LOCUS       sequence-168466-        9582 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 9582)
  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 9582)
  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-AcrB/AcrD/AcrFa, ABO_0964
FEATURES             Location/Qualifiers
     source          1..9582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             5925..5942
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5978..6049
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6065..6092
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6120..6137)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6254..6799)
                     /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(6334..6353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6913..7007)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7038..7832)
                     /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(7149..7168)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7759..7778
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(8017..9099)
                     /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     9061..9080
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9100..9177)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9383..9402
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9488..9493,9494..9511,9512..9518)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9526..9542
                     /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 atgtatatac tgaacctgat tcatttttca tgcaagcacc acaagccgtc tttaatggac
       61 aagctgatgg cgtgctatag cgtgtttcca agcactgagg tgattatgcg cagcggtttc
      121 attctccccc tgatagcagg cgtactcgca ctgcagctaa gtggctgcgg cgatgccggc
      181 caatccggcc aacaacaaca gcaacaagca cccgaagtcg gctttgtcac cgtcaaggcg
      241 gccaccgtca ccttgaaccg ggaactgccc gggcgaacca ctgcacacca ggttgccgaa
      301 gtgcggcccc aagtcagtgg tgtgatagaa aagcgcctct ttgaagaagg ccaagaagta
      361 agcgcggggc aaccgctgta caaaatagac tcacgcacct atcaagcagc ggtggctagc
      421 gcccgagcgg agctggcccg tgccactgcc accctgaatt ccaacgactt gcgagccaaa
      481 cgcttcaaga aactgttgga ttacaaagcg gtgagccagc aagaatacga cgaagctcag
      541 gcccagctgg atgagaacaa ggccgccgta gccgctgcac gtgccaatct gcaaagtgcc
      601 cagatcaatc tggattacgc cacgattaaa gcgccaatca gcgggcgcat tggccgctcc
      661 agcgtcaccg ccggtgcttt ggtcaccgcc aatcaggcaa acgccctggc caccattcgc
      721 caactcgacc cgatctacgt agacctgacc cagtcaagca acgagctgcg tcagctgcgc
      781 caagccatgc aggccggtga gctgcaacag gtcagcgacg acgaagcacg cattacccta
      841 atactcgaag atggctccca ctataaggaa gccggtgccc tgcaattttc cgaattcgcc
      901 gtggatgaaa gcaccggctc agtcaccttg cgtgccctat ttcccaaccc ggacggcgaa
      961 ctgctgcccg gcatgttcgt tcgcggccgc ctaccggaag gtcaacgcaa ggatgccatt
     1021 ctggttcccc aaaaaggcat tacccgcgac ccgaccggcc aagcctacgc catgctgatc
     1081 gacgacaata atcaggtaaa aaaagtgaaa gtccgcactg agcgcgcagt cggcaaccag
     1141 tggcttattg ccgacggact gaaagacggt gatcgcctga ttgtggacgg cctgcaacgg
     1201 attcagcccg gcgctccggt ttccccagtg gacacagaaa aagccaacgt cgagcccgca
     1261 cccgcacagc aagaacaacc ccagtccccc gccagcgacg ctggcgaggc acaaagcgca
     1321 gcgcaataat acgcaacgac gagaaacggg attcccatgg ccagattttt tattgaccgt
     1381 ccaatttttg cctgggtgat tgccatcatc atgatgatgg ccggtgcctt ggccatttac
     1441 accctgccca ttgagcagta ccccaccgtt gcgcctccgc aagtctccat tgccggtaac
     1501 taccccggcg cttcggcgaa aaccgtggaa gattcggtca cccaggtgat cgagcaacaa
     1561 atgaacggga tcgacaacct gctgtacatg agctccagca gtgactcttt cggtaatgcc
     1621 gcagtgaacc tgacgttcgc ccccggcacc gacccggaca ttgcgcaagt acaagtacag
     1681 aacaagctac agctggcgac ccccctgctg ccccaggaag tgcagcaaca aggaatgcag
     1741 gtcaccaaat cctcagactc tttcctgatg gtggcgggct ttacctccga agatggcagc
     1801 ctatcccgtg ctgacctagc cgattacgtg gcctccaatg tgcaggaccc ggtcagccgg
     1861 gtgcccggcg tgggtcagat ccagctgttc ggctctccct atgccatgcg cgtctggctg
     1921 gacccgaaca aactgaataa atttgattta accccccaag atgttaccca gacaatcgag
     1981 gttcaaaata atcaggtagc cagcggtcaa ttgggcggcg caccggccgt agaaggccag
     2041 caattgaacg ccaccatcat cgcccagacc cgcctggaag acgtggatca attcgagaat
     2101 atcctgctga aagtaaaccc ggacggctct cgagtgttcc ttaaagacgt ggcccgagtc
     2161 gaactggcag cgcagaacta cgacgtgcag ggccgttata acggccagcc cgccgccggc
