LOCUS Exported 15243 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Catalytically inactive Cas9 from S. pyogenes with P2A-BlastR under the hPGK promoter, and cloning backbone for sgRNA. Contains BsmBI sites for insertion of spacer sequences.. ACCESSION . VERSION . KEYWORDS Lenti-(BB)-hPGK-KRAB-dCas9-P2A-BlastR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15243) AUTHORS Miguel-Escalada I, Bonas-Guarch S, Cebola I, Ponsa-Cobas J, Mendieta-Esteban J, Atla G, Javierre BM, Rolando DMY, Farabella I, Morgan CC, Garcia-Hurtado J, Beucher A, Moran I, Pasquali L, Ramos-Rodriguez M, Appel EVR, Linneberg A, Gjesing AP, Witte DR, Pedersen O, Grarup N, Ravassard P, Torrents D, Mercader JM, Piemonti L, Berney T, de Koning EJP, Kerr-Conte J, Pattou F, Fedko IO, Groop L, Prokopenko I, Hansen T, Marti-Renom MA, Fraser P, Ferrer J TITLE Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes. JOURNAL Nat Genet. 2019 Jun 28. pii: 10.1038/s41588-019-0457-0. doi: 10.1038/s41588-019-0457-0. PUBMED 31253982 REFERENCE 2 (bases 1 to 15243) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15243 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 14..524 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 443..461 /label=hPGK-F /note="Human PGK promoter, forward primer" regulatory 537..546 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 573..758 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP" CDS 780..2498 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 4887..4913 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 4932..4952 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4959..4979 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5025..5081 /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 5082..5480 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 5505..6093 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5558..5578) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5976..5987) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6165..6398 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(6424..6441) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6430..6654 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 6700..7128 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6787..6806) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6997..7018 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7137..7157) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 7142..7471 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 7322..7457 /label=SV40 ori /note="SV40 origin of replication" primer_bind 7384..7403 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 7519..7566 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 7585..7959 /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 8089..8210 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8126..8145) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 8180..8199 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(8259..8275) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8259..8275) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(8272..8294) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 8283..8299 /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(8307..8337) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8352..8373 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(8490..8507) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(8661..9249) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8741..8760) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9420..10280) /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 10043..10062 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10281..10385) /gene="bla" /label=AmpR promoter primer_bind complement(10460..10479) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 10651..11030 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 11032..11230 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 11185..11205 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 11248..11428 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 11475..11600 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 12093..12326 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 12511..12555 /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 12853..12970 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 13021..13261 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 13021..13041 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 13192..13211 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 15151..15226 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 ctaggtcttg aaaggggttg gggttgcgcc ttttccaagg cagccctggg tttgcgcagg 61 gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggtctcgca 121 cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc 181 cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc 241 ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga cagcgccagg 301 gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc 361 gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt 421 gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg 481 cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccaggaccgg tgatccgcca 541 ccatggatgc taagtcacta actgcctggt cccggacact ggtgaccttc aaggatgtat 601 ttgtggactt caccagggag gagtggaagc tgctggacac tgctcagcag atcgtgtaca 661 gaaatgtgat gctggagaac tataagaacc tggtttcctt gggttatcag cttactaagc 721 cagatgtgat cctccggttg gagaagggag aagagcccgg cggttccggc ggagggtcga 781 tggacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtcggc tgggccgtga 841 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc 901 acagcatcaa gaagaacctg atcggcgccc tgctgttcga cagcggagaa acagccgagg 961 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct 1021 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac 1081 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca 1141 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga 1201 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca 1261 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg 1321 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca 1381 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac 1441 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc 1501 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg 1561 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc 1621 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc 1681 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta 1741 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc 1801 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg 1861 gctacatcga tggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg 1921 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga 1981 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg 2041 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg 2101 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca 2161 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag 2221 tggtggacaa gggcgccagc gcccagagct tcatcgagcg gatgaccaac ttcgataaga 2281 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt 2341 acaacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga 2401 gcggcgagca gaaaaaagcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg 2461 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct 2521 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta 2581 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc 2641 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc 2701 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca 2761 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg 2821 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca 2881 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc 2941 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag 3001 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga 3061 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa 3121 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg 3181 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg 3241 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggacgcta 3301 tcgtgcctca gagctttctg aaggacgact ccatcgataa caaagtgctg actcggagcg 3361 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga 3421 actactggcg ccagctgctg aatgccaagc tgattaccca gaggaagttc gacaatctga 3481 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc 3541 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca 3601 ctaagtacga cgagaacgac aaactgatcc gggaagtgaa agtgatcacc ctgaagtcca 3661 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact 3721 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt 3781 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga 3841 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca 3901 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc 3961 ctctgatcga gacaaacggc gaaacaggcg agatcgtgtg ggataagggc cgggactttg 4021 ccaccgtgcg gaaagtgctg tctatgcccc aagtgaatat cgtgaaaaag accgaggtgc 4081 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgac aagctgatcg 4141 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct 4201 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga 4261 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact 4321 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt 4381 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc 4441 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc 4501 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac 4561 agcacaaaca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga 4621 tcctggccga cgctaatctg gacaaggtgc tgagcgccta caacaagcac agagacaagc 4681 ctatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccagt ctgggagccc 4741 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag 4801 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg 4861 acctgtctca gctgggaggc gacgcctatc cctatgacgt gcccgattat gccagcctgg 4921 gcagcggctc ccccaagaaa aaacgcaagg tggaagatcc taagaaaaag cggaaagtgg 4981 acggcattgg tagtgggagc aacggcagca gcggggatcc ggccgcaaca aacttctctc 5041 tgctgaaaca agccggagat gtcgaagaga atcctggacc gatggccaag cctttgtctc 5101 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg 5161 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca 5221 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg 5281 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct 5341 