LOCUS       sequence-64010-e        9456 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 9456)
  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 9456)
  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-EPL42
FEATURES             Location/Qualifiers
     source          1..9456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             5799..5816
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5852..5923
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5939..5966
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5994..6011)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6128..6673)
                     /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(6208..6227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6787..6881)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6912..7706)
                     /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(7023..7042)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7633..7652
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7891..8973)
                     /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     8935..8954
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8974..9051)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9257..9276
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9362..9367,9368..9385,9386..9392)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9400..9416
                     /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 ttgtgcgcat cttgcgtgcc tgacccgagg attttcatgc aactcaagcc agctgttacc
       61 gctctggtca ctgccgtcgc cctggcatcg ctgctcagcg gatgtaaaaa ggaagaggcg
      121 gcacctgccc ctcaagcccc tcaggtcggc gtcgttaccc tgcaaccaca agcctttacc
      181 ttgacttccg aactcccggg ccggaccact gcgttccgca tcgccgaagt tcgcccacag
      241 gtaaacggga tcattctcaa gcgtctgttc aaagaaggtg gagacgtcaa ggaaggtcag
      301 cagctttatc agatcgatcc atcggtctat gaggcaacgc tgaagagtgc cgaggctaat
      361 ctgcaatcga ccaagtcgat ttctgatcgc tacaagcaac tggtcaacga gcaggccgta
      421 agccgccagg aatacgacac tgccgtggcc aaccgcctgc agtcggaagc ctcgctgcaa
      481 agcgcccaga tcaacctgcg ctacaccaaa gtgctgtcgc cactgaccgg ccgaatcggc
      541 cgttcctcgg tgaccgaagg cgccctggtg agcaatggcc aggccgatgc catggccgtg
      601 atccagcagc tggacccaat ctacgtcgac gttacccagt cctcggtgga actgctggaa
      661 ctgcgtcgcg aactggaaag cggccgcctg cagaaagtcg gcgacaatac cgccaaggtc
      721 aaactgaccc tggaagacgg cagccagtac aagctcgacg gcaagctgga attctccgaa
      781 gtctcggtgg acgaaaccac cggctcggtg accctgcgcg cagtgttccc gaaccctgag
      841 cacaccctgc tgcctggcat gttcgtccac gcccagctgc aagccggggt gaacagcgcg
      901 gcgatcctgg ctccccagca aggcgtgacg cgcgacctga aaggcacccc gaccgccctg
      961 gtggttggcc cggataacaa ggtcgagctg cgtcaactca aggccagccg cacggtcggc
     1021 agccagtggt tggtggaaga tgggctgaaa gccggcgatc gcctgatcac cgaaggcctg
     1081 cagttcgtca agccgggcat tgaagtcaag gccagcgaag caaccaacgt tccagcgaag
     1141 aacccggccc ctgcacaggc aactgacaaa gctgcaggcg gcaaagggga gtaaaccatg
     1201 tcgaaatttt ttatcgaccg tccgatcttc gcctgggtaa tcgccctggt gatcatgttg
     1261 gtcggggctc tatcgatcct caaattgccg atcaaccagt acccgagcat cgcgccgcca
     1321 gcgatcgcca tctccgtgac ctacccaggc gcttccgcac agaccgtgca ggacaccgtg
     1381 gttcaggtga tcgagcagca gctcaacggt atcgacaacc tgcgttatgt gtcctcggaa
     1441 agtaactccg acggcagcat gaccatcacc gcgaccttcg aacagggcac caactcggac
     1501 accgcgcagg ttcaggtaca gaacaagctc aacctggcca ctccgctgct gcctcaagaa
     1561 gtgcagcagc agggtatccg cgtgaccaag gcagtgaaga acttcctgct ggtgatcggc
     1621 gtggtttccg aggacggcag catgtccaag gacgacctgt cgaactacat cgtgtcgaac
     1681 atgcaggacc cgatctcgcg gaccgcgggc gtcggtgact tccaggtatt cggtgcccag
     1741 tacgcgatgc ggatctggct cgacccggcc aagctgaaca actacaacct gaccccggtg
     1801 gacgtgaaga ccgcggttgc cgcgcagaac gtccaggtat cgtccggcca gctcggcggc
     1861 ctgcctgccc tgcccggcca gcaactcaac gccaccatca tcggcaagac ccgcctgcag
     1921 actgccgagc agttcaaggc gatcctgctg aaggtcaaca aggacggctc gcaagtgcgt
     1981 ctgggcgatg tcgcggacgt cggcctgggt ggcgagaact acgccatcag cgcccagttc
     2041 aacggcaagc cggcgtccgg cctggcagtg aaactggcca acggcgccaa cgccctggac
     2101 accgccaagg ccctgcgcaa caccatcaag gacctggaac ccttcttccc gcaagggatg
     2161 aaggtggtgt tcccgtatga caccaccccg gtggtgaccg aatccatcaa gggtgtggtt
