质粒名称: | pEJS578_DD-dSpyCas9-mCherry-APEX2 |
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出品公司: | addgene |
目录编号: | 108570 |
存储实验室: | Erik Sontheimer |
载体骨架基本信息 |
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载体名称: | pHAGE-DEST |
空载体大小: | 6000bp |
完整质粒大小: | 12774bp |
载体类型: | 哺乳细胞表达 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | NEB Stable |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | dSpyCas9 |
物种来源: | Synthetic |
插入大小: | 4113 bp |
标签/融合蛋白: |
(C terminal on insert)
APEX2 (C terminal on insert)
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克隆方法: | 限制性内切酶 |
5’克隆位点: | MluI(未破坏) |
3’克隆位点: | SpeI(未破坏) |
5’测序引物 | cgcaaatgggcggtaggcgtg |
文献引用方法 |
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材料和方法部分: | pEJS578_DD-dSpyCas9-mCherry-APEX2 was a gift from Erik Sontheimer (Addgene plasmid # 108570 ; http://n2t.net/addgene:108570 ; RRID:Addgene_108570) |
参考文献部分: | C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2. Gao , Xin D., Li-Chun Tu, Aamir Mir, Tomás Rodriguez, Yuehe Ding, John Leszyk, Job Dekker, Scott A. Shaffer, Lihua Julie Zhu, Scot A. Wolfe & Erik J. Sontheimer. Nature Methods (2018) 10.1038/s4159 |
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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 pEJS578_DD-dSpyCas9-mCherry-APEX2 12774 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of drug-inducible dSpyCas9-mCherry-APEX2 for biotin-labeling genomic element-associated protein factors. ACCESSION . VERSION . KEYWORDS pEJS578_DD-dSpyCas9-mCherry-APEX2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12774) AUTHORS Gao , Xin D., Li-Chun Tu, Aamir Mir, Tomas Rodriguez, Yuehe Ding, John Leszyk, Job Dekker, Scott A. Shaffer, Lihua Julie Zhu, Scot A. TITLE C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2 JOURNAL Nature Methods (2018) REFERENCE 2 (bases 1 to 12774) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..12774 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 1..634 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 681..806 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1303..1536 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1721..1765 /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 2058..2175 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" enhancer 2299..2602 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 2603..2806 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2757..2777 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 2808..2826 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2829..2847 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 2857..2881 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 2965..2974 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2977..2997 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3004..3051 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 3064..3384 /codon_start=1 /product="destabilization domain that can be stabilized by Shield1 in the ProteoTuner(TM) system" /label=DD /note="L106P mutant of FKBP12" /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL KPE" CDS 3409..7509 /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 7528..7548 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7561..8268 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(7706..7724) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 7974..7993 /label=mCherry-F /note="mCherry, forward primer" CDS 8296..8319 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 8320..9066 /codon_start=1 /product="soybean ascorbate peroxidase, engineered to serve as a monomeric tag for electron microscopy and live-cell proteomics (Lam et al., 2015)" /label=APEX2 /note="more active than the original APEX due to the A134P mutation" /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK YAADEDAFFADYAEAHQKLSELGFADA" CDS 9073..9093 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9100..9147 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" misc_feature 9164..9752 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9217..9237) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9635..9646) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 9827..10060 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(10179..10197) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 10265..10369 /gene="bla" /label=AmpR promoter CDS 10370..11230 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(10588..10607) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 11401..11989 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 11890..11909 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 12143..12160 /label=L4440 /note="L4440 vector, forward primer" primer_bind 12252..12271 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 12400..12421 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 12436..12466 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12474..12490 /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)." primer_bind 12479..12501 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 12498..12514 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 12498..12514 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 12564..12583 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg 2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat 2221 gctagctgat gcgggcccta gttattaata gtaatcaatt acggggtcat tagttcatag 2281 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2341 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2401 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2461 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 2521 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 2581 