LOCUS Exported 7830 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pBbA5k-EPL64 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7830) AUTHORS Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD, Hadi MZ, Mukhopadhyay A TITLE Engineering microbial biofuel tolerance and export using efflux pumps. JOURNAL Mol Syst Biol. 2011 May 10;7:487. doi: 10.1038/msb.2011.21. PUBMED 21556065 REFERENCE 2 (bases 1 to 7830) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7830 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 4173..4190 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 4226..4297 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4313..4340 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(4368..4385) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4502..5047) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(4582..4601) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 5161..5255 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5286..6080) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5397..5416) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 6007..6026 /label=Neo-R /note="Neomycin resistance gene, reverse primer" CDS complement(6265..7347) /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 7309..7328 /label=LacI-R /note="LacI, reverse primer" promoter complement(7348..7425) /gene="lacI" /label=lacI promoter primer_bind 7631..7650 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 7736..7766 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind 7774..7790 /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)." ORIGIN 1 ttgcttgctc tcgccagtgt tctgccggct gtcctgaccg gctgcaacga aaaaacgcca 61 gcggacccgc gcacagaagc gccattggtg cgcgttgcga gcctccagga ggccggcctg 121 gagccccgtt ccttcaccgg aaccgtagcg gcgcgggtgc aaagcgatct tggttttcgg 181 gtgtccggca aggtgctgga gcggtttgtc gataccgggc aggccgtcaa ccgcggtcaa 241 ccgttgatgc gtatcgaccc agccgacctg aagctcgctg ccaacgccca acgcgaagca 301 gtgaatgcgg ccagagccaa ggcaaagcag gccgcagacg aagagatacg ttaccgaagc 361 ctgcgcagca gtggcaccat ttcagcgtca agctacgacc agatcaagtc gacggcggat 421 tcggccaagg ctcgactcag tgcagccgaa gcgcaggcag aagttgctct caatgcaact 481 cgctacgcag accttgtggc ggatgccgac ggcattgtca tggaaaccct cgttgagcca 541 ggccaggtcg tcagtgcagg gcaagccgtg gtaagcgtgg cacacgccgg accgcgtgaa 601 gcggttattc aacttccgga aaacctgcgc ccccgtatcg gttctaccgc ccaggccacg 661 ctgttcggta atcaggacct caacgccaca acacggttgc gacagctgtc ggacgttgcg 721 gaccgtcaga cccgcacctt tgaagctcgc tggatactcg agggcgagat ggcaaatacc 781 ccactgggca ccaccatcac agtgaaaatc cccagcgcag atagcgccgg gcaaagcacg 841 ttgcgggtgc caatcggcgc gctgtatgac gcaggcaagg gaccgggcat ctgggtcatt 901 caaggggagc cagcacaggt ttactggacg ccagtagtgg ttcagcagct tgatgacgag 961 caagcccgca tcagtacgca gctcaagcca ggcacgcgaa tagtcgcact tggcgcgcac 1021 cttctgcacg aaggccagca agtaagagtg accgctcaag cggccttggc acagagcgcg 1081 cgcccatgag taatgaccgt ttcaatctgt cagcgctggc cgtccgcgag cgctcgatta 1141 ccctgttcct tgtttgcctg atttcgcttg ccgggatcat tgcctttttc aaactgggcc 1201 gggccgaaga cccggcattt acagtcaagg taatgaccgt ggtgtccgtc tggccgggag 1261 caaccgccca ggagatgcag gatcaggtgg cggagaagat cgaaaaacgc cttcaggagc 1321 tgcgctggta cgaccgcgct gaaacctaca ccagaccggg catggcattc acaaccctga 1381 ctctgctcga cagcacgccg ccgtcgcaag tgccggatga gttttaccag gcgcgcaaga 1441 aaatcggtga cgaggccatg ccccttccgt ccggggttat cgggccgatg gtcaacgacg 1501 agtattcgga cgtcaccttc gcgctattcg cgctgaaggc caaaggcgag ccgcagcgcg 1561 tactggcgcg tgacgccgaa tcgctgcgcc agcgcttgct gcatgtgccg ggagtgaaga 1621 aggtcaatat cgtgggtgag cagcccgaac gagtctacgt cgagttctct cacgagcgac 1681 tggcaacgct gggaataagc cctcagacgg tattcgccgc gctgaatgac cagaacgcgc 1741 tgactgccgc gggctcggtc gaaacccgag gaccgcaggt atttatcagg ctcgacggcg 1801 ctttcgatga gctgcagaaa attcgcgata cgcctgttgt ggcacagggc cggacattga 1861 agctggcgga cattgccaca gtaaaacgcg gttacgaaga tccggcgacg ttcatgattc 1921 gcaacggcgg cgagcctgcg ctgctgctgg ggattgtcat gcgcgatggc tggaacgggc 1981 tggacctggg aaaggcactg gaccaggaag tgggcgcgat caacgccgaa ctgccactgg 2041 gcatgagcct gagcaaggtc acggatcagg ccgtcaatat cagttcggcg gtcgatgagt 2101 tcatgatcaa gttcttcgtc gcactgctgg ttgtcatgct ggtctgtttt atcagcatgg 2161 gctggcgtgt gggtgtggtg gttgctgccg cagtcccgct gaccctggcg gtggtcttcg 2221 tgatcatggc catgagcggc aagaatttcg accggatcac gctgggctcg ctgatcctgg 2281 cactcgggct gctggtagat gatgcgatca tcgccatcga aatgatggtg gtgaagatgg 2341 aagaaggata cgaccgcgtc gctgcctctg cgtatgcctg gagccacacc gccgcgccga 2401 tgttatccgg cacgctggtc accgccgtcg gcttcatgcc caacggcttc gcgcgctcca 2461 cggcaggcga atacaccagc aacatgttct ggatcgtcgg tatcgcgctg attgcctcgt 2521 gggtggtcgc cgtgtttttc accccgtatc tgggcgtcaa attgttgcct gatgtgaagc 2581 agatcgaagg tggacatcag gcgctttaca acacgccgcg ctacaaccgc ttccgtcgga 2641 ttctggcgcg cgtcattgca ggcaaatggc tggtagcagg ctcggtcatc gggctctttg 2701 tgctggccgt gctgggcatg ggactggtca agaaacagtt ctttccagtg tccgatcgtc 2761 ctgaagtatt gatcgaactg cagatgcctt atggcacctc gatttctcaa accagtgcag 2821 ccgctgcaaa agtggaaagc tggctggccg aacaggcgga agccgggatc gtcaccgcct 2881 acattggtca gggtgcgcca cgcttctaca tggcgatggg gccggaattg cctgatccgt 2941 catttgccaa gatcgtggta cgtaccgaca gccaggaaca gcgtgaaacg ctgaaacacc 3001 gattgcgcca ggctatttcc gaggggctgg ctgcagaagc gcgggtaagg gtcacgcaac 3061 tggtcttcgg cccctattcg ccctaccccg tcgcttaccg tattgccgga cgcgacccgg 3121 aaacactgcg cagcattgcg gcgcaggtgc aacaggttct gaacgccagc ccgatgatgc 3181 gcaccgtcaa taccgactgg ggcacacgta ctccggcgct gcatttcact ttgcaacagg 3241 accgtatgca ggccatgggg ctgagttcca gtcaggtggc tcagcaactg cagtttctcc 3301 tgacaggtct gcccgttacg gcagtgcgcg aggatattcg caccgtgcag gtggttgcac 3361 gctccgctgg cgacacccga cttgatccgg caaggataat ggacttcacc ctcaccggcg 3421 tcgacgggca acgtgttccg ctgtcgcaga ttgataccgt tgatgtgcgt atggaggaac 3481 cggtgatgcg acggcgcgac cgcacgccga ccatcactgt gcggggcaac atagccgacg 3541 gcctgcaacc accggatgtg tcgacagcca taacccgaca gttacagccg attatcgaca 3601 agctgcccag cggttaccgg atcgagcagg caggttcaat cgaggagtcc ggcaaggcca 3661 tgacagcgat gttgccattg tttccgatca tgctggcggt cacgctgatc attctgattc 3721 ttcaggtgcg ttcgatatcc gccatggtca tggtcttcct gaccagtccg ctggggctga 3781 tcggtgtggt gcctacgttg attctctttc agcagccatt cggcatcaat gcactggtcg 3841 ggctgatcgc actgtccggc attctgatgc gcaatacgct gattctgatt ggccagatac 3901 accacaacga gcaggcgggg ctcgacccgt ttcaagcggt ggtcgaggct accgtacaac 3961 gtgcgcgtcc cgtgatactg acggcgctgg ccgccattct ggcctttatc ccgctcaccc 4021 attcggtgtt ctggggcacg ctggcctaca cgctgatcgg cggcaccttt gcgggtacgg 4081 tgctgaccct ggtgtttctg ccggccatgt actcgatctg gttcaggatc agggcggatg 4141 gcagcgggcg acgactggag acacagcccg cccatcatca ccatcaccac tgaggatcca 4201 aactcgagta aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 4261 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc 4321 ttcgggtggg cctttctgcg tttataccta gggatatatt ccgcttcctc gctcactgac 4381 tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat 4441 ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt 4501 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt 4561 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc 4621 gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt 4681 ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg 4741 cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4801 aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga 4861 gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg 4921 cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa 4981 accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg 5041 atctcaagaa gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat 5101 ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgttac tagtgcttgg 5161 attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag 5221 ttctgaggtc attactggat ctatcaacag gagtccaagc gagctctcga accccagagt 5281 cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg 5341 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata 5401 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg 5461 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg 5521 gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct 5581 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc 5641 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga 5701 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca 5761 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc 5821 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat 5881 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa 5941 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc 6001 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc 6061 aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatcatgat 6121 cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc 6181 ccaaccttac cagagggcgc cccagctggc aattccgacg tcggtgccta atgagtgagc 6241 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 6301 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag 6361 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc 6421 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt 6481 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac 6541 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc 6601 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat 6661 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat 6721 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact 6781 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc 6841 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac 6901 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc 6961 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc 7021 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag 7081 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact 7141 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt 7201 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac 7261 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc 7321 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg 7381 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct 7441 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca 7501 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg 7561 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat 7621 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga 7681 tgccggccac gatgcgtccg gcgtagagga tcgagatcgt ttaggcaccc caggctttac 7741 actttatgct tccggctcgt ataatgtgtg gaattgtgag cggataacaa tttcagaatt 7801 caaaagatct tttaagaagg agatatacat //