     2221 gaaaccctgg tggaagcgat cgtcctggtg ttcctggtga tgttcctgtt cctgcagaac
     2281 ttccgcgcca cggtgatcac caccatgacg gtgccggtgg tactgctggg taccttcggc
     2341 atccttgcgg cggccggttt cagcatcaac accctgacca tgttcggcat ggtgctggcc
     2401 atcggcttgc tggtggacga cgccatcgtc gtggtggaga acgtcgagcg ggtgatgcac
     2461 gaggaagggc tgtcgcccaa ggaggcgacg aaaaagtcca tgggccagat ccagggagcc
     2521 ctggtgggta tcgccctggt gctgtccgcg gtactgctgc caatggcctt cttcagcggc
     2581 tccaccgggg tgatctacaa gcagttctcg atcaccgtgg tctcggccat ggccctgtcg
     2641 gtgctggttg ccctgatctt cactccggcc ctgtgcgcca ccatgctcaa ggccgtaccg
     2701 caaggcgaac acgccaccga gaagcgcggc ttcttcggct ggttcaaccg caccttcgat
     2761 cgcagcgtga agagctacga acgcggcgtg ggcaacatgc tgcgccacaa ggttccgtac
     2821 ctgctggcct acgtgctgat cgtcctgggc atgatctggc tgttcactcg catcccgacc
     2881 gcgttcctgc cggaagaaga ccagggcgtt ctgttcgccc aggtacagac gccggccggc
     2941 tccagtgccg agcggaccca ggtggtggtg gacgaaatgc gcgcctacct gctggacaag
     3001 gaaaaggaca ccgtggcttc ggtgttcacc gtgaacggct tcaacttcgc cggtcgcggc
     3061 cagagctcgg gcatggcctt catcatgctc aagccgtggg gcgagcgttc cgcggaaaac
     3121 aacgtgttca acctggcaac ccgcgcccag caacacttct tcagtttccg tgacgcgatg
     3181 gtgttcgcct tcgcccctcc ggcggtactg gagctgggta acgccaccgg tttcgacgtg
     3241 ttcctccagg accgcgccgg tatcggccat gagaaactga tggctgcacg caaccagttc
     3301 ctcggcatgg cagcccagag caaggtgctg tcccaggtac gtccgaacgg cctgaacgat
     3361 gaaccgcagt accagttgac catcgacgac gagcgcgcca gtgccctggg cgtgaccctc
     3421 accgacatca acaacaccct gtcgattgcc ctgggcagta gctacgtcaa cgacttcatc
     3481 gaccgtggtc gggtgaagaa ggtgtacatc cagggtcagg ccggtgcccg catgagcccc
     3541 gaagacctga agaagtggta cgtacgcaac agcgccggaa ccatggtgcc gttctcctcc
     3601 ttcgccaagg gcgagtggat ctacggctcg cccaagctgg cccgttacaa cggcgtggaa
     3661 gccatggaag tgctcggtgc cccggcccct ggctacagta ccggtgaagc catggccgaa
     3721 gtcgaggcca ttgccgcgaa gctgccggcg ggtgtcggta tctcctggac tggcctgtcc
     3781 tacgaggaac gtctgtcggg ctcccaggca cctgcgctgt acgccttgtc cctgctgatg
     3841 gtgttcctgt gtctggcagc gctgtatgaa agctggtcga ttccgatcgc agtgatgctg
     3901 gttgtgcccc tggggatcat cggtgcactc atggcaacca gcatgcgcgg cctgtccaac
     3961 gacgtgtact tccaggtggg tctgttggtg accattggtc tggcggcgaa aaacgccatt
     4021 ctgatcgtcg agttcgccaa ggaactgcac gagcagggcc gcagcctggt agacgccgcg
     4081 gtggaagcct gccggatgcg tctgcggccg atcatcatga cctccctggc cttcgtcctc
     4141 ggggtcgtgc cactggccat ctccacgggc gccggctcgg gtagccagca tgcgatcggt
     4201 accggggtga tcggcggtat gctcaccgcg actatcctgg cgatcttctg ggtgccattg
     4261 ttcttcgtca ccgtatcgtc catgggccgc aggaaagcgg acctgtcaga aactactgca
     4321 acccctaatg aggctggcca atgagcaagt cgctactctc cctggcaatc accgccatcg
     4381 cgctaagtgg ttgctcgctg atcccggatt accagcgccc ggaagcgccg gtggcggcgc
     4441 agttccccca aggcccagcc tactcgccaa aggaggccgc caacgtggcg gccgccgaac
     4501 agggctggcg gcagttcttc catgacccgg ctctgcagca actgatccag acgtccctgg
     4561 aaaacaaccg cgacctcagg gtcgcggcgc tgaacatcga tgcctacgcc gcgcagtacc
     4621 agatccagcg ggctgacctg ttccctgcgg tttcggccaa cgccagcggc agccgccagc
     4681 gcactccggc acgcatgtcg cagaccggcg aagccgggat caccagccag tactcggcca
     4741 ccgtgggcgt cagtgcctat gaactggacc tgttcggccg cgttcgcagc ctgagcgagc
     4801 aggccctgca gacctacttc tccagcgaag aagcccggcg cagcacccag atcagcctgg
     4861 tggccagcgt ggccaacgcc tacatgacct ggcaggccga caaggaactg ctgaagctga
     4921 cccaggacac cctcgccgcc tacgaggaaa gctaccggct gacctcgcgc agcaacgaag
     4981 tgggtgtggc gtcggccctg gacctgagcc agtcgcggac ctcggtggaa aacgcccggg
     5041 tgcaactggc ccgttacacc cgccaggtgg cccaggacga aaacagcctg accctgctgc
     5101 tgggtgcggc gattccggcc aatctgccga aggcccagcc gctgtcggct gacctgctga
     5161 gcgacctgcc ggcaggcttg ccgtcggagc tgctgcaacg gcgtccggac atcctcgaag
     5221 ccgagtacaa gctgaaagcg gccaacgcca atatcggcgc ggcgcgagca gcgttcttcc
     5281 cgagcatcag cctgaccgcc aatgccggta ccttgagccc ggatctctcg ggcctgttca
     5341 aggggggctc gggcacctgg ctgttccagc cgcagatcaa cctgccgatc ttcaacgccg
     5401 gcagcctgcg cgccagcctg gactactcga aaatccagaa agacatcacc gtcgcgcagt
     5461 acgagaaatc catccagacc gccttccagg aagtctccga tggcctggcg gcacgccaga
     5521 cctacaacca gcagttgcag gcccagcgtg atttcgtcaa cgccaaccag gactactatc
     5581 gtctggccga gcgccgctac cgtatcgggg tcgacagcaa cctgaccttc ctcgatgccc
     5641 agcggtcgct gttcagcgcc cagcaggcac tgatcaccga tcgcctgtcg cagctcacca
     5701 gcgaggtcaa cctgtacaag gccctcggcg gtggctggag cgaacagaca gcgaagaacg
     5761 agccactggc ggaaaaagcc ccggcgctgc acctgttcca tcatcaccat caccactgag
     5821 gatccaaact cgagtaagga tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     5881 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg
     5941 ctcaccttcg ggtgggcctt tctgcgttta tacctaggga tatattccgc ttcctcgctc
     6001 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
     6061 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
     6121 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
     6181 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
     6241 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
     6301 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
     6361 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
     6421 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
     6481 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
     6541 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
     6601 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
     6661 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca
     6721 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgttactagt
     6781 gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag
     6841 atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc tctcgaaccc
     6901 cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
     6961 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
     7021 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
     7081 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
     7141 ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt
     7201 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     7261 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     7321 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     7381 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     7441 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     7501 cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg
     7561 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
     7621 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
     7681 gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat
     7741 catgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag
     7801 ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccgacgtcgg tgcctaatga
     7861 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     7921 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     7981 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     8041 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     8101 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     8161 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     8221 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     8281 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     8341 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     8401 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     8461 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     8521 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     8581 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     8641 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     8701 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     8761 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     8821 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     8881 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     8941 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     9001 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     9061 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     9121 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     9181 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     9241 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     9301 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgtttag gcaccccagg
     9361 ctttacactt tatgcttccg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
     9421 agaattcaaa agatctttta agaaggagat atacat
//
