LOCUS Exported 8508 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION D16A+H588A mutant of Cas9 from Nm8013 cloned into pET28b with a C-terminal Flag tag for protein expression. ACCESSION . VERSION . KEYWORDS pET28b-NmCas9-Flag D16A+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 99..118 /label=T7 /note="T7 promoter, forward primer" promoter 99..117 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 118..142 /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 157..179 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 186..3431 /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="MAAFKPNSINYILGLAIGIASVGWAMVEIDEEENPIRLIDLGVRV FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG RLNEKGYVEIDAALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR" CDS 3432..3455 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 3468..3488 /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" primer_bind complement(3541..3559) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3555..3602 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3639..4094 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3726..3745) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3936..3957 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(4186..5001) /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 4909..4928 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4993..5012 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 5123..5711 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5612..5631 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5865..5882 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5897..6036 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5962..5981) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6122..6144 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(6138..6329) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(7138..8220) /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 8182..8201 /label=LacI-R /note="LacI, reverse primer" promoter complement(8221..8298) /gene="lacI" /label=lacI promoter primer_bind 8504..15 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc 61 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga 121 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat 181 ataccatggc tgccttcaaa cctaattcaa tcaactacat cctcggcctc gctatcggca 241 tcgcatccgt cggctgggcg atggtagaaa ttgacgaaga agaaaacccc atccgcctga 301 ttgatttggg cgtgcgcgta tttgagcgtg ccgaagtacc gaaaacaggc gactcccttg 361 ccatggcaag gcgtttggcg cgcagtgttc gccgcctgac ccgccgtcgc gcccaccgcc 421 tgcttcggac ccgccgccta ttgaaacgcg aaggcgtatt acaagccgcc aattttgacg 481 aaaacggctt gattaaatcc ttaccgaata caccatggca acttcgcgca gccgcattag 541 accgcaaact gacgccttta gagtggtcgg cagtcttgtt gcatttaatc aaacatcgcg 601 gctatttatc gcaacggaaa aacgagggcg aaactgccga taaggagctt ggcgctttgc 661 ttaaaggcgt agccggcaat gcccatgcct tacagacagg cgatttccgc acaccggccg 721 aattggcttt aaataaattt gagaaagaaa gcggccatat ccgcaatcag cgcagcgatt 781 attcgcatac gttcagccgc aaagatttac aggcggagct gattttgctg tttgaaaaac 841 aaaaagaatt tggcaatccg catgtttcag gcggccttaa agaaggtatt gaaaccctac 901 tgatgacgca acgccctgcc ctgtccggcg atgccgttca aaaaatgttg gggcattgca 961 ccttcgaacc ggcagagccg aaagccgcta aaaacaccta cacagccgaa cgtttcatct 1021 ggctgaccaa gctgaacaac ctgcgtattt tagagcaagg cagcgagcgg ccattgaccg 1081 ataccgaacg cgccacgctt atggacgagc catacagaaa atccaaactg acttacgcac 1141 aagcccgtaa gctgctgggt ttagaagata ccgccttttt caaaggcttg cgctatggta 1201 aagacaatgc cgaagcctca acattgatgg aaatgaaggc ctaccatgcc atcagccgtg 1261 cactggaaaa agaaggattg aaagacaaaa aatccccatt aaacctttct cccgaattac 1321 aagacgaaat cggcacggca ttctccctgt tcaaaaccga tgaagacatt acaggccgtc 1381 tgaaagaccg tatacagccc gaaatcttag aagcgctgtt gaaacacatc agcttcgata 1441 agttcgtcca aatttccttg aaagcattgc gccgaattgt gcctctaatg gaacaaggca 1501 aacgttacga tgaagcctgc gccgaaatct acggagacca ttacggcaag aagaatacgg 1561 aagaaaagat ttatctgccg ccgattcccg ccgacgaaat ccgcaacccc gtcgtcttgc 1621 gcgccttatc tcaagcacgt aaggtcatta acggcgtggt acgccgttac ggctccccag 1681 ctcgtatcca tattgaaact gcaagggaag taggtaaatc gtttaaagac cgcaaagaaa 1741 ttgagaaacg ccaagaagaa aaccgcaaag accgggaaaa agccgccgcc aaattccgag 1801 agtatttccc caattttgtc ggagaaccca aatccaaaga tattctgaaa ctgcgcctgt 1861 acgagcaaca acacggcaaa tgcctgtatt cgggcaaaga aatcaactta ggccgtctga 1921 acgaaaaagg ctatgtcgaa atcgacgctg ccctgccgtt ctcgcgcaca tgggacgaca 1981 gtttcaacaa taaagtactg gtattgggca gcgaaaacca aaacaaaggc aatcaaaccc 2041 cttacgaata cttcaacggc aaagacaaca gccgcgaatg gcaggaattt aaagcgcgtg 2101 tcgaaaccag ccgtttcccg cgcagtaaaa aacaacggat tctgctgcaa aaattcgatg 2161 aagacggctt taaagaacgc aatctgaacg acacgcgcta cgtcaaccgt ttcctgtgtc 2221 aatttgttgc cgaccgtatg cggctgacag gtaaaggcaa gaaacgtgtc tttgcatcca 2281 acggacaaat taccaatctg ttgcgcggct tttggggatt gcgcaaagtg cgtgcggaaa 2341 acgaccgcca tcacgccttg gacgccgtcg tcgttgcctg ctcgaccgtt gccatgcagc 2401 agaaaattac ccgttttgta cgctataaag agatgaacgc gtttgacggt aaaaccatag 2461 acaaagaaac aggagaagtg ctgcatcaaa aaacacactt cccacaacct tgggaatttt 2521 tcgcacaaga agtcatgatt cgcgtcttcg gcaaaccgga cggcaaaccc gaattcgaag 2581 aagccgatac cctagaaaaa ctgcgcacgt tgcttgccga aaaattatca tctcgccccg 2641 aagccgtaca cgaatacgtt acgccactgt ttgtttcacg cgcgcccaat cggaagatga 2701 gcgggcaagg gcatatggag accgtcaaat ccgccaaacg actggacgaa ggcgtcagcg 2761 tgttgcgcgt accgctgaca cagttaaaac tgaaagactt ggaaaaaatg gtcaatcggg 2821 agcgcgaacc taagctatac gaagcactga aagcacggct ggaagcacat aaagacgatc 2881 ctgccaaagc ctttgccgag ccgttttaca aatacgataa agcaggcaac cgcacccaac 2941 aggtaaaagc cgtacgcgta gagcaagtac agaaaaccgg cgtatgggtg cgcaaccata 3001 