质粒名称: | pC014-LwCas13a-msfGFP |
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出品公司: | 爱迪基因 |
目录编号: | 91902 |
存储实验室: | Feng Zhang |
载体骨架基本信息 |
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载体名称: | lenti(AMP) |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 9204 bp |
完整质粒大小: | 13584 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: | LwCas13a-msfGFP,LwCas13a,LwC2c2,Cas13a |
物种来源: | H. sapiens (human); Leptotrichia wadei |
插入大小: | 4380 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | EF1a |
标签/融合蛋白: |
msfGFP(插入件上的C端子)
HA(插入件上的C端子)
NLS(插入件上的N端子)
NLS(插入件上的C端子)
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克隆方法: | Gibson克隆 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | EF-1a Forward |
3' 测序引物 | WPRE-R |
文献引用方法 |
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材料和方法部分: | pC014 - LwCas13a-msfGFP was a gift from Feng Zhang (Addgene plasmid # 91902 ; http://n2t.net/addgene:91902 ; RRID:Addgene_91902) |
参考文献部分: | RNA targeting with CRISPR-Cas13. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F. Nature. 2017 Oct 4. doi: 10.1038/nature24049. 10.1038/nature24049 PubMed 28976959 |
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载体基本信息
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载体简介
载体序列
LOCUS pC014 - LwCas13a-msfGFP 13585 bp ds-DNA circular SYN 07-NOV-2018 DEFINITION Expresses active LwCas13a for mammalian RNA targeting. ACCESSION . VERSION . KEYWORDS pC014 - LwCas13a-msfGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13585) AUTHORS Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F TITLE RNA targeting with CRISPR-Cas13. JOURNAL Nature. 2017 Oct 4. doi: 10.1038/nature24049. PUBMED 28976959 REFERENCE 2 (bases 1 to 13585) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 7, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13585 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..66) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 772..792 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /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 2440..2557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2682..3860 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2913..3851 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3808..3828 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 3867..3876 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3882..3902 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3909..7364 /codon_start=1 /product="Leptotrichia wadei CRISPR-Cas effector that acts as an RNA-guided RNAse (Abudayyeh et al., 2017)" /label=LwCas13a /note="previously called C2c2" /translation="MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIY IKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEY DLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINEN NVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEK YKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLD KEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLL NKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETE NENGITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNE IEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF ELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNY IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE" CDS 8139..8159 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8169..8249 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" misc_feature 8277..8865 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8330..8350) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(8748..8759) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(8868..8884) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8869..8885) /label=pBluescriptKS /note="For pBluescript vector" LTR 9390..9570 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(9596..9613) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 9602..9826 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9872..10300 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9959..9978) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10169..10190 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(10309..10329) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 10314..10643 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 10494..10629 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10556..10575 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 10691..10738 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10757..11131 /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 11261..11382 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11298..11317) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 11352..11371 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(11431..11447) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11431..11447) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11444..11466) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11455..11471 /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(11479..11509) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11524..11545 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11662..11679) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11833..12421) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11913..11932) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12592..13452) /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 13215..13234 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13453..13557) /gene="bla" /label=AmpR promoter ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2581 agatccagtt tggttaatta atgcaaagat ggataaagtt ttaaacagag aggaatcttt 2641 gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt 2701 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa 2761 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 2821 