LOCUS Exported 7345 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNRB65 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7345) TITLE Bishop Lab Plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 7345) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7345 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 1622..2029 /gene="S. cerevisiae LEU2" /label=LEU2 promoter CDS 2030..3124 /codon_start=1 /gene="S. cerevisiae LEU2" /product="3-isopropylmalate dehydrogenase, required for leucine biosynthesis" /label=LEU2 /note="yeast auxotrophic marker" /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG DLGGSNSTTEVGDAVAEEVKKILA" primer_bind 5037..5053 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 5037..5053 /label=pBluescriptSK /note="For pBluescript vector" primer_bind complement(5107..5123) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5108..5124) /label=pBluescriptKS /note="For pBluescript vector" primer_bind complement(5157..5177) /label=T3 /note="T3 promoter, forward primer" promoter complement(5157..5175) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5196..5212) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5196..5212) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5209..5231) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5220..5236 /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)." promoter complement(5244..5274) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5289..5310 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5427..5444) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5598..6186) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5678..5697) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6357..7217) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6980..6999 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7218..7322) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca 661 ccgcggtggc ggccgctcta gaatatgaaa cctataggta cggtggccat tctatgtctg 721 atcccggtac tacctacaga actagagacg agattcagca tatgagatcc aagaacgatc 781 caattgctgg tcttaagatg catttgattg atctaggtat tgccactgaa gctgaagtca 841 aagcttacga caagtccgct agaaaatacg ttgacgaaca agttgaatta gctgatgctg 901 ctcctcctcc agaagccaaa ttatccatct tgtttgaaga cgtctacgtg aaaggtacag 961 aaactccaac cctaagaggt aggatccctg aagatacttg ggacttcaaa aagcaaggtt 1021 ttgcctctag ggattaatta aatcgtaagg aaaaataaaa taatagtgct gtgatcgcat 1081 gatattcttc cctggaagcg ccattttata gcaagaaatg taagtcaagt atattttaac 1141 tgtatatcaa caatatagct cttttttatg ccttgttgtt tttcttcggg ttttccccac 1201 acattgtgtg gagagatagt tattaacaga ccggaaaata gccgcccaag gataaacttt 1261 tatataaagg gaagggtagt tgacccaaaa aatttggatt ctacttttcc agatttactt 1321 tcaccctttt atatttgctg tagtctgtta tgccaatcag gaaagcattt gaacaaatag 1381 gtcgaggaga acttctagta tatctacata cctaatatta ttgccttatt aaaaatggaa 1441 tcccaacaat tacatcaaaa tccacattct cttcaaaatc aattgtcctg tacttccttg 1501 ttcatgtgtg ttcaaaaacg ttatatttat aggataatta tactctattt ctcaacaagt 1561 aattggttgt ttggccgagc ggtctaaggc gcctgattca agaaatatct tgaccgcagt 1621 taactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt agcaaccatt 1681 atttttttcc tcaacataac gagaacacac aggggcgcta tcgcacagaa tcaaattcga 1741 tgactggaaa ttttttgtta atttcagagg tcgcctgacg catatacctt tttcaactga 1801 aaaattggga gaaaaaggaa aggtgagagc cgcggaaccg gcttttcata tagaatagag 1861 aagcgttcat gactaaatgc ttgcatcaca atacttgaag ttgacaatat tatttaagga 1921 cctattgttt tttccaatag gtggttagca atcgtcttac tttctaactt ttcttacctt 1981 ttacatttca gcaatatata tatatatatt tcaaggatat accattctaa tgtctgcccc 2041 taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag ccgaagccat 2101 taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt tcgaaaatca 2161 tttaattggt ggtgctgcta tcgatgctac aggtgtccca cttccagatg aggcgctgga 2221 agcctccaag aaggttgatg ccgttttgtt aggtgctgtg ggtggtccta aatggggtac 2281 cggtagtgtt agacctgaac aaggtttact aaaaatccgt aaagaacttc aattgtacgc 2341 caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc caatcaagcc 2401 acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag gtatttactt 2461 tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac aatacaccgt 2521 tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac atgagccacc 2581 attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat tatggagaaa 2641 aactgtggag gaaaccatca agaacgaatt ccctacattg aaggttcaac atcaattgat 2701 tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta ttataatcac 2761 cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag gttccttggg 2821 tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat ttggtttgta 2881 cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggtcaacc ctatcgccac 2941 tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag aaggtaaggc 3001 cattgaagat