LOCUS Exported 14332 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses dead Cas9 (dCas9) fused to inactive DNMT3A catalytic domain (CD) under the control of the CMV promoter. ACCESSION . VERSION . KEYWORDS pCMV-dCas9-mD3A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14332) 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 14332) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14332 /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." CDS 2098..2142 /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 2440..2557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2611..3821 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3658..3677 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" regulatory 3856..3865 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3862..7965 /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 3862..7965 /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 7978..7998 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8008..8031 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 8050..8982 /codon_start=1 /product="catalytic domain of DNA methyltransferase 3A" /label=DNMT3A catalytic domain /translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFANVVAMGVS DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR LARQRLLGRSWSVPVIRHLFAPLKEYFACV" misc_feature 9024..9612 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9077..9097) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9495..9506) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(9615..9631) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9616..9632) /label=pBluescriptKS /note="For pBluescript vector" LTR 10137..10317 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(10343..10360) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 10349..10573 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 10619..11047 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(10706..10725) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10916..10937 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(11056..11076) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 11061..11390 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 11241..11376 /label=SV40 ori /note="SV40 origin of replication" primer_bind 11303..11322 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 11438..11485 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 11504..11878 /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 12008..12129 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(12045..12064) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 12099..12118 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(12178..12194) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12178..12194) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(12191..12213) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 12202..12218 /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(12226..12256) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12271..12292 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12409..12426) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(12580..13168) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(12660..12679) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(13339..14199) /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 13962..13981 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14200..14304) /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 agggtgcagc ggcctccgcg ccgggttttg gcgcctcccg 2641 cgggcgcccc cctcctcacg gcgagcgctg ccacgtcaga cgaagggcgc aggagcgttc 2701 ctgatccttc cgcccggacg ctcaggacag cggcccgctg ctcataagac tcggccttag 2761 aaccccagta tcagcagaag gacattttag gacgggactt gggtgactct agggcactgg 2821 ttttctttcc agagagcgga acaggcgagg aaaagtagtc ccttctcggc gattctgcgg 2881 agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccg ggtgtggcac 2941 agctagttcc gtcgcagccg ggatttgggt cgcggttctt gtttgtggat cgctgtgatc 3001 gtcacttggt gagttgcggg ctgctgggct ggccggggct ttcgtggccg ccgggccgct 3061 cggtgggacg gaagcgtgtg gagagaccgc caagggctgt agtctgggtc cgcgagcaag 3121 gttgccctga actgggggtt ggggggagcg cacaaaatgg cggctgttcc cgagtcttga 3181 atggaagacg cttgtaaggc gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg 3241 gcggcaagaa cccaaggtct tgaggccttc gctaatgcgg gaaagctctt attcgggtga 3301 gatgggctgg ggcaccatct ggggaccctg acgtgaagtt tgtcactgac tggagaactc 3361 gggtttgtcg tctggttgcg ggggcggcag ttatgcggtg ccgttgggca gtgcacccgt 3421 acctttggga gcgcgcgcct cgtcgtgtcg tgacgtcacc cgttctgttg gcttataatg 3481 cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt tctccgtcgc aggacgcagg 3541 gttcgggcct agggtaggct ctcctgaatc gacaggcgcc ggacctctgg tgaggggagg 3601 gataagtgag gcgtcagttt ctttggtcgg ttttatgtac ctatcttctt aagtagctga 3661 agctccggtt ttgaactatg cgctcggggt tggcgagtgt gttttgtgaa gttttttagg 3721 caccttttga aatgtaatca tttgggtcaa tatgtaattt tcagtgttag actagtaaat 3781 tgtccgctaa attctggccg tttttggctt ttttgttaga cgaagcttgg gctgcaggtc 3841 gactctagag gatccgccac catggacaag aagtactcca ttgggctcgc tatcggcaca 3901 aacagcgtcg gctgggccgt cattacggac gagtacaagg tgccgagcaa aaaattcaaa 3961 gttctgggca ataccgatcg ccacagcata aagaagaacc tcattggcgc cctcctgttc 4021 gactccgggg agacggccga agccacgcgg ctcaaaagaa cagcacggcg cagatatacc 4081 cgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat agctaaggtg 4141 gatgactctt tcttccatag gctggaggag tcctttttgg tggaggagga taaaaagcac 4201 gagcgccacc caatctttgg caatatcgtg gacgaggtgg cgtaccatga aaagtaccca 4261 accatatatc atctgaggaa gaagcttgta gacagtactg ataaggctga cttgcggttg 4321 atctatctcg cgctggcgca tatgatcaaa tttcggggac acttcctcat cgagggggac 4381 ctgaacccag acaacagcga tgtcgacaaa ctctttatcc aactggttca gacttacaat 4441 cagcttttcg aagagaaccc gatcaacgca tccggagttg acgccaaagc aatcctgagc 4501 gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg cacagctccc tggggagaag 4561 aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc caactttaaa 4621 tctaacttcg acctggccga agatgccaag cttcaactga gcaaagacac ctacgatgat 4681 gatctcgaca atctgctggc ccagatcggc gaccagtacg cagacctttt tttggcggca 4741 aagaacctgt cagacgccat tctgctgagt gatattctgc gagtgaacac ggagatcacc 4801 aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccacca agacttgact 4861 ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat tttcttcgat 4921 cagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga ggaattttac 4981 aaatttatta agcccatctt ggaaaaaatg gacggcaccg aggagctgct ggtaaagctt 5041 aacagagaag atctgttgcg caaacagcgc actttcgaca atggaagcat cccccaccag 5101 attcacctgg gcgaactgca cgctatcctc aggcggcaag aggatttcta cccctttttg 5161 aaagataaca gggaaaagat tgagaaaatc ctcacatttc ggatacccta ctatgtaggc 5221 cccctcgccc ggggaaattc cagattcgcg tggatgactc gcaaatcaga agagaccatc 5281 actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc cttcatcgaa 5341 aggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa acactctctg 5401 ctgtacgagt acttcacagt ttataacgag ctcaccaagg tcaaatacgt cacagaaggg 5461 atgagaaagc cagcattcct gtctggagag cagaagaaag ctatcgtgga cctcctcttc 5521 aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag actatttcaa aaagattgaa 5581 tgtttcgact ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc cctgggaacg 5641 tatcacgatc tcctgaaaat cattaaagac aaggacttcc tggacaatga ggagaacgag 5701 gacattcttg aggacattgt cctcaccctt acgttgtttg aagataggga gatgattgaa 5761 gaacgcttga aaacttacgc tcatctcttc gacgacaaag tcatgaaaca gctcaagagg 5821 cgccgatata caggatgggg gcggctgtca agaaaactga tcaatgggat ccgagacaag 5881 cagagtggaa agacaatcct ggattttctt aagtccgatg gatttgccaa ccggaacttc 5941 atgcagttga tccatgatga ctctctcacc tttaaggagg acatccagaa agcacaagtt 6001 tctggccagg gggacagtct tcacgagcac atcgctaatc ttgcaggtag cccagctatc 6061 aaaaagggaa tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt aatgggaagg 6121 cataagcccg agaatatcgt tatcgagatg gcccgagaga accaaactac ccagaaggga 6181 cagaagaaca gtagggaaag gatgaagagg attgaagagg gtataaaaga actggggtcc 6241 caaatcctta aggaacaccc agttgaaaac acccagcttc agaatgagaa gctctacctg 6301 tactacctgc agaacggcag ggacatgtac gtggatcagg aactggacat caatcggctc 6361 tccgactacg acgtggatgc tatcgtgccc cagtcttttc tcaaagatga ttctattgat 6421 aataaagtgt tgacaagatc cgataaaaat agagggaaga gtgataacgt cccctcagaa 6481 gaagttgtca agaaaatgaa aaattattgg cggcagctgc tgaacgccaa actgatcaca 6541 caacggaagt tcgataatct gactaaggct gaacgaggtg gcctgtctga gttggataaa 6601 gccggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca cgtggcccaa 6661 attctcgatt cacgcatgaa caccaagtac gatgaaaatg acaaactgat tcgagaggtg 6721 aaagttatta ctctgaagtc taagctggtc tcagatttca gaaaggactt tcagttttat 6781 aaggtgagag agatcaacaa ttaccaccat gcgcatgatg cctacctgaa tgcagtggta 6841 ggcactgcac ttatcaaaaa atatcccaag cttgaatctg aatttgttta cggagactat 6901 aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaatagg caaggccacc 6961 gctaagtact tcttttacag caatattatg aattttttca agaccgagat tacactggcc 7021 aatggagaga ttcggaagcg accacttatc gaaacaaacg gagaaacagg agaaatcgtg 7081 tgggacaagg gtagggattt cgcgacagtc cggaaggtcc tgtccatgcc gcaggtgaac 7141 atcgttaaaa agaccgaagt acagaccgga ggcttctcca aggaaagtat cctcccgaaa 7201 aggaacagcg acaagctgat cgcacgcaaa aaagattggg accccaagaa atacggcgga 7261 ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga gaaagggaag 7321 tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatgga gcgatcaagc 7381 ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat ataaagaggt caaaaaagac 7441 ctcatcatta agcttcccaa gtactctctc tttgagcttg aaaacggccg gaaacgaatg 7501 ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg cactgccctc taaatacgtt 7561 aatttcttgt atctggccag ccactatgaa aagctcaaag ggtctcccga agataatgag 7621 cagaagcagc tgttcgtgga acaacacaaa cactaccttg atgagatcat cgagcaaata 7681 agcgaattct ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt gctttctgct 7741 tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattat ccacttgttt 7801 actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac catagacaga 7861 aagcggtaca cctctacgaa ggaggtcctg gacgccacac tgattcatca gtcaattacg 7921 gggctctatg aaacaagaat cgacctctct cagctcggtg gagacagcag ggctgacccc 7981 aagaagaaga ggaaggtggg agctagcgac tacaaagacg atgacgacaa gggaagccga 8041 gaggactggc cctcccggct ccagatgttc ttcgctaata accacgacca ggaatttgac 8101 cctccaaagg tttacccacc tgtcccagct gagaagagga agcccatccg ggtgctgtct 8161 ctctttgatg gaatcgctac agggctcctg gtgctgaagg acttgggcat tcaggtggac 8221 cgctacattg cctcggaggt gtgtgaggac tccatcacgg tgggcatggt gcggcaccag 8281 gggaagatca tgtacgtcgg ggacgtccgc agcgtcacac agaagcatat ccaggagtgg 8341 ggcccattcg atctggtgat tgggggcagt ccctgcaatg acctctccat cgtcaaccct 8401 gctcgcaagg gcctctacga gggcactggc cggctcttct ttgagttcta ccgcctcctg 8461 catgatgcgc ggcccaagga gggagatgat cgccccttct tctggctctt tgcgaatgtg 8521 gtggccatgg gcgttagtga caagagggac atctcgcgat ttctcgagtc caaccctgtg 8581 atgattgatg ccaaagaagt gtcagctgca cacagggccc gctacttctg gggtaacctt 8641 cccggtatga acaggccgtt ggcatccact gtgaatgata agctggagct gcaggagtgt 8701 ctggagcatg gcaggatagc caagttcagc aaagtgagga ccattactac gaggtcaaac 8761 tccataaagc agggcaaaga ccagcatttt cctgtcttca tgaatgagaa agaggacatc 8821 ttatggtgca ctgaaatgga aagggtattt ggtttcccag tccactatac tgacgtctcc 