LOCUS Exported 8490 bp ds-DNA circular SYN 21-APR-2017 DEFINITION . ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 8490) AUTHORS Trial User TITLE Direct Submission JOURNAL Exported Jan 14, 2019 from SnapGene 4.1.9 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8490 /organism="recombinant plasmid" /mol_type="other DNA" misc_feature 1..25 /label=overlap with backbone /note="color: #a6acb3" primer_bind 1..21 /label=pMSCG7_BB_FW /note="color: black; sequence: AACATTGGAAGTGGATAACGG" CDS 66..83 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /note="color: #cc99b2" /translation="HHHHHH" terminator 150..197 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" /note="color: #ffffff" promoter 507..610 /gene="bla" /label=AmpR promoter /note="color: #ffffff; direction: RIGHT" CDS 611..1471 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /note="This feature has 2 segments: 1: 611 .. 679 / #ccffcc / signal sequence 2: 680 .. 1471 / #ccffcc Cleavage site after base 679" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 1642..2230 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" misc_feature 2416..2558 /label=bom /note="basis of mobility region from pBR322" /note="color: #a6acb3" CDS complement(2660..2851) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /note="color: #993366" /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)." /note="color: #993366" /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" promoter complement(4743..4820) /gene="lacI" /label=lacI promoter /note="color: #ffffff; direction: LEFT" promoter 5133..5151 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" /note="color: #ffffff; direction: RIGHT" 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)." /note="color: #31849b" RBS 5191..5213 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" /note="color: #a6acb3" CDS 5224..5241 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /note="color: #cc99b2" /translation="HHHHHH" CDS 5266..5286 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /note="color: #cc99b2 Cleavage site after base 5283" /translation="ENLYFQS" primer_bind 5268..5317 /label=SmuCas9_Gib_FW /note="color: black; sequence: GAACCTGTACTTCCAATCCAATGCCATGATGATGGAAAAATTTCACTATG" misc_feature 5268..5292 /label=Overlap with backbone /note="color: #a6acb3" primer_bind complement(5271..5292) /label=pMSCG7_BB_RV /note="color: black; sequence: GGCATTGGATTGGAAGTACAGG" CDS 5293..8490 /codon_start=1 /label=Simonsiella muelleri Cas9 /note="color: #ff6600" /translation="MMMEKFHYVLGLDLGIASVGWAAIEIDKETETSIGLLDCGVRTFE RAEVPKTGDSLAKARREARSTRRLIRRRSHRLLRLKRLLKREIFRQPETFKDLPINAWQ LRVKGLDSRLNEYEWAAVLLHLVKHRGYLSQRKSEMSETDSKSEMGRLLAGVAENHQLL QQEQYRTPAELALKKFVKHFRNKGGDYAHTFNRLDLQAELHLLFQKQRELGNPFTSPEL ERQVDDLLMTQRSALQGDAILKMLGHCGFEPEQFKAAKNTFSAERFIWLTKLNNLRIQD QGKERALTADERTKLLDEPYKKSKLTYAQVRKLLSLPQTAIFKGLRYDLEHDKKAENST LMEMKSYHNIRQTLEKSGLKTEWQSIATQPEILDAIGTAFSIYKTDEDISHELKTCRLP ENVLNELLKNINFDGFIQLSLTALRKILPLMEQGYRYDEACTQIYGNHHSGSLQQESKQ FLPHIPIDDVRNPVVFRTLTQARKVVNAIIRRYGSPARVHIEMARELGKSKSDRDRIEK QQQKNKKERENAVAKFKEDFPDFVGEPRGKDILKMRLYEQQHGKCLYSGHDIDINRLNE KGYVEIDHALPFSRTWDDSQNNKVLVLGSENQNKRNQTPDEYLDGANNSQRWLEFQARV QTCHFSYGKKQRIQLAKLDDETEKGFLERNLNDTRYIARFMCQFVQENLYLTGKGKRLV FASNGGMTATLRNLWGLRKVREDNDRHHALDAIVVACSTASMQQKITKAFQRHESIEYV DTETGEVKFRIPQPWDFFRQEVMIRVFSDQPCEDLVEKLSARPEALHDNVTPLFVSRAP NRKMSGQGHLETIKSAKRLSEENSMVKKPLTTLKLKDIPEIVGYPSREPQLYAALKTRL ETHDDDPIKAFAKPFYKPNKNGELGALVRSVRVKGVQNTGVMVHDGKGIADNATMVRVD VYTKAGKNYLVPVYVWQVAQGILPNRAVTSGKSEADWDLIDESFEFKFSLSRGDLVEMI SNKGRIFGYYNGLDRANGSIGIREHDLEKSKGKDGVHRVGVKTATAFNKYHVDPLGKEI HRCSSEPRPTLKIKSKK" misc_feature 5310..5317 /label=A13 /note="color: #ff9900" primer_bind complement(join(8461..8490,1..25)) /label=SmuCas9_Gibs_RV /note="color: black; sequence: GGATCCGTTATCCACTTCCAATGTTTTATTTCTTGGATTTGATTTTTAATGTGGG" 