LOCUS       sequence-64028-e        9529 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 9529)
  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 9529)
  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-EPL70
FEATURES             Location/Qualifiers
     source          1..9529
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      5645..5654
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5872..5889
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5925..5996
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6012..6039
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6067..6084)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6201..6746)
                     /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(6281..6300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6860..6954)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6985..7779)
                     /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(7096..7115)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7706..7725
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7964..9046)
                     /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     9008..9027
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9047..9124)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9330..9349
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9435..9440,9441..9458,9459..9465)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9473..9489
                     /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 atgaaacgga tgtgtgctgc cctgtgcctc tgtcttgccg cggtcccagc ttcggctgcg
       61 gaaggccaga tctcactgcc ccttgcggag gcagtcagga gtgccgtcga aaagaatctt
      121 gacgttaaag ccgagcttta caatcccgcc atggcccagg ccgatatccg gaagaacgaa
      181 gggatctatg acacaaccct ccgtttgacc acggatttca actattcagt tactgaacca
      241 gccagctcat ttcttaccgg cagtctcact aaccggcaga aaaccttcga tatcaatcct
      301 ggcataagcc agctcatacc gataggcgga accctcgggc tgaccttcaa caacacgtac
      361 aacagcaata attctacgag atccttcaat aattactgga attccgacct taccgtgaac
      421 cttacccagc cgttgttgaa aaatttcggc agggaaccga ccgagcttgc catcatggtg
      481 gcgcgaaaca gcaaggggga gtccctggag cgcttcagga ccacgctttc tgacacggtt
      541 gcgcgggtga ggaatgagta cttcaagctc tacagcctgc gcggggatct ggaggtgaaa
      601 accacctctt tgctcctggc ccggaagatt ctcgacgaca ccaaggctcg ggtaaaggcg
      661 ggggttctgc ctgccatgga gatccttaat gccgagtttg gggtggcgag ccgggagaag
      721 gatctgatcg atgcggaaaa ggcggtgcgc gaccagaatg acgtgctgag ggtgctcctg
      781 cagcttacgg gcagggacga gattgtcccg gtcgacatgc cgacgaaaga acccttcgcc
      841 gtcaacgaga aggagatggt caagcgcgcc ctggacaatc gctccgaaat cagagagcag
      901 ttggccgccc tcaaatcgag ggagcttgaa atgcgggtgg cccacaacag gacgcttccc
      961 gacctgaacc tgagcgccag cgctgcgctg accggccttg attcccacta caaccgggat
     1021 atggaaaaga tcggttcggc tgattatccg gtttggggcg taggattgca gttcgcgtat
     1081 cccttgggca acaatgccgc cgagaacgat tacatcagaa gcaagctccg cttggagcaa
     1141 gccaggaccc aggtgcgcag cctcgaagcg aatgtggaga acgatgtcaa ggcagccatc
     1201 cggggggtgg agtcgggata caagcaaatt gatgttaccg agcggggcag ggcctttgcc
     1261 gaagagcggc ttcggtcctt catcaagaag aacgaagtgg ggcttgccac cacaaaggat
     1321 gtactcgatg tggaaaacga tctggccgca gccaagatca accagatcaa tgccttggtc
     1381 gggtacaccg atgccataac ccagctctgg cggaccacgg gagaaatcct cgaccggaca
     1441 ggggttcagt tgtcggacac ggagggtgat tccctctacg agcggcacgt ccacaactga
     1501 aaccatcctc tcatcgctcc aaacccaccg gagatacacc atgaatctat ctctctcgcc
     1561 ccgcgacccg tcgagaagcg gcatcctgtc ccgtcgcgtt cgccgactgg tcgtgcaggg
     1621 ggcagcttat tctctcattg tcacacttgc cgcctgcgcc aagaaagagc agaaacctgc
     1681 tgccaagccg ccggtcccgg tagcgatcgc cacggtcgag cgaaagaccg tcccggtcca
     1741 gatcaaggcc atcggcaacg tggagcccta cgctaccgtg gccgtcaagg ctcaggtgaa
     1801 cggcgaggtg ctgaaggtcc atttcaccga gggacaggac gtgaaacggg gtgacctcct
     1861 tttcgccatt gacccgcgca cctatcaggc agcagtgaag aaagccgaag ccaatctttc
     1921 ccgtaacctc gtccaggcac ggaacgcccg gcaggacgcg gagcgctacg cccaactggt
     1981 gaaggagggc atcgtgactt cggagcagta cgagcagtac cggaccaggg ccgaggcctt
     2041 tgccgccgat gtgtccgccg accgggccgc cgtcgagaac gcgaaggtgg agctttccta
     2101 ctgcttcatc cgctccccct tgaccggccg caccgggaat ctggcggtcc atgcggggaa
