LOCUS Exported 8508 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 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 12, 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 74..93 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 177..196 /label=T7 /note="T7 promoter, forward primer" promoter 177..195 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 196..220 /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 235..257 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 264..3509 /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 3510..3533 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 3546..3566 /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" primer_bind complement(3619..3637) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3633..3680 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3717..4172 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3804..3823) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4014..4035 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(4264..5079) /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 4987..5006 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 5071..5090 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 5201..5789 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5690..5709 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5943..5960 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5975..6114 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(6040..6059) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6200..6222 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(6216..6407) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(7216..8298) /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 8260..8279 /label=LacI-R /note="LacI, reverse primer" promoter complement(8299..8376) /gene="lacI" /label=lacI promoter ORIGIN 1 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc 61 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg 121 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat 181 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt 241 taactttaag aaggagatat accatggctg ccttcaaacc taattcaatc aactacatcc 301 tcggcctcga tatcggcatc gcatccgtcg gctgggcgat ggtagaaatt gacgaagaag 361 aaaaccccat ccgcctgatt gatttgggcg tgcgcgtatt tgagcgtgcc gaagtaccga 421 aaacaggcga ctcccttgcc atggcaaggc gtttggcgcg cagtgttcgc cgcctgaccc 481 gccgtcgcgc ccaccgcctg cttcggaccc gccgcctatt gaaacgcgaa ggcgtattac 541 aagccgccaa ttttgacgaa aacggcttga ttaaatcctt accgaataca ccatggcaac 601 ttcgcgcagc cgcattagac cgcaaactga cgcctttaga gtggtcggca gtcttgttgc 661 atttaatcaa acatcgcggc tatttatcgc aacggaaaaa cgagggcgaa actgccgata 721 aggagcttgg cgctttgctt aaaggcgtag ccggcaatgc ccatgcctta cagacaggcg 781 atttccgcac accggccgaa ttggctttaa ataaatttga gaaagaaagc ggccatatcc 841 gcaatcagcg cagcgattat tcgcatacgt tcagccgcaa agatttacag gcggagctga 901 ttttgctgtt tgaaaaacaa aaagaatttg gcaatccgca tgtttcaggc ggccttaaag 961 aaggtattga aaccctactg atgacgcaac gccctgccct gtccggcgat gccgttcaaa 1021 aaatgttggg gcattgcacc ttcgaaccgg cagagccgaa agccgctaaa aacacctaca 1081 cagccgaacg tttcatctgg ctgaccaagc tgaacaacct gcgtatttta gagcaaggca 1141 gcgagcggcc attgaccgat accgaacgcg ccacgcttat ggacgagcca tacagaaaat 1201 ccaaactgac ttacgcacaa gcccgtaagc tgctgggttt agaagatacc gcctttttca 1261 aaggcttgcg ctatggtaaa gacaatgccg aagcctcaac attgatggaa atgaaggcct 1321 accatgccat cagccgtgca ctggaaaaag aaggattgaa agacaaaaaa tccccattaa 1381 acctttctcc cgaattacaa gacgaaatcg gcacggcatt ctccctgttc aaaaccgatg 1441 aagacattac aggccgtctg aaagaccgta tacagcccga aatcttagaa gcgctgttga 1501 aacacatcag cttcgataag ttcgtccaaa tttccttgaa agcattgcgc cgaattgtgc 1561 ctctaatgga acaaggcaaa cgttacgatg aagcctgcgc cgaaatctac ggagaccatt 1621 acggcaagaa gaatacggaa gaaaagattt atctgccgcc gattcccgcc gacgaaatcc 1681 gcaaccccgt cgtcttgcgc gccttatctc aagcacgtaa ggtcattaac ggcgtggtac 1741 gccgttacgg ctccccagct cgtatccata ttgaaactgc aagggaagta ggtaaatcgt 1801 ttaaagaccg caaagaaatt gagaaacgcc aagaagaaaa ccgcaaagac cgggaaaaag 1861 ccgccgccaa attccgagag tatttcccca attttgtcgg agaacccaaa tccaaagata 1921 ttctgaaact gcgcctgtac gagcaacaac acggcaaatg cctgtattcg ggcaaagaaa 1981 tcaacttagg ccgtctgaac gaaaaaggct atgtcgaaat cgaccatgcc ctgccgttct 2041 cgcgcacatg ggacgacagt ttcaacaata aagtactggt attgggcagc gaaaaccaaa 2101 acaaaggcaa tcaaacccct tacgaatact tcaacggcaa agacaacagc cgcgaatggc 2161 aggaatttaa agcgcgtgtc gaaaccagcc gtttcccgcg cagtaaaaaa caacggattc 2221 tgctgcaaaa attcgatgaa gacggcttta aagaacgcaa tctgaacgac acgcgctacg 2281 tcaaccgttt cctgtgtcaa tttgttgccg accgtatgcg gctgacaggt aaaggcaaga 2341 aacgtgtctt tgcatccaac ggacaaatta ccaatctgtt gcgcggcttt tggggattgc 2401 gcaaagtgcg tgcggaaaac gaccgccatc acgccttgga cgccgtcgtc gttgcctgct 2461 cgaccgttgc catgcagcag aaaattaccc gttttgtacg ctataaagag atgaacgcgt 2521 ttgacggtaa aaccatagac aaagaaacag gagaagtgct gcatcaaaaa acacacttcc 2581 cacaaccttg ggaatttttc gcacaagaag tcatgattcg cgtcttcggc aaaccggacg 2641 gcaaacccga attcgaagaa gccgataccc tagaaaaact gcgcacgttg cttgccgaaa 2701 aattatcatc tcgccccgaa gccgtacacg aatacgttac gccactgttt gtttcacgcg 2761 cgcccaatcg gaagatgagc gggcaagggc atatggagac cgtcaaatcc gccaaacgac 2821 tggacgaagg cgtcagcgtg ttgcgcgtac cgctgacaca gttaaaactg aaagacttgg 2881 aaaaaatggt caatcgggag cgcgaaccta agctatacga agcactgaaa gcacggctgg 2941 aagcacataa agacgatcct gccaaagcct ttgccgagcc gttttacaaa tacgataaag 3001 caggcaaccg cacccaacag gtaaaagccg tacgcgtaga gcaagtacag aaaaccggcg 3061 tatgggtgcg caaccataac ggtattgccg acaacgcaac catggtgcgc