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata 2641 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 2701 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 2761 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga 2821 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat 2881 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag 2941 cctaccggtt aaggtgaacg cgtggccacc atggccccta aaaagaaacg caaggtcgaa 3001 gataaacgcc cagcagctac aaagaaggca ggtcaagcca agaaaaagaa ggaagatgca 3061 tgcggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 3121 cagacctgtg tggtgcacta caccgggatg cttgaagatg gaaagaaagt cgattcctcc 3181 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 3241 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 3301 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 3361 gatgtggagc ttctaaaacc ggaaggcagt gaattcggaa gcggaagcga caagaagtac 3421 tccattgggc tcgctatcgg taccaacagc gtcggctggg ccgtcattac ggacgagtac 3481 aaggtgccga gcaaaaaatt caaagttctg ggcaataccg atcgccacag cataaagaag 3541 aacctcattg gagccctcct gttcgactcc ggggagacgg ccgaagccac gcggctcaaa 3601 agaacagcac ggcgcagata tacccgcaga aagaatcgga tctgctacct gcaggagatc 3661 tttagtaatg agatggctaa ggtggatgac tctttcttcc ataggctgga ggagtccttt 3721 ttggtggagg aggataaaaa gcacgagcgc cacccaatct ttggcaatat cgtggacgag 3781 gtggcgtacc atgaaaagta cccaaccata tatcatctga ggaagaagct ggtagacagt 3841 actgataagg ctgacttgcg gttgatctat ctcgcgctgg cgcacatgat caaatttcgg 3901 ggacacttcc tcatcgaggg ggacctgaac ccagacaaca gcgatgtcga caaactcttt 3961 atccaactgg ttcagactta caatcagctt ttcgaggaga acccgatcaa cgcatccggc 4021 gttgacgcca aagcaatcct gagcgctagg ctgtccaaat cccggcggct cgaaaacctc 4081 atcgcacagc tccctgggga gaagaagaac ggcctgtttg gtaatcttat cgccctgtca 4141 ctcgggctga cccccaactt taaatctaac ttcgacctgg ccgaagatgc caagctgcaa 4201 ctgagcaaag acacctacga tgatgatctc gacaatctgc tggcccagat cggcgaccag 4261 tacgcagacc tttttttggc ggcaaagaac ctgtcagacg ccattctgct gagtgatatt 4321 ctgcgagtga acacggagat caccaaagct ccgctgagcg ctagtatgat caagcgctat 4381 gatgagcacc accaagactt gactttgctg aaggcccttg tcagacagca actgcctgag 4441 aagtacaagg aaattttctt cgatcagtct aaaaatggct acgccggata cattgacggc 4501 ggagcaagcc aggaggaatt ttacaaattt attaagccca tcttggaaaa aatggacggc 4561 accgaggagc tgctggtaaa gctgaacaga gaagatctgt tgcgcaaaca gcgcactttc 4621 gacaatggaa gcatccccca ccagattcac ctgggcgaac tgcacgctat cctcaggcgg 4681 caagaggatt tctacccctt tttgaaagat aacagggaaa agattgagaa aatcctcaca 4741 tttcggatac cctactatgt aggccccctc gctcggggaa attccagatt cgcgtggatg 4801 actcgcaaat cagaagagac catcactccc tggaacttcg aggaagtcgt ggataagggg 4861 gcctctgccc agtccttcat cgaaaggatg actaactttg ataaaaatct gcctaacgaa 4921 aaggtgcttc ctaaacactc tctgctgtac gagtacttca cagtttataa cgagctcacc 4981 aaggtcaaat acgtcacaga agggatgaga aagccagcat tcctgtctgg agagcagaag 5041 aaagctatcg tggacctcct cttcaagacg aaccggaaag ttaccgtgaa acagctcaaa 5101 gaagactatt tcaaaaagat tgaatgtttc gactctgttg aaatcagcgg agtggaggat 5161 cgcttcaacg catccctggg aacgtatcac gatctcctga aaatcattaa agacaaggac 5221 ttcctggaca atgaggagaa cgaggacatt cttgaggaca ttgtcctcac ccttacgttg 5281 tttgaagata gggagatgat tgaagaacgc ttgaaaactt acgctcatct cttcgacgac 5341 aaagtcatga aacagctcaa gagacgccga tatacaggat gggggcggct gtcaagaaaa 5401 ctgatcaatg gcatccgaga caagcagagt ggaaagacaa tcctggattt tcttaagtcc 5461 gatggatttg ccaaccggaa cttcatgcag ttgatccatg atgactctct cacctttaag 5521 gaggacatcc agaaagcaca agtttctggc cagggggaca gtcttcacga gcacatcgct 5581 aatcttgcag gtagcccagc tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat 5641 gaactcgtca aagtaatggg aaggcataag cccgagaata tcgttatcga gatggcccga 5701 gagaaccaaa ctacccagaa gggacagaag aacagtaggg aaaggatgaa gaggattgaa 5761 gagggtataa aagaactggg gtcccaaatc cttaaggaac acccagttga aaacacccag 5821 cttcagaatg agaagctcta cctgtactac ctgcagaacg gcagggacat gtacgtggat 5881 caggaactgg acatcaaccg gttgtccgac tacgacgtgg atgctatcgt gccccaaagc 5941 tttctcaaag atgattctat tgataataaa gtgttgacaa gatccgataa aaatagaggg 6001 aagagtgata acgtcccctc agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag 6061 ctgctgaacg ccaaactgat cacacaacgg aagttcgata atctgactaa ggctgaacga 6121 ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc 6181 cagatcacca agcacgtggc ccaaattctc gattcacgca tgaacaccaa gtacgatgaa 6241 aatgacaaac tgattcgaga ggtgaaagtt attactctga agtctaagct ggtctcagat 6301 ttcagaaagg actttcagtt ttataaggtg agagagatca acaattacca ccatgcgcat 6361 gatgcctacc tgaatgcagt ggtaggcact gcacttatca aaaaatatcc caagctggaa 6421 tctgaatttg tttacggaga ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct 6481 gagcaggaaa taggcaaggc caccgctaag tacttctttt acagcaatat tatgaatttt 6541 ttcaagaccg agattacact ggccaatgga gagattcgga agcgaccact tatcgaaaca 6601 aacggagaaa caggagaaat cgtgtgggac aagggtaggg atttcgcgac agtccgcaag 6661 gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg aagtacagac cggaggcttc 6721 tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat 6781 tgggacccca agaaatacgg cggattcgat tctcctacag tcgcttacag tgtactggtt 6841 gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc 6901 atcacaatca tggagcgatc cagcttcgag aaaaacccca tcgactttct cgaagcgaaa 6961 ggatataaag aggtcaaaaa agacctcatc attaagctgc ccaagtactc tctctttgag 7021 cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg agctgcagaa aggtaacgag 7081 ctggcactgc cctctaaata cgttaatttc ttgtatctgg ccagccacta tgaaaagctc 7141 aaagggtctc ccgaagataa tgagcagaag cagctgttcg tggaacaaca caaacactac 7201 cttgatgaga tcatcgagca aataagcgag ttctccaaaa gagtgatcct cgccgacgct 7261 aacctcgata aggtgctttc tgcttacaat aagcacaggg ataagcccat cagggagcag 7321 gcagaaaaca ttatccactt gtttactctg accaacttgg gcgcgcctgc agccttcaag 7381 tacttcgaca ccaccataga cagaaagcgg tacacctcta caaaggaggt cctggacgcc 7441 acactgattc atcagtcaat tacggggctc tatgaaacaa gaatcgacct ctctcagctc 7501 ggtggagacg gctctactag tggctctccc aagaagaaga ggaaggtagg atccggaagt 7561 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 7621 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 7681 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 7741 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 7801 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 7861 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc actgcaggac 7921 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 7981 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 8041 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 8101 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 8161 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 8221 cgcgctgagg gccgccactc caccggcggc atggacgagc tgtacaagtc tggcggccgc 8281 ggcggtggag gtagcgacta caaggatgac gacgataagg gaaagtctta cccaactgtg 8341 agtgctgatt accaggacgc cgttgagaag gcgaagaaga agctcagagg cttcatcgct 8401 gagaagagat gcgctcctct aatgctccgt ttggcattcc actctgctgg aacctttgac 8461 aagggcacga agaccggtgg acccttcgga accatcaagc accctgccga actggctcac 8521 agcgctaaca acggtcttga catcgctgtt aggcttttgg agccactcaa ggcggagttc 8581 cctattttga gctacgccga tttctaccag ttggctggcg ttgttgccgt tgaggtcacg 8641 ggtggaccta aggttccatt ccaccctgga agagaggaca agcctgagcc accaccagag 8701 ggtcgcttgc ccgatcccac taagggttct gaccatttga gagatgtgtt tggcaaagct 8761 atggggctta ctgaccaaga tatcgttgct ctatctgggg gtcacactat tggagctgca 8821 cacaaggagc gttctggatt tgagggtccc tggacctcta atcctcttat tttcgacaac 8881 tcatacttca cggagttgtt gagtggtgag aaggaaggtc tccttcagct accttctgac 8941 aaggctcttt tgtctgaccc tgtattccgc cctctcgttg acaaatatgc agcggacgaa 9001 gatgccttct ttgctgatta cgctgaggct caccaaaagc tttccgagct tgggtttgct 9061 gatgccctcg agcctaaaaa gaaacgcaag gtcgaagata aacgcccagc agctacaaag 9121 aaggcaggtc aagccaagaa aaagaagggc tcttaatcta gataatcaac ctctggatta 9181 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg 9241 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc 9301 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca 9361 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac 9421 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact 9481 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc 9541 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg 9601 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc 9661 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac 9721 gagtcggatc tccctttggg ccgcctcccc gcctgagatc ctttaagacc aatgacttac 9781 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt 9841 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag 9901 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 9961 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 10021 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt 10081 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gcccgggtta 10141 attaaggaaa gggctagatc attcttgaag acgaaagggc ctcgtgatac gcctattttt 10201 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 10261 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 10321 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 10381 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 10441 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 10501 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 10561 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc 10621 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 10681 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 10741 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 10801 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 10861 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 10921 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 10981 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 11041 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 11101 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 11161 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 11221 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 11281 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 11341 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 11401 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 11461 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 11521 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 11581 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 11641 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 11701 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 11761 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 11821 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 11881 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 11941 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 12001 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 12061 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 12121 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 12181 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcaagc tcatggctga 12241 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 12301 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctccccgt ggcacgacag 12361 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 12421 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 12481 cggataacaa tttcacacag gaaacagcta tgacatgatt acgaatttca caaataaagc 12541 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 12601 ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca ccccataccc 12661 tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt cctctgaatt 12721 attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag tagt //
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