LOCUS Exported 14796 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral expression plasmid encoding dCas9-VPR and a P2A linked mCherry marker. ACCESSION . VERSION . KEYWORDS dCas9-VPR_P2A_mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14796) AUTHORS Umkehrer C, Holstein F, Formenti L, Jude J, Froussios K, Neumann T, Cronin SM, Haas L, Lipp JJ, Burkard TR, Fellner M, Wiesner T, Zuber J, Obenauf AC TITLE Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters. JOURNAL Nat Biotechnol. 2020 Jul 27. pii: 10.1038/s41587-020-0614-0. doi: 10.1038/s41587-020-0614-0. PUBMED 32719478 REFERENCE 2 (bases 1 to 14796) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14796 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(630..1490) /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 1253..1272 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1491..1595) /gene="bla" /label=AmpR promoter primer_bind complement(1670..1689) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 1861..2240 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 2242..2440 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2395..2415 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 2458..2638 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 2685..2810 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 3303..3536 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 3721..3765 /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 4063..4180 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 4273..4484 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 4508..8611 /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 NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 8624..8644 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind 8681..8705 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 8735..8884 /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 8909..8929 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9359..9715 /codon_start=1 /product="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /label=RelA (p65) AD /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALL" CDS 9734..10303 /codon_start=1 /product="transcriptional activation domain from the human herpesvirus 4 (Epstein-Barr virus) replication and transcription activator Rta/BRLF1 (Hardwick et al., 1992; Chavez et al., 2015)" /label=Rta AD /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL DTFLNDECLLHAMHISTGLSIFDTSLF" CDS 10319..10375 /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 10376..11086 /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(10521..10539) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 10789..10808 /label=mCherry-F /note="mCherry, forward primer" misc_feature 11111..11699 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(11164..11184) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(11582..11593) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(11702..11718) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11703..11719) /label=pBluescriptKS /note="For pBluescript vector" LTR 12224..12404 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(12430..12447) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 12436..12660 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 12706..13134 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(12793..12812) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 13003..13024 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(13143..13163) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 13148..13477 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 13328..13463 /label=SV40 ori /note="SV40 origin of replication" primer_bind 13390..13409 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 13525..13572 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 13591..13965 /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 14095..14216 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(14132..14151) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 14186..14205 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(14265..14281) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14265..14281) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(14278..14300) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 14289..14305 /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(14313..14343) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14358..14379 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(14496..14513) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(14667..459) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(14747..14766) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 61 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 121 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 181 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 241 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 301 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 361 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 421 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 481 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 541 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 601 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 661 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 721 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 781 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 841 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 901 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 961 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1021 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1081 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1141 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1201 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1261 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1321 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1381 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1441 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1501 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 1561 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 1621 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct 1681 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt 1741 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga 1801 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt 1861 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 1921 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 1981 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 2041 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 2101 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 2161 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 2221 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 2281 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 2341 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 2401 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 2461 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 2521 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 2581 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 2641 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 2701 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 2761 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 2821 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 2881 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 2941 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 3001 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 3061 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 3121 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 3181 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 3241 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 3301 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 3361 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 3421 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 3481 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 3541 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 3601 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 3661 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 3721 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 3781 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 3841 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 3901 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 3961 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 4021 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 4081 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 4141 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 4201 cagagatcca gtttggttaa ttagctagct aggtcttgaa aggagtggga attggctccg 4261 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg 4321 tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg 4381 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg 4441 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg ttctagagcg 4501 ctccaccatg gacaagaagt actccattgg gctcgctatc ggcacaaaca gcgtcggctg 4561 ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac 4621 cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac 4681 ggccgaagcc acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg 4741 gatctgctac ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt 4801 ccataggctg gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat 4861 ctttggcaat atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct 4921 gaggaagaag cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct 4981 ggcgcatatg atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa 5041 cagcgatgtc gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga 5101 gaacccgatc aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa 5161 atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt 5221 tggtaatctt atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct 5281 ggccgaagat gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct 5341 gctggcccag atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga 5401 cgccattctg ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag 5461 cgctagtatg atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct 5521 tgtcagacag caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg 5581 ctacgccgga tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc 5641 catcttggaa aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct 5701 gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga 5761 actgcacgct atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga 5821 aaagattgag aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg 5881 aaattccaga ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt 5941 cgaggaagtc gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt 6001 tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt 6061 cacagtttat aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc 6121 attcctgtct ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa 6181 agttaccgtg aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt 6241 tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct 6301 gaaaatcatt aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga 6361 cattgtcctc acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac 6421 ttacgctcat ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg 6481 atgggggcgg ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac 6541 aatcctggat tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca 6601 tgatgactct ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga 6661 cagtcttcac gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact 6721 gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa 6781 tatcgttatc gagatggccc gagagaacca aactacccag aagggacaga agaacagtag 6841 ggaaaggatg aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga 6901 acacccagtt gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa 6961 cggcagggac atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt 7021 ggctgctatc gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac 7081 aagatccgat aaagctagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa 7141 aatgaaaaat tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga 7201 taatctgact aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa 7261 aaggcagctt gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg 7321 catgaacacc aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct 7381 gaagtctaag ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat 7441 caacaattac caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat 7501 caaaaaatat cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt 7561 taggaaaatg atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt 7621 ttacagcaat attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg 7681 gaagcgacca cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag 7741 ggatttcgcg acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac 7801 cgaagtacag accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa 7861 gctgatcgca cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac 7921 agtcgcttac agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa 7981 aagcgtcaag gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc 8041 catcgacttt ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct 8101 tcccaagtac tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg 8161 cgagctgcag aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct 8221 ggccagccac tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt 8281 cgtggaacaa cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa 8341 aagagtgatc ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag 8401 ggataagccc atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt 8461 gggcgcgcct gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc 8521 tacaaaggag gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac 8581 aagaatcgac ctctctcagc tcggtggaga cagcagggct gaccccaaga agaagaggaa 8641 ggtgtcgcca gggatccgtc gacttgacgc gttgatatca acaagtttgt acaaaaaagc 8701 aggctacaaa gaggccagcg gttccggacg ggctgacgca ttggacgatt ttgatctgga 8761 tatgctggga agtgacgccc tcgatgattt tgaccttgac atgcttggtt cggatgccct 8821 tgatgacttt gacctcgaca tgctcggcag tgacgccctt gatgatttcg acctggacat 8881 gctgattaac tctagaagtt ccggatctcc gaaaaagaaa cgcaaagttg gtagccagta 8941 cctgcccgac accgacgacc ggcaccggat cgaggaaaag cggaagcgga cctacgagac 9001 attcaagagc atcatgaaga agtccccctt cagcggcccc accgacccta gacctccacc 9061 tagaagaatc gccgtgccca gcagatccag cgccagcgtg ccaaaacctg ccccccagcc 9121 ttaccccttc accagcagcc tgagcaccat caactacgac gagttcccta ccatggtgtt 9181 ccccagcggc cagatctctc aggcctctgc tctggctcca gcccctcctc aggtgctgcc 9241 tcaggctcct gctcctgcac cagctccagc catggtgtct gcactggctc aggcaccagc 9301 acccgtgcct gtgctggctc ctggacctcc acaggctgtg gctccaccag cccctaaacc 9361 tacacaggcc ggcgagggca cactgtctga agctctgctg cagctgcagt tcgacgacga 9421 ggatctggga gccctgctgg gaaacagcac cgatcctgcc gtgttcaccg acctggccag 9481 cgtggacaac agcgagttcc agcagctgct gaaccagggc atccctgtgg cccctcacac 9541 caccgagccc atgctgatgg aataccccga ggccatcacc cggctcgtga caggcgctca 9601 gaggcctcct gatccagctc ctgcccctct gggagcacca ggcctgccta atggactgct 9661 gtctggcgac gaggacttca gctctatcgc cgatatggat ttctcagcct tgctgggctc 9721 tggcagcggc agccgggatt ccagggaagg gatgtttttg ccgaagcctg aggccggctc 9781 cgctattagt gacgtgtttg agggccgcga ggtgtgccag ccaaaacgaa tccggccatt 9841 tcatcctcca ggaagtccat gggccaaccg cccactcccc gccagcctcg caccaacacc 9901 aaccggtcca gtacatgagc cagtcgggtc actgaccccg gcaccagtcc ctcagccact 9961 ggatccagcg cccgcagtga ctcccgaggc cagtcacctg ttggaggatc ccgatgaaga 10021 gacgagccag gctgtcaaag cccttcggga gatggccgat actgtgattc cccagaagga 10081 agaggctgca atctgtggcc aaatggacct ttcccatccg cccccaaggg gccatctgga 10141 tgagctgaca accacacttg agtccatgac cgaggatctg aacctggact cacccctgac 10201 cccggaattg aacgagattc tggatacctt cctgaacgac gagtgcctct tgcatgccat 10261 gcatatcagc acaggactgt ccatcttcga cacatctctg tttggatccg gcagcggcgc 10321 caccaacttc agcctgctga agcaggccgg cgacgtggag gagaaccccg gccccatggt 10381 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca 10441 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc 10501 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc 10561 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc 10621 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaaatggg agcgcgtgat 10681 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga 10741 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca 10801 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct 10861 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt 10921 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat 10981 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc 11041 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtgagaat tcgatatcaa 11101 gcttatcggt aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 11161 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat 11221 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta 11281 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc 11341 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt 11401 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg 11461 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc 11521 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc 11581 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct 11641 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca 11701 tcgataccgt cgacctcgag acctagaaaa acatggagca atcacaagta gcaatacagc 11761 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc 11821 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 11881 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat 11941 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc 12001 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga 12061 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag 12121 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct 12181 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga 12241 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 12301 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 12361 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc 12421 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 12481 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 12541 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 12601 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg 12661 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg 12721 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 12781 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 12841 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 12901 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 12961 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 13021 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 13081 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct 13141 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 13201 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 13261 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 13321 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 13381 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta 13441 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc 13501 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata 13561 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg 13621 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc 13681 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc 13741 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc 13801 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc 13861 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac 13921 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat 13981 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 14041 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 14101 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 14161 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 14221 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 14281 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 14341 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 14401 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 14461 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 14521 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 14581 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 14641 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 14701 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 14761 tttccccctg gaagctccct cgtgcgctct cctgtt //