LOCUS Exported 12843 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Constitutive dCas9-KRAB 2nd generation lentiviral expression vector. ACCESSION . VERSION . KEYWORDS pHAGE EF1-alpha dCas9-KRAB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12843) AUTHORS Kearns NA, Genga RM, Enuameh MS, Garber M, Wolfe SA, Maehr R TITLE Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. JOURNAL Development. 2014 Jan;141(1):219-23. doi: 10.1242/dev.103341. PUBMED 24346702 REFERENCE 2 (bases 1 to 12843) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12843 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 67..93 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" promoter 126..625 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(161..184) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 558..577 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 646..1245 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(646..665) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1142..1162 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 1258..1846 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1311..1331) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(1729..1740) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 1921..2154 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(2273..2291) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2359..2463 /gene="bla" /label=AmpR promoter CDS 2464..3324 /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(2682..2701) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3495..4083 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3984..4003 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4237..4254 /label=L4440 /note="L4440 vector, forward primer" primer_bind 4346..4365 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 4494..4515 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4530..4560 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4568..4584 /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 4573..4595 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4592..4608 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4592..4608 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 4658..4677 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" LTR 4869..5502 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 5549..5674 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 6171..6404 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6589..6633 /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 6926..7043 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 7088..8266 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 7319..8257 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 8214..8234 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 8301..8325 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" regulatory 8329..8338 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 8335..12438 /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 12451..12471 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 12472..12561 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" CDS 12598..12792 /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 QLTKPDVILRLEKGEEPWLV" protein_bind complement(12833..14) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" ORIGIN 1 ttgtacaaag tggttgatat ccagcacagt ggcggccgct cgagtctaga gggcccgcgg 61 ttcgaatacc catacgacgt cccagactac gcttagtaat gattaattaa actagaaatt 121 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc 181 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 241 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 301 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 361 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 421 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg 481 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag 541 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc 601 ctcttcctca tctccgggcc tttcgacctg cagcccaagc ttaccatgac cgagtacaag 661 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc 721 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg 781 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg 841 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg 901 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg 961 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg 1021 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc 1081 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc 1141 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc 1201 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgagcgat agatcctaat 1261 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct 1321 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg 1381 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg 1441 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt 1501 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt 1561 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg 1621 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc 1681 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat 1741 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc 1801 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctga gatcctttaa 1861 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac 1921 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc 1981 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 2041 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 2101 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta 2161 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg 2221 agaggcccgg gttaattaag gaaagggcta gatcattctt gaagacgaaa gggcctcgtg 2281 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 2341 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 2401 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 2461 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 2521 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 2581 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 2641 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 2701 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 2761 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 2821 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 2881 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 2941 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 3001 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 3061 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 3121 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 3181 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 3241 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 3301 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 3361 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 3421 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 3481 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 3541 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 3601 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 3661 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 3721 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 3781 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 3841 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 3901 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 3961 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 4021 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 4081 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 4141 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 4201 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 4261 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 4321 aagctcatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 4381 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc 4441 ccgtggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg 4501 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg 4561 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat gattacgaat 4621 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 4681 gtatcttatc atgtctggat caactggata actcaagcta accaaaatca tcccaaactt 4741 cccaccccat accctattac cactgccaat tacctgtggt ttcatttact ctaaacctgt 4801 gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg tactacaaac 4861 ttagtagttg gaagggctaa ttcactccca aagaagacaa gatatccttg atctgtggat 4921 ctaccacaca caaggctact tccctgatta gcagaactac acaccagggc caggggtcag 4981 atatccactg acctttggat ggtgctacaa gctagtacca gttgagccag ataaggtaga 5041 agaggccaat aaaggagaga acaccagctt gttacaccct gtgagcctgc atgggatgga 5101 tgacccggag agagaagtgt tagagtggag gtttgacagc cgcctagcat ttcatcacgt 5161 ggcccgagag ctgcatccgg agtacttcaa gaactgctga tatcgagctt