质粒名称: | pHSN401 |
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出品公司: | 爱迪基因 |
目录编号: | 50588 |
存储实验室: | Qi-Jun Chen |
载体骨架基本信息 |
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载体名称: | pGreen-like binary vector |
载体出品公司: | Mullineaux Lab, see http://www.pgreen.ac.uk/ |
载体抗性: | 卡那霉素和大观霉素 |
载体类型: | CRISPR;植物表达 |
筛选标记: | 潮霉素 |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | 3×FLAG-NLS-zCas9-NLS,Cas9 |
物种来源: | 合成的 |
基因突变: | maize codon optimized |
启动子 | 2×35Sp |
标签/融合蛋白: |
3xFLAG(插入N端子)
NLS(插入N端子)
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5' 测序引物 | hSpCas9-R1, CGCTCGTGCTTCTTATCCTC |
文献引用方法 |
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材料和方法部分: | pHSN401 was a gift from Qi-Jun Chen (Addgene plasmid # 50588 ; http://n2t.net/addgene:50588 ; RRID:Addgene_50588) |
参考文献部分: | A CRISPR/Cas9 toolkit for multiplex genome editing in plants. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. BMC Plant Biol. 2014 Nov 29;14(1):327. 10.1186/s12870-014-0327-y PubMed 25432517 |
买家导航
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 pHSN401 12729 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION CRISPR/Cas based plant genome editing and gene regulation; expresses 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Hyg resistance. ACCESSION . VERSION . KEYWORDS pHSN401 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12729) AUTHORS Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ TITLE A CRISPR/Cas9 toolkit for multiplex genome editing in plants. JOURNAL BMC Plant Biol. 2014 Nov 29;14(1):327. PUBMED 25432517 REFERENCE 2 (bases 1 to 12729) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12729 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 1..25 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" CDS 810..1601 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" misc_RNA 1753..1828 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 2442..2786 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" primer_bind 2730..2750 /label=35S promoter /note="CaMV 35S promoter, forward primer" CDS 2969..3034 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 3041..3061 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3086..7186 /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" RBS 7099..7107 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 7187..7234 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" terminator 7285..7537 /label=NOS terminator /note="nopaline synthase terminator and poly(A) signal" promoter 7653..8330 /label=CaMV 35S promoter (enhanced) /note="cauliflower mosaic virus 35S promoter with a duplicated enhancer region" CDS 8398..9423 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK K" polyA_signal 9463..9637 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" misc_feature 9787..9811 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" primer_bind complement(10059..10082) /label=pENTR-F /note="pENTR vectors, forward primer" terminator 10090..10117 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 10209..10295 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" rep_origin 10464..10899 /label=pSa ori /note="origin of replication from bacterial plasmid pSa" rep_origin complement(11014..11602) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11094..11113) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(11692..12501) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 12415..12434 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 12493..12512 /label=pENTR-R /note="pENTR vectors, reverse primer" ORIGIN 1 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 61 aatctgatcc aagctcaagc taagctcacg tgacggaatt aagcttcgac ttgccttccg 121 cacaatacat catttcttct tagctttttt tcttcttctt cgttcataca gttttttttt 181 gtttatcagc ttacattttc ttgaaccgta gctttcgttt tcttcttttt aactttccat 241 tcggagtttt tgtatcttgt ttcatagttt gtcccaggat tagaatgatt aggcatcgaa 301 ccttcaagaa tttgattgaa taaaacatct tcattcttaa gatatgaaga taatcttcaa 361 aaggcccctg ggaatctgaa agaagagaag caggcccatt tatatgggaa agaacaatag 421 tatttcttat ataggcccat ttaagttgaa aacaatcttc aaaagtccca catcgcttag 481 ataagaaaac gaagctgagt ttatatacag ctagagtcga agtagtgatt gggagaccaa 541 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac 601 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca 661 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag 721 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg 781 gcagtcgccc taaaacaaag ttaaacatca tgggggaagc ggtgatcgcc gaagtatcga 841 