LOCUS       sequence-63999-e        9501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9501)
  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 9501)
  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"; journalName: "Mol Syst 
            Biol."; date: "2011-05-10"; volume: "7:487. doi"; pages: " 
            10.1038/msb.2011.21"
COMMENT     Sequence Label: pBbA5k-EPL14
FEATURES             Location/Qualifiers
     source          1..9501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             5841..5861
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     terminator      5897..5968
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5984..6011
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6039..6056)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6173..6718)
                     /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(6253..6272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6832..6926)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6957..7751)
                     /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(7068..7087)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7678..7697
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7936..9018)
                     /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     8980..8999
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9019..9096)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9302..9321
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9407..9412,9413..9430,9431..9437)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9445..9461
                     /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 atggaacaat cactcaaacc tttgcgcttc cccctcgctg ccctggcagt ggtggtgatc
       61 agcgcttgcg gtcgaacccc cgacgccgtg caggctcccg ctgcgccgaa ggtaagtgtg
      121 gccaaggtga tcgaacaacc gatcaccgaa tgggacgagt tcaccggccg cctcgaagcc
      181 ccggaaaccg tcgaagtgcg cccgcgggtc tccggccaga tcgaccaggt cgccttcacc
      241 gaaggcgccc aggtcaagaa aggcgacctg ctgttccaga tcgacccgcg cccgttccag
      301 gctgaagtcc gccgcctcga agcccaactg caacaggcca aggccaccgc catccgcagc
      361 gccaacgaag cccgccgtgg cgagcgcctg cgtgacagca acgccatctc cgccgaactg
      421 gccgaatcgc gcagcagtgc cgccgccgaa gcccgtgccg gggtcgatgc catccaggcc
      481 cagctcgacc tggcgcgcct gaacctcagc ttcacccgcg tcaccgcgcc catcagcggc
      541 cgagtcagcc gcgccgagtt caccgccggc aacatcgtca ccgccgatgt caccccgctg
      601 accagcgtgg tgtccaccga caaggtctac gcctatttcg acgccgacga gcgcgtgtac
      661 ctcaagtaca cccagctggc ccgtgaaggc cagcgtagcc agagcactcc ggtatacctg
      721 ggcctgacca acgaaagcgg caacccgcac ctgggccaga tgaacttcgt cgacaaccag
      781 gtcaacccgc gcaccggcac catccgcggc cgcgcggtat tcgacaacag caacggccag
      841 ttcaccccgg gcctgtatgc gcgcctgaag ctggtcggca gcgcccagta cgacgccatg
      901 ctcatcaacg acgaagccgt gggcaccgac cttggcaaga agtttgtgct ggtcatggac
      961 aaggacaaca aggccaccta ccgcgccgtg gaactgggac cgaagctgga aggcttgcgc
     1021 atcgtgcgca gcggcctggg caaggacgac cgcatcgtgg tcaagggcct gcagcgcgta
     1081 cgccctggtt cgccggtaac cccggaagaa acacagatgg ccagcgagca aaccctcgcc
     1141 gccctcgccc agcagcgcca ggccctggag gccagcaacc cggcgccgaa ggtggcgggc
     1201 aacaacgtga aagtcgccag cgcccaggcg ccacgcggtt aagggaccct accgatgaac
     1261 ttctcgaaat tcttcattac ccggccgatc ttcgccgcgg tgctgtcgct ggtgctgctg
     1321 atcgcgggtt ccatctcgct gttccagctg ccgatcagcg aataccccga agtggtgccg
     1381 cccaccgtgg tggtgcgcgc caacttcccc ggcgccaacc ccaaggtcat cggcgaaacc
     1441 gttgccgcgc cgctggagca ggccattacc ggtgtggaga acatgctgta catgtcctcc
     1501 cagtccactg cggacggcaa gctgacgctg accatcacct tcgccctggg taccgacctg
     1561 gacaacgccc aggtgcaggt gcagaaccgc gtcacccgta cccagcccaa gctgcccgag
     1621 gaagtgaccc gtatcggtat caccgtcgac aaggcctcgc ctgacctgac catggtcgtg
     1681 cacctgacct cgccggacaa ccgctacgac atgctctatc tgtccaacta cgccatcctc
     1741 aacatcaagg acgagctggc gcgcctgggc ggcgtaggcg acgtgcagct gtttggcatg
     1801 ggcgactact cgctgcgcgt ctggctggac ccgaacaaga ccgcttcgcg caacctgacg
     1861 gccagtgatg tggtcgcggc aatccgcgag cagaaccgcc aggtggccgc cggccagctg
     1921 ggcgcgccgc ctgccccagg ctcgaccagc ttccagctgt cgatcaatac ccaaggccgc
     1981 ctggtcaacg aggaagagtt cgaaaacatc atcatccgcg ccggtgccga tggcgagatc
     2041 acccgcctga aagacatcgc ccgggtcgaa ctcggctcca gccagtacgc cctgcgctcg
     2101 ctgctgaaca accagccggc ggtggccatc ccgattttcc agcgcccagg ctccaacgcc
