LOCUS Exported 7198 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Provides the human codon optimized CDS of Cas9 protein as a level 0 GoldenBraid part. ACCESSION . VERSION . KEYWORDS phumanCas9 (GB0575) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7198) AUTHORS Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D TITLE GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. JOURNAL Plant Physiol. 2013 Jul;162(3):1618-31. PUBMED 23669743 REFERENCE 2 (bases 1 to 7198) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7198 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(99..117) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(100..117) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(135..151) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(135..151) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(148..170) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 159..175 /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(183..213) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 228..249 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(366..383) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(537..1125) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(617..636) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1296..2156) /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 1919..1938 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2157..2261) /gene="bla" /label=AmpR promoter rep_origin complement(2339..2794) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2476..2497) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2688..2707 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2920..2942 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2934..2951 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2935..2951 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 2958..2977 /label=T7 /note="T7 promoter, forward primer" promoter 2958..2976 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 3007..3016 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 3011..3020 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3055..7158 /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 7171..7191 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" ORIGIN 1 cgagaccact catcgcccaa tcactagtga attcgcggcc gcctgcaggt cgaccatatg 61 ggagagctcc caacgcgttg gatgcatagc ttgagtattc tatagtgtca cctaaatagc 121 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 181 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 241 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 301 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 361 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 421 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 481 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 541 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 601 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 661 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 721 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 781 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 841 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 901 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 961 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 1021 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 1081 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1141 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 1201 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 1261 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 1321 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 1381 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgcgac 1441 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1501 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1561 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1621 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1681 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 1741 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 1801 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 1861 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 1921 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 1981 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 2041 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 2101 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 2161 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 2221 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 2281 ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa 2341 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt 2401 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata 2461 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac 2521 gtcaaagggc gaaaaaccgt ctatcagggc gctggcccac tacgtgaacc atcaccctaa 2581 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc 2641 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg 2701 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca 2761 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact 2821 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat 2881 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa 2941 cgacggccag tgaattgtaa tacgactcac tatagggcga attgggcccg acgtcgcatg 3001 ctcccggccg ccatggcggc cgcgggaatt cgattgggcg atgagtggtc tcgaatggac 3061 aagaagtact ccattgggct cgatatcggc acaaacagcg tcggctgggc cgtcattacg 3121 gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga tcgccacagc 3181 ataaagaaga acctcattgg cgccctcctg ttcgactccg gggaaacggc cgaagccacg 3241 cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat ctgctacctg 3301 caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca taggctggag 3361 gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt tggcaatatc 3421 gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag gaagaagctt 3481 gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc gcatatgatc 3541 aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag cgatgtcgac 3601 aaactcttta tccaactggt tcagacttac aatcagcttt tcgaagagaa cccgatcaac 3661 gcatccggag ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc ccggcggctc 3721 gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg taatcttatc 3781 gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc cgaagatgcc 3841 aagcttcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct ggcccagatc 3901 ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc cattctgctg 3961 agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc tagtatgatc 4021 aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt cagacagcaa 4081 ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta cgccggatac 4141 attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat cttggaaaaa 4201 atggacggca ccgaggagct gctggtaaag cttaacagag aagatctgtt gcgcaaacag 4261 cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact gcacgctatc 4321 ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa gattgagaaa 4381 atcctcacat ttcggatacc ctactatgta ggccccctcg cccggggaaa ttccagattc 4441 gcgtggatga ctcgcaaatc agaagagact atcactccct ggaacttcga ggaagtcgtg 4501 gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga taaaaatctg 4561 cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac agtttataac 4621 gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt cctgtctgga 4681 gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt taccgtgaaa 4741 cagctcaaag aagattattt caaaaagatt gaatgtttcg actctgttga aatcagcgga 4801 gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa aatcattaaa 4861 gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat tgtcctcacc 4921 cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta cgctcatctc 4981 ttcgacgaca aagtcatgaa acagctcaag aggcgccgat atacaggatg ggggcggctg 5041 tcaagaaaac tgatcaatgg gatccgagac aagcagagtg gaaagacaat cctggatttt 5101 cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga tgactctctc 5161 acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag tctccacgag 5221 cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca gaccgttaag 5281 gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat cgttatcgag 5341 atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga aaggatgaag 5401 aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca cccagttgaa 5461 aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg cagggacatg 5521 tacgtggatc aggaactgga catcaatcgg ctctccgact acgacgtgga tcatatcgtg 5581 ccccagtctt ttctcaaaga tgattctatt gataataaag tgttgacaag atccgataaa 5641 aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat gaaaaattat 5701 tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa tctgactaag 5761 gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag gcagcttgtt 5821 gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat gaacaccaag 5881 tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa gtctaagctg 5941 gtttcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa caattaccac 6001 catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa aaaatatccc 6061 aagcttgaat ctgaatttgt ttacggagac tataaagtgt acgatgttag gaaaatgatc 6121 gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta cagcaatatt 6181 atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa gcgaccactt 6241 atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga tttcgcgaca 6301 gtccggaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga agtacagacc 6361 ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct gatcgcacgc 6421 aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt cgcttacagt 6481 gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag cgtcaaggaa 6541 ctgctgggca tcacaatcat ggagcgatca agcttcgaaa aaaaccccat cgactttctc 6601 gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc caagtactct 6661 ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga gctgcagaaa 6721 ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc cagccactat 6781 gaaaagctca aaggatctcc cgaagataat gagcagaagc agctgttcgt ggaacaacac 6841 aaacactacc ttgatgagat catcgagcaa ataagcgaat tctccaaaag agtgatcctc 6901 gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga taagcccatc 6961 agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg cgcgcctgca 7021 gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac aaaggaggtc 7081 ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag aatcgacctc 7141 tctcagctcg gtggagacag cagggctgac cccaagaaga agaggaaggt gtgagctt //