     2221 ctggccatca gcctagccac cggcgccaac gccctggaca ccgcagaagc actgcgtgcc
     2281 cgcctagccg aactgcagcc ctacttccct gataagatgg aaatggtgtt tccctacgac
     2341 accacgccct tcgtcagtat ctccatcgaa gaagtggtac ataccctgtt cgaagccatc
     2401 atcttggtct tcctggtcat gtatctgttc ttacaaaact tccgcgccac cctcatcccc
     2461 acactagcgg tgcccgtggt tctgctaggc acctttgcgg tgctggccgc cttcggcttt
     2521 tccatcaata ccctaaccat gtttggcatg gtgctggcca tcggcctact ggtagatgac
     2581 gccatcgtgg tggtggaaaa cgtggaacgg gtgatgcacg aggagggcct cccgcccaaa
     2641 gaggctaccc gtaaatccat ggggcagatt acgggcgcgc tggtgggcat cgccttggta
     2701 ctgtcagccg tattcgttcc catggccttt ttcccgggct ccaccggagc catttaccgc
     2761 cagttctcca tcaccattgt gtccgccatg gtgctgtccg tgctggtggc cttgatcctc
     2821 actccggccc tgtgtgccac catgctcaag gccaaggaca gcgagcacca aaccgatcat
     2881 ggcttcttcg gctggtttaa ccgcaccttc gacaagagca gccgtcgtta tcagggcagc
     2941 gtggaaaaaa tcctcggccg ccggggccgc tacctgttta tctatgtggt gatcgtcggt
     3001 gtgctcggct tcagcttcat gcgccttcca tccagcttcc tgccggaaga agatcagggg
     3061 attttgttca ccctagtgac cctgccagcg ggctcaaccc aagagcagac agtcaaagtg
     3121 ctggaaaaaa tggaggacta ttatctaaat gaagaggcct ccgctgtgga tggcctgttc
     3181 acggtggccg gcttcagttt caccggccgc ggccagaacg ccggcatggc gttcgtgaac
     3241 ctgaaggact ggtcagaacg ggatctgtcc gtggacggcg ccgacaatgt ggttgcccgc
     3301 gccatgggct atttctccac cattcgcgaa gcgatgatgt ttgcactgaa tccgccatcg
     3361 attcctgaac tgggcaacgc cagcgggttc gacttccagc tgcttgacca gtctggtcaa
     3421 gggcacgaag cgctgattca ggctcgtaac cagatgctcg gcatggccgc ccaagatcct
     3481 cgactggccg gggtacgccc caatggtttg gaagacagcc cgcagtacca aatcgatatt
     3541 gaccagcaga aagccaaagc gctagggctg tccatcagcg acattaacag cactctgcaa
     3601 atcgcctggg gctccagcta cgtcaataac tttgtcgacc ggggccgggt aaaacgagtg
     3661 tatgtgcagg ccgatgcccc ctaccgcatg ctaccggaaa acgtcaacga ctggtttgtg
     3721 cgtaataacc aagggaaaat ggtgccgttc tccacctttg ccacgggcca ctggacgtat
     3781 ggttctccca agctagaacg ctataacggt gtgtcgtcgg tgaacatcca aggcaacgcg
     3841 gcacccggct acagtacagg cgacgccatg gatgccatgg aagaactatc cgctaagctg
     3901 cccgccgggt tcggcttcga atggaccgga atgtcctacc aggaacgtca gtctggtgac
     3961 caggcccctg cgctgtatgt catctcacta ttagtagtat tcctctgtct ggcggccctg
     4021 tatgaaagct ggtcgattcc attcgcggta atgctggtgg tccctctggg cattctcggt
     4081 gcggtactgg ccgctacctt ccgtgacctg aacaacgacg tcttcttcca ggtggggctg
     4141 ttaaccacca tcggcctctc ggcgaaaaac gccattctga ttgcggaatt cgcgctggag
     4201 ctggagcaaa aaggcgagca cttgctcaag gccaccctag aagccgtgcg catgcggctg
     4261 cgcccgatcc tgatgacctc actggccttc atgctcgggg taaccccact gatgatcagt
     4321 actggtgctg gtgccggtgc tcgtaacgcc atcggtaccg gggtattcgg cggaatgctc
     4381 accgccaccg tcctcgccat cttcttcatt ccgctgttct acgtggcggt gcgcaaacta
     4441 tccggcgtgc ccctcgacgg caagaagaaa ggcaaagaat aagtccacgc ggagtaatac
     4501 tgatgacccg taacccactg acgctcgccc ttctgggcgg gctgatgctg ggtggctgca
     4561 ccctcgcccc cagctatcaa caaccactct ctcccgtccc tgcgcaactg ggcgaagcca
     4621 acggcaatag tgccagcgag ctaaccctgc ccagctggtc cagcctttat cagagcgcgg
     4681 aattgcaaca gctgattcaa agcgcattgg ataacaaccg agatctgcgc atcgccgtac
     4741 tcgatgtggc cgccctgcgc gcccagtacc aaatccagcg tgcagacctg tacccaaacc
     4801 tatccgtgga cgccagcggt tcccgtcagc gtctgccctc agacctgtcc ggcaccggcc
     4861 attccacatt atcggatcaa tacggtgtgc aagttggcgt taccgcctgg gaactggatc
     4921 tgtttggccg ggtacgcagc ctgaaagatg cgtcgctgga aaaatatctg gccagcgaag
     4981 aagcccagcg cagcacccaa ctggcgctgg ttgcccaagt ggcacaaagc tacttaaccc
     5041 tggttagtga tcgccaaaac ttggcaatca gtcgcgaaac cgtggtcgca caggaggact
     5101 ctctggcatt gattgaacgc cgatacgaga gcggtttggg atccgagctg gatgtgcgcc
     5161 aagcacaaat tgccttgcag gaagcccgcg ccaatctggc cagctttgaa cgccaggtgg
     5221 atcaagacta taacgcctta gccgtactgg tcggcgggcc actgcccacc ctgaaatcgg
     5281 tgaacttgga caacgatgcc ttggccctcg tcgcaccagt tccgcaaggc ctaccctcag
     5341 cactactcac ccagcgaccg gacgtacaac aagccgagca tcagctgcgg gccgccaatg
     5401 ccaatattgg tgccgcccga gccgccttct tcccaagcat tcaactcacc ggcgcctatg
     5461 gcaccatgag tagcgaactg tccggcctgt tcgacagcgg ctccaaagct tggtcattca
     5521 tccccagtat ttccctaccc attttctccg ggggccgtaa cagggccaat ctggatctag
     5581 ccgaagttcg tcaagatcaa gccgtggcca gctacgagaa agccatacaa accgcctttc