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 2881 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc 2941 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt 3001 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg 3061 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc 3121 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca 3181 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg 3241 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc 3301 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg 3361 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta 3421 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat 3481 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc 3541 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat 3601 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga 3661 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt 3721 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc 3781 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc 3841 ttccatttca ggtgtcgtga cgtacggcca ccatggctag ccccaaaaag aagaggaaag 3901 tgggatccat gaaagtgacc aaggtcgacg gcatcagcca caagaagtac atcgaagagg 3961 gcaagctcgt gaagtccacc agcgaggaaa accggaccag cgagagactg agcgagctgc 4021 tgagcatccg gctggacatc tacatcaaga accccgacaa cgcctccgag gaagagaacc 4081 ggatcagaag agagaacctg aagaagttct ttagcaacaa ggtgctgcac ctgaaggaca 4141 gcgtgctgta tctgaagaac cggaaagaaa agaacgccgt gcaggacaag aactatagcg 4201 aagaggacat cagcgagtac gacctgaaaa acaagaacag cttctccgtg ctgaagaaga 4261 tcctgctgaa cgaggacgtg aactctgagg aactggaaat ctttcggaag gacgtggaag 4321 ccaagctgaa caagatcaac agcctgaagt acagcttcga agagaacaag gccaactacc 4381 agaagatcaa cgagaacaac gtggaaaaag tgggcggcaa gagcaagcgg aacatcatct 4441 acgactacta cagagagagc gccaagcgca acgactacat caacaacgtg caggaagcct 4501 tcgacaagct gtataagaaa gaggatatcg agaaactgtt tttcctgatc gagaacagca 4561 agaagcacga gaagtacaag atccgcgagt actatcacaa gatcatcggc cggaagaacg 4621 acaaagagaa cttcgccaag attatctacg aagagatcca gaacgtgaac aacatcaaag 4681 agctgattga gaagatcccc gacatgtctg agctgaagaa aagccaggtg ttctacaagt 4741 actacctgga caaagaggaa ctgaacgaca agaatattaa gtacgccttc tgccacttcg 4801 tggaaatcga gatgtcccag ctgctgaaaa actacgtgta caagcggctg agcaacatca 4861 gcaacgataa gatcaagcgg atcttcgagt accagaatct gaaaaagctg atcgaaaaca 4921 aactgctgaa caagctggac acctacgtgc ggaactgcgg caagtacaac tactatctgc 4981 aagtgggcga gatcgccacc tccgacttta tcgcccggaa ccggcagaac gaggccttcc 5041 tgagaaacat catcggcgtg tccagcgtgg cctacttcag cctgaggaac atcctggaaa 5101 ccgagaacga gaacggtatc accggccgga tgcggggcaa gaccgtgaag aacaacaagg 5161 gcgaagagaa atacgtgtcc ggcgaggtgg acaagatcta caatgagaac aagcagaacg 5221 aagtgaaaga aaatctgaag atgttctaca gctacgactt caacatggac aacaagaacg 5281 agatcgagga cttcttcgcc aacatcgacg aggccatcag cagcatcaga cacggcatcg 5341 tgcacttcaa cctggaactg gaaggcaagg acatcttcgc cttcaagaat atcgccccca 5401 gcgagatctc caagaagatg tttcagaacg aaatcaacga aaagaagctg aagctgaaaa 5461 tcttcaagca gctgaacagc gccaacgtgt tcaactacta cgagaaggat gtgatcatca 5521 agtacctgaa gaataccaag ttcaacttcg tgaacaaaaa catccccttc gtgcccagct 5581 tcaccaagct gtacaacaag attgaggacc tgcggaatac cctgaagttt ttttggagcg 5641 tgcccaagga caaagaagag aaggacgccc agatctacct gctgaagaat atctactacg 5701 gcgagttcct gaacaagttc gtgaaaaact ccaaggtgtt ctttaagatc accaatgaag 5761 tgatcaagat taacaagcag cggaaccaga aaaccggcca ctacaagtat cagaagttcg 5821 agaacatcga gaaaaccgtg cccgtggaat acctggccat catccagagc agagagatga 5881 tcaacaacca ggacaaagag gaaaagaata cctacatcga ctttattcag cagattttcc 5941 tgaagggctt catcgactac ctgaacaaga acaatctgaa gtatatcgag agcaacaaca 6001 acaatgacaa caacgacatc ttctccaaga tcaagatcaa aaaggataac aaagagaagt 6061 acgacaagat cctgaagaac tatgagaagc acaatcggaa caaagaaatc cctcacgaga 6121 tcaatgagtt cgtgcgcgag atcaagctgg ggaagattct gaagtacacc gagaatctga 6181 acatgtttta cctgatcctg aagctgctga accacaaaga gctgaccaac ctgaagggca 6241 gcctggaaaa gtaccagtcc gccaacaaag aagaaacctt cagcgacgag ttggaactga 6301 tcaacctgct gaacctggac aacaacagag tgaccgagga cttcgagctg gaagccaacg 6361 agatcggcaa gttcctggac ttcaacgaaa acaaaatcaa ggaccggaaa gagctgaaaa 6421 agttcgacac caacaagatc tatttcgacg gcgagaacat catcaagcac cgggccttct 6481 acaatatcaa gaaatacggc atgctgaatc tgctggaaaa gatcgccgat aaggccaagt 6541 ataagatcag cctgaaagaa ctgaaagagt acagcaacaa gaagaatgag attgaaaaga 6601 actacaccat gcagcagaac ctgcaccgga agtacgccag acccaagaag gacgaaaagt 6661 tcaacgacga ggactacaaa gagtatgaga aggccatcgg caacatccag aagtacaccc 6721 acctgaagaa caaggtggaa ttcaatgagc tgaacctgct gcagggcctg ctgctgaaga 6781 tcctgcaccg gctcgtgggc tacaccagca tctgggagcg ggacctgaga ttccggctga 6841 agggcgagtt tcccgagaac cactacatcg aggaaatttt caatttcgac aactccaaga 6901 atgtgaagta caaaagcggc cagatcgtgg aaaagtatat caacttctac aaagaactgt 6961 acaaggacaa tgtggaaaag cggagcatct actccgacaa gaaagtgaag aaactgaagc 7021 aggaaaaaaa ggacctgtac atccggaact acattgccca cttcaactac atcccccacg 7081 ccgagattag cctgctggaa gtgctggaaa acctgcggaa gctgctgtcc tacgaccgga 7141 agctgaagaa cgccatcatg aagtccatcg tggacattct gaaagaatac ggcttcgtgg 7201 ccaccttcaa gatcggcgct gacaagaaga tcgaaatcca gaccctggaa tcagagaaga 7261 tcgtgcacct gaagaatctg aagaaaaaga aactgatgac cgaccggaac agcgaggaac 7321 tgtgcgaact cgtgaaagtc atgttcgagt acaaggccct ggaaggagga ggtggaagcg 7381 gaggaggagg aagcggagga ggaggtagcg tgagtaaagg tgaagaactc ttcactggag 7441 tagtgcccat tctggtagag cttgatggag atgtaaatgg acataaattc tccgtcaggg 7501 gcgaaggcga aggggacgcc acgaatggta agctgactct gaaattcatc tgtacgacgg 7561 gcaaactgcc cgtcccatgg cctacactcg taacgaccct cacctacggc gtgcaatgct 7621 tttctcgata tcccgaccac atgaaacagc atgacttttt caagtctgca atgcctgaag 7681 gttatgttca agaaaggacc atcagcttta aggatgatgg tacatataaa acccgagccg 7741 aggttaaatt tgaaggggac actctggtta atcgaattga actgaaaggt attgatttta 7801 aggaggacgg taacatactg gggcacaagt tggagtacaa ctttaacagc cataatgtgt 7861 atattaccgc tgataagcag aaaaatggga taaaggccaa ctttaagatc cgacataatg 7921 tcgaagatgg tagtgttcaa ctggctgatc attaccaaca aaatacgccc atcggagatg 7981 gacctgtact cttgcctgac aatcattatc tctccacgca atcaaagctt tccaaggacc 8041 caaacgaaaa gagagatcac atggtccttc tggaatttgt gactgccgca ggcatcactc 8101 tcggtatgga tgagctgtac aagggatccg agggagctcc caagaaaaag cgcaaggtag 8161 gtagttccta cccatacgat gttccagatt acgcttatcc ctacgacgtg cctgattatg 8221 catacccata tgatgtcccc gactatgcct aagaattcga tatcaagctt atcgataatc 8281 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt 8341 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg 8401 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc 8461 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt 8521 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg 8581 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg 8641 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct 8701 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc 8761 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc 8821 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac 8881 ctcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg 8941 attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg 9001 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa 9061 aggggggact ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga 9121 tctaccacac acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca 9181 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag 9241 aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg 9301 atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca 9361 tggcccgaga gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct 9421 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag 9481 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac 9541 ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg 9601 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 9661 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 9721 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 9781 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 9841 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg 9901 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 9961 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 10021 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 10081 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 10141 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 10201 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 10261 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc 10321 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 10381 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 10441 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 10501 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 10561 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 10621 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 10681 atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 10741 ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 10801 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 10861 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 10921 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 10981 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 11041 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 11101 cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc 11161 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 11221 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 11281 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 11341 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc 11401 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 11461 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 11521 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 11581 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 11641 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 11701 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 11761 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 11821 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 11881 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 11941 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 12001 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 12061 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 12121 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 12181 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 12241 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 12301 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 12361 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 12421 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 12481 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 12541 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 12601 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 12661 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 12721 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 12781 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 12841 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 12901 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 12961 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 13021 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 13081 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 13141 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 13201 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 13261 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 13321 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 13381 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 13441 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 13501 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 13561 atttccccga aaagtgccac ctgac //
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