     2161 atcgagatct ccgacgaagt gcgggcgaaa atggccgagc tgaaaaagga cttcccggaa
     2221 gggatggact acagcatcgt ctatgacccg accatcttcg tgcgcggctc catcgaagcg
     2281 gtggtgcaca ccctgttcga agccctggtg ctggtggtgc tggtggtgat cctgttcctg
     2341 cagacctggc gcgcctcgat catcccgctg ctggcggtgc cggtctcgct gatcggtacc
     2401 ttcgcggtca tgcacctgtt cggtttttca cttaacgcgc tgtcgttatt cgggctggtg
     2461 ctcgccatcg gtatcgtggt ggacgatgcc atcgtcgtgg tggagaacgt cgaacgtaac
     2521 atcgggctgg gcctgaaacc gctggaagcc acgcaaaagg ccatgagcga agtgaccggc
     2581 ccgatcatcg ccaccgccct ggtgctgtgc gcggtgttcg tacctgcagc gttcatttcc
     2641 ggccttaccg ggcagttcta caagcagttc gccctgacca tcgccatctc gaccgtcatt
     2701 tcggcgttca actcgctgac cctgtcgcca gccttggccg cggtgctgct caaggatcat
     2761 cacgcaccca aggaccgttt ctcgcggttc ctcgacaagc tgctgggcag ctggctgttc
     2821 tcccctttca accggttctt cgaccgggcc agccacagct acgtcggtgg cgtgcgccgg
     2881 gtcatccgct ccagcggcat cgccctgttc gtgtatgccg gcctgatggg tctgacctac
     2941 ctcggcttct cgtccacgcc gaccggtttc gtgccggccc aggacaagca gtacctggtg
     3001 gccttcgccc aactgccgga cgcggccagc ctcgaccgta ccgaagcggt gatcaagcgc
     3061 atgagcgaaa tcgccctgaa gcagcctggc gtggccgact cggtagcctt cccgggcctg
     3121 tcgatcaacg gtttcaccaa cagccccaac agcggcatcg tgttcacccc gctcaaacca
     3181 ttcgatgagc gcaaggaccc gagccagtcg gcggcagcca tcgcagccgc gctgaatgcc
     3241 cagttcgccg acatccagga cgcctacatc gccatcttcc caccgccgcc ggtacagggc
     3301 ctgggcacga ttggcgggtt ccgtctgcaa atcgaagacc gtggcaacct gggttacgaa
     3361 gcgctgtaca aggaaaccca gaacatcatc gccaagagcc acaacgtccc agagctggca
     3421 ggcctgttca ccagctacca ggtcaacgtg ccacaagtcg atgccgccat cgaccgggaa
     3481 aaggccaaga cccacggtgt ggcgatcacc gacatcttcg acaccctgca ggtttacctg
     3541 ggctcgctgt acaccaacga cttcaaccgc tttggccgca cttaccaggt caacgtccag
     3601 gccgagcagc agttccgcct tgatgccgag cagatcggcc aactgaaagt gcgcaacaac
     3661 cttggcgaga tgatcccgct ggcgaccttc ctcaaggtca gcgacacctc cgggccggac
     3721 cgggtcatgc actacaacgg cttcatcact gccgagatca acggcgctgc cgccccgggc
     3781 tacagctctg gtcaggcgga agctgcgatc gagaagctgc tgaaggaaga actgcccaac
     3841 ggcatgacct tcgagtggac cgacctgacc taccagcaga tcctctcggg caataccgca
     3901 ttgctggtgt tcccgctgtg cgtactgctg gcgttcctgg tgctggccgc ccagtacgaa
     3961 agctggagcc tgccgctggc ggtgatcctg atcgtgccga tgaccctgct gtcggccatc
     4021 accggtgtga tcgtatcggg tggcgacaac aacatcttca cccagatcgg cctgatcgta
     4081 ctggtgggcc tggcgtgcaa gaacgcgatc ctgatcgtcg agttcgccaa ggacgaacag
     4141 gccaagggcc tcgacccgct ggctgcggta ctggaagcct gccgcctgcg cctgcggccg
     4201 atcctgatga cctcgatcgc cttcatcatg ggtgtggtgc ccctggtgtt cagctccggc
     4261 gcaggttcgg aaatgcgcca tgccatgggg gtggcggtgt tctcggggat gatcggcgtg
     4321 accgtgttcg gcctgttcct caccccggtg ttcttcttcc tgatccgccg tttcgtcgag
     4381 cgtcgccagg cccgcaaggc tgaacacgct caagtgctgg agaaccacgc atgaacctgc
     4441 tcaaaccctt gaccccaagc ctgctggcac tggccctggc ggcctgtgcg gtaggcccgg
     4501 actaccaggc gccagccacc gaacctgcgc aattgagcag cgatgtgcag gccaaggcct
     4561 acgaccgtag ccgcttcgag agcctgtggt ggaagcagtt cgacgacccg gtgctgaacc
     4621 agctggtgca ggcttcgctg gacggcaacc gtgacttgcg cgtggccttc gcccgcctga
     4681 aatcggcccg ttcgatccgc gaagacgccg aaaacgaccc gttcccggtg gtcaccagcc
     4741 gtgccagcag cgatctcggc aaaggccaga tccccggcca gaccaccgag cgggtcaata
     4801 gcgagcgcta cgacctgggc ctggacatgg cctgggagct tgacctgttc gggcgcatcc
     4861 agcgccagat cgaagccagc gaagcccagg aagcggttgc cgccgccgac ctgcaacagt
     4921 tgcaggtcag cctgatcgcc gaactggtcg atgcctacgg gcaactgcgc ggtgcccagt
     4981 tgcgcgaaaa gatcgccctg gccaacctga agacccagca ggaatcacgc accattaccg
     5041 aaaccctgcg cgatgcgggc gtgggcaatg acctcgacgt ggtgcgtgcc gatgcgcgcc
     5101 tggccggggt cgaagccacc gtgccacaac tgcaagccga acaggcacgg gcgcggcatc
     5161 gcatcgccac cctgctcggc cagcgccctg acgcgctcag cgtagacctg tcgcccaagg
     5221 ccctgccggc catcgccaag gcgctgccgg tcggtgaccc gggtgagctg ctgcgccgcc
     5281 gcccggacat ccgcagcgcc gagcgccagc tggcggcggc caccgccaac gttggcgtgg
     5341 ccaccgccga cctgttcccg cgcgtcagcc tcagtggctt cctcggcttt accgccgccc
     5401 gcggctcgca gatcggctct tcggcggcca acgcctgggc actgggcccg agcattacct
     5461 gggcggcctt cgacctgggc agcgtgcggg cccgcctgcg cggcgccaag gccgatgccg
     5521 aaggggcact ggccaactac gaacagcagg tgctgctggc actggaagag tcggccaacg
