatgc 241 aaggaaagca gctgcacaga ataatataga tattctatgc atcaactata agaataagat 301 ttcttgggag gacataggaa aacaaagtat tgaagatgta gctattgaaa taaagggcat 361 tataaaaaat gttattgaaa aaaagtatga taacatttat tttttaggga aaagtgttgg 421 aacagaaata gctggatgta catcttctaa attaaatata gataatataa agtttcttta 481 cttaacacct attgaagaga ctttaaagta tatagttgac agaaagtgca ttgtggtgtc 541 aggcactaag gatgaatttt ttaaaagtga atgtataaat aaaatcagag aaaataaaaa 601 tgttaaattg atgttgtttg agggagctaa tcattctctt gagattaagg atgatattat 661 aagaagttta agaattttag ataacgtagc acaattgtat ttggacttct ttaaataaaa 721 acatggaggg ctaaaaatgg aacaatacaa acaagaattt atagaattta tggtggagag 781 taatgtactt acctttgggg atttcataac taaaagcggc agaagaacac cattttttat 841 aaatacaggt aactacaaga caggtaatca attaaataag ttggctaagt tttatgctaa 901 agcaatatat gataattttg gagatgattt tgatatttta tttgggcctg catataaagg 961 aataccttta agtgtttcag tagctatggc acttgataat atttatggaa ttaatgcagc 1021 ttattgttca aatagaaaag aagttaaaga tcacggtgat aagggaatac ttcttggagc 1081 aaagcttgaa gaaggagaca gagttataat tgtagaagat gtcacaacag ctggtacatc 1141 agtatacgaa acaatgccga tatcaggttg atgtaaaggg aatcataata tcagtggata 1201 gaatggaaag aggtaaggga gataagagtg ccttaactga acttaaagaa aagtttggat 1261 ttaaaacatg ttctattgtt actatggaag aggtagtaga atatttgtat aagaaaaata 1321 tcaatggcaa agtaatcata gatgataaaa tgaaagatag aattaatgag tactataaag 1381 agtatggagt aaaatagtaa aataaatgtg cctcaactta gatgttaagg cacatttatt 1441 ttatatatta ttcatgtttt gaaacatttt tatcttttgt gtattttacg tgtagtaatt 1501 tgtgagcaag tccttcacct ggttttccaa agtagctatc atacatttta ataatagctg 1561 gattatcatg tgactttctc tttgaaagaa catttttatc ttggttgtat aatactgatg 1621 ctcttagttt tctgtaatca acattttctc tatcaagagc atttacgtga ggttgacctc 1681 caccatttat acatccacca gggcaagcca ttacttctat aaagtgatat tgtttttcgt 1741 tcatttttcc agatttcata aactcgaaga agttagaagc accatttata acagcaacgt 1801 ttagtttatt tccagcaatt tcaacttccg cttcttttat gcctttaaag cctcttactt 1861 cagtgtaatc aacattttca agttctttat tttcagcaaa gtctttagct gatcttattg 1921 cagcttccat aacgccaccg gttgcaccaa agatagctcc agcaccactg taagtaccca 1981 tagcaggatc aacttcacca tcttcaagat ctgcaaattt aatttttgca tctttaatca 2041 tttttgcaag ctctcttgta gttaaggatg catcaatatc tcttaagctg ttagtttcca 2101 tgaaaggaat atctgcttca tattttttat cattacaagg catgatagta actgtataaa 2161 catcctcgag ctgcagtata aaatataaat aattttctaa aaaacttaac ttcatgtgaa 2221 aagtttgtta aaatataaat gagcacgtta atcatttaac atagataatt aaatagtaaa 2281 agggagtgtc gaggatcccc gggatggata agaaatactc aataggctta gctatcggca 2341 caaatagcgt cggatgggcg gtgatcactg atgaatataa ggttccgtct aaaaagttca 2401 aggttctggg aaatacagac cgccacagta tcaaaaaaaa tcttataggg gctcttttat 2461 ttgacagtgg agagacagcg gaagcgactc gtctcaaacg gacagctcgt agaaggtata 2521 cacgtcggaa gaatcgtatt tgttatctac aggagatttt ttcaaatgag atggcgaaag 2581 tagatgatag tttctttcat cgacttgaag agtctttttt ggtggaagaa gacaagaagc 2641 atgaacgtca tcctattttt ggaaatatag tagatgaagt tgcttatcat gagaaatatc 2701 caactatcta tcatctgcga aaaaaattgg tagattctac tgataaagcg gatttgcgct 2761 taatctattt ggccttagcg catatgatta agtttcgtgg tcattttttg attgagggag 2821 atttaaatcc tgataatagt gatgtggaca aactatttat ccagttggta caaacctaca 2881 atcaattatt tgaagaaaac cctattaacg caagtggagt agatgctaaa gcgattcttt 2941 ctgcacgatt gagtaaatca agacgattag aaaatctcat tgctcagctc cccggtgaga 3001 agaaaaatgg cttatttggg aatctcattg ctttgtcatt gggtttgacc cctaatttta 3061 aatcaaattt tgatttggca gaagatgcta aattacagct ttcaaaagat acttacgatg 3121 atgatttaga taatttattg gcgcaaattg gagatcaata tgctgatttg tttttggcag 3181 ctaagaattt atcagatgct attttacttt cagatatcct aagagtaaat actgaaataa 3241 ctaaggctcc cctatcagct tcaatgatta aacgctacga tgaacatcat caagacttga 3301 ctcttttaaa agctttagtt cgacaacaac ttccagaaaa gtataaagaa atcttttttg 3361 atcaatcaaa aaacggatat gcaggttata ttgatggggg agctagccaa gaagaatttt 3421 ataaatttat caaaccaatt ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac 3481 taaatcgtga agatttgctg cgcaagcaac ggacctttga caacggctct attccccatc 3541 aaattcactt gggtgagctg catgctattt tgagaagaca agaagacttt tatccatttt 3601 taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct tattatgttg 3661 gtccattggc gcgtggcaat agtcgttttg catggatgac tcggaagtct gaagaaacaa 