LOCUS       sequence-64030-e        9793 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 9793)
  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 9793)
  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-EPL84
FEATURES             Location/Qualifiers
     source          1..9793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             6136..6153
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      6189..6260
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6276..6303
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6331..6348)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6465..7010)
                     /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(6545..6564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(7124..7218)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7249..8043)
                     /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(7360..7379)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7970..7989
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(8228..9310)
                     /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     9272..9291
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9311..9388)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9594..9613
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9699..9704,9705..9722,9723..9729)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9737..9753
                     /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 atgcgttttt cgcctcagcg tgtctcggct ttcggtcttg ttgcgacgct ttcagtgttg
       61 ttggcagcat gcggcgaaaa gccccagttc aatcctggca tgccgcaggt cagcgtgatc
      121 acggtgcagc cgcagcgcgc cgcggtggtg tccgagctgc ccggccgcgt ggatgcagtg
      181 cgcgatgcac agatccgggc gcgggtaacc ggcatcgtcc agaagatcgc cttcgagcag
      241 ggcggcgacg tccaggccga ccagttgctg ttcaagatcg acccggcgcc ctaccaggcc
      301 gcctacgacc aggccaccgc gcagctcaag caggcgcagg ccgacctgtt cagcgccaag
      361 ctgctggccg agcgctacgc gccgctggtc aaggccaatg ccgtcagcaa gcaggaatac
      421 gacaacgccg tggcccgcta ccggcaggcc gacgccagcg tggcggcggc caaggccaac
      481 ctgaccaatg ccaagatcaa cctgggctat accgacgtga cctcgcccat caaggggcgc
      541 atcggcaagc cgctggtgac cgagggcgcg ctggtcgagg cgacctcggc cacccagatg
      601 gcgacggtgc agcagctcga cccgatctat gtcgacttca cgcaatccac cgccgacctg
      661 gcggcgctcc gcgcgttcgc cgccggcaag ctgcagcagg tgggcaagga cgcggtcaag
      721 gtccaggtgg tgctggaaga cggctcgatc taccccgaag cgggcaagct gctgtttacc
      781 ggcgtgacgg tggatccgac caccgggcag gtgaacctgc gggccgaggt gcccaatccc
      841 gacaacatcc tgctgcccgg catgtacgtg cgcgtgcggg tggaccaggg cgtcgacgac
      901 aaggcgctgc tggtgccgca gcaggccctg cagcgcaccg ccgacggcat gcagagcctg
      961 atgctggtca aggacaacaa ggtcgagcag gtgccggtga ccacgggcag cgtgatccac
     1021 aaccagtggg tcattaacag cggcctggcg gcgggagacg tggtggtggt cgaaggcttc
     1081 cagaagatcc gtccgggggc gccggtgcag gcttcgccct ggaacaaggg gggcgcggcg
     1141 cctgccggcc agcagcccgc tcctggccag caggcgccgg cccagcagcc cgccaagcag
     1201 cagcccgcca agcaggaacc cgccaagcag gaacccgcca agcaggaacc cgccaagcag
     1261 gaacccgcca agcaggaacc cgccaagcag gagcccgcca agcaggaacc cgccaagcag
     1321 gcgccggccg caggccagca gtcctaagcg gcgcgcggcg cccgacggcg ccgcgttccc
     1381 ttttcgtgag catcgctttc agagtcagcc cacatgccgc aattttttat tgatagaccg
     1441 atttttgcct gggtagtcgc actgttcatc ctgctgacag ggatattggc cattcccaat
     1501 atgcccgtgt cgcagtatcc ggacgtggcc ccgcctgcca tcacgatcac cgcgacctac
     1561 ccgggcgctt ccgccaagga agtggccgag tcggtcacca gtatcatcga ggaccagctc
     1621 aacggcgcca agggcctgct gtactacgaa tcggtcagcg actcgtacgg ccagtcgcag
     1681 atcaccgcga cgttcgctcc gggcaccgat cccgacctgg cccaggtgga cgtgcagaac
     1741 cgcgtgtcca acgtgacggc gcagctgccc gtggcggtga tgcagcaggg cctgcagtac
     1801 gagcagacca gcgccggctt cctgatgatc gtgacgctgt cgtccaccga cggcacgctc
     1861 gaccagacgg cgctggccga ctacatcacc cgcaacgtca agaacccggt gtcgcgcgtg
     1921 cccggcgtgg gccagttcca gctgttcgcc gcgccgcgcg ccatgcgcat ctgggtcgac
     1981 ccggacaagc tggtgggctt caacctcagc atggccgagg tcaaccaggc catcagccag
     2041 cagaacgtgc tgatttcggc cggcagcatc ggcgcgccgc ccaatccgga cagccagcgc
     2101 gccacggcca ccgtgatcgt caatggccag ttgagcacga tcgaaggctt cggccagatc
     2161 gtgctgcgcg ctaacaccga tggctcgaag gtgttgctgc gcgacgtggc ccgcatcgag
     2221 gtgggcgcgg acaactacca gttcggcgcg cgcctgaacg gcaagcccac ggcggcgttc
     2281 gccatcgtgc tctcgcccga cgccaacgcg ctggccaccg ccgagggcgt gcgccagcag