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca 5401 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg 5461 gttatgtgtg ggagggctaa gaattcgata tcaagcttat cggtaatcaa cctctggatt 5521 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 5581 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 5641 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 5701 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 5761 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 5821 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 5881 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 5941 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 6001 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 6061 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa 6121 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 6181 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat 6241 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 6301 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 6361 cctcagaccc ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa acccgctgat 6421 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 6481 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 6541 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 6601 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 6661 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 6721 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6781 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6841 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6901 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6961 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 7021 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 7081 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 7141 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 7201 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 7261 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 7321 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 7381 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 7441 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 7501 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa 7561 tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc 7621 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac 7681 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg 7741 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac 7801 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg 7861 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc 7921 ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat 7981 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg 8041 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt 8101 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 8161 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt 8221 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga 8281 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 8341 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 8401 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 8461 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 8521 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 8581 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 8641 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 8701 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 8761 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 8821 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 8881 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 8941 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 9001 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 9061 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 9121 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 9181 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 9241 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 9301 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 9361 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 9421 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 9481 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 9541 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 9601 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 9661 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 9721 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 9781 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 9841 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 9901 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 9961 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 10021 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 10081 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 10141 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 10201 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 10261 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 10321 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 10381 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc 10441 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc 10501 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa 10561 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc 10621 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt 10681 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 10741 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 10801 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 10861 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta 10921 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg 10981 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 11041 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 11101 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 11161 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 11221 atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat ctgagcctgg 11281 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg 11341 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc 11401 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag 11461 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag 11521 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga 11581 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa 11641 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca 11701 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac 11761 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat 11821 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg 11881 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg 11941 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat 12001 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga 12061 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga 12121 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta 12181 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat 12241 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa 12301 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc 12361 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat 12421 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc 12481 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat 12541 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta 12601 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata 12661 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg 12721 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga 12781 tccattcgat tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac aaatggcagt 12841 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat 12901 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat 12961 tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttaa ttaaggtacc 13021 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 13081 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 13141 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 13201 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 13261 cgaaacaccg gagacggttg taaatgagca cacaaaatac acatgctaaa atattatatt 13321 ctatgacctt tataaaatca accaaaatct tctttttaat aactttagta tcaataatta 13381 gaatttttat gttccttttt gcaaactttt aataaaaatg agcaaaataa aaaaacgcta 13441 gttttagtaa ctcgcgttgt tttcttcacc tttaataata gctactccac cacttgttcc 13501 taagcggtca gctcctgctt caatcatttt ttgagcatct tcaaatgttc taactccacc 13561 agctgcttta actaaagcat tgtctttaac aactgacttc attagtttaa catcttcaaa 13621 tgttgcacct gattttgaaa atcctgttga tgttttaaca aattctaatc cagcttcaac 13681 agctatttca caagctttca tgatttcttc ttttgttaat aaacaatttt ccataataca 13741 tttaacaaca tgtgatccag ctgctttttt tacagctttc atgtcttcta aaactaattc 13801 ataatttttg tcttttaatg caccaatatt taataccata tcaatttctg ttgcaccatc 13861 tttaattgct tcagaaactt cgaatgcttt tgtagctgtt gtgcatgcac ctagaggaaa 13921 acctacaaca tttgttattc ctacatttgt gccttttaat aattctttac aatagcttgt 13981 tcaatatgaa ttaacacaaa ctgttgcaaa atcaaattca attgcttcat cacataattg 14041 tttaatttca gctttcgtag catcttgttt taataatgtg tgatctatat atttgtttag 14101 tttcattttt tctcctatat attcattttt aattttaatt ctttaataat ttcgtctact 14161 ttaactttag cgttttgaac agattcacca acacctataa aataaatttt tagtttaggt 14221 tcagttccac ttgggcgaac agcaaatcat gacttatctt ctaaataaaa ttttagtaag 14281 tcttgtcctg gcatattata cattccatcg atgtagtctt caacattaac aactttaagt 14341 ccagcaattt gagttaaggg tgttgctctc aatgatttca ttaatggttc aatttttaat 14401 ttcttttctt ctggtttaaa attcaagttt aaagtgaaag tgtaatatgc acccatttct 14461 ttaaataaat cttctaaata gtctactaat gttttatttt gttttttata aaatcaagca 14521 gcctctgcta ttaatataga agcttgtatt ccatctttat ctctagctga gtcatcaatt 14581 acatatccat aactttcttc ataagcaaaa acaaaattta atccgttatc ttcttcttta 14641 gcaatttctc tacccattca tttaaatcca gttaaagttt ttacaatatt aactccatat 14701 ttttcatgag cgattctatc acccaaatca cttgttacaa aacttgaata tagagccgga 14761 ttttttggaa tgctatttaa gcgttttaga tttgataatt ttcaatcaat taaaattggt 14821 cctgtttgat ttccatctaa tcttacaaaa tgaccatcat gttttattgc cattccaaat 14881 ctgtcagcat ctgggtcatt cataataata atatctgcat catgtttaat accatattca 14941 agcggtattt ttcatgcagg atcaaattct ggatttggat ttacaacatt tttaaatgtt 15001 tcatcttcaa atgcatgctc ttcaacctca ataacgttat atcctgattc acgtaatatt 15061 tttggggtaa atttagttcc tgttccatta actgcgctaa aaataatttt taaatctttt 15121 ttagcttctt gctctttttt gtacgtctct gttttagagc tagaaatagc aagttaaaat 15181 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcgc 15241 tag //