LOCUS Exported 15520 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of gRNA and dCas9-VP64 from the same plasmid for CRISPRa. ACCESSION . VERSION . KEYWORDS pLentiV2-dCas9-VP64 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15520) TITLE pLentiV2-dCas9-VP64 JOURNAL Unpublished REFERENCE 2 (bases 1 to 15520) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15520 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 382..580 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 598..778 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 825..950 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1443..1676 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1861..1905 /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 2203..2320 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2371..2611 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2371..2391 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2542..2561 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4501..4576 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 4638..4849 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 4866..4913 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4914..9014 /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 N863A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 9069..9089 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9114..9263 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 9285..9341 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 9342..9938 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 9835..9855 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 9975..10563 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(10028..10048) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(10446..10457) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(10566..10582) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(10567..10583) /label=pBluescriptKS /note="For pBluescript vector" LTR 11088..11268 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(11294..11311) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 11300..11524 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 11570..11998 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11657..11676) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 11867..11888 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(12007..12027) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 12012..12341 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 12192..12327 /label=SV40 ori /note="SV40 origin of replication" primer_bind 12254..12273 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 12389..12436 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 12455..12829 /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 12959..13080 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(12996..13015) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 13050..13069 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(13129..13145) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(13129..13145) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(13142..13164) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 13153..13169 /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(13177..13207) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 13222..13243 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(13360..13377) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(13531..14119) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(13611..13630) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(14290..15150) /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 14913..14932 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(15151..15255) /gene="bla" /label=AmpR promoter primer_bind complement(15330..15349) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat 2401 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca 2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt 2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat 2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagacggttg taaatgagca 2641 cacaaaatac acatgctaaa atattatatt ctatgacctt tataaaatca accaaaatct 2701 tctttttaat aactttagta tcaataatta gaatttttat gttccttttt gcaaactttt 2761 aataaaaatg agcaaaataa aaaaacgcta gttttagtaa ctcgcgttgt tttcttcacc 2821 tttaataata gctactccac cacttgttcc taagcggtca gctcctgctt caatcatttt 2881 ttgagcatct tcaaatgttc taactccacc agctgcttta actaaagcat tgtctttaac 2941 aactgacttc attagtttaa catcttcaaa tgttgcacct gattttgaaa atcctgttga 3001 tgttttaaca aattctaatc cagcttcaac agctatttca caagctttca tgatttcttc 3061 ttttgttaat aaacaatttt ccataataca tttaacaaca tgtgatccag ctgctttttt 3121 tacagctttc atgtcttcta aaactaattc ataatttttg tcttttaatg caccaatatt 3181 taataccata tcaatttctg ttgcaccatc tttaattgct tcagaaactt cgaatgcttt 3241 tgtagctgtt gtgcatgcac ctagaggaaa acctacaaca tttgttattc ctacatttgt 3301 gccttttaat aattctttac aatagcttgt tcaatatgaa ttaacacaaa ctgttgcaaa 3361 atcaaattca attgcttcat cacataattg tttaatttca gctttcgtag catcttgttt 3421 taataatgtg tgatctatat atttgtttag tttcattttt tctcctatat attcattttt 3481 aattttaatt ctttaataat ttcgtctact ttaactttag cgttttgaac agattcacca 3541 acacctataa aataaatttt tagtttaggt tcagttccac ttgggcgaac agcaaatcat 3601 gacttatctt ctaaataaaa ttttagtaag tcttgtcctg gcatattata cattccatcg 3661 atgtagtctt caacattaac aactttaagt ccagcaattt gagttaaggg tgttgctctc 3721 aatgatttca ttaatggttc aatttttaat ttcttttctt ctggtttaaa attcaagttt 3781 aaagtgaaag tgtaatatgc acccatttct ttaaataaat cttctaaata gtctactaat 3841 gttttatttt gttttttata aaatcaagca gcctctgcta ttaatataga agcttgtatt 3901 ccatctttat ctctagctga gtcatcaatt acatatccat aactttcttc ataagcaaaa 3961 acaaaattta atccgttatc ttcttcttta gcaatttctc tacccattca tttaaatcca 4021 gttaaagttt ttacaatatt aactccatat ttttcatgag cgattctatc acccaaatca 4081 cttgttacaa aacttgaata tagagccgga ttttttggaa tgctatttaa gcgttttaga 4141 tttgataatt ttcaatcaat taaaattggt cctgtttgat ttccatctaa tcttacaaaa 4201 tgaccatcat gttttattgc cattccaaat ctgtcagcat ctgggtcatt cataataata 4261 atatctgcat catgtttaat accatattca agcggtattt ttcatgcagg atcaaattct 4321 ggatttggat ttacaacatt tttaaatgtt tcatcttcaa atgcatgctc ttcaacctca 4381 ataacgttat atcctgattc acgtaatatt tttggggtaa atttagttcc tgttccatta 4441 actgcgctaa aaataatttt taaatctttt ttagcttctt gctctttttt gtacgtctct 4501 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 4561 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg 4621 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg 4681 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga 4741 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt 4801 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg cgtacggcca 4861 ccatgaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aaggacaaga 4921 agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg 4981 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 5041 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc 5101 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 5161 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 5221 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 5281 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 5341 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 5401 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 5461 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 5521 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 5581 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc 5641 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac 5701 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 5761 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 5821 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga 5881 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 5941 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 6001 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 6061 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 6121 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc 6181 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 6241 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 6301 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca 6361 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 6421 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 6481 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc 6541 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 6601 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 6661 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 6721 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 6781 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 6841 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc 6901 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 