质粒名称: | pZLCv2-3xFLAG-dCas9-HA-2xNLS |
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出品公司: | 爱迪基因 |
目录编号: | 106357 |
存储实验室: | Stephen Tapscott |
载体骨架基本信息 |
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载体名称: | pZLCv2 |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 15000 bp |
完整质粒大小: | -- |
载体修饰: | 催化死亡(d)Cas9克隆到带有3xFLAG和HA标签的载体中。 |
载体类型: | 哺乳动物表达,慢病毒,CRISPR |
筛选标记: | 嘌呤霉素 |
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: | -- |
物种来源: | -- |
插入大小: | -- |
基因突变: | -- |
GenBank编号 | -- |
启动子 | U6 |
标签/融合蛋白: |
3XFLAG(插入件上的N端子)
HA(插入件上的C端子)
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克隆方法: | 限制性内切酶 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | -- |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pZLCv2-3xFLAG-dCas9-HA-2xNLS was a gift from Stephen Tapscott (Addgene plasmid # 106357 ; http://n2t.net/addgene:106357 ; RRID:Addgene_106357) |
参考文献部分: | NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins. Campbell AE, Shadle SC, Jagannathan S, Lim JW, Resnick R, Tawil R, van der Maarel SM, Tapscott SJ. Elife. 2018 Mar 13;7. pii: 31023. doi: 10.7554/eLife.31023. 10.7554/eLife.31023 PubMed 29533181 |
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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 pZLCv2-3xFLAG-dCas9-HA-2xNLS 14962 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Empty backbone for CRISPR/Cas9 engineered chromatin immunoprecipitation. ACCESSION . VERSION . KEYWORDS pZLCv2-3xFLAG-dCas9-HA-2xNLS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14962) AUTHORS Campbell AE, Shadle SC, Jagannathan S, Lim JW, Resnick R, Tawil R, van der Maarel SM, Tapscott SJ TITLE NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins. JOURNAL Elife. 2018 Mar 13;7. pii: 31023. doi: 10.7554/eLife.31023. PUBMED 29533181 REFERENCE 2 (bases 1 to 14962) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14962 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 382..580 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 598..778 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 825..950 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1443..1676 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1861..1905 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 2203..2320 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2371..2611 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2371..2391 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2542..2561 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4501..4576 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 4638..4849 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 4870..4935 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4942..9042 /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 H840A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 9046..9072 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 9091..9111 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9118..9138 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9148..9204 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 9205..9801 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 9698..9718 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 9817..10405 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9870..9890) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(10288..10299) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 10477..10710 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(10736..10753) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 10742..10966 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 11012..11440 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11099..11118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 11309..11330 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(11449..11469) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 11454..11783 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 11634..11769 /label=SV40 ori /note="SV40 origin of replication" primer_bind 11696..11715 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 11831..11878 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 11897..12271 /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 12401..12522 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(12438..12457) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 12492..12511 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(12571..12587) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12571..12587) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(12584..12606) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 12595..12611 /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(12619..12649) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12664..12685 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12802..12819) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(12973..13561) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(13053..13072) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(13732..14592) /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 14355..14374 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14593..14697) /gene="bla" /label=AmpR promoter primer_bind complement(14772..14791) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat 2401 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca 2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt 2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat 2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagacggttg taaatgagca 2641 cacaaaatac acatgctaaa atattatatt ctatgacctt tataaaatca accaaaatct 2701 tctttttaat aactttagta tcaataatta gaatttttat gttccttttt gcaaactttt 2761 aataaaaatg agcaaaataa aaaaacgcta gttttagtaa ctcgcgttgt tttcttcacc 2821 tttaataata gctactccac cacttgttcc taagcggtca gctcctgctt caatcatttt 2881 ttgagcatct tcaaatgttc taactccacc agctgcttta actaaagcat tgtctttaac 2941 aactgacttc attagtttaa catcttcaaa tgttgcacct gattttgaaa atcctgttga 3001 tgttttaaca aattctaatc cagcttcaac agctatttca caagctttca tgatttcttc 3061 ttttgttaat aaacaatttt ccataataca tttaacaaca tgtgatccag ctgctttttt 3121 tacagctttc atgtcttcta aaactaattc ataatttttg tcttttaatg caccaatatt 3181 taataccata tcaatttctg ttgcaccatc tttaattgct tcagaaactt cgaatgcttt 3241 tgtagctgtt gtgcatgcac ctagaggaaa acctacaaca tttgttattc ctacatttgt 3301 gccttttaat aattctttac aatagcttgt tcaatatgaa ttaacacaaa ctgttgcaaa 3361 atcaaattca attgcttcat cacataattg tttaatttca gctttcgtag catcttgttt 3421 taataatgtg tgatctatat atttgtttag tttcattttt tctcctatat attcattttt 3481 aattttaatt ctttaataat ttcgtctact ttaactttag cgttttgaac agattcacca 3541 acacctataa aataaatttt tagtttaggt tcagttccac ttgggcgaac agcaaatcat 3601 gacttatctt ctaaataaaa ttttagtaag tcttgtcctg gcatattata cattccatcg 3661 atgtagtctt caacattaac aactttaagt ccagcaattt gagttaaggg tgttgctctc 3721 aatgatttca ttaatggttc aatttttaat ttcttttctt ctggtttaaa attcaagttt 3781 aaagtgaaag tgtaatatgc acccatttct ttaaataaat cttctaaata gtctactaat 3841 gttttatttt gttttttata aaatcaagca gcctctgcta ttaatataga agcttgtatt 3901 ccatctttat ctctagctga gtcatcaatt acatatccat aactttcttc ataagcaaaa 3961 acaaaattta atccgttatc ttcttcttta gcaatttctc tacccattca tttaaatcca 4021 gttaaagttt ttacaatatt aactccatat ttttcatgag cgattctatc acccaaatca 4081 cttgttacaa aacttgaata tagagccgga ttttttggaa tgctatttaa gcgttttaga 4141 tttgataatt ttcaatcaat taaaattggt cctgtttgat ttccatctaa tcttacaaaa 4201 tgaccatcat gttttattgc cattccaaat ctgtcagcat ctgggtcatt cataataata 4261 atatctgcat catgtttaat accatattca agcggtattt ttcatgcagg atcaaattct 4321 ggatttggat ttacaacatt tttaaatgtt tcatcttcaa atgcatgctc ttcaacctca 4381 ataacgttat atcctgattc acgtaatatt tttggggtaa atttagttcc tgttccatta 4441 actgcgctaa aaataatttt taaatctttt ttagcttctt gctctttttt gtacgtctct 4501 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 4561 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg 4621 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg 4681 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga 4741 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt 4801 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggtgccg 4861 cccaccatgg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag 4921 gatgacgatg acaaggaatt cgacaagaag tacagcatcg gcctggccat cggcaccaac 4981 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg 5041 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggcgccct gctgttcgac 5101 agcggagaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga 5161 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac 5221 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag 5281 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc 5341 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc 5401 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg 5461 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag 5521 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc 5581 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag 5641 aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc 5701 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac 5761 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag 5821 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag 5881 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg 5941 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag 6001 agcaagaacg gctacgccgg ctacatcgat ggcggagcca gccaggaaga gttctacaag 6061 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac 6121 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc 6181 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag 6241 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct 6301 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc 6361 ccctggaact tcgaggaagt ggtggacaag ggcgccagcg cccagagctt catcgagcgg 6421 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg 6481 tacgagtact tcaccgtgta caacgagctg accaaagtga aatacgtgac cgagggaatg 6541 agaaagcccg ccttcctgag cggcgagcag aaaaaagcca tcgtggacct gctgttcaag 6601 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc 6661 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac 6721 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac 6781 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa 6841 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg 6901 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag 6961 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg 7021 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc 7081 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag 7141 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac 7201 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag 7261 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag 7321 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac 7381 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc 7441 gactacgatg tggacgctat cgtgcctcag agctttctga aggacgactc catcgataac 7501 aaagtgctga ctcggagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag 7561 gtcgtgaaga agatgaagaa ctactggcgc cagctgctga atgccaagct gattacccag 7621 aggaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc 7681 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc 7741 ctggactccc ggatgaacac taagtacgac gagaacgaca aactgatccg ggaagtgaaa 7801 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa 7861 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga 7921 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag 7981 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc 8041 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac 8101 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaacaggcga gatcgtgtgg 8161 gataagggcc gggactttgc caccgtgcgg aaagtgctgt ctatgcccca agtgaatatc 8221 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg 8281 aacagcgaca agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc 8341 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc 8401 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc 8461 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg 8521 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg 8581 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac 8641 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag 8701 aaacagctgt ttgtggaaca gcacaaacac tacctggacg agatcatcga gcagatcagc 8761 gagttctcca agagagtgat cctggccgac gctaatctgg acaaggtgct gagcgcctac 8821 aacaagcaca gagacaagcc tatcagagag caggccgaga atatcatcca cctgtttacc 8881 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag 8941 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc 9001 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acgcctatcc ctatgacgtg 9061 cccgattatg ccagcctggg cagcggctcc cccaagaaaa aacgcaaggt ggaagatcct 9121 aagaaaaagc ggaaagtggg atccggcgca acaaacttct ctctgctgaa acaagccgga 9181 gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct cgccacccgc 9241 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg 9301 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc 9361 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg 9421 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg 9481 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg 9541 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac 9601 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc 9661 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg 9721 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg 9781 acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt 9841 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 9901 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 9961 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 10021 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 10081 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 10141 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 10201 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg 10261 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 10321 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 10381 atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg 10441 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac 10501 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct 10561 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag 10621 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac 10681 ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg 10741 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 10801 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 10861 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 10921 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 10981 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg 11041 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 11101 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 11161 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 11221 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 11281 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 11341 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 11401 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc 11461 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 11521 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 11581 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 11641 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 11701 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 11761 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 11821 atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 11881 ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 11941 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 12001 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 12061 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 12121 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 12181 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 12241 cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc 12301 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 12361 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 12421 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 12481 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc 12541 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 12601 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 12661 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 12721 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 12781 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 12841 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 12901 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 12961 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 13021 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 13081 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 13141 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 13201 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 13261 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 13321 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 13381 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 13441 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 13501 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 13561 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 13621 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 13681 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 13741 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 13801 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 13861 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 13921 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 13981 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 14041 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 14101 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 14161 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 14221 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 14281 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 14341 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 14401 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 14461 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 14521 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 14581 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 14641 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 14701 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg 14761 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct 14821 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc 14881 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat 14941 gtacgggcca gatatacgcg tt //
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