     2341 atggcctcgc tgtcgcagta cttcccgtcc aacatccgct acgacatccc gtacgacacc
     2401 gcgccctacg tgaaggtgtc gatcgagcag gtgatccaca ccctggtcga agccatggtg
     2461 ctggtgttcc tggtgatgtt cctgttcctg cagaacgtgc gctataccct gatcccggcc
     2521 ctggtggtgc cggtggccat gctgggcgcg ttcgtggtga tgctggcgct gggcttctcg
     2581 atcaacgtgc tgaccatgtt cgccatggtg cttgccatcg ggatcctggt cgacgacgcc
     2641 atcgtggtgg tcgagaacgt ggagcggatc atggccagcg agggcctgtc gccgaaggag
     2701 gccaccaaga aggccatgcc gcagatcagc ggcgccatcg tcggcatcac gctggtgctg
     2761 gtcacggtgt tcctgccgct ggccttcatg accggctcgg tcggcgtgat ctatcgccag
     2821 ttctcggtcg cgatggcggt gtcgatcttc ttctcggcat tcctcgcgct cagcttcacg
     2881 ccggcgctgt gcgcgacgat cctcaagccc atccccaagg gccatcacca ggaaaagaaa
     2941 ggcttcttcg gctggttcaa ccgcaagttc gacgccacca cgcacaacta ccagaactgg
     3001 gtctcgcgca tcctgcacaa gggcgggcgc atgatgctgg cgttcgcgct gctggtggtg
     3061 ctgctgggct ggctgtacat gcgcctgccg tcgtcgttcc tgcccgagga agaccagggc
     3121 tacgtggtca gcaacatcga actgcccacc ggcgccagcg ccaaccgcac catcgaggtg
     3181 atcgaggaag tcgagaatta cttccgcaac atccccgcgg tggagaacgt catcacggtg
     3241 cagggctaca gcttcaacgg caacggcctg aacgcggcca tcgcgttcac cacgctcaag
     3301 gatttctccg agcgcaagag ccgcgccgac tcggccggcg cgattgcctt cacggctttt
     3361 tccaagcagc tgatgggcat tcacgacgcg caggtgttca ccctggtgcc gccggcgatt
     3421 tcgtcgctgg gcaacgcttc gggcttcgac ttccgcctgc aggaccgcgg cggcgccggc
     3481 accgaggcgc tgggcgcggc caccgccgag ctgatgggca tggcggccaa gagcccggtg
     3541 ctgtcgcagg tgcgcattac cgggctggga ccgggttcgc agctgagcct gacgatcgac
     3601 cgcgacaagg cggcggcgct gggggtcaac ttcgacgagg cggccaccct gatctccacc
     3661 gcggtgggct cggccttcct gaacaagttc cccaacatgg gccggatgca gaacatctgg
     3721 gtgcaggccg accagcagta ccgcatgcag gtcgaagacc tgctcaagct gaacgcccgt
     3781 aacaaccagg gcggcatggt gccgctgtcg accttcgtgt cggccaagtg ggtgcaaggg
     3841 ccggtccagg tggtgcgcta caacagctac gagtcggtgc gtatcggcgg cgacgcggcg
     3901 gcgggctaca ccaccggcga agccatgcag gaaatgcagg acctggtggc ccgcttgccg
     3961 cagggcttcg gctacgaatg gaacggcctg tcctaccagg agcggctggc cggcaaccag
     4021 gcgcccatcc tgatggggct ggcgctgctg gtcgtgttcc tggtgctggc cgccctgtat
     4081 gagagctggg ccatcccgct gtcggtgatg ctggtcgtgc cgctgggcat gctgggcgcc
     4141 gtcgcgctgg tgacggtgct gggcatgtcc aacgacgtgt acttccaggt gggcatggtg
     4201 acggtgatcg ggctggcggc caagaacgcc atcctgatcg tggagttcgc caaggaccag
     4261 tacgcgcgcg ggcagggcct gtacgagtcg gcggtggagg cggcgcgcct gcgtttccgc
     4321 cccatcctga tgacgtcgct ggcgttcatc ctgggcgtgg tgccgctggc catcgccacc
     4381 ggcgccggcg ccgccagcca gcgcgccgtc ggtatcggcg tgctgggcgg gatgctggcg
     4441 gccaccccgt tcgcggtgat tttcgtgccg accttcttcg tggtggtgct gggcctgttc
     4501 aagacccggc cgcgcctgct gggcgccgaa ctgcgcgcct tcgaggaaga gcaggcggcc
     4561 aagcgcgccg ccgagcaaca gcagccgcag gacggcgccg cgacgccggc gccgcagacc
     4621 ggcggtcagg agaacaagga atgatggccg tgatgtgcaa gcgcagtgcg ctctcggtgc
     4681 tggtggcggc cgcgctcgcg ggctgctcgc tggcgcccga ctacaagcga ccggaggcgc
     4741 cggtgcaggc gaactggccg gaccagccca aggtgcagta cggcgcctac gccagcccga
     4801 cctcgctggg cgagcagccg gccgcggcgg tggcgccgca gggcgccacg ccggccgacg
     4861 agatcggctg gtgcgagttc ttccgcgacg cgcgcctgca gaagctgatc gaactggccc
     4921 tggtcaacaa ccgcgatctg cgcgtggcgg tgcagcgcgt cgaagaggcg cgtgcgcagt
     4981 acggcgtgca gcgcggcgcg caatggccca gcattggcgc gggcatccag gggcagcgcc
     5041 agcatctgcc gcccgacatg cgtccgctcg gcccggattc gacctccatc tccagccagt
     5101 accaggccgg cctggggctg accacgttcg agatcgacct gttcggccgc ctgcgcaatc
     5161 tgtccgaggc ggcctaccag cagtacctgt cgaccgagca ggcgcagcgc agcgtgcaca
     5221 tcacgctggt cggagcggtg gcgcagtcgt acttcaacct gcgcgccgcc gaggtgcagc
     5281 tggagttgac ccagaagacg ctggcctcgc gccaggagtc gtacaacctg gtcaagacgc
     5341 gctttgacgg cggcgtggct tccgagctcg acctgaacca atccaagtcg ctgctcgaca
     5401 gcgcctccgc ggacctggcg gcgctggcgc gggcgcgcgc aggccgccaa cgccctggtg
     5461 gtgctggtcg ggctgccggg cctgcccgag gacctgccgg cgccggccgt attcggccgc
     5521 gaccagttgc tggccagcgt gccggccggg ctgccgtccg acctgctgac gcgccggccc
     5581 gacatcctgg cggcggagaa ccagctcaag gcggccaatg ccaatatcgg cgcggcgcgc
     5641 gcggcgttct tcccgaccat ttcgctgacc ggcctgttgg gcgtggccag cacgtcgctg
     5701 gacaccctgt tcaagggcgg gcagggctat tggagcttct cgccgtcgat caccacgccc
     5761 ctgttcgccg gcggcagcat ccgcgaaggc ctgaacctgg ccaaggcgcg cgacaacatc
     5821 gcggtggccc agtacgaaca gagcatccag caagcgttcc gcgaggtgtc ggatacgctg
     5881 gccggcgagg cgacctacag cgcgcagctc gacgcgcagc gcgcgttgca ggtggcggcc
     5941 gggcgcacgc tggagctgtc caacctgcgc tacaacaatg gcatcgacag ctatctgcag
     6001 gtgcagacgg cccaggtcga cttcttcaac gcgcagctcg cgctggtgca gaccgggctg
     6061 gcggcattga tcaaccgcgt cgaactctac aaggcgctcg gcggcggctg gcaggaatca
     6121 acgacgaagc aaccacatca tcaccatcac cactgaggat ccaaactcga gtaaggatct
     6181 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     6241 gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct
     6301 gcgtttatac ctagggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt
     6361 tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag
     6421 gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc
     6481 ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga
     6541 ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc
     6601 tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac
     6661 actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca
     6721 gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc
     6781 aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat
     6841 gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt
     6901 tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg
     6961 ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc
     7021 ttattaatca gataaaatat ttctagattt cagtgcaatt tatctcttca aatgtagcac
     7081 ctgaagtcag ccccatacga tataagttgt tactagtgct tggattctca ccaataaaaa
     7141 acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg
     7201 gatctatcaa caggagtcca agcgagctct cgaaccccag agtcccgctc agaagaactc
     7261 gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac
     7321 gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc
     7381 tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg
     7441 gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc
     7501 gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg
     7561 ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc
     7621 gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg
     7681 ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag
     7741 atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc
     7801 gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc
     7861 ctgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg
     7921 cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata
     7981 gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat
     8041 catgcgaaac gatcctcatc ctgtctcttg atcagatcat gatcccctgc gccatcagat
     8101 ccttggcggc aagaaagcca tccagtttac tttgcagggc ttcccaacct taccagaggg
     8161 cgccccagct ggcaattccg acgtcggtgc ctaatgagtg agctaactta cattaattgc
     8221 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     8281 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     8341 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     8401 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     8461 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
     8521 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     8581 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     8641 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     8701 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     8761 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     8821 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     8881 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     8941 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     9001 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     9061 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     9121 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     9181 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     9241 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     9301 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     9361 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     9421 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     9481 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     9541 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     9601 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     9661 ccggcgtaga ggatcgagat cgtttaggca ccccaggctt tacactttat gcttccggct
     9721 cgtataatgt gtggaattgt gagcggataa caatttcaga attcaaaaga tcttttaaga
     9781 aggagatata cat
//
