LOCUS Exported 7923 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pBbA5k-EPL65 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7923) 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 7923) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7923 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 4266..4283 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 4319..4390 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4406..4433 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(4461..4478) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4595..5140) /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(4675..4694) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 5254..5348 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5379..6173) /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(5490..5509) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 6100..6119 /label=Neo-R /note="Neomycin resistance gene, reverse primer" CDS complement(6358..7440) /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 7402..7421 /label=LacI-R /note="LacI, reverse primer" promoter complement(7441..7518) /gene="lacI" /label=lacI promoter primer_bind 7724..7743 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 7829..7859 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind 7867..7883 /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 atgcgcagac agaccctgac cgtcaccgct gtagtcctgc tgatagcctg cgcggccatt 61 gccggatgga aactgacacg ccccgctaca ctcccgaaaa actccgtgac tgcggtgcct 121 gtgaaggtga tcacggtcag cattgaggac gtaccacgtt tcgtcacagg tatcggttcg 181 gtgctgtcac tgcaaagtgt ggtgatccgt cctcaggtag atggcgtact gacgcgcgtg 241 ctggtcaatg aaggtcaaca ggtcaaggcc ggcgagttgc tggcgacgct ggatgatcga 301 tcgatccgcg cctcgctgga gcaggcacgc gcgcagctgg cgcagagcaa ggcgcagctt 361 gatgtcgcgc aactggacct gaagcgctat cgccaactca ctcaggacaa tggcatttcg 421 cgccagacat tcgatcagca gcaggcactg gtcaggcaac tcgaagccac ggcgaaaggc 481 aacgaagcaa gcatcaacgc ctcacaggtg cagctctcct acacccagat tcgctcgccg 541 gttacgggca gggtcggcat ccgcaatgtc gatgaaggca acttcctgcg cgtcggcgat 601 gctaccgggc tgttttcggt gacacagatc gacccgattg cggtggaatt ctctctgcct 661 cagcagatgt taccgaccct gcaggggctg attgccgagc gtagcgctgc gaccgtcaag 721 gcgtatcagg gcgacggcgc agcaaatggc ctgctactgg gcgaaggcac gctttcgctg 781 atcgacaatc aggtttcggc aaccaccggc acgatccgcg ccaaggctca attcaaaaac 841 cccggcgaac aactctggcc tgggcagttg gtcacggtga agatccagac cggtgtcgaa 901 ctcaactcgc tcaaagtacc gccacaagta gtgcagcgcg ggatcaatca gcacttcgtc 961 tatcgactca ctgctgataa aaaagtggaa gtggtaccgg tcaaggtgct ttatcaggac 1021 agcgagcaga cgttgatcag cgggccaaac gccggagatg tactggtcag cgacggccag 1081 tcgcggctgc ggccgggggc aactgtcgaa gtggtcagcg cacccgcccc ggatgtggca 1141 agcgcgcaag tggagcacgc acgatgagtg gccgaggctc ggtatcggcg tggtgcatcg 1201 accacccggt ggccaccttg ttattgacct tcgcactggt actgctaggg gtcattgcct 1261 ttccacgact gccgattgcg ccgttgccgg aagcggagtt cccgaccata caggttaccg 1321 cgcagctgcc cggtgccagc ccggaaacca tggcgtcttc ggttgccaca ccgctggaag 1381 tgcagttcag cgcgatcccc ggcatgaccc agatgacctc cagcagcgcg ctggggtcca 1441 ccaacctgac gctgcaattc accctcaaca aaagcatcga cactgccgcc caggaagtcc 1501 aggctgccat caacactgct gcgggacgcc tgccagccga catgccgagc ttgccgacct 1561 ggcgcaaggt caacccggct gacagtccgg tgctgatcct cagtgtcagc tccagcctga 1621 tgcccggcac ggagctgagc gatgtcaccg aaaccatcct ggcgcgccag ctcagccaga 1681 tcgaaggcgt gggccaggtg ttcatcaccg gtcagcaaag gccagcgatt cgtgtgcagg 1741 ccgcccccga aaaactggcc gcgctggggc tgaccctggc cgacattcgc ctggcggtgc 1801 aacagaccag ccttaacctg gccaaaggtg ccttatacgg caaggacagc atttcgacac 1861 tgtcgtccaa cgaccagttg ttcaagccgc aggactacgc gcaactgatc gtttcctaca 1921 agaacggcgc ccccgtccag ctcaaggatg tggcgcgggt ggtcgcgggt tcggaaaacg 1981 cctacgtcaa ggcctggtcc ggcgaccagc aaggcgtcaa catcgctatt tttcgtcagc 2041 cgggcgccaa cattgtcgac accgtggacc gcattcagcg tgaactgcca aggctgcagg 2101 aaatgctccc ggccaccgtt gaggtctcgg tactcaacga ccggacccgc accattcgtg 2161 cctcgctgca cgaagtggaa ctgaccctga tgatcgcagt gctgctggtg gtggcggtca 2221 tggcgctgtt tctgcgccag ctgtcggcca cgctgatcgt cagcgcggtg ctcggggtat 2281 cgctgatcgc cagctttgcg atgatgtacc tgttcggctt cagcttgaac aacctgacat 2341 tggtagccat cgtggtcgcg gtcgggttcg tggtggacga cgctatcgtg gtggtggaga 2401 acatccatcg ccaccttgaa gccggacaag gcacgcgcga ggccgccata aagggcgcgg 2461 gggaaatcgg ttttactgtg gtgtcgatca gtttctcgct ggtggcggcg ttcattccgc 2521 tgttgttcat gggcggcgtg gtcgggcggc tgttcaagga attcgccctg accgccacgg 2581 caaccatcct gatttcggtg atcgtctccc tgaccctggc gccaaccctt gcggcattgt 2641 tcatgcgcgc ccccacgcac aacccgcatc agaagccggg cttcggtgaa cgtttgctgg 2701 cttcctacga gcgaggcttg cgtaaggcgc tggcccatca gcgactgatg ctcggcgtgt 2761 tcggcctgac cctggcgctt gcggtggtcg gctacatcct catacccaag ggcttctttc 2821 cggttcagga cactgctttt gcactcggca ccaccgaggc cgctgccgac atttcctacc 2881 cggacatggt ggaaaaacac ctgcaactgg caaaaatcgt cggtgcagac ccggcagtgc 2941 tggcgttctc gcattcagtc ggtgtgtcag gcagcaacca gaccatcgcc aacggtcgct 3001 tctggatctc gctcaagccg cgctctgaac gcgatgtgtc ggtcagtgag ttcattgacc 3061 gcctgcgccc caaactggca aaggtccccg gcatcgttct gtatctgcgc gccgggcagg 3121 acatcaacct gagttccggg ccgagccgca gtcagtatca gtacgtgctc aagagcaacg 3181 acggcgaatt gctcaacacc tggacgcaac ggctgactga aaagctgcgc agcaatccgg 3241 cgtttcgcga tatgtccaac gacttgcaac tgggcggcag cgtcacccat atcgatattg 3301 atcgcagtgc tgccgcgcga ttcggcctga ccaccgccga cgtcgatcag gcgctgtacg 3361 atgcgttcgg ccagcggcag atcagcgaat accagaccga agtcaaccag tacaaagtga 3421 tcctcgaact ggatgcccag cagcgtggca aggccgaaag cctggcttac ttctacctgc 3481 gctcaccgct gaccaatgaa atggtgccgc tgtcggcact ggccaaagtc agtgcgccgc 3541 gcatgggccc gctgtcgatc agccacgacg gtatgttccc ggctgccaac ctgtcattca 3601 acctggcgtc cggagtcgca ctgggcgacg cggtaagcat gctcgatcag gccaagaaag 3661 agatcggcat gcctgcctcg atcatcggca gttttcaggg tgcggcgcag gcattccaga 3721 gttcgctggc caaccagccg tggctgattc tcgccgcgct ggtggcggtg tacatcattc 3781 tgggtgtgct ttatgaaagc ttcgtgcacc cgctgaccat tatctccacc ctcccctcgg 3841 caggtatcgg cgcgctgttg ctgctatgga tgatggggca ggacttctcg atcatggccc 3901 tgatcggcat cgtgctgctg atcggcatcg tcaagaagaa cggcattctg ctcgtggact 3961 ttgccctgca agcgcagcgt gagcagggcc tgtcgtcaca ggaggccatt tatgaggcct 4021 gcattacccg cttccgaccg attatcatga ccacccttgc cgccctgctc ggcgcactgc 4081 cgctgatgct gggtttcggg gtcggtgccg aactgcgtca accgttgggt atcgcagtgg 4141 tcggtggcct gctggtgagc cagatgctga ccctgttcac tacgccggtc atttacctgc 4201 aactggagcg gctgtttcat aaacgccacc cggcgcccgc cgcacccgcg ctggcccttc 4261 acaagcatca tcaccatcac cactgaggat ccaaactcga gtaaggatct ccaggcatca 4321 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt 4381 gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct gcgtttatac 4441 ctagggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg 4501 cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt 4561 aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca 4621 agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat 4681 accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta 4741 ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc 4801 gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc 4861 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca 4921 ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta 4981 aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt 5041 caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt 5101 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca 5161 gataaaatat ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag 5221 ccccatacga tataagttgt tactagtgct tggattctca ccaataaaaa acgcccggcg 5281 gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa 5341 caggagtcca agcgagctct cgaaccccag agtcccgctc agaagaactc gtcaagaagg 5401 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg 5461 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga 5521 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc 5581 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc 5641 atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc 5701 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt 5761 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca 5821 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc 5881 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct 5941 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca 6001 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc 6061 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc 6121 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac 6181 gatcctcatc ctgtctcttg atcagatcat gatcccctgc gccatcagat ccttggcggc 6241 aagaaagcca tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct 6301 ggcaattccg acgtcggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca 6361 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 6421 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac 6481 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac 6541 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca 6601 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc 6661 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc 6721 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact 6781 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca 6841 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg 6901 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa 6961 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt 7021 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc 7081 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg 7141 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc 7201 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga 7261 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc 7321 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga 7381 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac 7441 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt 7501 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga 7561 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca 7621 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac 7681 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat 7741 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga 7801 ggatcgagat cgtttaggca ccccaggctt tacactttat gcttccggct cgtataatgt 7861 gtggaattgt gagcggataa caatttcaga attcaaaaga tcttttaaga aggagatata 7921 cat //