LOCUS Exported 8508 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION H588A mutant of Cas9 from Nm8013 cloned into pET28b with a C-terminal Flag tag for protein expression. ACCESSION . VERSION . KEYWORDS pET28b-NmCas9-Flag H588A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8508) AUTHORS Rousseau BA, Hou Z, Gramelspacher MJ, Zhang Y TITLE Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis. JOURNAL Mol Cell. 2018 Mar 1;69(5):906-914.e4. doi: 10.1016/j.molcel.2018.01.025. Epub 2018 Feb 15. PUBMED 29456189 REFERENCE 2 (bases 1 to 8508) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8508 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 18..37 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 121..140 /label=T7 /note="T7 promoter, forward primer" promoter 121..139 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 140..164 /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 179..201 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 208..3453 /codon_start=1 /product="Cas9 endonuclease from the Neisseria meningitidis Type II CRISPR/Cas system (Esvelt et al., 2013)" /label=NmCas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRV FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG RLNEKGYVEIDAALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR" CDS 3454..3477 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 3490..3510 /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" primer_bind complement(3563..3581) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3577..3624 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3661..4116 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3748..3767) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3958..3979 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(4208..5023) /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 4931..4950 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 5015..5034 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 5145..5733 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5634..5653 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5887..5904 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5919..6058 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5984..6003) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6144..6166 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(6160..6351) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(7160..8242) /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" primer_bind 8204..8223 /label=LacI-R /note="LacI, reverse primer" promoter complement(8243..8320) /gene="lacI" /label=lacI promoter ORIGIN 1 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg 61 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat 121 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 181 tgtttaactt taagaaggag atataccatg gctgccttca aacctaattc aatcaactac 241 atcctcggcc tcgatatcgg catcgcatcc gtcggctggg cgatggtaga aattgacgaa 301 gaagaaaacc ccatccgcct gattgatttg ggcgtgcgcg tatttgagcg tgccgaagta 361 ccgaaaacag gcgactccct tgccatggca aggcgtttgg cgcgcagtgt tcgccgcctg 421 acccgccgtc gcgcccaccg cctgcttcgg acccgccgcc tattgaaacg cgaaggcgta 481 ttacaagccg ccaattttga cgaaaacggc ttgattaaat ccttaccgaa tacaccatgg 541 caacttcgcg cagccgcatt agaccgcaaa ctgacgcctt tagagtggtc ggcagtcttg 601 ttgcatttaa tcaaacatcg cggctattta tcgcaacgga aaaacgaggg cgaaactgcc 661 gataaggagc ttggcgcttt gcttaaaggc gtagccggca atgcccatgc cttacagaca 721 ggcgatttcc gcacaccggc cgaattggct ttaaataaat ttgagaaaga aagcggccat 781 atccgcaatc agcgcagcga ttattcgcat acgttcagcc gcaaagattt acaggcggag 841 ctgattttgc tgtttgaaaa acaaaaagaa tttggcaatc cgcatgtttc aggcggcctt 901 aaagaaggta ttgaaaccct actgatgacg caacgccctg ccctgtccgg cgatgccgtt 961 caaaaaatgt tggggcattg caccttcgaa ccggcagagc cgaaagccgc taaaaacacc 1021 tacacagccg aacgtttcat ctggctgacc aagctgaaca acctgcgtat tttagagcaa 1081 ggcagcgagc ggccattgac cgataccgaa cgcgccacgc ttatggacga gccatacaga 1141 aaatccaaac tgacttacgc acaagcccgt aagctgctgg gtttagaaga taccgccttt 1201 ttcaaaggct tgcgctatgg taaagacaat gccgaagcct caacattgat ggaaatgaag 1261 gcctaccatg ccatcagccg tgcactggaa aaagaaggat tgaaagacaa aaaatcccca 1321 ttaaaccttt ctcccgaatt acaagacgaa atcggcacgg cattctccct gttcaaaacc 1381 gatgaagaca ttacaggccg tctgaaagac cgtatacagc ccgaaatctt agaagcgctg 1441 ttgaaacaca tcagcttcga taagttcgtc caaatttcct tgaaagcatt gcgccgaatt 1501 gtgcctctaa tggaacaagg caaacgttac gatgaagcct gcgccgaaat ctacggagac 1561 cattacggca agaagaatac ggaagaaaag atttatctgc cgccgattcc cgccgacgaa 1621 atccgcaacc ccgtcgtctt gcgcgcctta tctcaagcac gtaaggtcat taacggcgtg 1681 gtacgccgtt acggctcccc agctcgtatc catattgaaa ctgcaaggga agtaggtaaa 1741 tcgtttaaag accgcaaaga aattgagaaa cgccaagaag aaaaccgcaa agaccgggaa 1801 aaagccgccg ccaaattccg agagtatttc cccaattttg tcggagaacc caaatccaaa 1861 gatattctga aactgcgcct gtacgagcaa caacacggca aatgcctgta ttcgggcaaa 1921 gaaatcaact taggccgtct gaacgaaaaa ggctatgtcg aaatcgacgc tgccctgccg 1981 ttctcgcgca catgggacga cagtttcaac aataaagtac tggtattggg cagcgaaaac 2041 caaaacaaag gcaatcaaac cccttacgaa tacttcaacg gcaaagacaa cagccgcgaa 2101 tggcaggaat ttaaagcgcg tgtcgaaacc agccgtttcc cgcgcagtaa aaaacaacgg 2161 attctgctgc aaaaattcga tgaagacggc tttaaagaac gcaatctgaa cgacacgcgc 2221 tacgtcaacc gtttcctgtg tcaatttgtt gccgaccgta tgcggctgac aggtaaaggc 2281 aagaaacgtg tctttgcatc caacggacaa attaccaatc tgttgcgcgg cttttgggga 2341 ttgcgcaaag tgcgtgcgga aaacgaccgc catcacgcct tggacgccgt cgtcgttgcc 2401 tgctcgaccg ttgccatgca gcagaaaatt acccgttttg tacgctataa agagatgaac 2461 gcgtttgacg gtaaaaccat agacaaagaa acaggagaag tgctgcatca aaaaacacac 2521 ttcccacaac cttgggaatt tttcgcacaa gaagtcatga ttcgcgtctt cggcaaaccg 2581 gacggcaaac ccgaattcga agaagccgat accctagaaa aactgcgcac gttgcttgcc 2641 gaaaaattat catctcgccc cgaagccgta cacgaatacg ttacgccact gtttgtttca 2701 cgcgcgccca atcggaagat gagcgggcaa gggcatatgg agaccgtcaa atccgccaaa 2761 cgactggacg aaggcgtcag