     2161 catcgtaaag gccaatgaga acccccccct ggtcaccatc aaccagatta cgccggtcta
     2221 cgtggcgttt tccatccccg aaaaggacct cgccgaggtc aagcgccgca tggctgcagg
     2281 aaagctggcg gtggaggcga ccattcccaa cgatgccggt cccgctgagc aggggatgat
     2341 ctccttcctc gacaacacgg ttgacgtggc caccggcacc atcaaactca aggggacctt
     2401 cgagaacagg gcacgtcgcc tctggcccgg gcagttcgcc aacgtggttg tgaccctcgc
     2461 ggccaggccc aatgcgattg tggtcccggc ccaggcgatt cagaccggcc agagcggtcc
     2521 ctatgtcttt gtggtaaagg ctgactcttc ggtagaaatc cggccggtga cgccgggcat
     2581 tgcctacgaa ggggtcacca ttatcgagca ggggctcgcg cccggtgaaa gggttgtgac
     2641 cgacggccag atgaggctca cgcccaaggc cagggtgacg gaaaaaccgg gaccggggac
     2701 cagggaccaa gggccggcaa acccccggca agcttctggc cagcccaagg gccagcaacc
     2761 atgagcatcg ccgaggtttt catccggcgg ccggtcatga cgagcctcgt catgctggct
     2821 gtcatgctct tcggtctcct cgcctacagc aagctgccgg tcaacgacct tcccaccgtc
     2881 gactatccga ccatccaggt ttccgccagc cttcccggcg ccaaccccga caccatggcc
     2941 gcgtcggtgg cgctccccct ggagcgggag ttctcaacca tagccggcgt ggactccatg
     3001 aactccacca acggtctcgg aacgtcccgc atcaccatca aattcaccct ggagcgcgac
     3061 atcgacgccg ccgcccagga tgtccaggca gccatctccc gggcccaacg ccagctcccc
     3121 caagacatgc cggctccacc gtcgttccag aaggttaacc cggcggacca ggcagttctc
     3181 tatctggccc tcagctcgcc gacgcttccc ctgtcaacgg tcaatgacta tgccgaagcc
     3241 atgatcgccc agcggatttc catggttaac ggcgtggcct cggtgcaggt caacggttcc
     3301 cagaagtacg cggtgcgggt ccaggtggac cccaaggcgc ttgcggcccg caagatcggc
     3361 atcgacgagg tggcccaggc cctggagcgc ggaaacgtga acctccccac cggcaccctc
     3421 cagggaaagc acgaggccct aacgatccag accagcggcc agctctatag cgccaaggag
     3481 tatgagccgc tcatcgttgc ctaccgcaac ggttcgccgg tccgcctcgg ggagatcggc
     3541 aaggtcatcg acagtgtgga gaacgacaag gttgccggct ggtacaagca gacccgcgcc
     3601 atcatcctcg ccatccagcg ccagcccggc accaacacca tcgaggtggt gaaccgtatc
     3661 aaggagctga ttccccactt ccgctcgcaa ctccccgcct cggtggacct gaacatcctt
     3721 ttcgaccgga ccggggggat caaggagtcg gtggccgacg tgaagttcac catgatgctc
     3781 accatctgtc tcgtcatcct ggtgattttc ctctttctgc gcaacctccc cgccacactg
     3841 attccgagcc tcgccctgcc actctccatc gtcggagcct tttcagccat gtacctgatg
     3901 gggttctcgg tcaacaacat aaccctcatg gccctgaccc tttcggtggg cttcgtggtg
     3961 gacgacgcca ttgtcatgct ggagaacatc gttcgacacc tggagaaggg agaggacccc
     4021 agggaggcgt cgttgcgcgg ctcccgggag atcggcttca ccattgtctc catgaccatt
     4081 tcgctggtgg cggtcttcat tccggttctc ttcatgcagg ggatgctcgg tcggctcctc
     4141 cacgaattcg ccgtcaccat cagcctagcg atcctcatct ccggtttcgt ttccctctcg
     4201 ctcaccccca tgctctgcag ccgcttcctc cgtccccatg gcgagcagcg ccacggccgg
     4261 ttctacaatt tcatggagcg cttcttcaac ggcatgtacc gcttctatga agtgaccctg
     4321 gcgcgagtcc tgaggcttcg ccggacggtg ctggccggca cgctggtctt gacgctcctc
     4381 accatctggc tctttaccaa ggtacccacg ggcctgttgc cgagcgacga catcggtgcc
     4441 atcttcgcca tgaccgaagg ggcccaggga atctccttcg aggagatgaa ggccaagcag
     4501 cagcaactgg cggccatcgt gctccaggac ccgaacgtgg aggcgttcat gtcgacggcg
     4561 ggcgccagcg ggagccgtgt gtcgtccaac tcgggtttca tgttcatccg cctcaagccc
     4621 cgccaggagc ggaagctgaa cgccgatcag gtgattcagc agttgcgtcc caaggtaatg
     4681 ggggtgccgg ggattgtcat gttcctccag aacccgccgc cgatccgcct ggaggcccgg
     4741 ctggcgaaaa gccagtacca gttcgtgctc cagagccccg acaccgagga tctctacgcg
     4801 cgcgcggccg aactggaggc aaagctgaag caggtgccgt tgctgctgga cgtgacgagc
     4861 gaccttcagc ttcagaatcc acaggttcag ctggacatcg accgggacaa ggcatcggcc
     4921 ctcggggtca ccgcccatca ggtagaggac gcactgtact acgcctacgg tgcacggcag
     4981 gtcaccacca tcttcgcccc cgacaatcag tataaggtga ttctggaact ggagccccaa
     5041 tatcagaccg atcctacagc cctctccatg ctctacatcc gctcccagaa gggggatctg
     5101 gtccccctga atacgctggc aaacctgcgg cgcaccctcg gccccctgtc ggtgaaccat
     5161 ctggggcaga tcccggcggt gaccatctcc ttcaacctgc ggcccgacac ccccctgagc
     5221 gaggcggtgt cagccatcga aaaggtgagc cgcgagacgc ttcccgccac cttcaccaca
     5281 agcttccagg gggttgccca ggcctatcag cagtcaacca cggggctcac ggttctgatt
     5341 atcatggcga ttgttgttat ctacatcgtt ctcggcatcc tctacgagag ctacatccat
     5401 ccgatcacca tcctgtcggg attgccttca gccggtttcg gggcgctgat caccctcatg
     5461 attttcggca aggagctgaa cctctatggt ttcgtgggcc ttatcatgct gatcggcatc
     5521 gtcaagaaga acgccatcat gatgatcgac ttcgcgctgg aggcccagcg cagtgaaggg
     5581 atgccccccc tggaggcgat ccaccaaggc gcccttgtcc gttttcgtcc cattatgatg
     5641 accaccatgg cggctctcat gggaaccctt cccattgccc tcggcatcgg ggcaggggcc
     5701 gaggcccggc gcaccctcgg gctttcagtc gtgggggggc ttgtcgtgtc gcagctgctg
     5761 accctctaca tcacgccggt catctactac tatatggatc gcatgcagga gtgggtaaag
     5821 acgcgtttcg ggatcgggca gagttcccga aaggagcgac agtcggcagg gcatcatcac
     5881 catcaccact gaggatccaa actcgagtaa ggatctccag gcatcaaata aaacgaaagg
     5941 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     6001 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatacctag ggatatattc
     6061 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     6121 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     6181 agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc
     6241 tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6301 cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
     6361 ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg
     6421 ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg
     6481 taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac
     6541 tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa
     6601 ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
     6661 ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga
     6721 ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct
     6781 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     6841 agttgttact agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc
     6901 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg
     6961 agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     7021 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     7081 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     7141 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     7201 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     7261 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     7321 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     7381 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     7441 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     7501 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     7561 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     7621 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     7681 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     7741 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     7801 ctcttgatca gatcatgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
     7861 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccgacgt
     7921 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     7981 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     8041 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     8101 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     8161 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     8221 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     8281 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     8341 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     8401 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     8461 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     8521 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     8581 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     8641 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     8701 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     8761 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     8821 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     8881 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     8941 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     9001 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     9061 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     9121 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     9181 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     9241 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     9301 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     9361 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgtt
     9421 taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg aattgtgagc
     9481 ggataacaat ttcagaattc aaaagatctt ttaagaagga gatatacat
//
