LOCUS Exported 10731 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of increased fidelity SpCas9-HF1-plus in bacterial cells. Cleaves both 20nt and 5'-extended 21nt spacer containing sgRNAs with close to the same fidelity as SpCas9-HF1.. ACCESSION . VERSION . KEYWORDS pET-FLAG-SpCas9-HF1-plus SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10731) AUTHORS Kulcsar , P.I., Talas, A., Toth, E., Nyeste A, Ligeti, Z., Welker, Z., Welker E. TITLE Blackjack mutations improve the on-target activities of all increased fidelity variants of SpCas9 without compromising their fidelities JOURNAL Nature Communications, 1223, 2020 REFERENCE 2 (bases 1 to 10731) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10731 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 538..557 /label=LacI-R /note="LacI, reverse primer" promoter complement(577..654) /gene="lacI" /label=lacI promoter primer_bind 860..879 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 963..982 /label=T7 /note="T7 promoter, forward primer" promoter 963..981 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 982..1006 /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)." RBS 1021..1043 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1054..1071 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1075..2172 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQT" primer_bind 2146..2169 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 2203..2223 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2236..2301 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 2308..2328 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2353..4068 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 6433..6480 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(6508..6525) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" CDS 6558..6575 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6628..6646) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6642..6689 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 6726..7181 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6813..6832) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7023..7044 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(7273..8088) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 7996..8015 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 8080..8099 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 8210..8798 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8699..8718 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8952..8969 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8984..9123 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(9049..9068) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 9209..9231 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(9225..9416) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(10225..576) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" ORIGIN 1 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg 61 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg 121 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc 181 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt 241 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct 301 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag 361 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc 421 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt 481 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc 541 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc 601 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat 661 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc 721 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc 781 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc 841 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg 901 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa 961 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat 1021 tttgtttaac tttaagaagg agatatacat atgcaccatc accatcacca tggaaaaatc 1081 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa 1141 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa 1201 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg 1261 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac 1321 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag 1381 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg 1441 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa 1501 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct 1561 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc 1621 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa 1681 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca 1741 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat 1801 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg 1861 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac 1921 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta 1981 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa 2041 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc 2101 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa 2161 gacgcgcaga ctaattcgag ctcgaacaac aacaacacta gtgaaaacct gtatttccag 2221 ggagcagcct cgatggacta taaggaccac gacggagact acaaggatca tgatattgat 2281 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga 2341 gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc 2401 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac 2461 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa 2521 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac 2581 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc 2641 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc 2701 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac 2761 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc 2821 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac 2881 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag 2941 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc 3001 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg 3061 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac 3121 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac 3181 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc 3241 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg 3301 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct 3361 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac 3421 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag 3481 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac 3541 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga 3601 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg 3661 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg 3721 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac 3781 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccgcc 3841 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac 3901 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc 3961 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg 4021 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc 4081 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg 4141 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa 4201 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa 4261 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc 4321 ggctggggca ggctgagccg gaagctgatc aacggcatcc gcgacaagca gtccggcaag 4381 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat ggcgctgatc 4441 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc 4501 gatagcctgc acgagcacat tgccaatctc gccggcagcc ccgccattaa gaagggcatc 4561 ctccagacag tgaaggtggt ggacgagctg gtgaaagtga tgggccggca caagcccgag 4621 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagt 4681 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa 4741 gaacaccccg tggaaaacac ccagctccag aacgagaagc tgtacctgta ctacctccag 4801 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat 4861 gtggaccata tcgtgcctca gagctttctt aaggacgact ccatcgacaa caaggtgctg 4921 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag 4981 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc 5041 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc 5101 aagagacagc tggtggaaac ccgggcgatc acaaagcacg tggcacagat cctggactcc 5161 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc 5221 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag 5281 atcaacaact accaccacgc ccacgacgcg tacctgaacg ccgtcgtggg aaccgccctg 5341 atcaaaaagt accctaagct gggtggcaag gtgtacgatg tacggaagat gatcgccaag 5401 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 5461 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 5521 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 5581 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 5641 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 5701 gactgggatc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 5761 gtggtggcca aagtggaaaa gggcaagtcc aagaagctta agagtgtgaa agagctgctg 5821 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 5881 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctcgctcttc 5941 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 6001 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 6061 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 6121 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 6181 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 6241 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 6301 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 6361 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 6421 ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 6481 taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 6541 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc 6601 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg 6661 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga 6721 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 6781 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 6841 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 6901 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 6961 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 7021 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 7081 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 7141 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga 7201 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 7261 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc 7321 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc 7381 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac 7441 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc 7501 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg 7561 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt 7621 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca 7681 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc 7741 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag 7801 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc 7861 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc 7921 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc 7981 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga 8041 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt 8101 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt 8161 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 8221 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 8281 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 8341 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 8401 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 8461 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 8521 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 8581 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 8641 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 8701 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 8761 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 8821 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 8881 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 8941 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc 9001 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc 9061 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct 9121 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 9181 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 9241 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat 9301 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat 9361 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat 9421 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg 9481 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt 9541 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag 9601 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag 9661 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg 9721 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt 9781 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct 9841 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg 9901 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg 9961 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga 10021 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg 10081 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag 10141 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc 10201 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 10261 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag 10321 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc 10381 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt 10441 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac 10501 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc 10561 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat 10621 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat 10681 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg a //