LOCUS       sequence-176877-        7971 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 7971)
  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 7971)
  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-PP_3456
FEATURES             Location/Qualifiers
     source          1..7971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4314..4331
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4367..4438
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4454..4481
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4509..4526)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4643..5188)
                     /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(4723..4742)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5302..5396)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5427..6221)
                     /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(5538..5557)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6148..6167
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6406..7488)
                     /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     7450..7469
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7489..7566)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7772..7791
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7877..7882,7883..7900,7901..7907)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    7915..7931
                     /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 atgcctacta ccctctcccc gtgcctgcgc ccgttggcgc tcatggcctt gctgagcctg
       61 tccctggccg gctgcagcga cagcgccgag ccagacgaac aggcccccac cgcgcaagtg
      121 cgcgtagaaa ccctgcaact gcagccactg gccatcagca gtgaactcag cggccgtatc
      181 ctcgcgccac gcaccgccga agtgcgtgcg cgggtcgcag gcgtggtgct caagcgggtg
      241 taccgcgaag gcagcgacgt caagcagggt gatgtactgt tcctgatcga cccggcgccg
      301 ttcaaggccg accatgacag cgcccgcgcc acactggcca aggccgaagc cacccggtac
      361 caggcgcgcc tgcaagagca gcgctaccgc gagctggtcg acgacaaagc cgtcagccgc
      421 caggaatacg acaacgccaa ggccagtttc ctgcaggccg atgccgaagt ggccgaagcc
      481 agggcggcgc tggagcgcgc ccggctgaac ctcggctatg ccaccgtgac cgcgccgatc
      541 tccggccgta tcggccgcgc ccaggtcacc gaaggcgcgc tggtcgggca aaacgaaacc
      601 accccgctgg cgaccatcca gcagttggac ccgatccatg ccgatgtcac ccagtctacc
      661 cgcgagctca acgccctgcg ccgcgccctg cgcgccggcg aattgcagca ggtgggcgac
      721 ggccaggcac gcgccacgct gatccaggat gacggcagcg cctacccgct gccgggcaag
      781 ctgctgttct ccgacatcag cgtcgacccc agcaccaacc agatcaccct gcgcagcgag
      841 ttccccaacc cggacctcga cctgctgcca ggcagctacg tgcgggtacg cctggagcaa
      901 gcggtgcaac ccaagggcct gagcgtgccg caacgcgcca tcctgcgcga cagcgccggt
      961 gtgcccaagg tgctggtggt agaccagcac gcccgggtca gcgaccgcca ggttgtactg
     1021 ggcagcgccc agggtgaccg ctggatcgtc agcgagggcc tggcggccgg tgagcgggtg
     1081 gtggtcgagg gcctgcagca cgtcaaggcc ggtgaccagg tgcaggtcga caacacgccc
     1141 gctgccccac ccatcgcaca gcataccggc cagtgagggc ctgattcatg ccgcaattct
     1201 tcatcgatcg cccggtattc gcctgggtgg tcgcactgtt catcctgctg gctggcgcgc
     1261 tggccatccc gcaactgccg gtggcccagt accccaacgt ggcaccgccg caggtggaaa
     1321 tctacgccgt gtacccgggt gcctcggcgg cgaccatgga cgaaagcgtg gtcagcctga
     1381 tcgaacagga gctcaacggc gccgacaacc tgctgtactt cgagtcgcag agcagcctgg
     1441 gcagcgccac catcaccgcc accttcgccc cgggtaccca cccggacctg gcccaggtcg
     1501 acgtgcagaa ccgcctgaaa gtggtcgagt cgcgcctgcc gcgcccggtc acccagcagg
     1561 gcctgcaggt ggagaaagtg tccaccggct tcctgctgtt ggcgaccctc acctccgaag
     1621 acggcaagct cgatgaaacc gcgctgtcgg acatccttgc acgtaacgtg atggacgaga
     1681 tccgccgcct caagggcgtc ggcaaggccc agttgtatgg ctccgaacgg gccatgcgca
     1741 tctggatcga cccgcgcaag ctgatcggtt tcaacctcac gcccaacgat gtagccgaag
     1801 ccatcgccgc gcagaacgcc caggtcgccc ctggcagcat cggcgatctg cccagccgca
     1861 gcacccagga aatcaccgcc aacgtggtgg tcaaaggcca gttgagcagc cccgacgagt
     1921 ttgctgccat tgtcctgcgt gccaacccgg acggctccac ggtcaccatc ggggatgtcg
     1981 cccgggttga aatcggtgcc caggaatacc agtacggcac gcgcctgaac ggcaaaccgg
     2041 ccacggcctt cagcgtgcag ttgtcacccg gcgccaacgc catggaaacc gctaccctgg
     2101 tacgggcgaa gatgcaggac ctggcgcgct acttcccgga aggcgtcaag tacgacatcc
     2161 cctacgacac ctcgccgttc gtcaaggtgt cgatcgagca ggtcatcaac accctgttcg
     2221 aggccatgct gctggtgttc gcagtcatgt tcctgttcct gcagaacctg cgctacacgc
     2281 tgatcccgac cctggtcgtg ccagtggcat tgatgggcac cttcgccgtg atgctggcca
     2341 tgggcttctc ggtcaatgtg ctgacactgt tcggcatggt gctggccatc ggcattctgg
     2401 tcgacgatgc catcgtggtg gtggagaacg tcgagcgcat catggccgaa gaaggcttgc
     2461 cgcccaagca agccacgcgc aaggccatgg gccagatcag cggtgcaatc gtcggcatca
     2521 cgctggtgct ggtggcggta ttcctgccca tggccttcat gcaaggctcg gtgggggtga
     2581 tctaccagca gttctcgctg tcgatggcgg tgtcgatcct gttctcggcg tttctcgccc
     2641 tcagcctgac cccggcattg tgcgccaccc tgctcaagcc cgtggccaag ggcgagcacc
     2701 atgaacgcaa aggcttcttc ggctggttca atcgccgctt cgaaagcatg agcaacggct
     2761 accagcgctg ggtagtgcag gcgctcaagc gcagcggccg ctacctgctg gtgtacgcgg
     2821 tgctgctggc ggtgctgggc tacggtttca gccagctgcc tacggcgttc ctgcctaccg
     2881 aagaccaagg ctacaccatc accgacattc agctgccgcc gggcgccagc cgcatgcgca
     2941 ccgaacaggt agccgcgcag atcgaggcgc acaacgccga agaaccgggc gtgggcaaca
     3001 ccaccttgat cctcggcttc agcttctccg gtagcgggca gaacgcggcg ctggccttca
     3061 ccaccctcaa ggactggtcc gagcgcggcg ccgacgacag cgcccagtcg atcgccgacc