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg 901 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc 961 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt 1021 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg 1081 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag 1141 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc 1201 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg 1261 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa 1321 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt 1381 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact 1441 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt 1501 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc 1561 actacgtgaa aggcgagatc accaaggtag tcggcaaata atgtctagct agaaattcgt 1621 tcaagccgac gccgcttcgc ggcgcggctt aactcaagcg ttagatgcac taagcacata 1681 attgctcaca gccaaactat caggtcaagt ctgcttttat tatttttaag cgtgcataat 1741 aagccggtct cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca 1801 acttgaaaaa gtggcaccga gtcggtgctt ttttttgcaa aattttccag atcgatttct 1861 tcttcctctg ttcttcggcg ttcaatttct ggggttttct cttcgttttc tgtaactgaa 1921 acctaaaatt tgacctaaaa aaaatctcaa ataatatgat tcagtggttt tgtacttttc 1981 agttagttga gttttgcagt tccgatgaga taaaccaata agcttgcatg cctgcaggtc 2041 aacatggtgg agcacgacac acttgtctac tccaaaaata tcaaagatac agtctcagaa 2101 gaccaaaggg caattgagac ttttcaacaa agggtaatat ccggaaacct cctcggattc 2161 cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg tggctcctac 2221 aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc cgacagtggt 2281 cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg 2341 tcttcaaagc aagtggattg atgtgataac atggtggagc acgacacact tgtctactcc 2401 aaaaatatca aagatacagt ctcagaagac caaagggcaa ttgagacttt tcaacaaagg 2461 gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag 2521 atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc 2581 gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc 2641 gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc 2701 actgacgtaa gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa 2761 ggaagttcat ttcatttgga gaggacctcg acctcaacac aacatataca aaacaaacga 2821 atctcaagca atcaagcatt ctacttctat tgcagcaatt taaatcattt cttttaaagc 2881 aaaagcaatt ttctgaaaat tttcaccatt tacgaacgat actcgagggg gatccccaat 2941 acttgtatgg ccgcggccgc tctagatgga ttacaaggac cacgacgggg attacaagga 3001 ccacgacatt gattacaagg atgatgatga caagatggct ccgaagaaga agaggaaggt 3061 tggcatccac ggggtgccag ctgctgacaa gaagtactcg atcggcctcg atattgggac 3121 taactctgtt ggctgggccg tgatcaccga cgagtacaag gtgccctcaa agaagttcaa 3181 ggtcctgggc aacaccgatc ggcattccat caagaagaat ctcattggcg ctctcctgtt 3241 cgacagcggc gagacggctg aggctacgcg gctcaagcgc accgcccgca ggcggtacac 3301 gcgcaggaag aatcgcatct gctacctgca ggagattttc tccaacgaga tggcgaaggt 3361 tgacgattct ttcttccaca ggctggagga gtcattcctc gtggaggagg ataagaagca 3421 cgagcggcat ccaatcttcg gcaacattgt cgacgaggtt gcctaccacg agaagtaccc 3481 tacgatctac catctgcgga agaagctcgt ggactccaca gataaggcgg acctccgcct 3541 gatctacctc gctctggccc acatgattaa gttcaggggc catttcctga tcgaggggga 3601 tctcaacccg gacaatagcg atgttgacaa gctgttcatc cagctcgtgc agacgtacaa 3661 ccagctcttc gaggagaacc ccattaatgc gtcaggcgtc gacgcgaagg ctatcctgtc 3721 cgctaggctc tcgaagtctc ggcgcctcga gaacctgatc gcccagctgc cgggcgagaa 3781 gaagaacggc ctgttcggga atctcattgc gctcagcctg gggctcacgc ccaacttcaa 3841 gtcgaatttc gatctcgctg aggacgccaa gctgcagctc tccaaggaca catacgacga 3901 tgacctggat aacctcctgg cccagatcgg cgatcagtac gcggacctgt tcctcgctgc 3961 caagaatctg tcggacgcca tcctcctgtc tgatattctc agggtgaaca ccgagattac 4021 gaaggctccg ctctcagcct ccatgatcaa gcgctacgac gagcaccatc aggatctgac 4081 cctcctgaag gcgctggtca ggcagcagct ccccgagaag tacaaggaga tcttcttcga 4141 tcagtcgaag aacggctacg ctgggtacat tgacggcggg gcctctcagg aggagttcta 4201 caagttcatc aagccgattc tggagaagat ggacggcacg gaggagctgc tggtgaagct 4261 caatcgcgag gacctcctga ggaagcagcg gacattcgat aacggcagca tcccacacca 4321 gattcatctc ggggagctgc acgctatcct gaggaggcag gaggacttct accctttcct 4381 caaggataac cgcgagaaga tcgagaagat tctgactttc aggatcccgt actacgtcgg 4441 cccactcgct aggggcaact cccgcttcgc ttggatgacc cgcaagtcag aggagacgat 4501 cacgccgtgg aacttcgagg aggtggtcga caagggcgct agcgctcagt cgttcatcga 4561 gaggatgacg aatttcgaca agaacctgcc aaatgagaag gtgctcccta agcactcgct 4621 cctgtacgag tacttcacag tctacaacga gctgactaag gtgaagtatg tgaccgaggg 4681 catgaggaag ccggctttcc tgtctgggga gcagaagaag gccatcgtgg acctcctgtt 4741 caagaccaac cggaaggtca cggttaagca gctcaaggag gactacttca agaagattga 4801 gtgcttcgat tcggtcgaga tctctggcgt tgaggaccgc ttcaacgcct ccctggggac 4861 ctaccacgat ctcctgaaga tcattaagga taaggacttc ctggacaacg aggagaatga 4921 ggatatcctc gaggacattg tgctgacact cactctgttc gaggaccggg agatgatcga 4981 ggagcgcctg aagacttacg cccatctctt cgatgacaag gtcatgaagc agctcaagag 5041 gaggaggtac accggctggg ggaggctgag caggaagctc atcaacggca ttcgggacaa 5101 gcagtccggg aagacgatcc tcgacttcct gaagagcgat ggcttcgcga accgcaattt 5161 catgcagctg attcacgatg acagcctcac attcaaggag gatatccaga aggctcaggt 5221 gagcggccag ggggactcgc tgcacgagca tatcgcgaac ctcgctggct cgccagctat 5281 caagaagggg attctgcaga ccgtgaaggt tgtggacgag ctggtgaagg tcatgggcag 5341 gcacaagcct gagaacatcg tcattgagat ggcccgggag aatcagacca cgcagaaggg 5401 ccagaagaac tcacgcgaga ggatgaagag gatcgaggag ggcattaagg agctggggtc 5461 ccagatcctc aaggagcacc cggtggagaa cacgcagctg cagaatgaga agctctacct 5521 gtactacctc cagaatggcc gcgatatgta tgtggaccag gagctggata ttaacaggct 5581 cagcgattac gacgtcgatc atatcgttcc acagtcattc ctgaaggatg actccattga 5641 caacaaggtc ctcaccaggt cggacaagaa ccggggcaag tctgataatg ttccttcaga 5701 ggaggtcgtt aagaagatga agaactactg gcgccagctc ctgaatgcca agctgatcac 5761 gcagcggaag ttcgataacc tcacaaaggc tgagaggggc gggctctctg agctggacaa 5821 ggcgggcttc atcaagaggc agctggtcga gacacggcag atcactaagc acgttgcgca 5881 gattctcgac tcacggatga acactaagta cgatgagaat gacaagctga tccgcgaggt 5941 gaaggtcatc accctgaagt caaagctcgt ctccgacttc aggaaggatt tccagttcta 6001 caaggttcgg gagatcaaca attaccacca tgcccatgac gcgtacctga acgcggtggt 6061 cggcacagct ctgatcaaga agtacccaaa gctcgagagc gagttcgtgt acggggacta 6121 caaggtttac gatgtgagga agatgatcgc caagtcggag caggagattg gcaaggctac 6181 cgccaagtac ttcttctact ctaacattat gaatttcttc aagacagaga tcactctggc 6241 caatggcgag atccggaagc gccccctcat cgagacgaac ggcgagacgg gggagatcgt 6301 gtgggacaag ggcagggatt tcgcgaccgt caggaaggtt ctctccatgc cacaagtgaa 6361 tatcgtcaag aagacagagg tccagactgg cgggttctct aaggagtcaa ttctgcctaa 6421 gcggaacagc gacaagctca tcgcccgcaa gaaggactgg gatccgaaga agtacggcgg 6481 gttcgacagc cccactgtgg cctactcggt cctggttgtg gcgaaggttg agaagggcaa 6541 gtccaagaag ctcaagagcg tgaaggagct gctggggatc acgattatgg agcgctccag 6601 cttcgagaag aacccgatcg atttcctgga ggcgaagggc tacaaggagg tgaagaagga 6661 cctgatcatt aagctcccca agtactcact cttcgagctg gagaacggca ggaagcggat 6721 gctggcttcc gctggcgagc tgcagaaggg gaacgagctg gctctgccgt ccaagtatgt 6781 gaacttcctc tacctggcct cccactacga gaagctcaag ggcagccccg aggacaacga 6841 gcagaagcag ctgttcgtcg agcagcacaa gcattacctc gacgagatca ttgagcagat 6901 ttccgagttc tccaagcgcg tgatcctggc cgacgcgaat ctggataagg tcctctccgc 6961 gtacaacaag caccgcgaca agccaatcag ggagcaggct gagaatatca ttcatctctt 7021 caccctgacg aacctcggcg cccctgctgc tttcaagtac ttcgacacaa ctatcgatcg 7081 caagaggtac acaagcacta aggaggtcct ggacgcgacc ctcatccacc agtcgattac 7141 cggcctctac gagacgcgca tcgacctgtc tcagctcggg ggcgacaagc ggccagcggc 7201 gacgaagaag gcggggcagg cgaagaagaa gaagtgagct caggcctcca tacaagtatt 7261 ggggatccac tagcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga 7321 ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag 7381 catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga 7441 gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat 7501 aaattatcgc gcgcggtgtc atctatgtta ctagatcgga attcgaggcg gtttgcgtat 7561 tggctagagc agcttgccaa catggtggag cacgacactc tcgtctactc caagaatatc 7621 aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag ggtaatatcg 7681 ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcaaaag gacagtagaa 7741 aaggaaggtg gcacctacaa atgccatcat tgcgataaag gaaaggctat cgttcaagat 7801 gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa 7861 gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgataacat ggtggagcac 7921 gacactctcg tctactccaa gaatatcaaa gatacagtct cagaagacca aagggctatt 7981 gagacttttc aacaaagggt aatatcggga aacctcctcg gattccattg cccagctatc 8041 tgtcacttca tcaaaaggac agtagaaaag gaaggtggca cctacaaatg ccatcattgc 8101 gataaaggaa aggctatcgt tcaagatgcc tctgccgaca gtggtcccaa agatggaccc 8161 ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg 8221 gattgatgtg atatctccac tgacgtaagg gatgacgcac aatcccacta tccttcgcaa 8281 gaccttcctc tatataagga agttcatttc atttggagag gacacgctga aatcaccagt 8341 ctctctctac aaatctatct ctctcgagct ttcgcagatc ccggggggca atgagatatg 8401 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc 8461 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta 8521 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt 8581 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg 8641 gagtttagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa 8701 gacctgcctg aaaccgaact gcccgctgtt ctacaaccgg tcgcggaggc tatggatgcg 8761 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc 8821 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac 8881 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg 8941 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc 9001 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg 9061 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt 9121 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccacga 9181 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc 9241 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc 9301 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt 9361 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaagaaa 9421 tagagtagat gccgaccgga tctgtcgatc gacaagctcg agtttctcca taataatgtg 9481 tgagtagttc ccagataagg gaattagggt tcctataggg tttcgctcat gtgttgagca 9541 tataagaaac ccttagtatg tatttgtatt tgtaaaatac ttctatcaat aaaatttcta 9601 attcctaaaa ccaaaatcca gtactaaaat ccagatcccc cgaattaatt cggcgttaat 9661 tcagtacatt aaaaacgtcc gcaatgtgtt attaagttgt cactagtcag gttaactcaa 9721 ttcggcgtta attcagtaca ttaaaaacgt ccgcaatgtg ttattaagtt gtctaagcgt 9781 caatttgttt acaccacaat atatcctgcc accagccagc caacagctcc ccgaccggca 9841 gctcggcaca aaatcaccac tcgatacagg cagcccatca gtccgggacg gcgtcagcgg 9901 gagagccgtt gtaaggcggc agactttgct catgttaccg atgctattcg gaagaacggc 9961 aactaagctg ccgggtttga aacacggatg atctcgcgga gggtagcatg ttgattgtaa 10021 cgatgacaga gcgttgctgc ctgtgatcac ttaagtaact aactaacagg aagagtttgt 10081 agaaacgcaa aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt 10141 tatggcgggc gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct 10201 cccggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag 10261 gcccagtctt ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc 10321 ttagtagtta gacgtccccg agatccatgc tagaccatga atccagaagc ccgagaggtt 10381 gccgcctttc gggctttttc tttttcaaaa aaaaaaattt ataaaacgat ctgttgcggc 10441 cggccgccgg gttgtgggca aaggcgctcg acggtgggca accgcttgcg gttgtccacg 10501 ggcggagccg gtgcgcgtag cgcattgtcc acaagccaag ggcgaccaat aattgatata 10561 tatattcata attgaaaagc taattgaaca tactacttgc tgtaactact tgccggagcg 10621 aggggtgttt gcaagctgtt gatctgaaag ggctattagc gttctcacgt gcctttttga 10681 ttagcgattt cacgtgacct tattagcgat ttcacgtact ccgattagcg atttcacgta 10741 ccctgattag cgatttcacg tggatagttt ttggagcggg ccggaaagcc ccgtgaatca 10801 aggctttgcg gggcattagc ggtttcacgt ggataactac cctctatcca caggcttccg 10861 gggataaaaa agcccgctcg acggcgggct gttggatggg gatctagcgg taatacggtt 10921 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 10981 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 11041 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 11101 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 11161 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 11221 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 11281 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 11341 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 11401 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 11461 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 11521 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 11581 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctaccgggt ctgacgctca 11641 gtggaacggg gcccaatctg aataatgtta caaccaatta accaattctg attagaaaaa 11701 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt 11761 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc 11821 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt 11881 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg 11941 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg 12001 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc 12061 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg 12121 gcgcagggac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa 12181 tacctggaat gctgtttttc cggggatcgc agtggtgagt aaccatgcat catcaggagt 12241 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac 12301 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg 12361 cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga cattatcgcg 12421 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgacgt 12481 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt 12541 tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac 12601 gggccagagc tgcagtttga tcccgagggg aaccctgtgg ttgacatgca catacaaatg 12661 gacgaacgga taaacctttt cacgcccttt taaatatccg ttattctaat aaacgctctt 12721 ttctcttag //
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