gtagatgtgt 3121 ttgagaaagg cgacaagtat tatctggtac cgatttacag ttggcaggta gcgaaaggga 3181 ttttgccgga tagggctgtt gtacaaggaa aagatgaaga agattggcaa cttattgatg 3241 atagtttcaa ctttaaattc tcattacacc ctaatgattt agtcgaggtt ataacaaaaa 3301 aagctagaat gtttggttac tttgccagct gccatcgagg cacaggtaat atcaatatac 3361 gcattcatga tcttgatcat aaaattggca aaaatggaat actggaaggt atcggcgtca 3421 aaaccgccct ttcattccaa aaataccaaa ttgacgaact gggcaaagaa atcagaccat 3481 gccgtctgaa aaaacgcccg cctgtccgtg attataaaga tgatgatgat aaatgagcac 3541 tcgagcacca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag 3601 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac 3661 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc 3721 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 3781 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 3841 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 3901 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 3961 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 4021 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 4081 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 4141 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc 4201 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa 4261 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc 4321 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt 4381 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca 4441 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac 4501 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg 4561 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga 4621 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat 4681 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg 4741 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc 4801 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca 4861 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag 4921 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc 4981 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg 5041 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt 5101 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc 5161 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 5221 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 5281 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 5341 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5401 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5461 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5521 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5581 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 5641 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 5701 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 5761 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 5821 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 5881 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 5941 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc 6001 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc 6061 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga 6121 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 6181 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 6241 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg 6301 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt 6361 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg 6421 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg 6481 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg 6541 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt 6601 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac 6661 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag 6721 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc 6781 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc 6841 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc 6901 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc 6961 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc 7021 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag 7081 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg 7141 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca 7201 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 7261 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt 7321 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag 7381 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt 7441 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc 7501 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc 7561 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg 7621 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg 7681 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc 7741 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt 7801 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa 7861 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg 7921 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca 7981 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc 8041 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc 8101 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta 8161 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc 8221 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta 8281 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc 8341 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat 8401 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg 8461 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggcca //