LOCUS       sequence-173320-        7924 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 7924)
  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 7924)
  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_3494
FEATURES             Location/Qualifiers
     source          1..7924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      3886..3895
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4267..4284
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4320..4391
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4407..4434
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4462..4479)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4596..5141)
                     /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(4676..4695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5255..5349)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5380..6174)
                     /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(5491..5510)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6101..6120
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6359..7441)
                     /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     7403..7422
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7442..7519)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7725..7744
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7830..7835,7836..7853,7854..7860)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    7868..7884
                     /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 gtgatttcaa acaaacacct gcccgacagc cgcctgctgg cgctgctgct aaccagtatt
       61 tttgtacttt ccggatgcag cgatccggca ccgcaacaac aaaaccagcg cccggcgcct
      121 caggtggggg tgtatgaagt ggaagaacaa ccactggccc tgtcttccgc cctgcctggc
      181 cgcacccgcg cctggcggac tgccgaggtt cgcccacagg tgaatggcat tattgaagag
      241 cgactgttcg ttgaaggcag cgtagtggat gaggggtcag tgctgtacca gatcgatgac
      301 gctccctacc gggccgccct ggcccgggcc aatgccagcc tgaccaatgc ccgcaacctg
      361 gccaagcgct acgaaaacct ggtgggcacc cgttccatca gccagcagca gtatgacgac
      421 gccctggctg ccctgcgcag cgccgaggcg gaatacgaag ccgccagcat caacatgaac
      481 tacacccggg tgaaagcccc gatcaccggc aagatcagcc gttcgcgagt cagcgaaggc
      541 gccctggtga ccagcggcca gcagcaggaa atggccacca tcacccaact ggacccgatc
      601 tacgtggaca tcgtgcaacc ggtcacccgc cttctggacc tgcagagcgc tatcgaagcc
      661 gggctgattg aagccgatga gaacgacgtg gttgaagtct cgctggtgct ggagaacggc
      721 caggactatc cgcacaccgg tttgctgaag ttctccgaag tggtggtcga tgagggcacc
      781 ggctcggtca ccctgcgggc tgagttcccg aatccggatg gcaagctgct gccgggtatg
      841 tttgtgcgcg cgctggtgga agaaggcacc cgcccgaacg ccatcctggt gccccagcaa
      901 gcggtgtcac gcgatacccg cggcgtacca accgtctggg tgctggccga ggacaacacg
      961 gtaagcgtcc gccagattga aaccgagcgc accgtgggca acgcctggct ggtgcaaaag
     1021 gggctgcagg ccggtgaaca ggtggtgacc gagggcgttc agcgcctggc gccgggcatt
     1081 cccgtgcagc cggcaccggc agccaatgtc gacctgaaaa ccgaattcgc aacccaggcc
     1141 gaataaggag ctccggacat gccccgattt tttatcgaac gccccatatt tgcctgggtc
     1201 gtcgccctga tgctgatgct cggaggcgga ctggcagtaa agaacctcgc cgttaaccag
     1261 tttccggatg tcgccccgcc ggcgattgcc ttgtcggtga attaccccgg cgcttccgcc
     1321 cagaccgtgc aggacacggt ggtccaggtg atcgagcaac agctcaacgg cctggacggc
     1381 ttgcgctaca tctcctccga gagcaactcg gatggcagca tgaccatcat tgccaccttc
     1441 gaacagggca ccgacccaga catcgcccag gtgcaggtgc aaaacaaact gcagctggcc
     1501 aacccgctgc tgcccgaaga agtgcagcgt caaggcatcc gggtgtctaa gtacaaggtg
     1561 aacttcttca ccgtatttgc gctgaccagc cctgatggca agtacaccca gggcgacctg
     1621 gccgattaca tcgtgtccaa tatccaggac ccggttgcgc gaacccaggg cgtcggtgac
     1681 tttctgctgt tcgtctccca gtacgccatg cgcctgtggc tcgacccgga aaaactcaac
     1741 agctaccagc tgaccccgca ggacgtgatc aattcggtgc gcgcccagaa cgtgcaggtc
     1801 tctgccggcc agctcggtgg cctgcccacc gcagaaggcg tgcagctgca ggccaccgtt
     1861 atcggcaagc agcgcatgaa gaccgccgaa gaattcgaaa acatcctgct gaaagtgaac
     1921 ccggacggct cccaggtgcg cctggccgat gtcgccgagg tgaatctggg caacgagaac
     1981 tacgccacca ccggtaaata caacggcgcg ccggcagccg gtatggccct gcgcctggcc
     2041 accggcgcca accagctgga aaccgctggc cgggtaaaag aaaccctggc agaactggaa
     2101 cgctttctgc ctgaaggcgt agagatcgtc ttcccctacg acaccacacc ggtcgtgtcg
     2161 gcgtctatcg agaccgtcgc catgaccctg atcgaagccg tggtgctggt gttcctggtg
     2221 atgttcctgt tcctgcaaag ctggcgcgcc accatcattc ccacactggc ggtgccggtg
     2281 gttctgctgg ctacctttgg cgtactgtac gcctttggct tcaccgtcaa cgtgatgacc
     2341 atgtttgcca tggtcctggc catcggtttg ctggtggacg acgccatcgt agtggtggaa
     2401 aacgtcgagc gactgatgga agaagagggg ctgtcgccca aggaagccgc caagaaatcc
     2461 atggaccaga tctccggcgc actgctgggc atcggcctgg tgatttcggc ggtgttcctg
     2521 cccatggcct tttttggcgg ctccaccggg gtgatctacc ggcagttctc ggtcaccatc
     2581 atctcggcaa tgtcgttctc tgtgctggtg gcatttattt tcaccccggc cctgtgcgcc
     2641 accttgctga aacccggcga ccagcacgtc aggaaaggct ttttcggctg gtttaaccgc
     2701 acctttgacc gcagcgcaga ccgctacaaa tcaggcgtca gttacctgat caaacgtaag
     2761 ggccgcttta tgggcgtgta cctgttgctg gtcgtggctg tgggcttcct gttcaagggc
     2821 ctgcccaccg cattcctgcc ggacgaagat cagggcgtga tgattgtgat ggtgcagctg
     2881 cccaccaatg ccacgggcga gcgcaccgaa gccgtactgg cagaagctgg aaattatttg
     2941 ctggaagaag agtccgaggt ggtcaagagc gtgatgtctg tgcggggctt caacttcgcc
     3001 ggccgtggcc agaactccgg tattctgttc gtggacctga agccctttgc tgaccgggaa
     3061 agctttgccc agagcgtgtt cgccctcgcg ggccgctccg gggcaaggtt cgcccagatc
     3121 aaagacgcca tcgtgttccc gattgtgcca ccggccattc tggagctcgg caacgccacc
     3181 ggctttgacc tctacctgaa agacaacgga gctatcggcc accacgccct gatggccgcc
     3241 accaatgagt ttatcagcag agccaatgcc gcgccggagc tgaacatggt gcgccacaac
     3301 ggcctgccgg acgagcccca ataccaggtg atcatcgatg acgaaaaagc ccggctgttg
     3361 caggtgtcca ttgccgacat caatgccacc atgtctgccg cctggggttc aagctatgtg
     3421 aacgacttcc tgcacaacgg ccgggtgaag aaagtctatg tgcagggcaa accggattca
     3481 cgactggcac cggaagactt cgacaaatgg tttgtgcgca acgcccaggg tgagatggtg
     3541 ccctttgccg cctttgccac tggcgagtgg gtgttcggtt cgccgcgcct gcagcggtat
     3601 cagggcttac ccgctacgca aatccagggt gctccggcta acggttacag taccggtgat
     3661 gccatggccg cactggaacg cattgccgcc gacctgcccc agggcctggg cctggaatac
     3721 accggcctgt cgttcgagga aaaacaggcc ggcaaccagg cgatgatgct gtatctgctg
     3781 tcgatcctgg tggtgttcct gtgcctggca gccctgtatg aaagctggtc gattccgttt
     3841 gcggtgatca tgctggtgcc actgggcgtg cttggcgcgg tcctggccac catggccaga
     3901 gggctgtcca acgatgtgtt cttccaggtg ggtatgctga ctaccatggg gctggccgcc
     3961 aagaacgcca tcctgatcgt ggagttcgcc cgccagctct atgagcagga aggcaagccg
     4021 ctgctgcagg ccaccgccga ggccgcccgg ctgcgcctga ggccgatcat catgacctcg
     4081 ctggcgttca tcttcggggt actgcccatg gccatcgcca gcggtgcctc cagcgccagc
     4141 cagcatgcca tcggcaccgc ggtggtcggc ggcacactgg ccgcgaccat cctggcgatc
     4201 ttctttgtac cgctgttcta cgtgtttgtg gtgggcctga ccggaaagcg gaaatccgct
     4261 gacgatcatc atcaccatca ccactgagga tccaaactcg agtaaggatc tccaggcatc
     4321 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     4381 tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata
     4441 cctagggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg
     4501 gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact
     4561 taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac
     4621 aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga
     4681 taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt
     4741 accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc
     4801 cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg
     4861 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc
     4921 actggcagca gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt
     4981 aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt
     5041 tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt
     5101 tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc
     5161 agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca
     5221 gccccatacg atataagttg ttactagtgc ttggattctc accaataaaa aacgcccggc
     5281 ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca
     5341 acaggagtcc aagcgagctc tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     5401 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     5461 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     5521 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     5581 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     5641 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     5701 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     5761 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     5821 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     5881 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     5941 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
     6001 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     6061 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     6121 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     6181 cgatcctcat cctgtctctt gatcagatca tgatcccctg cgccatcaga tccttggcgg
     6241 caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
     6301 tggcaattcc gacgtcggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     6361 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6421 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     6481 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     6541 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     6601 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     6661 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     6721 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     6781 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     6841 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6901 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6961 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     7021 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     7081 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     7141 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     7201 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     7261 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     7321 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     7381 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     7441 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     7501 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     7561 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     7621 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7681 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     7741 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     7801 aggatcgaga tcgtttaggc accccaggct ttacacttta tgcttccggc tcgtataatg
     7861 tgtggaattg tgagcggata acaatttcag aattcaaaag atcttttaag aaggagatat
     7921 acat
//