3721 ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg 3781 aacgcatgac aaactttgat aaaaatcttc caaatgaaaa agtactacca aaacatagtt 3841 tgctttatga gtattttacg gtttataacg aattgacaaa ggtcaaatat gttactgaag 3901 gaatgcgaaa accagcattt ctttcaggtg aacagaagaa agccattgtt gatttactct 3961 tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga agattatttc aaaaaaatag 4021 aatgttttga tagtgttgaa atttcaggag ttgaagatag atttaatgct tcattaggta 4081 cctaccatga tttgctaaaa attattaaag ataaagattt tttggataat gaagaaaatg 4141 aagatatctt agaggatatt gttttaacat tgaccttatt tgaagatagg gagatgattg 4201 aggaaagact taaaacatat gctcacctct ttgatgataa ggtgatgaaa cagcttaaac 4261 gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt gattaatggt attagggata 4321 agcaatctgg caaaacaata ttagattttt tgaaatcaga tggttttgcc aatcgcaatt 4381 ttatgcagct gatccatgat gatagtttga catttaaaga agacattcaa aaagcacaag 4441 tgtctggaca aggcgatagt ttacatgaac atattgcaaa tttagctggt agccctgcta 4501 ttaaaaaagg tattttacag actgtaaaag ttgttgatga attggtcaaa gtaatggggc 4561 ggcataagcc agaaaatatc gttattgaaa tggcacgtga aaatcagaca actcaaaagg 4621 gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa 4681 gtcagattct taaagagcat cctgttgaaa atactcaatt gcaaaatgaa aagctctatc 4741 tctattatct ccaaaatgga agagacatgt atgtggacca agaattagat attaatcgtt 4801 taagtgatta tgatgtcgat cacattgttc cacaaagttt ccttaaagac gattcaatag 4861 acaataaggt cttaacgcgt tctgataaaa atcgtggtaa atcggataac gttccaagtg 4921 aagaagtagt caaaaagatg aaaaactatt ggagacaact tctaaacgcc aagttaatca 4981 ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata 5041 aagctggttt tatcaaacgc caattggttg aaactcgcca aatcactaag catgtggcac 5101 aaattttgga tagtcgcatg aatactaaat acgatgaaaa tgataaactt attcgagagg 5161 ttaaagtgat taccttaaaa tctaaattag tttctgactt ccgaaaagat ttccaattct 5221 ataaagtacg tgagattaac aattaccatc atgcccatga tgcgtatcta aatgccgtcg 5281 ttggaactgc tttgattaag aaatatccaa aacttgaatc ggagtttgtc tatggtgatt 5341 ataaagttta tgatgttcgt aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa 5401 ccgcaaaata tttcttttac tctaatatca tgaacttctt caaaacagaa attacacttg 5461 caaatggaga gattcgcaaa cgccctctaa tcgaaactaa tggggaaact ggagaaattg 5521 tctgggataa agggcgagat tttgccacag tgcgcaaagt attgtccatg ccccaagtca 5581 atattgtcaa gaaaacagaa gtacagacag gcggattctc caaggagtca attttaccaa 5641 aaagaaattc ggacaagctt attgctcgta aaaaagactg ggatccaaaa aaatatggtg 5701 gttttgatag tccaacggta gcttattcag tcctagtggt tgctaaggtg gaaaaaggga 5761 aatcgaagaa gttaaaatcc gttaaagagt tactagggat cacaattatg gaaagaagtt 5821 cctttgaaaa aaatccgatt gactttttag aagctaaagg atataaggaa gttaaaaaag 5881 acttaatcat taaactacct aaatatagtc tttttgagtt agaaaacggt cgtaaacgga 5941 tgctggctag tgccggagaa ttacaaaaag gaaatgagct ggctctgcca agcaaatatg 6001 tgaatttttt atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg 6061 aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa 6121 tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg 6181 catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat 6241 ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc 6301 gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca 6361 ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgactga cccgggaatt 6421 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 6481 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 6541 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc 6601 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 6661 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 6721 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 6781 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 6841 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 6901 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 6961 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 7021 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 7081 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 7141 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 7201 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 7261 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 7321 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 7381 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 7441 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 7501 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 7561 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 7621 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 7681 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 7741 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 7801 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 7861 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 7921 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 7981 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 8041 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 8101 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 8161 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 8221 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 8281 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 8341 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 8401 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 8461 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 8521 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 8581 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 8641 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 8701 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 8761 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 8821 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 8881 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 8941 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 9001 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctttggc 9061 taacacacac gccattccaa ccaatagttt tctcggcata aagccatgct ctgacgctta 9121 aatgcactaa tgccttaaaa aaacattaaa gtctaacaca ctagacttat ttacttcgta 9181 attaagtcgt taaaccgtgt gctctacgac caaaagtata aaacctttaa gaactttctt 9241 ttttcttgta aaaaaagaaa ctagataaat ctctcatatc ttttattcaa taatcgcatc 9301 agattgcagt ataaatttaa cgatcactca tcatgttcat atttatcaga gctcgtgcta 9361 taattatact aattttataa ggaggaaaaa ataaagaggg ttataatgaa cgagaaaaat 9421 ataaaacaca gtcaaaactt tattacttca aaacataata tagataaaat aatgacaaat 9481 ataagattaa atgaacatga taatatcttt gaaatcggct caggaaaagg gcattttacc 9541 cttgaattag tacagaggtg taatttcgta actgccattg aaatagacca taaattatgc 9601 aaaactacag aaaataaact tgttgatcac gataatttcc aagttttaaa caaggatata 9661 ttgcagttta aatttcctaa aaaccaatcc tataaaatat ttggtaatat accttataac 9721 ataagtacgg atataatacg caaaattgtt tttgatagta tagctgatga gatttattta 9781 atcgtggaat acgggtttgc taaaagatta ttaaatacaa aacgctcatt ggcattattt 9841 ttaatggcag aagttgatat ttctatatta agtatggttc caagagaata ttttcatcct 9901 aaacctaaag tgaatagctc acttatcaga ttaaatagaa aaaaatcaag aatatcacac 9961 aaagataaac agaagtataa ttatttcgtt atgaaatggg ttaacaaaga atacaagaaa 10021 atatttacaa aaaatcaatt taacaattcc ttaaaacatg caggaattga cgatttaaac 10081 aatattagct ttgaacaatt cttatctctt ttcaatagct ataaattatt taataagtaa 10141 gttaagggat gcataaactg catcccttaa cttgtttttc gtgtacctat tttttgtgaa 10201 tcgattatgt cttttgcgca ttcacttctt ttctatataa atatgagcga agcgaataag 10261 cgtcggaaaa gcagcaaaaa gtttcctttt tgctgttgga gcatgggggt tcagggggtg 10321 cagtatctga cgtcaatgcc gagcgaaagc gagccgaagg gtagcattta cgttagataa 10381 ccccctgata tgctccgacg ctttatatag aaaagaagat tcaactaggt aaaatcttaa 10441 tataggttga gatgataagg tttataagga atttgtttgt tctaattttt cactcatttt 10501 gttctaattt cttttaacaa atgttctttt ttttttagaa cagttatgat atagttagaa 10561 tagtttaaaa taaggagtga gaaaaagatg aaagaaagat atggaacagt ctataaaggc 10621 tctcagaggc tcatagacga agaaagtgga gaagtcatag aggtagacaa gttataccgt 10681 aaacaaacgt ctggtaactt cgtaaaggca tatatagtgc aattaataag tatgttagat 10741 atgattggcg gaaaaaaact taaaatcgtt aactatatcc tagataatgt ccacttaagt 10801 aacaatacaa tgatagctac aacaagagaa atagcaaaag ctacaggaac aagtctacaa 10861 acagtaataa caacacttaa aatcttagaa gaaggaaata ttataaaaag aaaaactgga 10921 gtattaatgt taaaccctga actactaatg agaggcgacg accaaaaaca aaaatacctc 10981 ttactcgaat ttgggaactt tgagcaagag gcaaatgaaa tagattgacc tcccaataac 11041 accacgtagt tattgggagg tcaatctatg aaaatgcgat ta //
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