LOCUS Exported 8957 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pBbA5k-EPL66 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8957) 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 8957) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8957 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 5297..5317 /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" terminator 5353..5424 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 5440..5467 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(5495..5512) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5629..6174) /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(5709..5728) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 6288..6382 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(6413..7207) /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(6524..6543) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 7134..7153 /label=Neo-R /note="Neomycin resistance gene, reverse primer" CDS complement(7392..8474) /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 8436..8455 /label=LacI-R /note="LacI, reverse primer" promoter complement(8475..8552) /gene="lacI" /label=lacI promoter primer_bind 8758..8777 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 8863..8893 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind 8901..8917 /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 atgcccgttc ttccttccag actggttaca ttcggtctcg ctgctgcctt ggcgatgtta 61 acggggtgtg gcgaggaaaa accggcggcg cccgaactgc ccagggttta tgtgcaaacg 121 gtaaaaagtg ccgacttcgc cgccagtgtc gccctgatcg gtgatgttca ggcgcgggtg 181 cagacccggc tttcattccg ggtcaatggc aagatcattc agcgtaatgt cgatgtgggc 241 gaccgggtaa cggcccggca ggtgctggcc cggcttgatc cgaaggattt gcagatcaat 301 gtcgattcgg ccgcagcctc tgtggccgcc gagcaggccc gcgtgaatca gacccgcgcc 361 gcttttgtcc ggcaacaaaa gctgttgccc aaaggctata ccagccgcag tgaatatgac 421 tcggcccagg ccgccttgcg cggcagcgaa agctcgttga aggctgcgca agcgcaactg 481 gccaatgccc gtgaacaatt ggggtatacc gcactggtcg cggatgcgcc gggtgtgatc 541 accgcgcgtc aggctgaagt cgggcaggtg gtgcaggcca cgatgccgat tttcgatctg 601 gcacgcgacg gtgatcggga tgcggtgttc aatgtctacg agtccctgtt cgttcagccg 661 ccgaccaatc agcctgtgca ggtcacgctg ctcgacaacc cggacatcaa ggtcagcggc 721 aaagtacgcg aagtgacccc ggcagtctcg gcgcaaaccg gcactttaca ggtcaagatc 781 gcactcgatc cgctgcctga aggcatggac ctgggctcgg tagtcagcgt ggcgctgagc 841 gcaccggcca atgccagtgt cgagctgcct tgggcggcgc tgaccaagga ccttggcgag 901 cagttgggca agcctgccgt gtgggtggtg gacgagcagg gcaaagtcaa tttgcgcagg 961 gtcaccgtta ctcgttatct gactgccaaa gtcatcatcg gcgacgggct taaaagtgga 1021 gagaaagtcg tggtggcagg cggacagttg ttacacccgg acatgcaggt cgagattgcc 1081 gaacagccca aacagcagaa cgcgcaggtg cagccatgaa gcgtctgtcg tcatgcctgt 1141 tgctgggcgg ccttctgtcc agtggctttc tgttgagtgg ctgcggcaag gacgaggccc 1201 ctccggagcc ggtgcgtcct gtggtgttcg tcgaagccag gcctgagtca aatcaggatt 1261 tcgggcgttt tgccggcaat attcaggcgc gttatcagag cgtgttgggt tttcgtgtgg 1321 cgggacgtat tgtccggcgc aacgtcgacg taggcgctga agtgaagaaa ggcgatttgc 1381 tcgccaccct tgatccgacc gatcagcaga atacagtgcg tgcccggcag ggcgacctgg 1441 ccaatgtgca ggcgcagttg atcaatgctc aggccaatgc tcgccgccag caggaactgt 1501 ttgatcgcgg tgtgggtgcc caggcgcaac tggacatcgc cctgaccgat ctgaagaccg 1561 ccagctcgtc gcaggaacag gccaaagccg ctgcacaaca ggcccgcgat caattgagct 1621 acagcgagct gcgcagtgat cacgatgctg tggtcaccga atggaaagtt gaagccggtc 1681 aggttgttac ggccgggcag gaagtcgtga ccctggcgcg gccggacatc aaggaagcgg 1741 tgatcgacct ccctgacaca ctggccgacc agctgccgtc cgacgtggta ttcaccgtcg 1801 ccagtcagtt gaacccggaa gtgaacacca ccgccaccat tcgtgaaatc gaacctcagg 1861 ccgaccgtac cacacgtacc cgccgtgcac gtctgacgct ggcgcaaacg ccagcggcgt 1921 tccggctggg cacggccatc agcgtgacgc tcagctcgac catcacgccg cgcatgcgtt 1981 tgccgatcaa tgccttgcag caggccgatg gcaaaagcca gatatggatc gtcgacccgc 2041 aaaaccagac ggtcaacccg agggctgtga acattatcag ccgtgacagc aacagcttcg 2101 tgctggccaa tggcgtcaag gcaggtgaaa aagtggtaag cgcgggcgtc aacagcctca 2161 agccgggcca gaaagtcaaa gtcgatgagg aaagcccgcg atgaaaggga atttcaatct 2221 gtccgagtgg gccatcaagc atcaatcgtt cgtctggtac ctgatgtttg tcgcgctgct 2281 gatgggcgtg ttttcctaca tgaagcttgg gcgcgaggaa gacccgtcct tcaccatcaa 2341 gaccatgatt atccagaccc gctggccggg cgcgacggtg gatgagaccc tcaagcaggt 2401 caccgaccgc atcgagaaga agctcgaaga actggactcg ctggactacg tgaagagcta 2461 cacacgaccg ggcgagtcca cagtcttcgt ctacctgcgt gacaccacca gtgccaaggc 2521 gataccggaa atctggtatc aggtgcgcaa gaaggtcgat gacatccgtg gccagttccc 2581 gcagggcctg caaggcccgt cgttcaacga cgagttcggc gatgtatacg gctcgatcta 2641 tgcgtttacc gcagacggtt tctcgatgcg tcagttgcgt gactacgtcg agaaggtccg 2701 cgccgacatc cgcgacgtgc ccgggctggg caaggtcgag atgatcggtc agcaggacga 2761 agtggtgtac ctgaacttct cgacacgcaa gctcgcagca ttgggcatcg accagagcca 2821 ggtggtgcaa agcctgcaat cgcaaaacgc ggtgacgcct gccggggtga tagaagccgg 2881 gccggagcgg atctcggtgc gtacctccgg ccagttcgcg tccgagaagg atctggctgc 2941 agtcaatctg cgcatcaatg accgttttta tcgtctgagc gacattgctg acataacccg 3001 tggctacacc gatccgccca agccgctgtt ccgcttcgac ggcaaaccgg cgataggcct 3061 ggccatcgcg atgcagaagg gcggcaatat ccagtcgttc ggcaaggcgc tgcacgaacg 3121 tatggacgct acaacagcgg agctgccagt cggcattggc gtgcacaagg tgtcggatca 3181 ggccgaggtg gtcaacaagg ctgtgggcgg cttcaccagc gcgttgttcg aggcagtgat 3241 tatcgtgctg ttggtcagct tcgtcagcct cgggtttcgc gcagggttgg tggtcgcgtg 3301 ttcgattccg ctggtgttgg cgatggtgtt cgtgttcatg gaatacagcg gcatcaccat 3361 gcaacggatt tccctcggcg cgctgatcat cgccctcggc ctgctggtgg atgacgccat 3421 gattaccgtg gaaatgatgg tcacacgttt ggaaatgggt gaatccaagg agcaggccgc 3481 gacgtacgcc tatacctcga cagcgttccc gatgctcacc ggtacgctgg tgaccgtggc 3541 cggctttgtg ccgatcggcc tgaacaacag ctctgcgggg gaatacacat tcacgctgtt 3601 tgccgtgata gccgtggcga tgctggtgtc gtggctggtc gccgttctgt tcgcaccggt 3661 gatcggcgtg cacattctga gcgccaacat caaaccgaaa tccgaagagc cgggccgcat 3721 cggccgggca ttcaacggca gcatgctctg ggccatgcgc catcgctggc tggcgattgc 3781 catcacggta gggttgttcg ccgcgtcgct gttctccatg cagttcgtgc agaaccagtt 3841 cttcccgtct tcggaccgcc cggaaattct ggtcgacctg aacctgccac agaacgcctc 3901 aatcaacgaa acgcgcaagg tggtggatcg ttttgaagcg agcctcaagg acgatccgga 3961 catcgagcgc tggagcacct acatcggcca gggcgcgttg cgtttctacc tgccactcga 4021 tcagcagctg gaaaatccgt tctacgccca gctggtgatt gtcagcaaag gcctggagga 4081 gcggagcgcg ttgactgccc gtctgcaaaa acgcttgcgt gatgacttcg tcggcatcgg 4141 ttcgtatgtg caggcgctgg agatgggccc tccggtgggt cgtccgttgc agtatcgcgt 4201 cagcggcgag aacatcgaca aggtgcgcca gcacgccatc gagctggcca aactgctcga 4261 tcacaactcg catgtgggcg aagtgatcta cgactggaac gagccgggca aggtgctgcg 4321 catcgatatc aatcaggaca aggcccggca gttgggcctg tcgtcggaag acgtcgccaa 4381 gctgatgaac agcgtggtca gcggttcaac cgtgacccag gtgcgtgatg atatctatct 4441 gatcaatgtg atcggacgtg ctgaagacgc agaacgcggc acgccggaaa ccctgcagaa 4501 cctgcaaatc gtgacccaaa ctggaacatc cattccgctg ctggcgttcg ccaccgttgg 4561 ttacgagctg gagcagccgt tggtctggcg ccgcgatcgc atgccgacca tcaccgtcaa 4621 gggcgcggta cgggacgcga ttcagccaac cgatctggtc aaacagctgc aacctgaaat 4681 cgacaagttc gccgctggtc tgccggtcgg ctacaaggtt gcaacgggcg gcacggtcga 4741 agaaagcagc aaggcgcagg ggccgattgc cagcgttgcg ccgctgatgc tgttcctcat 4801 ggcgacgttc ctgatgattc agttgcacag cattcagaag atgtttctgg tggccagtgt 4861 cgcgcctctg gggctaatcg gtgtggtgct ggcgctgatc ccgacgggga cgccactggg 4921 gttcgtggcg attctcgggg tgctggcgct gatcggcatc atcatccgca actcggtgat 4981 tctggtgaca cagatcgatg cctatgagcg cagtgggtat ctgccttggg acgctgtggt 5041 cgaagccacc gagcatcgcc gtcgtccgat tttgctgacc gcggccgcgg cgagcctggg 5101 catgatccct attgcgcggg aagtgttctg gggcccgatg gcctacgcca tgatcggtgg 5161 catcatcatc gccacgttgc tgaccctgct attcctgcct gcgttgtacg tggcctggta 5221 ccgcatcaag gaaccaaccg acgaacagcg tcgcgaagcg gctgacaagg gtgacggcga 5281 gcacgcgcag ccagcgcatc atcatcacca tcaccactga ggatccaaac tcgagtaagg 5341 atctccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc 5401 tgttgtttgt cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct 5461 ttctgcgttt atacctaggg atatattccg cttcctcgct cactgactcg ctacgctcgg 5521 tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg 5581 ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt tccataggct 5641 ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac 5701 aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc 5761 tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc 5821 tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg 5881 ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac 5941 atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag 6001 tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc 6061 cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag 6121 gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat 6181 catcttatta atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta 6241 gcacctgaag tcagccccat acgatataag ttgttactag tgcttggatt ctcaccaata 6301 aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt 6361 actggatcta tcaacaggag tccaagcgag ctctcgaacc ccagagtccc gctcagaaga 6421 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 6481 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca 6541 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa 6601 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat 6661 cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct 6721 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc 6781 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca 6841 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca 6901 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 6961 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 7021 cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 7081 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt 7141 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 7201 caatcatgcg aaacgatcct catcctgtct cttgatcaga tcatgatccc ctgcgccatc 7261 agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag 7321 agggcgcccc agctggcaat tccgacgtcg gtgcctaatg agtgagctaa cttacattaa 7381 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 7441 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt 7501 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc 7561 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac 7621 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca 7681 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg 7741 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa 7801 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg 7861 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct 7921 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg 7981 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac 8041 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag 8101 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct 8161 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga 8221 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg 8281 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc 8341 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg 8401 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac 8461 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata 8521 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga 8581 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag 8641 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat 8701 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt 8761 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat 8821 gcgtccggcg tagaggatcg agatcgttta ggcaccccag gctttacact ttatgcttcc 8881 ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cagaattcaa aagatctttt 8941 aagaaggaga tatacat //