     5641 aggaagtcgc taatgcgctg caagcccgcg acactctggc tcgccaacag caggctcaac
     5701 aggcactagt caccgccact gagcagtccc tgacgttgtc acagcaacgc tacgaacaag
     5761 gcgcagatga ttaccttgcg gtgttggacg cccaacgcac tctgctctcg gcccgccaac
     5821 agctagtgac gctaaaactg cagcagttga ccaacgaaac cgagctttac cgcgccttgg
     5881 gtggtggctg ggatgcgcaa cagcccaccc atgtggccag ccgccatcat caccatcacc
     5941 actgaggatc caaactcgag taaggatctc caggcatcaa ataaaacgaa aggctcagtc
     6001 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca
     6061 cactggctca ccttcgggtg ggcctttctg cgtttatacc tagggatata ttccgcttcc
     6121 tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa
     6181 cggggcggag atttcctgga agatgccagg aagatactta acagggaagt gagagggccg
     6241 cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct
     6301 caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg
     6361 gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat
     6421 ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag
     6481 ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat
     6541 cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat
     6601 tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag
     6661 ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag
     6721 aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg
     6781 cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc
     6841 agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgtt
     6901 actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa
     6961 atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctctc
     7021 gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     7081 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     7141 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     7201 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     7261 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg
     7321 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     7381 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     7441 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     7501 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     7561 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
     7621 gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg
     7681 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     7741 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     7801 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     7861 tcagatcatg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact
     7921 ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccga cgtcggtgcc
     7981 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     8041 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     8101 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     8161 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     8221 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     8281 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     8341 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     8401 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     8461 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     8521 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     8581 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     8641 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     8701 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     8761 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     8821 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     8881 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     8941 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     9001 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     9061 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     9121 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     9181 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     9241 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     9301 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     9361 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     9421 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gtttaggcac
     9481 cccaggcttt acactttatg cttccggctc gtataatgtg tggaattgtg agcggataac
     9541 aatttcagaa ttcaaaagat cttttaagaa ggagatatac at
//
