质粒名称: | lenti-EF1a-dCas9-KRAB-Puro |
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出品公司: | 爱迪基因 |
目录编号: | 99372 |
存储实验室: | Kristen Brennand |
载体骨架基本信息 |
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载体名称: | pLenti |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 9192 bp |
完整质粒大小: | 14337 bp |
载体修饰: | -- |
载体类型: | 哺乳细胞表达 |
筛选标记: | -- |
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: | dCas9-KRAB-T2A-Puro |
物种来源: | -- |
插入大小: | 5145 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | -- |
克隆方法: | 吉布森克隆 |
5' 克隆位点: | AgeI(未破坏) |
3' 克隆位点: | NotI (未破坏) |
5' 测序引物 | CMV-F |
3' 测序引物 | SV40pA-R |
文献引用方法 |
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材料和方法部分: | lenti-EF1a-dCas9-KRAB-Puro was a gift from Kristen Brennand (Addgene plasmid # 99372 ; http://n2t.net/addgene:99372 ; RRID:Addgene_99372) |
参考文献部分: | Evaluating Synthetic Activation and Repression of Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and Astrocytes. Ho SM, Hartley BJ, Flaherty E, Rajarajan P, Abdelaal R, Obiorah I, Barretto N, Muhammad H, Phatnani HP, Akbarian S, Brennand KJ. Stem Cell Reports. 2017 Aug 8;9(2):615-628. doi: 10.1016/j.stemcr.2017.06.012. Epub 2017 Jul 27. 10.1016/j.stemcr.2017.06.012 PubMed 28757163 |
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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 lenti-EF1a-dCas9-KRAB-Puro 14337 bp ds-DNA circular SYN 07-AUG-2019 DEFINITION 3rd generation lenti vector encoding dCas9-KRAB with 2A puromycin resistance marker (EF1a-dCas9-KRAB-T2A-Puro-WPRE). ACCESSION . VERSION . KEYWORDS lenti-EF1a-dCas9-KRAB-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14337) AUTHORS Ho SM, Hartley BJ, Flaherty E, Rajarajan P, Abdelaal R, Obiorah I, Barretto N, Muhammad H, Phatnani HP, Akbarian S, Brennand KJ TITLE Evaluating Synthetic Activation and Repression of Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and Astrocytes. JOURNAL Stem Cell Reports. 2017 Aug 8;9(2):615-628. doi: 10.1016/j.stemcr.2017.06.012. Epub 2017 Jul 27. PUBMED 28757163 REFERENCE 2 (bases 1 to 14337) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 7, 2019 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14337 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 179..412 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." misc_feature 939..1056 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1186..2364 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 1417..2355 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 2312..2332 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 2380..2427 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 2428..6528 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the N863A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6583..6603 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6628..6822 /codon_start=1 /gene="ZNF10" /gene_synonym="KOX1" /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEPWLV" CDS 6844..6897 /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 6910..7503 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="EYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVT ELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQ QQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAP RNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 7400..7420 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 7528..8116 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(7581..7601) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(7999..8010) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(8119..8135) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8120..8136) /label=pBluescriptKS /note="For pBluescript vector" LTR 8641..8821 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(8847..8864) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 8853..9077 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9123..9551 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9210..9229) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 9420..9441 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(9560..9580) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 9565..9894 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 9745..9880 /label=SV40 ori /note="SV40 origin of replication" primer_bind 9807..9826 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 9942..9989 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10008..10382 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 10512..10633 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(10549..10568) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 10603..10622 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(10682..10698) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(10682..10698) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(10695..10717) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 10706..10722 /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(10730..10760) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 10775..10796 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(10913..10930) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11084..11672) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11164..11183) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(11843..12703) /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 12466..12485 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(12704..12808) /gene="bla" /label=AmpR promoter primer_bind complement(12883..12902) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 13074..13453 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 13455..13653 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 13608..13628 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 13671..13851 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 13898..14023 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" ORIGIN 1 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga 61 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag 121 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag 181 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga 241 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc 301 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc 361 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt 421 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta 481 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta 541 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga 601 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa 661 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa 721 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat 781 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag 841 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg 901 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt 961 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa 1021 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga 1081 gatccagttt ggttaattag ctagctgcaa agatggataa agttttaaac agagaggaat 1141 ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt 1201 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat 1261 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg 1321 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac 1381 gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc 1441 gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg 1501 cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct 1561 tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc 1621 cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta 1681 gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta 1741 aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg 1801 gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga 1861 atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg 1921 tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa 1981 agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga 2041 gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct 2101 tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt 2161 tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact 2221 gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt 2281 gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt 2341 tttcttccat ttcaggtgtc gtgacgtacg gccaccatga aaaggccggc ggccacgaaa 2401 aaggccggcc aggcaaaaaa gaaaaaggac aagaagtaca gcatcggcct ggccatcggc 2461 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc 2521 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg 2581 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac 2641 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag 2701 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag 2761 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac 2821 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg 2881 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc 2941 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac 3001 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg 3061 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag 3121 aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc 3181 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac 3241 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc 3301 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc 3361 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg 3421 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc 3481 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc 3541 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag 3601 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac 3661 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc 3721 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg 3781 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc 3841 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc 3901 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc 3961 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag 4021 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg 4081 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc 4141 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc 4201 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac 4261 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc 4321 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag 4381 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac 4441 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac 4501 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag 4561 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc 4621 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc 4681 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag 4741 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc 4801 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac 4861 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg 4921 ctgtccgact acgatgtgga ccacatcgtg cctcagagct ttctgaagga cgactccatc 4981 gacaacaagg tgctgaccag aagcgacaag gcccggggca agagcgacaa cgtgccctcc 5041 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt 5101 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat 5161 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca 5221 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa 5281 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt 5341 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc 5401 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac 5461 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct 5521 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg 5581 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc 5641 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg 5701 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc 5761 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc 5821 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc 5881 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc 5941 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag 6001 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga 6061 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat 6121 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat 6181 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag 6241 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc 6301 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg 6361 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac 6421 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc 6481 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacag cgctggagga 6541 ggtggaagcg gaggaggagg aagcggagga ggaggtagcg gacctaagaa aaagaggaag 6601 gtggcggccg ctggatccgg acgggctaga acactggtta cgttcaagga cgtgtttgtg 6661 gactttacac gtgaggagtg gaaattgctg gatactgcgc aacaaattgt gtatcgaaat 6721 gtcatgcttg agaattacaa gaacctcgtc agtctcggat accagttgac gaaaccggat 6781 gtgatcctta ggctcgaaaa gggggaagaa ccttggctgg taattaactg tacaggttca 6841 ggggagggaa gaggttccct gctcacatgc ggagacgtag aagagaatcc cggacccatg 6901 acctctagag agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc 6961 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg 7021 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc 7081 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg 7141 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc 7201 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag 7261 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg 7321 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc 7381 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc 7441 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc 7501 tgagaattcg atatcaagct tatcggtaat caacctctgg attacaaaat ttgtgaaaga 7561 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 7621 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 7681 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 7741 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 7801 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 7861 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 7921 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 7981 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 8041 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 8101 tgggccgcct ccccgcatcg ataccgtcga cctcgagacc tagaaaaaca tggagcaatc 8161 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag 8221 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca 8281 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc 8341 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat 8401 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac 8461 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc 8521 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg 8581 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact 8641 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 8701 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 8761 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 8821 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 8881 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 8941 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 9001 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 9061 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc 9121 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 9181 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 9241 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 9301 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 9361 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 9421 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 9481 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 9541 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc 9601 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag 9661 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 9721 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 9781 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct 9841 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc 9901 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg 9961 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga 10021 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg 10081 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg 10141 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct 10201 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga 10261 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg 10321 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact 10381 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 10441 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct 10501 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 10561 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 10621 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat 10681 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 10741 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 10801 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 10861 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 10921 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 10981 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 11041 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 11101 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 11161 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 11221 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 11281 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 11341 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 11401 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 11461 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 11521 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 11581 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 11641 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 11701 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 11761 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 11821 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 11881 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 11941 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 12001 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 12061 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 12121 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 12181 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 12241 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 12301 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 12361 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 12421 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 12481 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 12541 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 12601 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 12661 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 12721 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 12781 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg 12841 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc 12901 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc 12961 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct 13021 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg 13081 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 13141 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 13201 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 13261 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 13321 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 13381 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 13441 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 13501 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 13561 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 13621 taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact gggtctctct 13681 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc 13741 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg 13801 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc 13861 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct 13921 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 13981 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 14041 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 14101 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 14161 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 14221 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 14281 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaa //
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