LOCUS Exported 13668 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION single-vector system with EGFP reporter for use in CROPseq knockout screens. ACCESSION . VERSION . KEYWORDS CROPseq-Guide-EFS-SpCas9-P2A-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13668) AUTHORS Hewitt TITLE CROPseq-Guide-EFS-SpCas9-P2A-EGFP JOURNAL Unpublished REFERENCE 2 (bases 1 to 13668) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13668 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..17 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 1..17 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 36..56 /label=T3 /note="T3 promoter, forward primer" promoter 38..56 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 84..310 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 311..491 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 538..663 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1156..1389 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1574..1618 /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 1799..1916 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2105..2316 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 2335..2344 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2341..6444 /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 6445..6492 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 6493..6516 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 6526..6582 /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 6583..7299 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(6625..6646) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6886..6905) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7233..7254 /label=EGFP-C /note="EGFP, forward primer" misc_feature 7315..7903 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(7368..7388) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(7786..7797) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" promoter 8059..8299 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8059..8079 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8230..8249 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 10189..10264 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" LTR 10317..10497 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" polyA_signal 10575..10696 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(10612..10631) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 10666..10685 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 10736..10871 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10798..10817 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(10891..10910) /label=T7 /note="T7 promoter, forward primer" promoter complement(10892..10910) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(10920..10937) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(10920..10936) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(10929..10951) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 11078..11533 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11165..11184) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 11375..11396 /label=F1ori-F /note="F1 origin, forward primer" promoter 11559..11663 /gene="bla" /label=AmpR promoter CDS 11664..12524 /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 complement(11882..11901) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 12695..13283 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 13184..13203 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 13437..13454 /label=L4440 /note="L4440 vector, forward primer" protein_bind 13571..13592 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 13607..13637 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 13645..13661 /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 13650..4 /label=M13/pUC Reverse /note="In lacZ gene" ORIGIN 1 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac 61 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat 121 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt 181 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg 241 gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg 301 atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact 361 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc 421 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa 481 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc 541 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg 601 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc 661 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg 721 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc 781 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta 841 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc 901 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata 961 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc 1021 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa 1081 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa 1141 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat 1201 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca 1261 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt 1321 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca 1381 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg 1441 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga 1501 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca 1561 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg 1621 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg 1681 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca 1741 gggatattca ccattatcgt ttcagaatct tgagacaaat ggcagtattc atccacaatt 1801 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag 1861 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg 1921 tttattacag ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggtgcaaa 1981 gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga 2041 aaggagtggg aattggctcc gtaggtcttg aaaggagtgg gaattggctc cggtgcccgt 2101 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat 2161 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg 2221 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac 2281 gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag cgctgccacc 2341 atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg 2401 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg 2461 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag 2521 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc 2581 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 2641 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc 2701 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 2761 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac 2821 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 2881 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc 2941 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga 3001 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac 3061 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 3121 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 3181 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc 3241 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct 3301 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg 3361 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc 3421 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg 3481 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg 3541 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac 3601 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc 3661 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc 3721 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa 3781 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 3841 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 3901 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg 3961 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc 4021 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc 4081 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt 4141 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg 4201 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc 4261 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc 4321 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg 4381 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 4441 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg 4501 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca 4561 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 4621 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga 4681 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc 4741 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg 4801 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat 4861 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc 4921 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 4981 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg 5041 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag 5101 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac 5161 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc 5221 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac 5281 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 5341 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag 5401 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc 5461 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg 5521 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 5581 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 5641 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc 5701 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc 5761 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 5821 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac 5881 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 5941 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg 6001 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc 6061 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa 6121 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg 6181 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag 6241 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc 6301 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa 6361 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc 6421 gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct 6481 aagaagaaga aagattacaa agacgatgac gataagggat ccggcgcaac aaacttctct 6541 ctgctgaaac aagccggaga tgtcgaagag aatcctggac cggtgagcaa gggcgaggag 6601 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 6661 ttcagcgtgt ctggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 6721 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 6781 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 6841 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 6901 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 6961 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 7021 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc gaacttcaag 7081 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 7141 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 7201 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 7261 gccgggatca ctctcggcat ggacgagctg tacaagtaaa cgcgttaagt cgacaatcaa 7321 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt 7381 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct 7441 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 7501 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg 7561 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc 7621 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc 7681 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt 7741 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca 7801 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt 7861 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa 7921 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag 7981 ggctaattca ctcccaacga agacaagatt tcaccattat cgtttcagac ccacctccca 8041 accccgaggg gacccagaga gggcctattt cccatgattc cttcatattt gcatatacga 8101 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta 8161 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt 8221 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta 8281 tatatcttgt ggaaaggacg aaacaccgga gacggttgta aatgagcaca caaaatacac 8341 atgctaaaat attatattct atgaccttta taaaatcaac caaaatcttc tttttaataa 8401 ctttagtatc aataattaga atttttatgt tcctttttgc aaacttttaa taaaaatgag 8461 caaaataaaa aaacgctagt tttagtaact cgcgttgttt tcttcacctt taataatagc 8521 tactccacca cttgttccta agcggtcagc tcctgcttca atcatttttt gagcatcttc 8581 aaatgttcta actccaccag ctgctttaac taaagcattg tctttaacaa ctgacttcat 8641 tagtttaaca tcttcaaatg ttgcacctga ttttgaaaat cctgttgatg ttttaacaaa 8701 ttctaatcca gcttcaacag ctatttcaca agctttcatg atttcttctt ttgttaataa 8761 acaattttcc ataatacatt taacaacatg tgatccagct gcttttttta cagctttcat 8821 gtcttctaaa actaattcat aatttttgtc ttttaatgca ccaatattta ataccatatc 8881 aatttctgtt gcaccatctt taattgcttc agaaacttcg aatgcttttg tagctgttgt 8941 gcatgcacct agaggaaaac ctacaacatt tgttattcct acatttgtgc cttttaataa 9001 ttctttacaa tagcttgttc aatatgaatt aacacaaact gttgcaaaat caaattcaat 9061 tgcttcatca cataattgtt taatttcagc tttcgtagca tcttgtttta ataatgtgtg 9121 atctatatat ttgtttagtt tcattttttc tcctatatat tcatttttaa ttttaattct 9181 ttaataattt cgtctacttt aactttagcg ttttgaacag attcaccaac acctataaaa 9241 taaattttta gtttaggttc agttccactt gggcgaacag caaatcatga cttatcttct 9301 aaataaaatt ttagtaagtc ttgtcctggc atattataca ttccatcgat gtagtcttca 9361 acattaacaa ctttaagtcc agcaatttga gttaagggtg ttgctctcaa tgatttcatt 9421 aatggttcaa tttttaattt cttttcttct ggtttaaaat tcaagtttaa agtgaaagtg 9481 taatatgcac ccatttcttt aaataaatct tctaaatagt ctactaatgt tttattttgt 9541 tttttataaa atcaagcagc ctctgctatt aatatagaag cttgtattcc atctttatct 9601 ctagctgagt catcaattac atatccataa ctttcttcat aagcaaaaac aaaatttaat 9661 ccgttatctt cttctttagc aatttctcta cccattcatt taaatccagt taaagttttt 9721 acaatattaa ctccatattt ttcatgagcg attctatcac ccaaatcact tgttacaaaa 9781 cttgaatata gagccggatt ttttggaatg ctatttaagc gttttagatt tgataatttt 9841 caatcaatta aaattggtcc tgtttgattt ccatctaatc ttacaaaatg accatcatgt 9901 tttattgcca ttccaaatct gtcagcatct gggtcattca taataataat atctgcatca 9961 tgtttaatac catattcaag cggtattttt catgcaggat caaattctgg atttggattt 10021 acaacatttt taaatgtttc atcttcaaat gcatgctctt caacctcaat aacgttatat 10081 cctgattcac gtaatatttt tggggtaaat ttagttcctg ttccattaac tgcgctaaaa 10141 ataattttta aatctttttt agcttcttgc tcttttttgt acgtctctgt tttagagcta 10201 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 10261 gtgctttttt aagcttggcg taactagatc ttgagacact gctttttgct tgtactgggt 10321 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 10381 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 10441 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta 10501 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat 10561 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 10621 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 10681 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc 10741 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 10801 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 10861 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca 10921 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 10981 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 11041 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 11101 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 11161 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 11221 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 11281 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 11341 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 11401 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 11461 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 11521 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 11581 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 11641 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 11701 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 11761 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 11821 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 11881 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 11941 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 12001 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 12061 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 12121 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 12181 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 12241 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 12301 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 12361 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 12421 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 12481 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 12541 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 12601 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 12661 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 12721 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 12781 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 12841 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 12901 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 12961 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 13021 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 13081 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 13141 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 13201 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 13261 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 13321 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 13381 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 13441 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 13501 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 13561 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 13621 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcaca //