质粒名称: | pFC334 |
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出品公司: | Addgene |
目录编号: | 87846 |
存储实验室: | Uffe Mortensen |
载体骨架基本信息 |
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载体名称: | pFC331 |
载体抗性: | 氨苄青霉素 |
载体类型: | 细菌表达,CRISPR;真菌表达 |
筛选标记: | argB |
高拷贝/低拷贝: | 未知 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Cas9 |
物种来源: | 合成;合成基因,黑曲霉密码子优化 |
插入大小: | 4131 bp |
GenBank编号 | KT031983 |
启动子 | Aspergillus nidulans tef1 promoter |
标签/融合蛋白: | SV40 NLS(C端子插入) |
5' 测序引物 | CTTCTCTGCTCAGCACCTCTACG |
文献引用方法 |
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材料和方法部分: | pFC334 was a gift from Uffe Mortensen (Addgene plasmid # 87846 ; http://n2t.net/addgene:87846 ; RRID:Addgene_87846) |
参考文献部分: | A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. PONE-D-15-11561 [pii] PubMed 26177455 |
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载体序列
LOCUS pFC334 17327 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION AMA1 plasmid with Aspergillus optimized Cas9, argB selection marker and yA specific sgRNA expressed with ribozymes. ACCESSION . VERSION . KEYWORDS pFC334 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 17327) AUTHORS Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH TITLE A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. JOURNAL PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. PUBMED 26177455 REFERENCE 2 (bases 1 to 17327) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..17327 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 622..641 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(860..951) /gene="bla" /label=AmpR promoter primer_bind 993..1012 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1018..1037 /label=pENTR-R /note="pENTR vectors, reverse primer" protein_bind 1244..1277 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" intron 7484..7599 /gene="gpdA" /label=gpdA intron /note="intron from the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene (Punt et al., 1990)" misc_RNA 7672..7747 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 8955..13058 /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 13062..13082 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind complement(15827..15860) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" primer_bind complement(16330..16347) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(16330..16346) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(16445..16462) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(16616..17204) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(16696..16715) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(17326..859) /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" ORIGIN 1 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 61 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 121 gcgctgcgat gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc 181 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 241 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 301 ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 361 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 421 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 481 tggttatggc agcgctacat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 541 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 601 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 661 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 721 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 781 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 841 ggaaatgttg aatactcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt 901 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca 961 gtgttacaac caattaacca attctgaaca ttatcgcgag cccatttata cctgaatatg 1021 gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg 1081 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga 1141 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg 1201 cccgggctaa ttatggggtg tcgcccttat tcgactctat agtgaagttc ctattctcta 1261 gaaagtatag gaacttctga agtggggatt taaatgcggc cgcgctgagg gtttaatcga 1321 cgaagcagct gacggccagt gccaagctta acgcgtaccg ggcccagtat atgttccgca 1381 gatgactgga gctctgccat acgtgccctc tcaagcacca tttgttccat ctacagagac 1441 tagtcaccaa ctagtctatc aagactcaca gggtacattg ctgagaccaa ctgaccagag 1501 gcagggtagc ggattgacgg ctccatctcc ttcacttaca aggtctattg aaagcccttt 1561 agcatcacca agcggagaat agattgttaa gcttattttt tgtatactgt tttgtgatag 1621 cacgaagttt ttccacggta tcttgttaaa aatatatatt tgtggcgggc ttacctacat 1681 caaattaata agagactaat tataaactaa acacacaagc aagctacttt agggtaaaag 1741 tttataaatg cttttgacgt ataaacgttg cttgtattta ttattacaat taaaggtgga 1801 tagaaaacct agagactagt tagaaactaa tctcaggttt gcgttaaact aaatcagagc 1861 ccgagaggtt aacagaacct agaaggggac tagatatccg ggtagggaaa caaaaaaaaa 1921 aancaagaca gccacatatt agggagacta gttagaagct agttccagga ctaggaaaat 1981 aaaagacaat gataccacag tctagttgac aactagatag attctagatt gaggccaaag 2041 tctctgagat ccaggttagt tgcaactaat actagttagt atctagtctc ctataactct 2101 gaagctagaa taacttacta ctattatcct caccactgtt cagctgcgca aacggagtga 2161 ttgcaaggtg ttcagagact agttattgac tagtcagtga ctagcaataa ctaacaaggt 2221 attaacctac catgtctgcc atcaccctgc acttcctcgg gctcagcagc cttttcctcc 2281 tcattttcat gctcattttc cttgtttaag actgtgacta gtcaaagact agtccagaac 2341 cacaaaggag aaatgtctta ccactttctt cattgcttgt ctcttttgca ttatccatgt 2401 ctgcaactag ttagagtcta gttagtgact agtccgacga ggacttgctt gtctccggat 2461 tgttggagga actctccagg gcctcaagat ccacaacaga gccttctaga agactggtca 2521 ataactagtt ggtctttgtc tgagtctgac ttacgaggtt gcatactcgc tccctttgcc 2581 tcgtcaatcg atgagaaaaa gcgccaaaac tcgcaatatg gctttgaacc acacggtgct 2641 gagactagtt agaatctagt cccaaactag cttggatagc ttacctttgc cctttgcgtt 2701 gcgacaggtc ttgcagggta tggttccttt ctcaccagct gatttagctg ccttgctacc 2761 ctcacggcgg atctgcataa agagtggcta gaggttataa attagcactg atcctaggta 2821 cggggctgaa tgtaacttgc cyttcctttc tcatcgcgcg gcaagacagg cttgctcaaa 2881 ttcctaccag tcacaggggt atgcacggcg tacggaccac ttgaactagt cacagattag 2941 ttagcaacta gtctgcattg aatggctgta cttacgggcc ctcgccattg tcctgatcat 3001 ttccagcttc accctcgttg ctgcaaagta gttagtgact agtcaaggac tagttgaaat 3061 gggagaagaa actcacgaat tctcgacacc cttagtattg tggtccttgg acttggtgct 3121 gctatatatt agctaataca ctagttagac tcacagaaac ttacgcagct cgcttgcgct 3181 tcttggtagg agtcggggtt gggagaacag tgccttcaaa caagccttca taccatgcta 3241 cttgactagt cagggactag tcaccaagta atctagatag gacttgcctt tggcctccat 3301 cagttccttc atagtgggag gtccattgtg caatgtaaac tccatgccgt gggagttctt 3361 gtccttcaag tgcttgacca atatgtttct gttggcagag ggaacctgtc aactagttaa 3421 taactagtca gaaactagta tagcagtaga ctcactgtac gcttgaggca tcccttcact 3481 cggcagtaga cttcatatgg atggatatca ggcacgccat tgtcgtcctg tggactagtc 3541 agtaactagg cttaaagcta gtcgggtcgg cttactatct tgaaatccgg cagcgtaagc 3601 tccccgtcct taactgcctc gagatagtga cagtactctg gggactttcg gagatcgtta 3661 tcgcgaatgc tcggcatact aatcgttgac tagtcttgga ctagtcccga gcaaaaagga 3721 ttggaggagg aggaggaagg tgagagtgag acaaagagcg aaataagagc ttcaaaggct 3781 atctctaagc agtatgaagg ttaagtatct agttcttgac tagatttaaa agagatttcg 3841 actagttatg tacctggagt ttggatatag gaatgtgttg tggtaacgaa atgtaaggga 3901 gaggaaagaa aaagtcggtc aagaggtaac tctaagtcgg ccattccttt ttgggaggcg 3961 ctaaccataa acggcatggt cgacttagag ttagctcagg gaatttaggg agttatctgc 4021 gaccaccgag gaacggcgga atgccaaaga atcccgatgg agctctagct ggcggttgac 4081 aaccccacct tttggcgttt ctgcggcgtt gcaggcggga ctggatactt cgtagaacca 4141 gaaaggcaag gcagaacgcg ctcagcaaga gtgttggaag tgatagcatg atgtgccttg 4201 ttaactaggt caaaatctgc agtatgcttg atgttatcca aagtgtgaga gaggaaggtc 4261 caaacataca cgattgggag agggcctagg tataagagtt tttgagtaga acgcatgtga 4321 gcccagccat ctcgaggaga ttaaacacgg gccggcattt gatggctatg ttagtacccc 4381 aatggaaacg gtgagagtcc agtggtcgca gataactccc taaattccct gagctaactc 4441 taagtcgacc atgccgttta tggttagcgc ctcccaaaaa ggaatggccg acttagagtt 4501 acctcttgac cgactttttc tttcctcccc cttacatttc gttaccacaa cacattccta 4561 tatccaaact ccaggtacat aactagtcga aatctctttt aaatctagtc aagaactaga 4621 tacttaacct tcatactgct tagagatagc ctttgaagct cttatttcgc tctttgtctc 4681 actctcacct tcctcctcct cctccaatcc tttttgctcg ggactagtcc aagactagtc 4741 aacgattagt atgccgagca ttcgcgataa cgatctccga aagtccccag agtactgtca 4801 ctatctcgag gcagttaagg acggggagct tacgctgccg gatttcaaga tagtaagccg 4861 acccgactag ctttaagcct agttactgac tagtccacag gacgacaatg gcgtgcctga 4921 tatccatcca tatgaagtct actgccgagt gaagggatgc ctcaagcgta cagtgagtct 4981 actgctatac tagtttctga ctagttatta actagttgac aggttccctc tgccaacaga 5041 aacatattgg tcaagcactt gaaggacaag aactcccacg gcatggagtt tacattgcac 5101 aatggacctc ccactatgaa ggaactgatg gaggccaaag gcaagtccta tctagattac 5161 ttggtgacta