LOCUS Exported 8512 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Wild type Cas9 from Nm8013 cloned into pET28b with a C-terminal Flag tag for protein expression. ACCESSION . VERSION . KEYWORDS pET28b-NmCas9-Flag SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8512) 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 8512) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8512 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 12..467 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(99..118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 309..330 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(560..1375) /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 1283..1302 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1367..1386 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 1497..2085 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1986..2005 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2239..2256 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2271..2413 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2339..2358) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2499..2521 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2515..2706) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3515..4597) /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 4559..4578 /label=LacI-R /note="LacI, reverse primer" promoter complement(4598..4675) /gene="lacI" /label=lacI promoter primer_bind 4881..4900 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4984..5003 /label=T7 /note="T7 promoter, forward primer" promoter 4984..5002 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5003..5027 /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 5042..5064 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5071..8316 /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 RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR" CDS 8317..8340 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 8353..8373 /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" primer_bind complement(8426..8444) /label=T7 Term /note="T7 terminator, reverse primer" terminator 8440..8487 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5041 ttttgtttaa ctttaagaag gagatatacc atggctgcct tcaaacctaa ttcaatcaac 5101 tacatcctcg gcctcgatat cggcatcgca tccgtcggct gggcgatggt agaaattgac 5161 gaagaagaaa accccatccg cctgattgat ttgggcgtgc gcgtatttga gcgtgccgaa 5221 gtaccgaaaa caggcgactc ccttgccatg gcaaggcgtt tggcgcgcag tgttcgccgc 5281 ctgacccgcc gtcgcgccca ccgcctgctt cggacccgcc gcctattgaa acgcgaaggc 5341 gtattacaag ccgccaattt tgacgaaaac ggcttgatta aatccttacc gaatacacca 5401 tggcaacttc gcgcagccgc attagaccgc aaactgacgc ctttagagtg gtcggcagtc 5461 ttgttgcatt taatcaaaca tcgcggctat ttatcgcaac ggaaaaacga gggcgaaact 5521 gccgataagg agcttggcgc tttgcttaaa ggcgtagccg gcaatgccca tgccttacag 5581 acaggcgatt tccgcacacc ggccgaattg gctttaaata aatttgagaa agaaagcggc 5641 catatccgca atcagcgcag cgattattcg catacgttca gccgcaaaga tttacaggcg 5701 gagctgattt tgctgtttga aaaacaaaaa gaatttggca atccgcatgt ttcaggcggc 5761 cttaaagaag gtattgaaac cctactgatg acgcaacgcc ctgccctgtc cggcgatgcc 5821 gttcaaaaaa tgttggggca ttgcaccttc gaaccggcag agccgaaagc cgctaaaaac 5881 acctacacag ccgaacgttt catctggctg accaagctga acaacctgcg tattttagag 5941 caaggcagcg agcggccatt gaccgatacc gaacgcgcca cgcttatgga cgagccatac 6001 agaaaatcca aactgactta cgcacaagcc cgtaagctgc tgggtttaga agataccgcc 6061 tttttcaaag gcttgcgcta tggtaaagac aatgccgaag cctcaacatt gatggaaatg 6121 aaggcctacc atgccatcag ccgtgcactg gaaaaagaag gattgaaaga caaaaaatcc 6181 ccattaaacc tttctcccga attacaagac gaaatcggca cggcattctc cctgttcaaa 6241 accgatgaag acattacagg ccgtctgaaa gaccgtatac agcccgaaat cttagaagcg 6301 ctgttgaaac acatcagctt cgataagttc gtccaaattt ccttgaaagc attgcgccga 6361 attgtgcctc taatggaaca aggcaaacgt tacgatgaag cctgcgccga aatctacgga 6421 gaccattacg gcaagaagaa tacggaagaa aagatttatc tgccgccgat tcccgccgac 6481 gaaatccgca accccgtcgt cttgcgcgcc ttatctcaag cacgtaaggt cattaacggc 6541 gtggtacgcc gttacggctc cccagctcgt atccatattg aaactgcaag ggaagtaggt 6601 aaatcgttta aagaccgcaa agaaattgag aaacgccaag aagaaaaccg caaagaccgg 6661 gaaaaagccg ccgccaaatt ccgagagtat ttccccaatt ttgtcggaga acccaaatcc 6721 aaagatattc tgaaactgcg cctgtacgag caacaacacg gcaaatgcct gtattcgggc 6781 aaagaaatca acttaggccg tctgaacgaa aaaggctatg tcgaaatcga ccatgccctg 6841 ccgttctcgc gcacatggga cgacagtttc aacaataaag tactggtatt gggcagcgaa 6901 aaccaaaaca aaggcaatca aaccccttac gaatacttca acggcaaaga caacagccgc 6961 gaatggcagg aatttaaagc gcgtgtcgaa accagccgtt tcccgcgcag taaaaaacaa 7021 cggattctgc tgcaaaaatt cgatgaagac ggctttaaag aacgcaatct gaacgacacg 7081 cgctacgtca accgtttcct gtgtcaattt gttgccgacc gtatgcggct gacaggtaaa 7141 ggcaagaaac gtgtctttgc atccaacgga caaattacca atctgttgcg cggcttttgg 7201 ggattgcgca aagtgcgtgc ggaaaacgac cgccatcacg ccttggacgc cgtcgtcgtt 7261 gcctgctcga ccgttgccat gcagcagaaa attacccgtt ttgtacgcta taaagagatg 7321 aacgcgtttg acggtaaaac catagacaaa gaaacaggag aagtgctgca tcaaaaaaca 7381 cacttcccac aaccttggga atttttcgca caagaagtca tgattcgcgt cttcggcaaa 7441 ccggacggca aacccgaatt cgaagaagcc gataccctag aaaaactgcg cacgttgctt 7501 gccgaaaaat tatcatctcg ccccgaagcc gtacacgaat acgttacgcc actgtttgtt 7561 tcacgcgcgc ccaatcggaa gatgagcggg caagggcata tggagaccgt caaatccgcc 7621 aaacgactgg acgaaggcgt cagcgtgttg cgcgtaccgc tgacacagtt aaaactgaaa 7681 gacttggaaa aaatggtcaa tcgggagcgc gaacctaagc tatacgaagc actgaaagca 7741 cggctggaag cacataaaga cgatcctgcc aaagcctttg ccgagccgtt ttacaaatac 7801 gataaagcag gcaaccgcac ccaacaggta aaagccgtac gcgtagagca agtacagaaa 7861 accggcgtat gggtgcgcaa ccataacggt attgccgaca acgcaaccat ggtgcgcgta 7921 gatgtgtttg agaaaggcga caagtattat ctggtaccga tttacagttg gcaggtagcg 7981 aaagggattt tgccggatag ggctgttgta caaggaaaag atgaagaaga ttggcaactt 8041 attgatgata gtttcaactt taaattctca ttacacccta atgatttagt cgaggttata 8101 acaaaaaaag ctagaatgtt tggttacttt gccagctgcc atcgaggcac aggtaatatc 8161 aatatacgca ttcatgatct tgatcataaa attggcaaaa atggaatact ggaaggtatc 8221 ggcgtcaaaa ccgccctttc attccaaaaa taccaaattg acgaactggg caaagaaatc 8281 agaccatgcc gtctgaaaaa acgcccgcct gtccgtgatt ataaagatga tgatgataaa 8341 tgagcactcg agcaccacca ccaccaccac cactgagatc cggctgctaa caaagcccga 8401 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 8461 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at //