LOCUS Exported 14784 bp ds-DNA circular SYN 17-JUN-2022 DEFINITION 3rd generation transfer vector. Co-expresses human optimized S. pyogenes Cas9 and GFP. ACCESSION . VERSION . KEYWORDS pLV hUbC-Cas9-T2A-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14784) AUTHORS Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA TITLE Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. JOURNAL Nucleic Acids Res. 2014 Aug 13. pii: gku749. PUBMED 25122746 REFERENCE 2 (bases 1 to 14784) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 17, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14784 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1..4104 /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 4117..4137 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4144..4197 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 4204..4917 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 4204..4917 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(4246..4267) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4507..4526) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4854..4875 /label=EGFP-C /note="EGFP, forward primer" protein_bind 4933..4966 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 4991..5579 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5044..5064) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5462..5473) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(5582..5598) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5583..5599) /label=pBluescriptKS /note="For pBluescript vector" LTR 6104..6284 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(6310..6327) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6316..6540 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 6586..7014 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6673..6692) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6883..6904 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7023..7043) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 7028..7357 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 7208..7343 /label=SV40 ori /note="SV40 origin of replication" primer_bind 7270..7289 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 7405..7452 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 7471..7845 /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 7975..8096 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8012..8031) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 8066..8085 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(8145..8161) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 8169..8185 /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(8193..8223) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8238..8259 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(8376..8393) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(8547..9135) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8627..8646) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9306..10166) /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 9929..9948 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10167..10271) /gene="bla" /label=AmpR promoter primer_bind complement(10346..10365) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 10537..10916 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 10918..11116 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 11071..11091 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 11134..11314 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 11361..11486 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 11979..12212 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 12397..12441 /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 12739..12856 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 12901..12934 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(12921..13247) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATTVSY HNFV" primer_bind 13097..13119 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 13111..13128 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 13112..13128 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 13138..13157 /label=T7 /note="T7 promoter, forward primer" promoter 13138..13156 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(13204..13224) /label=T3 /note="T3 promoter, forward primer" promoter complement(13204..13222) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(13243..13259) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 13267..13283 /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(13291..13321) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 13336..13357 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 13434..14645 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 14482..14501 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" regulatory 14674..14683 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 14683..14748 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 14755..14775 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" ORIGIN 1 atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 61 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 121 cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga aaccgccgaa 181 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 241 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 301 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 361 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 421 aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 481 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 541 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 601 atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 661 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 721 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 781 gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 841 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 901 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 961 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1021 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1081 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1141 gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1201 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1261 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1321 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1381 agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1441 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1501 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1561 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1621 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1681 gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1741 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1801 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1861 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1921 catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1981 cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2041 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2101 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2161 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2221 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2281 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2341 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2401 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2461 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2521 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2581 gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2641 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2701 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2761 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2821 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2881 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2941 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3001 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3061 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3121 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3181 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3241 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3301 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3361 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3421 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3481 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3541 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3601 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3661 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3721 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3781 caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3841 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3901 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3961 cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4021 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4081 gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtggct 4141 agcgagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctggt 4201 accgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 4261 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 4321 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 4381 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 4441 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 4501 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 4561 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 4621 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 4681 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 4741 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 4801 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 4861 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagacc 4921 ggttgataat agataacttc gtatagcata cattatacga agttatgaat tcgatatcaa 4981 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 5041 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat 5101 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta 5161 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc 5221 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt 5281 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg 5341 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc 5401 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc 5461 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct 5521 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca 5581 tcgataccgt cgacctcgag acctagaaaa acatggagca atcacaagta gcaatacagc 5641 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc 5701 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 5761 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat 5821 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc 5881 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga 5941 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag 6001 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct 6061 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga 6121 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 6181 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 6241 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc 6301 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 6361 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 6421 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 6481 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg 6541 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg 6601 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6661 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6721 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6781 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 6841 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6901 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 6961 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct 7021 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 7081 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 7141 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 7201 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 7261 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta 7321 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc 7381 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata 7441 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg 7501 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc 7561 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc 7621 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc 7681 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc 7741 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac 7801 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat 7861 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 7921 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 7981 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 8041 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 8101 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 8161 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 8221 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 8281 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 8341 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 8401 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 8461 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 8521 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 8581 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 8641 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 8701 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 8761 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 8821 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 8881 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 8941 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 9001 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 9061 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 9121 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 9181 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 9241 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 9301 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 9361 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 9421 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 9481 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 9541 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 9601 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 9661 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 9721 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 9781 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 9841 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 9901 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 9961 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 10021 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 10081 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 10141 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 10201 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 10261 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc 10321 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 10381 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta 10441 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 10501 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt 10561 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 10621 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 10681 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 10741 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 10801 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 10861 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 10921 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 10981 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 11041 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 11101 aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga 11161 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 11221 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 11281 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 11341 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 11401 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 11461 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 11521 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 11581 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 11641 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 11701 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 11761 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 11821 aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa 11881 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag 11941 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc 12001 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga 12061 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa 12121 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct 12181 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc 12241 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt 12301 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata 12361 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa 12421 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata 12481 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt 12541 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca 12601 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga 12661 gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat 12721 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga 12781 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac 12841 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggttaattaa 12901 ataacttcgt atagcataca ttatacgaag ttatgataag agacggtggt ggcgccgcta 12961 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 13021 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 13081 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa 13141 tacgactcac tatagggcga attgggtacc gggccccccc tcgaggtcct ccagcttttg 13201 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt 13261 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 13321 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 13381 tttccactgc atgacgtctc cacaattaat taacccgtgt cggctccaga tctggcctcc 13441 gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg ctgccacgtc 13501 agacgaaggg cgcagcgagc gtcctgatcc ttccgcccgg acgctcagga cagcggcccg 13561 ctgctcataa gactcggcct tagaacccca gtatcagcag aaggacattt taggacggga 13621 cttgggtgac tctagggcac tggttttctt tccagagagc ggaacaggcg aggaaaagta 13681 gtcccttctc ggcgattctg cggagggatc tccgtggggc ggtgaacgcc gatgattata 13741 taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt 13801 cttgtttgtg gatcgctgtg atcgtcactt ggtgagtagc gggctgctgg gctggccggg 13861 gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt gtggagagac cgccaagggc 13921 tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg gttgggggga gcgcagcaaa 13981 atggcggctg ttcccgagtc ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt 14041 gaaacaaggt ggggggcatg gtgggcggca agaacccaag gtcttgaggc cttcgctaat 14101 gcgggaaagc tcttattcgg gtgagatggg ctggggcacc atctggggac cctgacgtga 14161 agtttgtcac tgactggaga actcggtttg tcgtctgttg cgggggcggc agttatggcg 14221 gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt 14281 cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg 14341 cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg 14401 cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat 14461 gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga 14521 gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta 14581 attttcagtg ttagactagt aaattgtccg ctaaattctg gccgtttttg gcttttttgt 14641 tagacgaagc ttgggctgca ggtcgactct agagccacca tggactacaa agaccatgac 14701 ggtgattata aagatcatga catcgattac aaggatgacg atgacaagat ggcccccaag 14761 aagaagagga aggtgggccg cgga //