gtccctgact agtcaagtag catggtatga aggcttgttt gaaggcactg 5221 ttctcccaac cccgactcct accaagaagc gcaagcgagc tgcgtaagtt tctgtgagtc 5281 taactagtgt attagctaat atatagcagc accaagtcca aggaccacaa tactaagggt 5341 gtcgagaatt cgtgagtttc ttctcccatt tcaactagtc cttgactagt cactaactac 5401 tttgcagcaa cgagggtgaa gctggaaatg atcaggacaa tggcgagggc ccgtaagtac 5461 agccattcaa tgcagactag ttgctaacta atctgtgact agttcaagtg gtccgtacgc 5521 cgtgcatacc cctgtgactg gtaggaattt gagcaagcct gtcttgccgc gcgatgagaa 5581 aggaarggca agttacattc agccccgtac ctaggatcag tgctaattta taacctctag 5641 ccactcttta tgcagatccg ccgtgagggt agcaaggcag ctaaatcagc tggtgagaaa 5701 ggaaccatac cctgcaagac ctgtcgcaac gcaaagggca aaggtaagct atccaagcta 5761 gtttgggact agattctaac tagtctcagc accgtgtggt tcaaagccat attgcgagtt 5821 ttggcgcttt ttctcatcga ttgacgaggc aaagggagcg agtatgcaac ctcgtaagtc 5881 agactcagac aaagaccaac tagttattga ccagtcttct agaaggctct gttgtggatc 5941 ttgaggccct ggagagttcc tccaacaatc cggagacaag caagtcctcg tcggactagt 6001 cactaactag actctaacta gttgcagaca tggataatgc aaaagagaca agcaatgaag 6061 aaagtggtaa gacatttctc ctttgtggtt ctggactagt ctttgactag tcacagtctt 6121 aaacaaggaa aatgagcatg aaaatgagga ggaaaaggct gctgagcccg aggaagtgca 6181 gggtgatggc agacatggta ggttaatacc ttgttagtta ttgctagtca ctgactagtc 6241 aataactagt ctctgaacac cttgcaatca ctccgtttgc gcagctgaac agtggtgagg 6301 ataatagtag taagttattc tagcttcaga gttataggag actagatact aactagtatt 6361 agttgcaact aacctggatc tcagagactt tggcctcaat ctagaatcta tctagttgtc 6421 aactagactg tggtatcatt gtcttttatt ttcctagtcc tggaactagc ttctaactag 6481 tctccctaat atgtggctgt cttgtttttt ttttttgttt ccctacccgg atatctagtc 6541 cccttctagg ttctgttaac ctctcgggct ctgatttagt ttaacgcaaa cctgagatta 6601 gtttctaact agtctctagg ttttctatcc acctttaatt gtaataataa atacaagcaa 6661 cgtttatacg tcaaaagcat ttataaactt ttaccctaaa gtagcttgct tgtgtgttta 6721 gtttataatt agtctcttat taatttgatg taggtaagcc cgccacaaat atatattttt 6781 aacaagatac cgtggaaaaa cttcgtgcta tcacaaaaca gtatacaaaa aataagctta 6841 acaatctatt ctccgcttgg tgatgctaaa gggctttcaa tagaccttgt aagtgaagga 6901 gatggagccg tcaatccgct accctgcctc tggtcagttg gtctcagcaa tgtaccctgt 6961 gagtcttgat agactagttg gtgactagtc tctgtagatg gaacaaatgg tgcttgagag 7021 ggcacgtatg gcagagctcc agtcatctgc ggaacatata ctgggcccgg gaagatctca 7081 tggtcatagc tgtttccgct gagggtttaa tgcgtaagct ccctaattgg cccatccggc 7141 atctgtaggg cgtccaaata tcgtgcctct cctgctttgc ccggtgtatg aaaccggaaa 7201 ggccgctcag gagctggcca gcggcgcaga ccgggaacac aagctggcag tcgacccatc 7261 cggtgctcta cactcgacct gctgaggtcc ctcagtccct ggtaggcagc tttgccccgt 7321 ctgtccgccc ggtgtgtcgg cggggttgac aaggtcgttg cgtcagtcca acatttgttg 7381 ccatattttc ctgctctccc caccagctgc tcttttcttt tctctttctt ttcccatctt 7441 cagtatattc atcttcccat ccaagaacct ttatttcccc taagtaagta ctttgctaca 7501 tccatactcc atccttccca tcccttattc ctttgaacct ttcagttcga gctttcccac 7561 ttcatcgcag cttgactaac agctaccccg cttgagcaga catcaccgtc cgccctgatg 7621 agtccgtgag gacgaaacga gtaagctcgt cggcggagta tcataacatc ggttttagag 7681 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 7741 tcggtgcttt tggccggcat ggtcccagcc tcctcgctgg cgccggctgg gcaacatgct 7801 tcggcatggc gaatgggact gatttaatag ctccatgtca acaagaataa aacgcgtttc 7861 gggtttacct cttccagata cagctcatct gcaatgcatt aatgcattgg acctcgcaac 7921 cctagtacgc ccttcaggct ccggcgaagc agaagaatag cttagcagag tctattttca 7981 ttttcgggag acgagatcaa gcagatcaac ggtcgtcaag agacctacga gactgaggaa 8041 tccgctcttg gctcattaag acctcagccg agacagcaga atcaccgccc aagttaagcc 8101 tttgtgctga tcatgctctc gaacgggcca agttcgggaa aagcaaagga gcgtttagtg 8161 aggggcaatt tgactcacct cccaggcaac agatgagggg ggcaaaaaga aagaaatttt 8221 cgtgagtcaa tatggattcc gagcatcatt ttcttgcggt ctatcttgct acgtatgttg 8281 atcttgacgc tgtggatcaa gcaacgccac tcgctcgctc catcgcaggc tggtcgcaga 8341 caaattaaaa ggcggcaaac tcgtacagcc gcggggttgt ccgctgcaaa gtacagagtg 8401 ataaaagccg ccatgcgacc atcaacgcgt tgatgcccag ctttttcgat ccgagaatcc 8461 accgtagagg cgatagcaag taaagaaaag ctaaacaaaa aaaaatttct gcccctaagc 8521 catgaaaacg agatggggtg gagcagaacc aaggaaagag tcgcgctggg ctgccgttcc 8581 ggaaggtgtt gtaaaggctc gacgcccaag gtgggagtct aggagaagaa tttgcatcgg 8641 gagtggggcg ggttacccct ccatatccaa tgacagatat ctaccagcca agggtttgag 8701 cccgcccgct tagtcgtcgt cctcgcttgc ccctccataa aaggatttcc cctccccctc 8761 ccacaaaatt ttctttccct tcctctcctt gtccgcttca gtacgtatat cttcccttcc 8821 ctcgcttctc tcctccatcc ttctttcatc catctcctgc taacttctct gctcagcacc 8881 tctacgcatt actagccgta gtatctgagc acttctccct tttatattcc acaaaacata 8941 acacaacctt caccatggac aagaagtata gcatcgggct ggacattgga acgaactcgg 9001 ttggttgggc tgtgattacg gacgaataca aggtgccatc caagaagttt aaggtcctgg 9061 gaaacaccga ccgtcactca atcaagaaga atctcattgg agccctgctc ttcgatagtg 9121 gggagaccgc cgaagctact cgactgaagc gaacggctcg ccggcgttat acacgacgca 9181 agaatcgcat ctgctacctc caggagattt tcagcaacga aatggctaag gttgatgact 9241 cattctttca tcgactcgaa gaaagtttct tggtcgagga ggataagaag cacgagcgcc 9301 atccgatctt tggtaacatt gtggatgagg ttgcctatca cgaaaagtac ccaactatct 9361 atcatcttcg taagaagctg gtcgatagca cggacaaggc tgatttgcga cttatctacc 9421 tggcactcgc gcacatgatt aagttccgcg gccattttct tatcgagggt gacctgaacc 9481 ccgataattc tgacgttgat aagctcttca tccagttggt ccaaacctac aatcagctgt 9541 ttgaggaaaa ccctattaat gcatctggcg tggacgccaa ggctatcctt tcggcgcgcc 9601 tgtctaagtc gcggcgtttg gagaacctta tcgcacaact ccccggcgaa aagaagaacg 9661 gcctcttcgg taatttgatt gcgttgtcac ttggtctgac tcctaacttc aagagtaatt 9721 ttgacctggc agaggatgcg aagctccagt tgtctaagga tacgtatgat gacgatctcg 9781 acaacttgct tgcccaaatc ggtgaccagt acgctgatct tttcctggcc gctaagaatc 9841 tctcagatgc aatcctgctc agtgacattt tgcgggtcaa caccgagatt actaaggccc 9901 ccctgtcagc tagtatgatc aagcggtatg atgagcacca tcaggacctc accttgctta 9961 aggccctcgt gcgtcagcaa ttgcctgaga agtataagga aatcttcttt gaccaatcca 10021 agaacggata cgcagggtat attgatggcg gtgcgagcca ggaggaattc tacaagttta 10081 tcaagccgat tttggagaag atggacggca ctgaggaact gctcgtcaag ctgaatcgcg 10141 aagatttgct tcgtaagcaa cgaacgttcg acaacggctc catcccgcac cagattcatc 10201 tgggcgagct ccacgccatc cttcgacgcc aggaagattt ctacccattt ctgaaggaca 10261 accgtgagaa gatcgaaaag attcttacat tccgaatccc ctactatgtg ggacctttgg 10321 cccgtgggaa ttcccgattt gcttggatga cccgaaagag cgaggaaacc atcactccgt 10381 ggaacttcga ggaagtcgtg gacaagggtg catccgcgca gagcttcatt gagcggatga 10441 ccaattttga taagaacctt ccgaatgaaa aggtcctgcc aaagcattcg ctgctctacg 10501 agtatttcac cgtgtataac gaactgacta aggtcaagta cgtgacggag ggaatgcgga 10561 agccagcctt cctctcaggg gaacaaaaga aggctatcgt cgatttgctt tttaagacca 10621 atcgtaaagt gactgttaag cagctgaagg aggattattt caagaagatt gaatgtttcg 10681 actccgtcga gatcagcggc gtggaagatc gctttaacgc ttccctcggt acctaccacg 10741 acctgctcaa gatcattaag gacaaggatt tcctcgataa cgaggaaaat gaggacatct 10801 tggaagatat tgtcctcacg ttgacacttt ttgaggaccg cgaaatgatc gaggaacggc 10861 tcaagacata tgcccatttg ttcgacgata aggtgatgaa gcagctgaag cggcgtcgat 10921 acaccggatg gggtcgcctt agccggaagc tgatcaacgg cattcgagat aagcaatctg 10981 gtaagactat cttggatttc cttaagtcgg acggcttcgc caaccgcaat tttatgcagc 11041 ttattcacga cgattccctg acgttcaagg aggacatcca gaaggcacaa gtctcaggac 11101 aaggggattc cctgcacgag catatcgcca acctggctgg atccccggcg atcaagaagg 11161 ggattcttca gaccgtcaag gttgtcgacg agctggtcaa ggtgatgggc cgtcataagc 11221 cagaaaacat cgtgattgag atggcccgag aaaatcagac cactcaaaag ggtcagaaga 11281 acagccgcga gcggatgaag cggatcgagg aaggcattaa ggaacttggt tctcagatcc 11341 tgaaggagca ccctgttgaa aacacacagc tccaaaatga gaagctgtat ctctactatt 11401 tgcaaaatgg acgcgacatg tacgtcgatc aggagctcga cattaaccgg ttgtcggact 11461 acgatgttga ccatatcgtc ccgcaatcct tccttaagga cgatagcatt gataacaagg 11521 tgctgactcg ctcagataag aaccggggca agtccgacaa tgttccaagc gaggaagtgg 11581 ttaagaagat gaagaactac tggcgccaat tgcttaatgc caagctcatc acacagcgca 11641 agtttgacaa cttgaccaag gccgagcggg gagggctgag tgaactcgat aaggctggct 11701 tcatcaagcg tcaactcgtg gagacgcgac agatcacaaa gcacgttgct cagattctgg 11761 actcccggat gaacacaaag tacgacgaga atgataagct catccgtgaa gttaaggtca 11821 ttaccctcaa gtctaagttg gtgtcggatt tccgcaagga cttccaattt tataaggttc 11881 gggagatcaa caattatcac catgcacatg atgcgtacct caacgcagtc gtgggaactg 11941 cgctcatcaa gaagtatccc aagttggagt ccgaattcgt ctacggggat tataaggttt 12001 acgacgtccg caagatgatc gccaagagtg agcaggaaat tggcaaggcc acggctaagt 12061 atttctttta ctccaacatc atgaatttct ttaagacgga gatcacactc gccaatggag 12121 aaatccgtaa gcgacctttg attgagacca acggcgagac tggtgaaatc gtttgggata 12181 aggggcgcga cttcgctacc gtgcggaagg ttctgagcat gccgcaagtc aatatcgtca 12241 agaaaaccga ggtgcagaca ggcggtttct ctaaggaatc gattcttcca aagcgtaact 12301 ctgacaagct gatcgctcga aagaaggatt gggaccccaa gaagtatgga gggttcgatt 12361 ctcctacagt ggcatactcg gttctcgttg tcgcgaaggt tgagaaggga aagtctaaga 12421 agctgaagtc ggtcaaggaa ctgctcggga tcaccattat ggagcgctcc agcttcgaaa 12481 agaatcccat cgactttctc gaggccaagg gctataagga agtcaagaag gatcttatca 12541 ttaagctgcc taagtactct ttgttcgagc ttgaaaacgg tcgaaagcga atgctcgcat 12601 cggcaggaga gttgcagaag gggaatgaat tggcacttcc ctcaaagtac gtgaacttcc 12661 tgtatctcgc gtcccactac gagaagctga agggtagccc tgaggacaac gaacagaagc 12721 aactttttgt tgagcaacac aagcattatc tggatgagat cattgaacag atttcagagt 12781 tcagtaagcg cgtcatcctc gccgatgcta atctcgacaa ggtgttgtcg gcctacaaca 12841 agcaccgtga caagccgatc cgagagcagg ctgaaaatat cattcatctg ttcaccctca 12901 ctaacttggg agcaccagca gcgttcaagt attttgatac gacaatcgac cgtaagcgat 12961 acacgtccac aaaggaggtg cttgatgcga ccctgattca tcaatccatc actgggctct 13021 atgaaacccg tatcgacctt agtcaactgg ggggcgaccc tcccaagaag aagcgcaagg 13081 tctgagcgga cattcgattt atgccgttat gacttcctta aaaaagcctt tacgaatgaa 13141 agaaatggaa ttagacttgt tatgtagttg attctacaat ggattatgat tcctgaactt 13201 caaatccgct gttcattatt aatctcagct cttcccgtaa agccaatgtt gaaactattc 13261 gtaaatgtac ctcgttttgc gtgtaccttg cttatcacgt gatattacat gacctggaca 13321 gagttctgcg cgaaagtcat aacgtaaatc ccgggcggta ggtgcgtccc gggcggaagg 13381 tagttttctc gtccacccca acgcgtttat caacctcaac tttcaacaac catcatgcca 13441 ccaaaagcgc gtaaaacaaa gcgagatttg attgagcaag agggcaggat ccaatgcgcg 13501 attcaagaca ttaaaaatgg aaaatttcaa aaaattgcgc ccgcagcgcg tgcatacaaa 13561 attcatccca atacaagtgt aatactaggt aatatcgcgt gcattccgcg gttttttggg 13621 gtagtcatct aatgaaacag acccggacgc agcagaggaa gcccgcgatg actctatacc 13681 accgtacgcc gatatatcat catcgcggcg atggagaagt tgggttgact ccgaagacac 13741 ttcaaaggag cgacgctgtt gatttgtaga cgacgcttga tagggagaag cattattgtc 13801 gtgatgctcg cccaacagag gccgactcgc ctcatccgtc ataacgaacg ctgtgtaaag 13861 cggagtgggg gggaaagtgt ggattgtgga gagtatgcga tagtgttgag gctgatcaga 13921 cggcgaatcg ggccagatat gaccagttta gaggcctcat ttgactataa tttacataaa 13981 ttagataaat agagatgaac gcatgcaata attgcagcaa atattgatga agcgagaggt 14041 aggacgatga aggactgtga gcagttcaag gtatcagcag agtcaagggc ctgatgcaat 14101 ggcggtgatc cgtgatcagc gaacggaagg ggcgctaact ctgtttcttt accaatgatc 14161 ggaagctcct gctggcggac ttatgagtca ttcacgaatc atttctcagt tatttgtgga 14221 tgccctcgtt ctgtccacaa tttctttccg ccccaagtct tttaagttct ttaacatcta 14281 tattcttgca cttccaatgg catcccttcg ctccgtactc aagagccaga gcttgcgaca 14341 caccgtgcga tcctactcct cgcaaaccat gcctcccgcc tcaccctttg ctccccgcca 14401 cttcctctcc attgcggacc tctcgccctc cgagttcgca acccttgttc gcaatgcctc 14461 ctcacacaaa cgggctatca agtcggggtc aatgccccaa aacttgcagg gatcactcct 14521 tgggaaaact gtggccatga tcttcagcaa acgaagcacg aggacaaggg tatctacaga 14581 aggggccgtt gtgcagatgg gaggtcatcc gatgttcttg ggcaaggatg atatccaact 14641 aggtgtcaac gagtccctat acgacacctc cgttgtcatt tcgtccatgg tatcctgcat 14701 tgtagcccgt gtcggtaaac atgcagaggt cgcagatctg gcgaagcact cttcggttcc 14761 agtcatcaat gctttgtgtg actctttcca ccctctccaa gccgtggccg atttccagac 14821 catctatgaa gcattcaccc ccaaggcgca ccacctttca agtctagggt tggaaggatt 14881 gaagatcgct tgggtgggtg acgccaacaa cgtcctgttc gatatggcca ttgctgctac 14941 aaaaatgggt gtcgacattg ctgtcgctac tcccaagggg tacgaaatcc ctcctcacat 15001 gctggagctc atcaagtctg ctggagaggg tgtctcgaaa ccaggaaagc ttctgcaaac 15061 caatattccc gaagaagcgg tcaaggacgc cgatattctg gtcacagaca cctgggtctc 15121 tatgggccaa gaggaagaga aggctcagag gctgaaggag tttgatggtt tccaaatcac 15181 tgctgaactc gccaagcgag gaggagctaa ggagggctgg aagttcatgc actgtctccc 15241 gcgacaccct gaggaggtca gcgacgaggt tttctacagc aaccggtcac ttgtcttccc 15301 tgaggctgag aaccggttat gggctgcgat ttccgccttg gagggtttcg ttgtcaataa 15361 gggaaaaatt gaataaatat aaccaggctt ccatttaaat atatagaggc tggcgttatc 15421 aaactgatga gttgacgggt atgagatcat tccgtcccta aatatattta ctccgatcac 15481 gtaaaagcct gttagtagaa gcattttccc aattatcctg accaattctt tctagcatat 15541 atcaaataac taattgacat gttctctcgc ttccttatat tactcagagt attggaaatg 15601 gggcaaatcg cacccggtga ctttcacatg tcacgaatgc ggagtcgtcc tagccaaggt 15661 agatccaggc ctaacacacc ccaaccctcg actctcctca ttccatactc ttgacctcta 15721 tccagcacat tcttctgagg tttcgcaatg gctgtaggtg gcgctgaggc atccccacta 15781 ccgcattaag acctcagcgc ggccgcaaat ttaaataaaa tgaagtgaag ttcctatact 15841 ttctagagaa taggaacttc tatagtgagt cgaataaggg cgacacaaaa tttattctaa 15901 atgcataata aatactgata acatcttata gtttgtatta tattttgtat tatcgttgac 15961 atgtataatt ttgatatcaa aaactgattt tccctttatt attttcgaga tttattttct 16021 taattctctt taacaaacta gaaatattgt atatacaaaa aatcataaat aatagatgaa 16081 tagtttaatt ataggtgttc atcaatcgaa aaagcaacgt atcttattta aagtgcgttg 16141 cttttttctc atttataagg ttaaataatt ctcatatatc aagcaaagtg acaggcgccc 16201 ttaaatattc tgacaaatgc tctttcccta aactcccccc ataaaaaaac ccgccgaagc 16261 gggtttttac gttatttgcg gattaacgat tactcgttat cagaaccgcc cagggggccc 16321 gagcttaaga ctggccgtcg ttttacaaca cagaaagagt ttgtagaaac gcaaaaaggc 16381 catccgtcag gggccttctg cttagtttga tgcctggcag ttccctactc tcgccttccg 16441 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 16501 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 16561 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 16621 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 16681 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 16741 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 16801 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 16861 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 16921 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 16981 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tgggctaact 17041 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 17101 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 17161 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 17221 tttctacggg gtctgacgct cagtggaacg acgcgcgcgt aactcacgtt aagggatttt 17281 ggtcatgagc ttgcgccgtc ccgtcaagtc agcgtaatgc tctgctt //
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