gctacaaggg 5221 actttccgct ggggactttc cagggaggcg tggcctgggc gggactgggg agtggcgagc 5281 cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc 5341 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 5401 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 5461 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga 5521 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc 5581 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg 5641 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc 5701 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat 5761 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc 5821 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga 5881 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat 5941 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac 6001 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat 6061 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc 6121 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg 6181 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg 6241 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct 6301 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca 6361 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc 6421 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct 6481 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac 6541 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa 6601 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg 6661 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt 6721 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag 6781 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga 6841 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt 6901 cacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg 6961 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac 7021 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttgga 7081 ctacgatggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 7141 gttgtgggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 7201 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 7261 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 7321 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 7381 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 7441 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 7501 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 7561 cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 7621 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 7681 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 7741 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 7801 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 7861 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 7921 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 7981 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 8041 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 8101 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 8161 gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 8221 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaacta gtccagtgtg 8281 gtggaattct gcagatatca acaagtttgt acaaaaaagc aggctggcgc caccatggac 8341 aagaagtact ccattgggct cgctatcggt accaacagcg tcggctgggc cgtcattacg 8401 gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga tcgccacagc 8461 ataaagaaga acctcattgg agccctcctg ttcgactccg gggagacggc cgaagccacg 8521 cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat ctgctacctg 8581 caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca taggctggag 8641 gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt tggcaatatc 8701 gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag gaagaagctg 8761 gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc gcacatgatc 8821 aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag cgatgtcgac 8881 aaactcttta tccaactggt tcagacttac aatcagcttt tcgaggagaa cccgatcaac 8941 gcatccggcg ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc ccggcggctc 9001 gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg taatcttatc 9061 gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc cgaagatgcc 9121 aagctgcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct ggcccagatc 9181 ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc cattctgctg 9241 agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc tagtatgatc 9301 aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt cagacagcaa 9361 ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta cgccggatac 9421 attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat cttggaaaaa 9481 atggacggca ccgaggagct gctggtaaag ctgaacagag aagatctgtt gcgcaaacag 9541 cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact gcacgctatc 9601 ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa gattgagaaa 9661 atcctcacat ttcggatacc ctactatgta ggccccctcg ctcggggaaa ttccagattc 9721 gcgtggatga ctcgcaaatc agaagagacc atcactccct ggaacttcga ggaagtcgtg 9781 gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga taaaaatctg 9841 cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac agtttataac 9901 gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt cctgtctgga 9961 gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt taccgtgaaa 10021 cagctcaaag aagactattt caaaaagatt gaatgtttcg actctgttga aatcagcgga 10081 gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa aatcattaaa 10141 gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat tgtcctcacc 10201 cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta cgctcatctc 10261 ttcgacgaca aagtcatgaa acagctcaag agacgccgat atacaggatg ggggcggctg 10321 tcaagaaaac tgatcaatgg catccgagac aagcagagtg gaaagacaat cctggatttt 10381 cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga tgactctctc 10441 acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag tcttcacgag 10501 cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca gaccgttaag 10561 gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat cgttatcgag 10621 atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga aaggatgaag 10681 aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca cccagttgaa 10741 aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg cagggacatg 10801 tacgtggatc aggaactgga catcaaccgg ttgtccgact acgacgtgga tgctatcgtg 10861 ccccaaagct ttctcaaaga tgattctatt gataataaag tgttgacaag atccgataaa 10921 aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat gaaaaattat 10981 tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa tctgactaag 11041 gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag gcagcttgtt 11101 gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat gaacaccaag 11161 tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa gtctaagctg 11221 gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa caattaccac 11281 catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa aaaatatccc 11341 aagctggaat ctgaatttgt ttacggagac tataaagtgt acgatgttag gaaaatgatc 11401 gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta cagcaatatt 11461 atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa gcgaccactt 11521 atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga tttcgcgaca 11581 gtccgcaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga agtacagacc 11641 ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct gatcgcacgc 11701 aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt cgcttacagt 11761 gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag cgtcaaggaa 11821 ctgctgggca tcacaatcat ggagcgatcc agcttcgaga aaaaccccat cgactttctc 11881 gaagcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagctgcc caagtactct 11941 ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga gctgcagaaa 12001 ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc cagccactat 12061 gaaaagctca aagggtctcc cgaagataat gagcagaagc agctgttcgt ggaacaacac 12121 aaacactacc ttgatgagat catcgagcaa ataagcgagt tctccaaaag agtgatcctc 12181 gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga taagcccatc 12241 agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg cgcgcctgca 12301 gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac aaaggaggtc 12361 ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag aatcgacctc 12421 tctcagctcg gtggagacgg caccggcggg cccaagaaga agaggaaggt atacccatac 12481 gatgttcctg actatgcggg ctatccctat gacgtcccgg actatgcagg atcgtatcct 12541 tatgacgttc cagattacgc tggatccatg gacgcgaaat cacttacggc atggtcgaga 12601 acactggtta cgttcaagga cgtgtttgtg gactttacac gtgaggagtg gaaattgctg 12661 gatactgcgc aacaaattgt gtatcgaaat gtcatgcttg agaattacaa gaacctcgtc 12721 agtctcggat accagttgac gaaaccggat gtgatcctta ggctcgaaaa gggggaagaa 12781 ccttggctgg tataattcga atccggataa gcggccgcct cgagatatct agacccagct 12841 ttc //