     5581 ccttcagcga ttacgacaag acccaacagc gtctgctgtc gctgatgcgc cagagcgacg
     5641 ccagccgcaa ggctgcagaa ctggcctcga ttcgctatcg cgaaggtacc gtggactacc
     5701 tggtactgct cgatgccgaa cgtgagcggc taagtgctga agatgcacaa gctcagggtg
     5761 aagtcgacct ttatcgcggc atcgtctcga tctacaaggc cctgggtggc ggctggcaac
     5821 cggagacggt ggccagcgcg caccatcatc accatcacca ctgaggatcc aaactcgagt
     5881 aaggatctcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     5941 tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg
     6001 gcctttctgc gtttatacct agggatatat tccgcttcct cgctcactga ctcgctacgc
     6061 tcggtcgttc gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa
     6121 gatgccagga agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata
     6181 ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc
     6241 cgacaggact ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg
     6301 ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca
     6361 cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc
     6421 cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa
     6481 agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt
     6541 gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc
     6601 aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg
     6661 caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga
     6721 agatcatctt attaatcaga taaaatattt ctagatttca gtgcaattta tctcttcaaa
     6781 tgtagcacct gaagtcagcc ccatacgata taagttgtta ctagtgcttg gattctcacc
     6841 aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt
     6901 cattactgga tctatcaaca ggagtccaag cgagctctcg aaccccagag tcccgctcag
     6961 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
     7021 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
     7081 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
     7141 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
     7201 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
     7261 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
     7321 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
     7381 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
     7441 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
     7501 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
     7561 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg
     7621 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
     7681 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
     7741 tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatcatga tcccctgcgc
     7801 catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta
     7861 ccagagggcg ccccagctgg caattccgac gtcggtgcct aatgagtgag ctaacttaca
     7921 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     7981 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     8041 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     8101 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     8161 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     8221 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     8281 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     8341 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     8401 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     8461 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     8521 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     8581 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     8641 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     8701 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     8761 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     8821 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     8881 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     8941 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     9001 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     9061 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     9121 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     9181 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     9241 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     9301 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     9361 cgatgcgtcc ggcgtagagg atcgagatcg tttaggcacc ccaggcttta cactttatgc
     9421 ttccggctcg tataatgtgt ggaattgtga gcggataaca atttcagaat tcaaaagatc
     9481 ttttaagaag gagatataca t
//
