LOCUS Exported 13181 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses human codon-optimized S. pyogenes Cas9 protein and Venus reporter from EFS promoter. Lentiviral backbone.. ACCESSION . VERSION . KEYWORDS lentiCas9-Venus SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13181) AUTHORS Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan GC, Zhang F, Orkin SH, Bauer DE TITLE BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. JOURNAL Nature. 2015 Sep 16. doi: 10.1038/nature15521. PUBMED 26375006 REFERENCE 2 (bases 1 to 13181) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13181 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..66) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 772..792 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2098..2142 /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 2440..2557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2651..2862 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 2881..2890 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2887..6990 /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 6991..7038 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 7039..7062 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 7072..7128 /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 7129..7848 /codon_start=1 /product="yellow fluorescent protein (YFP) with fast and efficient maturation (Nagai et al., 2002)" /label=Venus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(7174..7195) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(7435..7454) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7782..7803 /label=EGFP-C /note="EGFP, forward primer" misc_feature 7873..8461 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(7926..7946) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(8344..8355) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(8464..8480) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8465..8481) /label=pBluescriptKS /note="For pBluescript vector" LTR 8986..9166 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(9192..9209) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 9198..9422 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9468..9896 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9555..9574) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 9765..9786 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(9905..9925) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 9910..10239 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 10090..10225 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10152..10171 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 10287..10334 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10353..10727 /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 10857..10978 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(10894..10913) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 10948..10967 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(11027..11043) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11027..11043) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11040..11062) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11051..11067 /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(11075..11105) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11120..11141 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11258..11275) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11429..12017) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11509..11528) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12188..13048) /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 12811..12830 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13049..13153) /gene="bla" /label=AmpR promoter ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2581 agatccagtt tggttaatta agctagctag gtcttgaaag gagtgggaat tggctccggt 2641 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc 2701 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg 2761 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc 2821 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct 2881 gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg 2941 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc 3001 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca 3061 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg 3121 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc 3181 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc 3241 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg 3301 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg 3361 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac 3421 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa 3481 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag 3541 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc 3601 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg 3661 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg 3721 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac 3781 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc 3841 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc 3901 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc 3961 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc 4021 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg 4081 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag 4141 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa 4201 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga 4261 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc 4321 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc 4381 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc 4441 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc 4501 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa 4561 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg 4621 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg 4681 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat 4741 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc 4801 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc 4861 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca 4921 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac 4981 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat 5041 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg 5101 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac 5161 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc 5221 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa 5281 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat 5341 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg 5401 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc 5461 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag 5521 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac 5581 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag 5641 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg 5701 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg 5761 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc 5821 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc 5881 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg 5941 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc 6001 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg 6061 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg 6121 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc 6181 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag 6241 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc 6301 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag 6361 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc 6421 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg 6481 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc 6541 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg 6601 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt 6661 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag 6721 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg 6781 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg 6841 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc 6901 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca 6961 cggatcgacc tgtctcagct gggaggcgac aagcgacctg ccgccacaaa gaaggctgga 7021 caggctaaga agaagaaaga ttacaaagac gatgacgata agggatccgg cgcaacaaac 7081 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat ggtgagcaag 7141 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 7201 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 7261 ctgaagctga tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 7321 ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc 7381 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 7441 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 7501 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 7561 aactacaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc 7621 aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag 7681 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac 7741 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 7801 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaga attcgatatc 7861 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 7921 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 7981 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 8041 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 8101 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 8161 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 8221 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt 8281 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 8341 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 8401 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 8461 catcgatacc gtcgacctcg agacctagaa aaacatggag caatcacaag tagcaataca 8521 gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt 8581 ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc 8641 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat 8701 atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca 8761 ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt 8821 gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg 8881 agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc 8941 ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta 9001 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa 9061 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac 9121 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac 9181 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 9241 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 9301 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 9361 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 9421 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag 9481 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 9541 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 9601 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 9661 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 9721 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 9781 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc 9841 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt 9901 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 9961 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 10021 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 10081 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 10141 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag 10201 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata 10261 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca 10321 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg 10381 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct 10441 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca 10501 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg 10561 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct 10621 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg 10681 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag 10741 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg 10801 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact 10861 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 10921 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 10981 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 11041 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 11101 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 11161 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 11221 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 11281 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 11341 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 11401 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 11461 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 11521 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 11581 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 11641 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 11701 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 11761 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 11821 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 11881 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 11941 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 12001 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 12061 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 12121 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 12181 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 12241 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 12301 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 12361 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 12421 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 12481 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 12541 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 12601 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 12661 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 12721 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 12781 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 12841 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 12901 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 12961 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 13021 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 13081 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 13141 taggggttcc gcgcacattt ccccgaaaag tgccacctga c //