LOCUS Exported 6765 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pCas9CR5 with mutations to change PAM site specificity to NG from Nishimasu et al.. ACCESSION . VERSION . KEYWORDS pCas9CR5-NG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6765) TITLE pCas9CR4 variants for increased targeting space JOURNAL Unpublished REFERENCE 2 (bases 1 to 6765) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6765 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(7..630) /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" primer_bind 538..557 /label=Tet-R /note="Tetracycline resistance gene, reverse primer" promoter 681..736 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 717..735 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 772..2490 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" terminator 4930..5001 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 5017..5044 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(5072..5089) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5206..5751) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(5286..5305) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 5865..5959 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5980..6639) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 6554..6573 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(6640..6742) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" ORIGIN 1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 601 aatcacttta cttttatcta atctagacat cattaattcc taattgctag cattgtacct 661 aggactgagc tagccataaa gttgacactc tatcgttgat agagttattt taccactccc 721 tatcagtgat agagaaaaga attcaaaaga tctaaagagg acttcggatc tatggataag 781 aaatactcaa taggcttaga tatcggcaca aatagcgtcg gatgggcggt gatcactgat 841 gaatataagg ttccgtctaa aaagttcaag gttctgggaa atacagaccg ccacagtatc 901 aaaaaaaatc ttataggggc tcttttattt gacagtggag agacagcgga agcgactcgt 961 ctcaaacgga cagctcgtag aaggtataca cgtcggaaga atcgtatttg ttatctacag 1021 gagatttttt caaatgagat ggcgaaagta gatgatagtt tctttcatcg acttgaagag 1081 tcttttttgg tggaagaaga caagaagcat gaacgtcatc ctatttttgg aaatatagta 1141 gatgaagttg cttatcatga gaaatatcca actatctatc atctgcgaaa aaaattggta 1201 gattctactg ataaagcgga tttgcgctta atctatttgg ccttagcgca tatgattaag 1261 tttcgtggtc attttttgat tgagggagat ttaaatcctg ataatagtga tgtggacaaa 1321 ctatttatcc agttggtaca aacctacaat caattatttg aagaaaaccc tattaacgca 1381 agtggagtag atgctaaagc gattctttct gcacgattga gtaaatcaag acgattagaa 1441 aatctcattg ctcagctccc cggtgagaag aaaaatggct tatttgggaa tctcattgct 1501 ttgtcattgg gtttgacccc taattttaaa tcaaattttg atttggcaga agatgctaaa 1561 ttacagcttt caaaagatac ttacgatgat gatttagata atttattggc gcaaattgga 1621 gatcaatatg ctgatttgtt tttggcagct aagaatttat cagatgctat tttactttca 1681 gatatcctaa gagtaaatac tgaaataact aaggctcccc tatcagcttc aatgattaaa 1741 cgctacgatg aacatcatca agacttgact cttttaaaag ctttagttcg acaacaactt 1801 ccagaaaagt ataaagaaat cttttttgat caatcaaaaa acggatatgc aggttatatt 1861 gatgggggag ctagccaaga agaattttat aaatttatca aaccaatttt agaaaaaatg 1921 gatggtactg aggaattatt ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg 1981 acctttgaca acggctctat tccccatcaa attcacttgg gtgagctgca tgctattttg 2041 agaagacaag aagactttta tccattttta aaagacaatc gtgagaagat tgaaaaaatc 2101 ttgacttttc gaattcctta ttatgttggt ccattggcgc gtggcaatag tcgttttgca 2161 tggatgactc ggaagtctga agaaacaatt accccatgga attttgaaga agttgtcgat 2221 aaaggtgctt cagctcaatc atttattgaa cgcatgacaa actttgataa aaatcttcca 2281 aatgaaaaag tactaccaaa acatagtttg ctttatgagt attttacggt ttataacgaa 2341 ttgacaaagg tcaaatatgt tactgaagga atgcgaaaac cagcatttct ttcaggtgaa 2401 cagaagaaag ccattgttga tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa 2461 ttaaaagaag attatttcaa aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt 2521 gaagatagat ttaatgcttc attaggtacc taccatgatt tgctaaaaat tattaaagat 2581 aaagattttt tggataatga agaaaatgaa gatatcttag aggatattgt tttaacattg 2641 accttatttg aagataggga gatgattgag gaaagactta aaacatatgc tcacctcttt 2701 gatgataagg tgatgaaaca gcttaaacgt cgccgttata ctggttgggg acgtttgtct 2761 cgaaaattga ttaatggtat tagggataag caatctggca aaacaatatt agattttttg 2821 aaatcagatg gttttgccaa tcgcaatttt atgcagctga tccatgatga tagtttgaca 2881 tttaaagaag acattcaaaa agcacaagtg tctggacaag gcgatagttt acatgaacat 2941 attgcaaatt tagctggtag ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt 3001 gttgatgaat tggtcaaagt aatggggcgg cataagccag aaaatatcgt tattgaaatg 3061 gcacgtgaaa atcagacaac tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga 3121 atcgaagaag gtatcaaaga attaggaagt cagattctta aagagcatcc tgttgaaaat 3181 actcaattgc aaaatgaaaa gctctatctc tattatctcc aaaatggaag agacatgtat 3241 gtggaccaag aattagatat taatcgttta agtgattatg atgtcgatca cattgttcca 3301 caaagtttcc ttaaagacga ttcaatagac aataaggtct taacgcgttc tgataaaaat 3361 cgtggtaaat cggataacgt tccaagtgaa gaagtagtca aaaagatgaa aaactattgg 3421 agacaacttc taaacgccaa gttaatcact caacgtaagt ttgataattt aacgaaagct 3481 gaacgtggag gtttgagtga acttgataaa gctggtttta tcaaacgcca attggttgaa 3541 actcgccaaa tcactaagca tgtggcacaa attttggata gtcgcatgaa tactaaatac 3601 gatgaaaatg ataaacttat tcgagaggtt aaagtgatta ccttaaaatc taaattagtt 3661 tctgacttcc gaaaagattt ccaattctat aaagtacgtg agattaacaa ttaccatcat 3721 gcccatgatg cgtatctaaa tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa 3781 cttgaatcgg agtttgtcta tggtgattat aaagtttatg atgttcgtaa aatgattgct 3841 aagtctgagc aagaaatagg caaagcaacc gcaaaatatt tcttttactc taatatcatg 3901 aacttcttca aaacagaaat tacacttgca aatggagaga ttcgcaaacg ccctctaatc 3961 gaaactaatg gggaaactgg agaaattgtc tgggataaag ggcgagattt tgccacagtg 4021 cgcaaagtat tgtccatgcc ccaagtcaat attgtcaaga aaacagaagt acagacaggc 4081 ggattctcca aggagtcaat tcgaccaaaa agaaattcgg acaagcttat tgctcgtaaa 4141 aaagactggg atccaaaaaa atatggtggt tttgttagtc caacggtagc ttattcagtc 4201 ctagtggttg ctaaggtgga aaaagggaaa tcgaagaagt taaaatccgt taaagagtta 4261 ctagggatca caattatgga aagaagttcc tttgaaaaaa atccgattga ctttttagaa 4321 gctaaaggat ataaggaagt taaaaaagac ttaatcatta aactacctaa atatagtctt 4381 tttgagttag aaaacggtcg taaacggatg ctggctagtg cccgattctt acaaaaagga 4441 aatgagctgg ctctgccaag caaatatgtg aattttttat atttagctag tcattatgaa 4501 aagttgaagg gtagtccaga agataacgaa caaaaacaat tgtttgtgga gcagcataag 4561 cattatttag atgagattat tgagcaaatc agtgaatttt ctaagcgtgt tattttagca 4621 gatgccaatt tagataaagt tcttagtgca tataacaaac atagagacaa accaatacgt 4681 gaacaagcag aaaatattat tcatttattt acgttgacga atcttggagc tccccgtgct 4741 tttaaatatt ttgatacaac aattgatcgt aaagtatatc ggtctacaaa agaagtttta 4801 gatgccactc ttatccatca atccatcact ggtctttatg aaacacgcat tgatttgagt 4861 cagctaggag gtgactgacg acgaaaacta cgctctggct gcttaactcg agtaaggatc 4921 tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 4981 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc 5041 tgcgtttata cctagggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg 5101 ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca 5161 ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg 5221 cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg 5281 actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc 5341 ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga 5401 cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc 5461 agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg 5521 caaaagcacc actggcagca gccactggta attgatttag aggagttagt cttgaagtca 5581 tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag 5641 ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg 5701 gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat 5761 cttattaatc agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca 5821 cctgaagtca gccccatacg atataagttg ttactagtgc ttggattctc accaataaaa 5881 aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact 5941 ggatctatca acaggagtcc aagcgagctc gatatcaaat tacgccccgc cctgccactc 6001 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc 6061 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc 6121 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt 6181 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa 6241 ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg 6301 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac 6361 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg 6421 aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 6481 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 6541 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 6601 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 6661 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 6721 ggaacctctt acgtgccgat caacgtctca ttttcgccag atatc //