gcagttaaaa aggttttgga tgcaggtatc agaactggtg atttaggtgg 3061 ttccaacagt accacggaag tcggtgatgc tgtcgccgaa gaagttaaga aaatccttgc 3121 ttaaaaagat tctctttttt tatgatattt gtacataaac tttataaatg aaattcataa 3181 tagaaacgac acgaaattac aaaatggaat atgttcatag ggtagacgaa actatatacg 3241 caatctacat acatttatca agaaggagaa aaaggaggat gtaaaggaat acaggtaagc 3301 aaattgatac taatggctca acgtgataag gaaaaagaat tgcactttaa cattaatatt 3361 gacaaggagg agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg aaactatgat 3421 tcctaattta tatattggag gattttctct aaaaaaaaaa aaatacaaca aataaaaaac 3481 actcaatgac ctgaccattt gatggagttt aagtcaatac cttcttgaac catttcccat 3541 aatggtgaaa gttccctcaa gaattttact ctgtcagaaa cggccttaac gacgtagtcg 3601 accaattggt gaaaaaggta ttaccgattc aagtgactag tttttgtata cttttttaat 3661 gaagatgaca ttgctccttt attaaacttt tcttttactt tgtgttacta atattattaa 3721 tatcttgtta tgattctttg ttttagcagc tccaaaagaa aggaatatta taatataatt 3781 gttgtaattc ggaaaaaaaa tattaaatta ctatggtgca tatgcgtata taaaattgag 3841 cattacctaa cgccatatat tactatctaa agtcctcaca gatgatgtgt tttcaaaatc 3901 aaatgattgg agctcctcat catcatcccc ggtaaactgc tcggtttcag atccattatc 3961 cagatacctc aacgggctag tgtacgcaat tgaagggtac ctataaaata aagtacgttc 4021 ctctttcaac tgaaaaaacc cccgataatc ccttgacaag aaataataca taatataatc 4081 tcgctttctg acaagacttt tcagttcttg gtactctaat tcccgttcga aaaccggata 4141 tgtgccagca atttcgtcag ttattatagc gtttataata gcctcgtagt catcgggctc 4201 aattctattt tcgtagttga acagacggta aaaagggcta cagtcataat tatgctcgca 4261 ttcctcgaga ataatatctg aatcagattg taaatgtatg atatttcttg gcgttacaac 4321 aatagaatcc tcaggccctg atattttctc tatagatggg gaatccggta tatagtcaag 4381 gttgatatct gttaattcat ttgaatttag gttagcgcga gcttcaattt cgttaggctt 4441 cttgaaattt gccttaactg ggatttgctt tgccaatttt ttgtcatcta gcttttttct 4501 atgattgaaa tttaggtcac gaagccttat atgttcaaga tccttggatt ttgtagcagc 4561 tttttccttt atcaagaaac tgcttgactt cttttgcgaa aaagggtggg tttgattttc 4621 atcttgatgc aacctctcat cgacatccat tttgtcactg tgtctgagaa tctatacttc 4681 agggcctgct acaagaagta gaaactaaaa tacaatgtta caaatgcgag aagggttttt 4741 tttatgatca tgagtgaccg caataaaatc tttcgaagct ttttcgaata ttttaacatt 4801 ttttctctat agcttctttg tggcatcgcc catgggatca aaccatactg gtttctttgt 4861 aaaacaggcg caaaatatag aaactcagaa aaagagaaaa cgagtagtgg aaaaatttat 4921 cagccattta tgcgacgcat ccgtttgact gatctaccta tttaaaacag aaacctacgt 4981 tgaaaagtta tttttccaac acattactgg atctcatcaa attgccaatg taatcttcta 5041 gaactagtgg atccggtccg gttaccttgg gggggggggg ctgcaggaat tcgatatcaa 5101 gcttatcgat accgtcgacc tcgaggccta tagggcccgg tacccagctt ttgttccctt 5161 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 5221 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 5281 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 5341 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 5401 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 5461 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 5521 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 5581 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 5641 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 5701 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 5761 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 5821 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 5881 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 5941 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 6001 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 6061 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 6121 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 6181 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 6241 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 6301 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 6361 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 6421 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 6481 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 6541 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 6601 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 6661 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 6721 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 6781 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 6841 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 6901 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 6961 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 7021 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 7081 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 7141 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 7201 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 7261 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 7321 cgcacatttc cccgaaaagt gccac //