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 ccatgatgat ggaaaaattt cactatgtat tgggtttgga tttgggtatc 5341 gcctctgtgg ggtgggctgc cattgaaatt gacaaggaaa ccgaaacatc aatcggttta 5401 ttggattgcg gtgtcagaac atttgaacgt gcagaagtac ccaaaacagg cgattctctt 5461 gccaaagctc gccgtgaagc cagaagtact cgccgtttaa ttcgcagacg ttcgcatcgc 5521 ttattacgtt taaaacgttt attgaaacgt gaaattttca ggcagcctga aacgtttaaa 5581 gacttaccaa tcaatgcttg gcaattgcgt gttaaaggct tggatagtcg gttgaatgaa 5641 tatgaatggg cggccgtttt attgcatttg gtgaagcatc gcggttattt atcgcaacgc 5701 aaaagcgaaa tgagcgaaac agacagcaaa tctgaaatgg gcagattact ggcaggtgtg 5761 gcggaaaatc accaactttt acaacaagaa caatatcgta caccagccga attagcactc 5821 aaaaaatttg tgaaacattt tcgcaataaa ggtggcgatt atgcacacac tttcaaccgt 5881 ttggatttgc aagccgaatt gcatttattg tttcaaaaac aacgtgaatt aggcaatcca 5941 ttcacttcac cagaattgga acggcaagtt gatgatttgt tgatgacgca gcgcagtgct 6001 ttacaaggtg atgcgatttt gaaaatgttg ggtcattgtg ggtttgaacc tgaacaattc 6061 aaagcagcga aaaacacatt cagtgccgaa cgttttattt ggttgacaaa actcaataat 6121 cttcgcattc aagaccaagg caaagaacgt gcgttaactg ccgatgagcg taccaaattg 6181 ttggacgagc cttataaaaa aagtaaattg acttacgcac aagttcgcaa attattaagc 6241 ttgcctcaaa ctgctatttt taaaggtttg cgttatgatt tggaacatga caaaaaagca 6301 gaaaacagta cgttgatgga aatgaaatcc tatcacaaca tccgccaaac attggaaaaa 6361 tcaggtttga aaacagaatg gcaaagtatt gccacgcagc ctgaaatttt agatgcaatt 6421 ggcacggcgt tttccattta taaaaccgat gaagatattt cgcatgaatt aaaaacgtgc 6481 aggctgcctg aaaacgtatt gaatgaatta ctgaaaaaca tcaattttga tggattcatt 6541 caattatcgt tgactgcatt acgcaaaatt ttgcccttga tggaacaagg ctaccgttat 6601 gatgaagcgt gtacccaaat ttacggtaat catcattcag gcagcttgca acaagaatca 6661 aagcaatttt tgccacatat tccgattgat gatgtccgaa atcctgtggt gttccgtact 6721 ttgacccaag caagaaaagt ggtgaatgcg attattcgtc ggtatggttc gccagctcgt 6781 gtgcatattg agatggcgcg tgaattgggt aagtctaaat cagaccgtga ccgaattgaa 6841 aaacaacaac aaaaaaataa aaaagaacgt gaaaacgcag tcgccaaatt caaagaagat 6901 ttccctgatt ttgtgggcga acccagaggg aaagatattt tgaaaatgcg tttgtatgaa 6961 caacaacacg gcaaatgttt gtattcgggt catgatattg atattaatcg attgaatgaa 7021 aaaggttatg ttgaaattga ccatgccctg ccattttcac ggacttggga tgatagtcaa 7081 aataataaag ttttggtact tggcagcgaa aaccaaaata aacgcaatca aacgcctgat 7141 gaatatttgg acggtgcaaa caatagccaa cgttggcttg aatttcaagc gcgtgtacaa 7201 acttgtcatt tttcttacgg taaaaaacaa cgcattcaat tagccaaatt agacgatgaa 7261 accgaaaaag gatttttaga acgcaatcta aatgatacgc ggtatattgc tcgttttatg 7321 tgtcaatttg tccaagaaaa tttatatttg acaggtaaag gaaaacgtct tgtttttgca 7381 tcaaacggcg gaatgaccgc aacattgaga aatttatggg gtttgagaaa agtccgtgaa 7441 gacaacgacc gccatcatgc tcttgatgcg attgtggtgg cgtgttccac tgcttctatg 7501 caacaaaaaa taaccaaagc atttcaacgc catgaaagca ttgaatatgt ggataccgaa 7561 acgggcgaag taaaatttcg tattccacag ccttgggatt ttttccgtca ggaagtgatg 7621 attcgtgtgt tcagcgacca accgtgtgaa gatttggtag aaaaattgtc ggctcgtccc 7681 gaagctttgc atgacaacgt aacgccttta tttgtctcgc gtgcaccaaa tcgcaaaatg 7741 tcggggcaag ggcatttgga aaccatcaaa tctgcaaaaa ggctgtctga agaaaacagt 7801 atggttaaaa aaccattaac cacattgaaa ttaaaagata ttccagaaat cgtaggctac 7861 ccgagtcgtg aacctcaatt gtatgccgca ttgaaaacac gtttagaaac gcatgatgat 7921 gacccaatta aagcctttgc caaacccttt tacaaaccca ataaaaatgg tgaattgggg 7981 gcgttggttc gatcggtgcg tgtgaaaggt gtacaaaata cgggtgtaat ggttcatgat 8041 ggcaaaggca ttgccgataa tgccacaatg gttcgtgttg atgtctatac caaagcgggc 8101 aaaaattacc ttgttcctgt gtatgtttgg caggtggctc aaggaatttt gccaaatcgg 8161 gcggttactt ctggcaaaag tgaagcagat tgggatttaa ttgatgaaag ttttgaattt 8221 aaattttcgc tgtctcgtgg ggatttagtg gaaatgatta gcaataaagg aagaattttt 8281 ggttattaca atgggttaga tcgtgcaaat ggaagtattg ggattcgtga acatgatttg 8341 gaaaagtcca aaggaaaaga tggtgttcat cgtgttggcg tgaaaaccgc caccgcattc 8401 aacaaatacc acgttgaccc acttggtaaa gaaattcatc ggtgttcatc tgaaccacgc 8461 cccacattaa aaatcaaatc caagaaataa //