cgtgttgcgc gtaccgctga cacagttaaa actgaaagac 2821 ttggaaaaaa tggtcaatcg ggagcgcgaa cctaagctat acgaagcact gaaagcacgg 2881 ctggaagcac ataaagacga tcctgccaaa gcctttgccg agccgtttta caaatacgat 2941 aaagcaggca accgcaccca acaggtaaaa gccgtacgcg tagagcaagt acagaaaacc 3001 ggcgtatggg tgcgcaacca taacggtatt gccgacaacg caaccatggt gcgcgtagat 3061 gtgtttgaga aaggcgacaa gtattatctg gtaccgattt acagttggca ggtagcgaaa 3121 gggattttgc cggatagggc tgttgtacaa ggaaaagatg aagaagattg gcaacttatt 3181 gatgatagtt tcaactttaa attctcatta caccctaatg atttagtcga ggttataaca 3241 aaaaaagcta gaatgtttgg ttactttgcc agctgccatc gaggcacagg taatatcaat 3301 atacgcattc atgatcttga tcataaaatt ggcaaaaatg gaatactgga aggtatcggc 3361 gtcaaaaccg ccctttcatt ccaaaaatac caaattgacg aactgggcaa agaaatcaga 3421 ccatgccgtc tgaaaaaacg cccgcctgtc cgtgattata aagatgatga tgataaatga 3481 gcactcgagc accaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag 3541 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 3601 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg 3661 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 3721 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 3781 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 3841 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 3901 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 3961 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 4021 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 4081 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt 4141 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa 4201 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat 4261 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc 4321 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa 4381 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag 4441 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa 4501 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc 4561 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag 4621 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat 4681 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc 4741 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca 4801 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt 4861 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt 4921 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta 4981 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac 5041 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg 5101 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 5161 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 5221 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 5281 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 5341 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 5401 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 5461 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 5521 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 5581 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 5641 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5701 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5761 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5821 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5881 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt 5941 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg 6001 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc 6061 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 6121 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 6181 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca 6241 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg 6301 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc 6361 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct 6421 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca 6481 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt 6541 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg 6601 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc 6661 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt 6721 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg 6781 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat 6841 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc 6901 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg 6961 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa 7021 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac 7081 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact 7141 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 7201 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg 7261 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag 7321 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg 7381 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga 7441 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct 7501 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt 7561 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat 7621 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg 7681 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc 7741 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa 7801 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca 7861 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt 7921 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat 7981 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg 8041 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa 8101 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc 8161 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat 8221 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc 8281 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc 8341 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc 8401 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct 8461 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcga //