LOCUS Exported 8538 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Nme2Cas9 bacterial expression plasmid. ACCESSION . VERSION . KEYWORDS Nme2Cas9_pMCSG7 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8538) AUTHORS Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ TITLE A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing. JOURNAL Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 10.1016/j.molcel.2018.12.003. PUBMED 30581144 REFERENCE 2 (bases 1 to 8538) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8538 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 66..83 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(136..154) /label=T7 Term /note="T7 terminator, reverse primer" terminator 150..197 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind complement(321..340) /label=F1ori-R /note="F1 origin, reverse primer" promoter 507..610 /gene="bla" /label=AmpR promoter CDS 611..1471 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(829..848) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1642..2230 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2131..2150 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2384..2401 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2416..2558 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2484..2503) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2644..2666 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2660..2851) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3660..4742) /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 4704..4723 /label=LacI-R /note="LacI, reverse primer" promoter complement(4743..4820) /gene="lacI" /label=lacI promoter primer_bind 5026..5045 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5133..5152 /label=T7 /note="T7 promoter, forward primer" promoter 5133..5151 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5152..5176 /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 5191..5213 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5224..5241 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5266..5286 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" ORIGIN 1 aacattggaa gtggataacg gatccgaatt cgagctccgt cgacaagctt gcggccgcac 61 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg 121 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg 181 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc 241 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 301 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 361 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 421 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacattaac gcttacaatt 481 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 541 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatttgaaa 601 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 661 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 721 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 781 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 841 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 901 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 961 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 1021 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 1081 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 1141 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 1201 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 1261 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 1321 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 1381 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 1441 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 1501 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 1561 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 1621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 1681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 1741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 1801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 1861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 1921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 1981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 2041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 2101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 2161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 2221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 2281 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 2341 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 2401 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 2461 ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat 2521 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg 2581 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 2641 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc 2701 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg 2761 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca 2821 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt 2881 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg 2941 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc 3001 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg 3061 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg 3121 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg 3181 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt 3241 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca 3301 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac 3361 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg 3421 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc 3481 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg 3541 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg 3601 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct 3661 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 3721 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag 3781 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc 3841 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa 3901 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc 3961 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc 4021 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca 4081 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc 4141 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt 4201 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag 4261 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta 4321 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc 4381 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt 4441 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc 4501 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac 4561 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc 4621 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg 4681 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc 4741 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt 4801 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag 4861 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg 4921 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa 4981 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat 5041 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta 5101 gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg 5161 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat 5221 atgcaccatc atcatcatca ttcttctggt gtagatctgg gtaccgagaa cctgtacttc 5281 caatccaatg ccatggccgc cttcaagcct aacccaatca attacatcct gggactggac 5341 atcggaatcg catccgtggg atgggctatg gtggagatcg acgaggagga gaatcctatc 5401 cggctgatcg atctgggcgt gagagtgttt gagagggccg aggtgccaaa gaccggcgat 5461 tctctggcta tggcccggag actggcacgg agcgtgaggc gcctgacacg gagaagggca 5521 cacaggctgc tgagggcacg ccggctgctg aagagagagg gcgtgctgca ggcagcagac 5581 ttcgatgaga atggcctgat caagagcctg ccaaacaccc cctggcagct gagagcagcc 5641 gccctggaca ggaagctgac accactggag tggtctgccg tgctgctgca cctgatcaag 5701 caccgcggct acctgagcca gcggaagaac gagggagaga cagcagacaa ggagctgggc 5761 gccctgctga agggagtggc caacaatgcc cacgccctgc agaccggcga tttcaggaca 5821 cctgccgagc tggccctgaa taagtttgag aaggagtccg gccacatcag aaaccagagg 5881 ggcgactata gccacacctt ctcccgcaag gatctgcagg ccgagctgat cctgctgttc 5941 gagaagcaga aggagtttgg caatccacac gtgagcggag gcctgaagga gggaatcgag 6001 accctgctga tgacacagag gcctgccctg tccggcgacg cagtgcagaa gatgctggga 6061 cactgcacct tcgagcctgc agagccaaag gccgccaaga acacctacac agccgagcgg 6121 tttatctggc tgacaaagct gaacaatctg agaatcctgg agcagggatc cgagaggcca 6181 ctgaccgaca cagagagggc caccctgatg gatgagcctt accggaagtc taagctgaca 6241 tatgcccagg ccagaaagct gctgggcctg gaggacaccg ccttctttaa gggcctgaga 6301 tacggcaagg ataatgccga ggcctccaca ctgatggaga tgaaggccta tcacgccatc 6361 tctcgcgccc tggagaagga gggcctgaag gacaagaagt cccccctgaa cctgagctcc 6421 gagctgcagg atgagatcgg caccgccttc tctctgttta agaccgacga ggatatcaca 6481 ggccgcctga aggacagggt gcagcctgag atcctggagg ccctgctgaa gcacatctct 6541 ttcgataagt ttgtgcagat cagcctgaag gccctgagaa ggatcgtgcc actgatggag 6601 cagggcaagc ggtacgacga ggcctgcgcc gagatctacg gcgatcacta tggcaagaag 6661 aacacagagg agaagatcta tctgccccct atccctgccg acgagatcag aaatcctgtg 6721 gtgctgaggg ccctgtccca ggcaagaaaa gtgatcaacg gagtggtgcg ccggtacgga 6781 tctccagccc ggatccacat cgagaccgcc agagaagtgg gcaagagctt caaggaccgg 6841 aaggagatcg agaagagaca ggaggagaat cgcaaggatc gggagaaggc cgccgccaag 6901 tttagggagt acttccctaa ctttgtgggc gagccaaagt ctaaggacat cctgaagctg 6961 cgcctgtacg agcagcagca cggcaagtgt ctgtatagcg gcaaggagat caatctggtg 7021 cggctgaacg agaagggcta tgtggagatc gatcacgccc tgcctttctc cagaacctgg 7081 gacgattctt ttaacaataa ggtgctggtg ctgggcagcg agaaccagaa taagggcaat 7141 cagacaccat acgagtattt caatggcaag gacaactcca gggagtggca ggagttcaag 7201 gcccgcgtgg agacctctag atttcccagg agcaagaagc agcggatcct gctgcagaag 7261 ttcgacgagg atggctttaa ggagtgcaac ctgaatgaca ccagatacgt gaaccggttc 7321 ctgtgccagt ttgtggccga tcacatcctg ctgaccggca agggcaagag aagggtgttc 7381 gcctctaatg gccagatcac aaacctgctg aggggatttt ggggactgag gaaggtgcgg 7441 gcagagaatg acagacacca cgcactggat gcagtggtgg tggcatgcag caccgtggca 7501 atgcagcaga agatcacaag attcgtgagg tataaggaga tgaacgcctt tgacggcaag 7561 accatcgata aggagacagg caaggtgctg caccagaaga cccacttccc ccagccttgg 7621 gagttctttg cccaggaagt gatgatccgg gtgttcggca agccagacgg caagcctgag 7681 tttgaggagg ccgatacccc agagaagctg aggacactgc tggcagagaa gctgtctagc 7741 aggccagagg cagtgcacga gtacgtgacc ccactgttcg tgtccagggc acccaatcgg 7801 aagatgtctg gcgcccacaa ggacacactg agaagcgcca agaggtttgt gaagcacaac 7861 gagaagatct ccgtgaagag agtgtggctg accgagatca agctggccga tctggagaac 7921 atggtgaatt acaagaacgg cagggagatc gagctgtatg aggccctgaa ggcaaggctg 7981 gaggcctacg gaggaaatgc caagcaggcc ttcgacccaa aggataaccc cttttataag 8041 aagggaggac agctggtgaa ggccgtgcgg gtggagaaga cccaggagag cggcgtgctg 8101 ctgaataaga agaacgccta cacaatcgcc gacaatggcg atatggtgag agtggacgtg 8161 ttctgtaagg tggataagaa gggcaagaat cagtacttta tcgtgcctat ctatgcctgg 8221 caggtggccg agaacatcct gccagacatc gattgcaagg gctacagaat cgacgatagc 8281 tatacattct gtttttccct gcacaagtat gacctgatcg ccttccagaa ggatgagaag 8341 tccaaggtgg agtttgccta ctatatcaat tgcgactcct ctaacggcag gttctacctg 8401 gcctggcacg ataagggcag caaggagcag cagtttcgca tctccaccca gaatctggtg 8461 ctgatccaga agtatcaggt gaacgagctg ggcaaggaga tcaggccatg tcggctgaag 8521 aagcgcccac ccgtgcgg //