6961 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct 7021 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 7081 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 7141 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 7201 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 7261 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 7321 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 7381 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccac atcgtgcctc 7441 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaaggccc 7501 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 7561 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 7621 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 7681 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 7741 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 7801 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 7861 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 7921 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 7981 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 8041 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 8101 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 8161 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 8221 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 8281 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 8341 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 8401 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 8461 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 8521 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa 8581 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 8641 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 8701 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 8761 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 8821 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 8881 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 8941 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 9001 agctgggagg cgacagcgct ggaggaggtg gaagcggagg aggaggaagc ggaggaggag 9061 gtagcggacc taagaaaaag aggaaggtgg cggccgctgg atccggacgg gctgacgcat 9121 tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt gaccttgaca 9181 tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt gacgcccttg 9241 atgatttcga cctggacatg ctgattaact gtacaggcag tggagcaaca aacttctctc 9301 tgctgaaaca agccggagat gtcgaagaga atcctggacc gaccgagtac aagcccacgg 9361 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg 9421 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg 9481 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg 9541 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt 9601 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac 9661 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg 9721 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag 9781 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc 9841 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac 9901 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gaattcgata 9961 tcaagcttat cggtaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc 10021 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg 10081 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc 10141 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg 10201 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg 10261 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga 10321 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct 10381 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg 10441 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc 10501 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc 10561 cgcatcgata ccgtcgacct cgagacctag aaaaacatgg agcaatcaca agtagcaata 10621 cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt 10681 ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct gtagatctta 10741 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag 10801 atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca 10861 caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag 10921 ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg 10981 tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc 11041 gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt 11101 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 11161 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 11221 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa 11281 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 11341 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 11401 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 11461 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 11521 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt 11581 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 11641 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 11701 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 11761 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 11821 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 11881 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 11941 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa 12001 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 12061 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 12121 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 12181 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 12241 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga 12301 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta 12361 tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg 12421 catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc 12481 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt 12541 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt 12601 catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg 12661 cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc 12721 ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg 12781 cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg 12841 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga 12901 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa 12961 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 13021 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 13081 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt 13141 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 13201 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 13261 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 13321 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 13381 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 13441 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 13501 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 13561 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 13621 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 13681 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 13741 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 13801 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 13861 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 13921 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 13981 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 14041 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 14101 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 14161 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 14221 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 14281 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 14341 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 14401 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 14461 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 14521 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 14581 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 14641 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 14701 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 14761 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 14821 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 14881 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 14941 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 15001 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 15061 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 15121 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 15181 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 15241 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga 15301 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 15361 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa 15421 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt 15481 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt //