LOCUS       sequence-173329-        9496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses efflux pump in E. coli to provide resistance to biofuel 
            (medium copy).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9496)
  AUTHORS   Turner WJ, Dunlop MJ
  TITLE     Trade-Offs in Improving Biofuel Tolerance Using Combinations of 
            Efflux Pumps.
  JOURNAL   ACS Synth Biol. 2015 Oct 16;4(10):1056-63. doi: 10.1021/sb500307w. 
            Epub 2014 Dec 12.
  PUBMED    25496359
REFERENCE   2  (bases 1 to 9496)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1021/sb500307w";
            journalName: "ACS Synth Biol"; date: "2015-10-16- 16"; volume: "4"; 
            issue: "10"; pages: "1056-63"
COMMENT     Sequence Label: pBbA5k-Maqu_0582
FEATURES             Location/Qualifiers
     source          1..9496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             5839..5856
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5892..5963
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5979..6006
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6034..6051)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6168..6713)
                     /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(6248..6267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6827..6921)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6952..7746)
                     /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(7063..7082)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7673..7692
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7931..9013)
                     /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     8975..8994
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9014..9091)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9297..9316
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9402..9407,9408..9425,9426..9432)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9440..9456
                     /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 atgcagacag agaagagttt gatggtgcgg ctatttgggg tccgcatgtt gccaatgtgg
       61 ttggcggttc ttttgctggc tggttgtgac aattcctccg gtcaggcaaa ggctgaacgg
      121 tcagcgccgc cgcctccgga agtcggggtt gttgaaatgc agccgcagcg tgtgaacctt
      181 tcaatggaac tgccgggcag gaccgccgct taccgtatct cggaagtgag gccgcaagtc
      241 acgggcattt tgcaggagcg cttgtttgaa caaggcgccc aggtcaaagc aggtgaggtc
      301 ctctaccaga tcgaccccaa ccgataccgt gctgcccatc aacgggccca ggcagaactg
      361 gagcgcgcac aggccgaatt gcgacaggct cagcgggagt gggcacgtac gtcagacttg
      421 tttgagaaga atgcggtcag caagcgtcag cgtgatgaag cgctatcggc cttggaggca
      481 gcccgggcgg atgtctcccg atccgaggcg atggtcgaaa cggcgggtat cgagctggcg
      541 tataccgagg tcaaggctcc catcgccggg cgcattggcc cgacactgta cacggagggt
      601 gcactggtaa ccgccaatca gccccaggtc ctggcgcgcg tggttcagct tgatcccatg
      661 tatgtcgata ttcagctacc ggtcgaaaaa ctggctcgaa ttcggtcgtc tctgaatgag
      721 ggcgcggctt ctaagcccgg cctaaatgaa gccgagactg ttttgttgcg cgatgacggt
      781 actctctatc cgcataaggg acgcgttgac gttgcggatg ttaccgtcaa ccaaagcacc
      841 agttcggtca ctgtgcgggc ggttttcccc aatcctgacg aagaccttct gcctggaatg
      901 tacgtgcgcg ttcgcctgag cgagggggtg cgggagaacg cccttctggc gccgcagcag
      961 ggcgtgagcc gtaatccgca aggcgaggcc acggcgttgc tggtcaaccc gcagggagaa
     1021 gttgttgccc gccagttaga ggcagagcgg gccattggcg ctttctggct ggtggagaac
     1081 gggttagagc cgggggatcg ccttatcgtc tccggtctgc aaaaggttcg gccaggggcc
     1141 aaggtgaagc cggttgaggc agatgttcca ttacatccca cccaaaacca gcaagccagt
     1201 ggcaaggata gttcacagaa aagcgcctcc gacgacgagg gccccgcaca atgattaatt
     1261 tcttcatttc caggcccgtt ttttcctggg ttctggccat tgtcgccatg ttggcgggca
     1321 tcatggcgat cttcgttttg ccggtccagc gatacccgtc ggtggcgccg ccggcggtgg
     1381 agattcaggc cgattatcct ggcgcctcgg cggacaccgt gtccaatacg gtggttcagg
     1441 tcattgaaca agagatgacc ggcctcgaca atttgctcta tatggggtca acagcggatt
     1501 cctcaggcca cgctacggtt actctcacct ttgcggcggg caccgatccg gacattgccc
     1561 aggtccaggt gcagaacaag ctcaaactgg cggaaccgcg gctcccggag gtggtgcgcc
     1621 agcaggggat cagcgtcgag aaatccagca ccagttttct gatggtaatg gcattcgtct
     1681 ctaccgacgg tcgcctcagc aagctggata tcgccgattt tatcagctcc gagttagccg
     1741 aacccattgg acgggtaact ggtatcggca gcgtgcaggt tttcggctct gaatatgcga
     1801 tgcggatttg gctggaccct tcggcgttga ccaattacgg cctgaccgtc gccgacgtga
     1861 gcgccgccat cgaggcgcaa aacgcccagg tcaccgccgg ccagctcggc gggctgcccg
     1921 cggtcgaggg tcagcagctc aatgccacgg tcaccgccca gaccttgctc acctcaaccg
     1981 aagagttccg caacatcctg ctcaagagta tgcccgacgg gtcccgggtg cgcctggggg
     2041 atgttgcccg cgttgagctg ggcggcggtg cagtccagat tgacactttc tacgatggcg
     2101 agccggcagc cggccttggc atcaatctgg cgccgggtgc caactcgctt gcggtcacag
     2161 aggctgttaa agcgcggctc caggagctcg agccgtattt tccggaaggg gtggagattc
     2221 ggtatcccta ccagacggcc cccttcgtgg aggcctccat cgacgcggtg gtcacaacca
     2281 tcatcgaggc aatcgccctg gtggtcctgg tcatgctggt atttctccag agttggcggg
     2341 ctaccctgat accagcgatc gcaatcccgg tggtgctgct gggcaccttt gcgatcatgg
     2401 cggccttcgg cttttccatc aacatgctca ctctcttcgg tctcgtattg gcgatcggcc
     2461 ttctggtgga tgacgccatc gtggtggttg agaacgtcga gcgtgtgatg catgaggacg
     2521 gcctgagccc gatggaggcg acgcgcaagt ccatggggca gatagccagc gcgctgattg
     2581 gcatcggcgt ggtgttgtct gcggtgttta tcccgatggc cttctttccg ggctccaccg
     2641 gggcaatcta tcggcagttt tccctgagta ttgctggtgc catggtgctt tcggtgctgg
     2701 tggctctgat tctgagcccc atgctgtgtg gtcggatcct caagcccacg catgaaagaa
     2761 ccttgctgca aaaactgttc ggctggtttg aggcaggcct ggggcgactg acccggggct
     2821 atgtccgcct ggttggccat acggcgcgcc acgcctggct ctacacactg gcgttcctgg
     2881 gcatcgtcgg cctggtggcc gttctctttg tgcggttgcc cggcgggttc ctgcccgcgg
     2941 aagaccaggg gttcgccgtg gtgcagtatc agttgcccgc cggtgcgacc cagcagcgca
     3001 caatcgatac gattcaggtt atcgaagatt acttcatgga ccaggatgag gtgcagggcc
     3061 tcttcacaat cgctggtttc agcttcgctg gccgggcgca gaacgcggga ctggcgttcg
     3121 tcaatctcaa gccatggagt gagcgcgatc ccgagacgca aagcgccgac gcgatcattc
     3181 agcgggcgaa ccgggcgctt gccgggctgg tgcgtgatgg gcgggctttt gctttcaatt
     3241 tgccaccgat accggaattg gggcaggccc tgggctttga attgcgttta caggatcggg
     3301 gtgccatcgg gcatgaagcg ctcatggcag cgcaaggtca attgctgcag ttggcggccg
     3361 aaagccccgt gctgacgagt gtgcgcccca acggtttggc ggacaatccc cgctacaaag
     3421 tggacattga ttacgaaaag gcgcaggccc ttggcataga gccgtccgag atcagtcgac
     3481 ttctgtcggt gacctggggc tcgcaatacg ttaacgattt tctccatgag gggcgggtca
     3541 agcgagtcta tgttcagggt gatgcccctt tccggatgct gcctgaggat tttgaggcgt
     3601 ggtatctgcg agccgctaac ggagagatga ccccgttgag cgaagtgacg aacggacgct
     3661 gggagtatgg ctcaccgcgc cttgagcgtt tcaacggtgt tccgtcccgt cagattcagg
     3721 gtgagccggc accgggctac agtaccggtg aggccatggc ggaggtcgaa cgtttgatcg
     3781 ctcaactccc ggacggtgtg gccggcgcct ggagcggcct gtcctatcag gagcgccagg
     3841 ccggtgccca ggcatccctg ctttacgcgc tgtcggcact ggtggtgttt ctggcgctcg
     3901 ccgcgctcta cgaaagttgg accataccca tttccgtgat gctggcggtg cccttgggcg
     3961 tactcggggc ggtacttgca gcgatggttc gcgggcttcc taacgacgtc ttcttccagg
     4021 ttggcattct gaccaccgtt ggtgtgactg cgcgcaacgc tatacttctg gtggagttcg
     4081 cccgctcgtt agaggatcag ggaatgaaat tgatcgaggc aaccaaggaa gcggcgcgag
     4141 tgcgcttgag gccgattctg atgacatcgg ttgctttcgg catgggtgtg ttgccgcttg
     4201 ctttcgccag tggtgccggt gcaaccaccc gtattgcgat aggaacggcg gttcttggcg
     4261 gcatgatttc ggcaacgata ctggcgacgt tctttattcc actgttctat gtggtcgtcc
     4321 gccgtatcac ggattttctg agtggctcca gagaccagga tgactcggct gcggccgctc
     4381 ccggacgggc aaatggaggt gtggctgata accgttaagg tattgctgac cactttgatg
     4441 gtgttgtcac ttggtggctg ctcactcgcg ccgactgtta agaagcccga gccgtcgatg
     4501 ccggaaaact ggacgtccgc acgactttcg gcggacgaga aactccctct ggactggtgg
     4561 cgtggctatg gcgatccggc acttgtcacg cttgttgagg aggcacttga tgccaacaac
     4621 gatctgcagc tcgccgctgc ccgggttgcc gaagcaaggg cgctccttac cggacagcag
     4681 gcagaacgat acccacttct ggaggtggag ggctccgcgg tgcgccaggg cccgagtgag
     4741 gagtccgtca acagtactgg tggtcaaccc ttcaatgata tccaggttgg cggtgttcta
     4801 tcgtacgagc tggatctctg ggggcggctt gccaatgcca gcgaagcagc ccgggcacgg
     4861 cttctggcca gtgcagcgaa tcgcgaggcc gtgcgactgg ccgtgattgg tgaggtggcc
     4921 aatggttact tcaatttgcg tgcgcttgat cgtcagatcg acatcgcgga acgcaccgtg
     4981 tcgtcccgcc gcgaatcggt cgccctgcaa cgggcccggt ttgaaggagg cgagattaat
     5041 gaattggccc tgcggcaggc agaatccgag cttgcggccg ctgaatccga acttccgagg
     5101 cttcgccaac agcgctccct tcagcgaaac gccttggcag tgcttctggg gcgagatcca
     5161 caacggattg tacgtagtga tatttccgta gcgaatacca ttggtgagat ggatgttccg
     5221 gccagtatcc ctgcaggccg accagcagat ctgatggttc ggcgacccga cattgccgca
     5281 gccgaacagg aattgatcgc ggccaacgcc gaaatcggtg ttgcccgggc ggcgtttctg
     5341 cccaggattt cattcgaggg gctgctgggc ctgcgaagtg cgtccggtgg cgacttgttc
     5401 cagggttcgg catccacgtg gcagttgggc ggctcgttag tcggcccgtt gctggacttc
     5461 gggcgttccg aggcactagt caatcaggcg gaggcgcgtc aacgccaggc cctgattgcg
     5521 tatcgtcagg ccattcaggt ggcgtttcgg gaggtgctcg acgcgatggc cggggtgcgt
     5581 ggtaccgaag atcgcctgga agcgcaggcc cggcaggtag cagcactgcg aagcacggct
     5641 aaacttgcgc gattgcgctt tgagggtggc gcgtccagtt acctggaggt gctggacgct
     5701 gaacgaagcc tgtttacaac cgaacttgat ctcgtggaaa cgcggcgcga tcacttgcag
     5761 tcgacggtta atttgtatcg cgcgctgggc gggggttggc agaattcatt gcaatcttct
     5821 gattatgaaa acactgacca tcatcaccat caccactgag gatccaaact cgagtaagga
     5881 tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     5941 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     6001 tctgcgttta tacctaggga tatattccgc ttcctcgctc actgactcgc tacgctcggt
     6061 cgttcgactg cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc
     6121 caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc
     6181 cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca
     6241 ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct
     6301 gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct
     6361 gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt
     6421 tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca
     6481 tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt
     6541 catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc
     6601 agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg
     6661 cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc
     6721 atcttattaa tcagataaaa tatttctaga tttcagtgca atttatctct tcaaatgtag
     6781 cacctgaagt cagccccata cgatataagt tgttactagt gcttggattc tcaccaataa
     6841 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
     6901 ctggatctat caacaggagt ccaagcgagc tctcgaaccc cagagtcccg ctcagaagaa
     6961 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     7021 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     7081 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     7141 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc
     7201 ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
     7261 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg
     7321 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
     7381 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
     7441 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
     7501 gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
     7561 gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc
     7621 ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc
     7681 atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc
     7741 aatcatgcga aacgatcctc atcctgtctc ttgatcagat catgatcccc tgcgccatca
     7801 gatccttggc ggcaagaaag ccatccagtt tactttgcag ggcttcccaa ccttaccaga
     7861 gggcgcccca gctggcaatt ccgacgtcgg tgcctaatga gtgagctaac ttacattaat
     7921 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     7981 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     8041 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     8101 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     8161 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     8221 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     8281 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     8341 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     8401 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     8461 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     8521 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     8581 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     8641 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     8701 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     8761 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     8821 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     8881 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     8941 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     9001 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     9061 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     9121 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     9181 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     9241 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     9301 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     9361 cgtccggcgt agaggatcga gatcgtttag gcaccccagg ctttacactt tatgcttccg
     9421 gctcgtataa tgtgtggaat tgtgagcgga taacaatttc agaattcaaa agatctttta
     9481 agaaggagat atacat
//