8881 aacatgagcc gcttggcgag gcagagactg ctgggccggt catggagcgt gccagtcatc 8941 cgccacctct tcgctccgct gaaggagtat tttgcgtgtg tgtaaaccgg tctagctaag 9001 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga 9061 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 9121 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 9181 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 9241 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 9301 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 9361 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat 9421 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct 9481 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 9541 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 9601 ccgcctcccc gcatcgatac cgtcgacctc gagacctaga aaaacatgga gcaatcacaa 9661 gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg 9721 aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg 9781 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac 9841 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc 9901 agaactacac accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc 9961 tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt 10021 tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt 10081 ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt 10141 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 10201 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 10261 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg 10321 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 10381 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 10441 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 10501 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 10561 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 10621 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 10681 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 10741 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 10801 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 10861 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 10921 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 10981 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 11041 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 11101 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 11161 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 11221 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 11281 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc 11341 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg 11401 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata 11461 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag 11521 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg 11581 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt 11641 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt 11701 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt 11761 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt 11821 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca 11881 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt 11941 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc 12001 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 12061 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 12121 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc 12181 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 12241 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 12301 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 12361 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 12421 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 12481 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 12541 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 12601 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 12661 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 12721 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 12781 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 12841 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 12901 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 12961 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 13021 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 13081 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 13141 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 13201 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 13261 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 13321 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 13381 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 13441 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 13501 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 13561 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 13621 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 13681 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 13741 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 13801 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 13861 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 13921 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 13981 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 14041 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 14101 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 14161 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 14221 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 14281 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ac //