acggtattgc cgacaacgca accatggtgc gcgtagatgt gtttgagaaa ggcgacaagt 3061 attatctggt accgatttac agttggcagg tagcgaaagg gattttgccg gatagggctg 3121 ttgtacaagg aaaagatgaa gaagattggc aacttattga tgatagtttc aactttaaat 3181 tctcattaca ccctaatgat ttagtcgagg ttataacaaa aaaagctaga atgtttggtt 3241 actttgccag ctgccatcga ggcacaggta atatcaatat acgcattcat gatcttgatc 3301 ataaaattgg caaaaatgga atactggaag gtatcggcgt caaaaccgcc ctttcattcc 3361 aaaaatacca aattgacgaa ctgggcaaag aaatcagacc atgccgtctg aaaaaacgcc 3421 cgcctgtccg tgattataaa gatgatgatg ataaatgagc actcgagcac caccaccacc 3481 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca 3541 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt 3601 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt 3661 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 3721 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 3781 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 3841 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 3901 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 3961 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 4021 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt 4081 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 4141 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc 4201 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa 4261 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc 4321 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc 4381 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa 4441 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc 4501 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg 4561 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag 4621 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg 4681 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat 4741 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc 4801 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc 4861 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca 4921 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt 4981 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt 5041 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 5101 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 5161 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 5221 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 5281 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 5341 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 5401 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 5461 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 5521 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 5581 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 5641 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 5701 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 5761 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 5821 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 5881 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 5941 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac 6001 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct 6061 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 6121 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg 6181 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg 6241 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg 6301 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc 6361 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat 6421 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg 6481 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt 6541 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct 6601 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct 6661 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca 6721 ttctgctaac cagtaaggca accccgccag cctagcctgg tcctcaacga caggagcacg 6781 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt 6841 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca 6901 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag 6961 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg 7021 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc 7081 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca 7141 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 7201 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac 7261 gggcaacagc tgattgccct tcaccgcctg gccctgagat agttgcagca agcggtccac 7321 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca 7381 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc 7441 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc 7501 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact 7561 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca 7621 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg 7681 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa 7741 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt 7801 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc 7861 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg 7921 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc 7981 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga 8041 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc 8101 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga 8161 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac 8221 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt 8281 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga 8341 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca 8401 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac 8461 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtga //