LOCUS Exported 14828 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses dead Cas9 (dCas9) fused to inactive DNMT3A catalytic domain (CD) linked to IRES-mCherry under the control of TetOp and a minimal CMV promoter. ACCESSION . VERSION . KEYWORDS TetO-dCas9-mD3A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14828) AUTHORS McDonald JI, Celik H, Rois LE, Fishberger G, Fowler T, Rees R, Kramer A, Martens A, Edwards JR, Challen GA TITLE Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. JOURNAL Biol Open. 2016 May 11. pii: bio.019067. doi: 10.1242/bio.019067. PUBMED 27170255 REFERENCE 2 (bases 1 to 14828) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14828 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 74..453 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 455..653 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 608..628 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 671..851 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 898..1023 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1516..1749 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1934..1978 /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 2276..2393 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2446..2716 /label=tetracycline response element /bound_moiety="tetracycline repressor TetR" /note="contains seven copies of the tetracycline operator tetO" promoter 2749..2786 /label=minimal CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2783..2807 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 2907..2916 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2913..7016 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9m4 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the mutations D10A, D839A, H840A, and N863A (Mali et al., 2013)" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEIAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 2913..7016 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEIAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 7029..7049 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7059..7082 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 7101..8033 /codon_start=1 /product="catalytic domain of DNA methyltransferase 3A" /label=DNMT3A catalytic domain /translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFANVVAMGVS DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR LARQRLLGRSWSVPVIRHLFAPLKEYFACV" primer_bind complement(8205..8222) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 8432..8451 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 8613..9323 /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(8758..8776) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 9026..9045 /label=mCherry-F /note="mCherry, forward primer" misc_feature 9356..9944 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9409..9429) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9827..9838) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(9947..9963) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9948..9964) /label=pBluescriptKS /note="For pBluescript vector" LTR 10469..10649 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(10675..10692) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 10681..10905 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 10951..11379 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11038..11057) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 11248..11269 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(11388..11408) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 11393..11722 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 11573..11708 /label=SV40 ori /note="SV40 origin of replication" primer_bind 11635..11654 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 11770..11817 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 11836..12210 /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 12340..12461 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(12377..12396) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 12431..12450 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(12510..12526) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12510..12526) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(12523..12545) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 12534..12550 /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(12558..12588) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12603..12624 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12741..12758) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(12912..13500) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(12992..13011) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(13671..14531) /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 14294..14313 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14532..14636) /gene="bla" /label=AmpR promoter primer_bind complement(14711..14730) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc 61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca 121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt 661 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact 721 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc 781 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa 841 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc 901 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg 961 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc 1021 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg 1081 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc 1141 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta 1201 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc 1261 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata 1321 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc 1381 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa 1441 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa 1501 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat 1561 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca 1621 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt 1681 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca 1741 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg 1801 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga 1861 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca 1921 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg 1981 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg 2041 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca 2101 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc 2161 cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa 2221 cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta 2281 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa 2341 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt 2401 attacaggga cagcagagat ccagtttggt tatcgagttt accactccct atcagtgata 2461 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga 2521 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca 2581 gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa 2641 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc 2701 ctatcagtga tagagaaaag tgaaagtcga gctcggtacc cgggtcgagt aggcgtgtac 2761 ggtgggaggc ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc 2821 atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc cgcggccccg 2881 aattcgccat acattctaga ggatccgcca ccatggacaa gaagtactcc attgggctcg 2941 ctatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag gtgccgagca 3001 aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac ctcattggcg 3061 ccctcctgtt cgactccggg gagacggccg aagccacgcg gctcaaaaga acagcacggc 3121 gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt agtaatgaga 3181 tagctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg gtggaggagg 3241 ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg 3301 aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact gataaggctg 3361 acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga cacttcctca 3421 tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc caactggttc 3481 agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt gacgccaaag 3541 caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc gcacagctcc 3601 ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc gggctgaccc 3661 ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg agcaaagaca 3721 cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac gcagaccttt 3781 ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg cgagtgaaca 3841 cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc 3901 aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag tacaaggaaa 3961 ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga gcaagccagg 4021 aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc gaggagctgc 4081 tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac aatggaagca 4141 tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa gaggatttct 4201 accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt cggataccct 4261 actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact cgcaaatcag 4321 aagagaccat cactccctgg aacttcgagg aagtcgtgga taagggggcc tctgcccagt 4381 ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag gtgcttccta 4441 aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag gtcaaatacg 4501 tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg 4561 acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa gactatttca 4621 aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat 4681 ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc ctggacaatg 4741 aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt gaagataggg 4801 agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac 4861 agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg atcaatggga 4921 tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat ggatttgcca 4981 accggaactt catgcagttg atccatgatg actctctcac ctttaaggag gacatccaga 5041 aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat cttgcaggta 5101 gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag 5161 taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag aaccaaacta 5221 cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag ggtataaaag 5281 aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt cagaatgaga 5341 agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag gaactggaca 5401 tcaatcggct ctccgactac gacgtggatg ctatcgtgcc ccagtctttt ctcaaagatg 5461 attctattga taataaagtg ttgacaagat ccgataaaaa tagagggaag agtgataacg 5521 tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg ctgaacgcca 5581 aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg 5641 agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag atcaccaagc 5701 acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat gacaaactga 5761 ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc agaaaggact 5821 ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat gcctacctga 5881 atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct gaatttgttt 5941 acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag caggaaatag 6001 gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc aagaccgaga 6061 ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac ggagaaacag 6121 gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc 6181 cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc aaggaaagta 6241 tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg gaccccaaga 6301 aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg gccaaagtgg 6361 agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg 6421 agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga tataaagagg 6481 tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt gaaaacggcc 6541 ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg gcactgccct 6601 ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa gggtctcccg 6661 aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt gatgagatca 6721 tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac ctcgataagg 6781 tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca gaaaacatta 6841 tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac ttcgacacca 6901 ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca ctgattcatc 6961 agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt ggagacagca 7021 gggctgaccc caagaagaag aggaaggtgg gagctagcga ctacaaagac gatgacgaca 7081 agggaagccg agaggactgg ccctcccggc tccagatgtt cttcgctaat aaccacgacc 7141 aggaatttga ccctccaaag gtttacccac ctgtcccagc tgagaagagg aagcccatcc 7201 gggtgctgtc tctctttgat ggaatcgcta cagggctcct ggtgctgaag gacttgggca 7261 ttcaggtgga ccgctacatt gcctcggagg tgtgtgagga ctccatcacg gtgggcatgg 7321 tgcggcacca ggggaagatc atgtacgtcg gggacgtccg cagcgtcaca cagaagcata 7381 tccaggagtg gggcccattc gatctggtga ttgggggcag tccctgcaat gacctctcca 7441 tcgtcaaccc tgctcgcaag ggcctctacg agggcactgg ccggctcttc tttgagttct 7501 accgcctcct gcatgatgcg cggcccaagg agggagatga tcgccccttc ttctggctct 7561 ttgcgaatgt ggtggccatg ggcgttagtg acaagaggga catctcgcga tttctcgagt 7621 ccaaccctgt gatgattgat gccaaagaag tgtcagctgc acacagggcc cgctacttct 7681 ggggtaacct tcccggtatg aacaggccgt tggcatccac tgtgaatgat aagctggagc 7741 tgcaggagtg tctggagcat ggcaggatag ccaagttcag caaagtgagg accattacta 7801 cgaggtcaaa ctccataaag cagggcaaag accagcattt tcctgtcttc atgaatgaga 7861 aagaggacat cttatggtgc actgaaatgg aaagggtatt tggtttccca gtccactata 7921 ctgacgtctc caacatgagc cgcttggcga ggcagagact gctgggccgg tcatggagcg 7981 tgccagtcat ccgccacctc ttcgctccgc tgaaggagta ttttgcgtgt gtgtaaaccg 8041 gtactcgacc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc 8101 gcttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa 8161 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat 8221 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac 8281 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcccctg 8341 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt 8401 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg 8461 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac 8521 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga 8581 cgtggttttc ctttgaaaaa cacgataata ccatggtgag caagggcgag gaggataaca 8641 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc 8701 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca 8761 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt 8821 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc 8881 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg 8941 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc 9001 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg 9061 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc 9121 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga 9181 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca 9241 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg 9301 gcatggacga gctgtacaag tagacgcgcc cgaattcgat atcaagctta tcgataatca 9361 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 9421 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 9481 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 9541 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 9601 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 9661 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 9721 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg 9781 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 9841 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 9901 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc 9961 tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga 10021 ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt 10081 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa 10141 ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat 10201 ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc cagggatcag 10261 atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga 10321 agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga 10381 tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat 10441 ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg 10501 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt 10561 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc 10621 cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga tcagcctcga 10681 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 10741 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 10801 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 10861 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 10921 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 10981 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 11041 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 11101 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 11161 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 11221 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 11281 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 11341 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 11401 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 11461 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 11521 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 11581 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 11641 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 11701 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga 11761 tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag 11821 gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac 11881 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag 11941 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac 12001 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac 12061 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc 12121 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc 12181 gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc 12241 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 12301 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 12361 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 12421 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg 12481 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 12541 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 12601 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 12661 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 12721 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 12781 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 12841 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 12901 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 12961 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 13021 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 13081 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 13141 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 13201 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 13261 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 13321 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 13381 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 13441 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 13501 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 13561 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 13621 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 13681 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 13741 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 13801 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 13861 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 13921 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 13981 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 14041 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 14101 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 14161 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 14221 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 14281 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 14341 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 14401 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 14461 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 14521 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 14581 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 14641 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt 14701 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt 14761 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct 14821 tgaccgac //