     3121 gcgcgaccat ggccttcacc cagctcaagg acgcgattgc ctattcggta ctgccgcccc
     3181 ccattgacgg cctcggtgag tcgaccggtt tcgaatttcg ccttcaggac cgtggcggca
     3241 tgggccacgc cgagctgatg gcagcccgtg accagctgct cgaaagcgcc agcaaaagca
     3301 aggtgctgac caacgtgcgc gaagcctcgc tggcggaaag cccgcaggtg caactggaaa
     3361 tcgaccgccg ccaggctaat gcactgggtg tgtcgtttgc cgatatcggc acggtgctgg
     3421 acgtggccgt gggctccagc tacgtcaacg atttccccaa ccagggccgc atgcagcggg
     3481 tggtggtgca ggccgaaggt gaccagcgca gccaggtcga agacttgctg aacattcacg
     3541 tgcgcaacga cagcggcaag atggtgccac tgggcgcgtt cgtgcaggcc aggtgggtca
     3601 gcggcccggt gcaactgacc cgctacaacg gttacccggc ggtgtcgatc tcaggcgagc
     3661 ctgccgccgg ctacagctcg ggcgaggcca tggccgaagt cgagcggctg gtggcgcagc
     3721 taccggccgg taccggtctg gaatggaccg gcctgtcact gcaggagcgg ctgtccggta
     3781 gccaggcccc cctgctgatg gcgctgtcgc tgctggtggt gttcctgtgc ctggcagcgc
     3841 tgtacgaaag ctggtcgatc ccgactgcgg tgctgctggt cgttccgctc ggtgtgctcg
     3901 gtgcggtgct ggcggtgacc ttgcgcggca tgcccaacga cgtgttcttc aaggttggcc
     3961 tgattaccct gatcggcctg tcggcgaaga acgccatcct gatcatcgag ttcgccaagc
     4021 atctggttga ccagggcgtg gacgccgccg acgccgccgt gcaagcggcc cgcctgcgcc
     4081 tgcggccgat cgtgatgacg tcgctggcgt tcatcctcgg cgtggtgccc ctggccatcg
     4141 ccagcggcgc aagctctgcc agccagcagg cgattggcac cggggtgatc ggcggcatgc
     4201 tcagcgccac cctggcggtg gtgttcgtgc cggtgttctt cgtggtggtg atgcgcctgt
     4261 ccgggcgacg ccaggcgcat gacagcgacg ggcaacccgt gccccgcgaa agccatcatc
     4321 accatcacca ctgaggatcc aaactcgagt aaggatctcc aggcatcaaa taaaacgaaa
     4381 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta
     4441 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttatacct agggatatat
     4501 tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg
     4561 gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg
     4621 agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa
     4681 tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4741 cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc
     4801 cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt
     4861 cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc
     4921 ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc
     4981 actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga
     5041 aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt
     5101 agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga
     5161 gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt
     5221 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
     5281 taagttgtta ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt
     5341 tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag
     5401 cgagctctcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     5461 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     5521 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     5581 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     5641 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     5701 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     5761 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     5821 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     5881 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     5941 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     6001 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     6061 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     6121 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     6181 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     6241 gtctcttgat cagatcatga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc
     6301 cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccgac
     6361 gtcggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     6421 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6481 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     6541 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     6601 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     6661 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     6721 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     6781 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     6841 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     6901 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     6961 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     7021 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     7081 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     7141 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     7201 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     7261 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     7321 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     7381 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     7441 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     7501 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     7561 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     7621 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     7681 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     7741 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     7801 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     7861 tttaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt ggaattgtga
     7921 gcggataaca atttcagaat tcaaaagatc ttttaagaag gagatataca t
//
