LOCUS Exported 14628 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION 2nd generation lentiviral vector expressing a KRAB-dCas9 fusion protein and GFP. ACCESSION . VERSION . KEYWORDS TRE-KRAB-dCas9-IRES-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14628) AUTHORS Fulco CP, Munschauer M, Anyoha R, Munson G, Grossman SR, Perez EM, Kane M, Cleary B, Lander ES, Engreitz JM TITLE Systematic mapping of functional enhancer-promoter connections with CRISPR interference. JOURNAL Science. 2016 Sep 29. pii: aag2445. PUBMED 27708057 REFERENCE 2 (bases 1 to 14628) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14628 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(21..40) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 834..1422 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1323..1342 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1576..1593 /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(1663..1683) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 1668..1997 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1848..1983 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1910..1929 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" intron 3131..3196 /label=small t intron /note="SV40 (simian virus 40) small t antigen intron" CDS 3326..3346 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 3771..3905 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3808..3827) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3862..3881 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" LTR 4073..4706 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 4753..4878 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 5375..5608 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 5793..5837 /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 6122..6239 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 6301..6650 /label=TRE3GV promoter /note="3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On(R) 3G, optimized for retroviral and lentiviral vectors" protein_bind 6309..6327 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6345..6363 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6381..6399 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6417..6435 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6453..6471 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6489..6507 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6525..6543 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 6589..6613 /label=LNCX /note="Human CMV promoter, forward primer" CDS 6732..6917 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP" CDS 6939..11042 /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 11046..11072 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 11091..11111 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 11118..11138 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" misc_feature 11237..11789 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(11381..11398) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 11608..11627 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 11801..12520 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(11846..11867) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(12107..12126) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 12454..12475 /label=EGFP-C /note="EGFP, forward primer" misc_feature 12564..13152 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(12617..12637) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(13035..13046) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(13155..13171) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(13156..13172) /label=pBluescriptKS /note="For pBluescript vector" LTR 13262..13495 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" promoter complement(13520..13538) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(13521..13538) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(13828..13847) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind complement(14061..14080) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 14180..14202 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(14240..14258) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 14326..14430 /gene="bla" /label=AmpR promoter CDS 14431..663 /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" ORIGIN 1 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 61 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 121 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 181 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 241 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 301 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 361 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 421 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 481 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 541 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 601 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 661 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 721 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 781 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 841 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 901 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 961 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 1021 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 1081 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 1141 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 1201 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 1261 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 1321 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 1381 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 1441 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 1501 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 1561 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 1621 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct gtggaatgtg tgtcagttag 1681 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 1741 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 1801 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 1861 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 1921 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 1981 gcctaggctt ttgcaaaaag cttggacaca agacaggctt gcgagatatg tttgagaata 2041 ccactttatc ccgcgtcagg gagaggcagt gcgtaaaaag acgcggactc atgtgaaata 2101 ctggttttta gtgcgccaga tctctataat ctcgcgcaac ctattttccc ctcgaacact 2161 ttttaagccg tagataaaca ggctgggaca cttcacatga gcgaaaaata catcgtcacc 2221 tgggacatgt tgcagatcca tgcacgtaaa ctcgcaagcc gactgatgcc ttctgaacaa 2281 tggaaaggca ttattgccgt aagccgtggc ggtctgtacc gggtgcgtta ctggcgcgtg 2341 aactgggtat tcgtcatgtc gataccgttt gtatttccag ctacgatcac gacaaccagc 2401 gcgagcttaa agtgctgaaa cgcgcagaag gcgatggcga aggcttcatc gttattgatg 2461 acctggtgga taccggtggt actgcggttg cgattcgtga aatgtatcca aaagcgcact 2521 ttgtcaccat cttcgcaaaa ccggctggtc gtccgctggt tgatgactat gttgttgata 2581 tcccgcaaga tacctggatt gaacagccgt gggatatggg cgtcgtattc gtcccgccaa 2641 tctccggtcg ctaatctttt caacgcctgg cactgccggg cgttgttctt tttaacttca 2701 ggcgggttac aatagtttcc agtaagtatt ctggaggctg catccatgac acaggcaaac 2761 ctgagcgaaa ccctgttcaa accccgcttt aaacatcctg aaacctcgac gctagtccgc 2821 cgctttaatc acggcgcaca accgcctgtg cagtcggccc ttgatggtaa aaccatccct 2881 cactggtatc gcatgattaa ccgtctgatg tggatctggc gcggcattga cccacgcgaa 2941 atcctcgacg tccaggcacg tattgtgatg agcgatgccg aacgtaccga cgatgattta 3001 tacgatacgg tgattggcta ccgtggcggc aactggattt atgagtgggc cccggatctt 3061 tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 3121 aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 3181 tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 3241 ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 3301 tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 3361 ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 3421 ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 3481 aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt 3541 tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 3601 ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac 3661 tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 3721 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 3781 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 3841 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 3901 ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc ccatacccta 3961 ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc tctgaattat 4021 tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta gttggaaggg 4081 ctaattcact cccaaagaag acaagatatc cttgatctgt ggatctacca cacacaaggc 4141 tacttccctg attagcagaa ctacacacca gggccagggg tcagatatcc actgaccttt 4201 ggatggtgct acaagctagt accagttgag ccagataagg tagaagaggc caataaagga 4261 gagaacacca gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa 4321 gtgttagagt ggaggtttga cagccgccta gcatttcatc acgtggcccg agagctgcat 4381 ccggagtact tcaagaactg ctgatatcga gcttgctaca agggactttc cgctggggac 4441 tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata 4501 taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga 4561 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct 4621 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt 4681 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg 4741 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc 4801 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga 4861 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt 4921 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg 4981 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa 5041 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat 5101 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa 5161 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc 5221 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat 5281 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa 5341 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg 5401 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat 5461 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc 5521 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg 5581 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt 5641 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga 5701 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact 5761 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag 5821 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat 5881 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta 5941 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc 6001 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca 6061 gatccattcg attagtgaac ggatctcgac ggtatcgcca aatggcagta ttcatccaca 6121 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa 6181 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc 6241 gggtttatta cagggacagc agagatccag tttatcgatg aggccctttc gtcttcactc 6301 gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag 6361 agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc 6421 tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta 6481 cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag 6541 agaacgtatg tcgaggtagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt 6601 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatactt acgcgtctgc 6661 aggatatcaa gcttgcggta ccgcgggccc gggatcgcac catggatgct aagtcactaa 6721 ctgcctggtc ccggacactg gtgaccttca aggatgtatt tgtggacttc accagggagg 6781 agtggaagct gctggacact gctcagcaga tcgtgtacag aaatgtgatg ctggagaact 6841 ataagaacct ggtttccttg ggttatcagc ttactaagcc agatgtgatc ctccggttgg 6901 agaagggaga agagcccggc ggttccggcg gagggtcgat ggacaagaag tacagcatcg 6961 gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc 7021 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga 7081 tcggcgccct gctgttcgac agcggagaaa cagccgaggc cacccggctg aagagaaccg 7141 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca 7201 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg 7261 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct 7321 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca 7381 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact 7441 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc 7501 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg 7561 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc 7621 agctgcccgg cgagaagaag aatggcctgt tcggcaacct gattgccctg agcctgggcc 7681 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca 7741 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg 7801 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag 7861 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc 7921 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca 7981 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacatcgat ggcggagcca 8041 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg 8101 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg 8161 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag 8221 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca 8281 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa 8341 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgccagcg 8401 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc 8461 tgcccaagca cagcctgctg tacgagtact tcaccgtgta caacgagctg accaaagtga 8521 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaagcca 8581 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact 8641 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca 8701 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg 8761 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg 8821 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga 8881 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca 8941 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct 9001 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca 9061 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg 9121 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg 9181 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc 9241 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca 9301 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga 9361 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac 9421 tggacatcaa ccggctgtcc gactacgatg tggacgctat cgtgcctcag agctttctga 9481 aggacgactc catcgataac aaagtgctga ctcggagcga caagaaccgg ggcaagagcg 9541 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgc cagctgctga 9601 atgccaagct gattacccag aggaagttcg acaatctgac caaggccgag agaggcggcc 9661 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca 9721 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaacgaca 9781 aactgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga 9841 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct 9901 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt 9961 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg 10021 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga 10081 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg 10141 aaacaggcga gatcgtgtgg gataagggcc gggactttgc caccgtgcgg aaagtgctgt 10201 ctatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag 10261 agtctatcct gcccaagagg aacagcgaca agctgatcgc cagaaagaag gactgggacc 10321 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca 10381 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca 10441 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca 10501 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa 10561 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc 10621 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct 10681 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaaacac tacctggacg 10741 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg 10801 acaaggtgct gagcgcctac aacaagcaca gagacaagcc tatcagagag caggccgaga 10861 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg 10921 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga 10981 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg 11041 acgcctatcc ctatgacgtg cccgattatg ccagcctggg cagcggctcc cccaagaaaa 11101 aacgcaaggt ggaagatcct aagaaaaagc ggaaagtgga cggcattggt agtgggagca 11161 acggcagcag cggatcctaa gcggccgctc gataagcttg atatcgaatt ccsccccccc 11221 cccycccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt 11281 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct 11341 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa 11401 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg 11461 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc 11521 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg 11581 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg 11641 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc 11701 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg 11761 gttttccttt gaaaaacacg atgataatat ggccacaacc atggtgagca agggcgagga 11821 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 11881 gttcagcgtg tctggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 11941 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 12001 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 12061 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 12121 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 12181 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 12241 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg cgaacttcaa 12301 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 12361 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 12421 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 12481 cgccgggatc actctcggca tggacgagct gtacaagtaa actagtcctg caggcatgca 12541 agcttgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga 12601 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 12661 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 12721 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 12781 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 12841 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 12901 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat 12961 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct 13021 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 13081 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 13141 ccgcctcccc gcatcgatac cgtcgacctc gagggaatta attcgagctc ggtaccttta 13201 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 13261 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 13321 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 13381 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 13441 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagcatc 13501 tagaattaat tccgtgtatt ctatagtgtc acctaaatcg tatgtgtatg atacataagg 13561 ttatgtatta attgtagccg cgttctaacg acaatatgta caagcctaat tgtgtagcat 13621 ctggcttact gaagcagacc ctatcatctc tctcgtaaac tgccgtcaga gtcggtttgg 13681 ttggacgaac cttctgagtt tctggtaacg ccgtcccgca cccggaaatg gtcagcgaac 13741 caatcagcag ggtcatcgct agccagatcc tctacgccgg acgcatcgtg gccggcatca 13801 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc 13861 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg 13921 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc 13981 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc 14041 gtcgaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga 14101 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 14161 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 14221 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 14281 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 14341 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 14401 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 14461 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 14521 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 14581 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatg //