LOCUS       sequence-64378-e        6769 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 6769)
  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 6769)
  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-EPL124
FEATURES             Location/Qualifiers
     source          1..6769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3112..3129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3165..3236
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3252..3279
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(3307..3324)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3441..3986)
                     /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(3521..3540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4100..4194)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4225..5019)
                     /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(4336..4355)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4946..4965
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(5204..6286)
                     /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     6248..6267
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6287..6364)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6570..6589
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6675..6680,6681..6698,6699..6705)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    6713..6729
                     /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 atggcgaatt tctttattga tcgccccatt tttgcctggg tgctggcaat cctgttgtgt
       61 ctgacaggta ccctggcgat tttttcattg cccgttgaac aataccccga tctcgcgcca
      121 ccgaatgtgc gagtgaccgc taactatccc ggcgcatcgg cccagacgct ggaaaacacc
      181 gtgacccagg ttatcgagca aaatatgacc ggcctcgata atctcatgta tatgtcatct
      241 cagagcagtg gcaccggtca ggcatctgtc actttaagtt ttaaagcagg caccgatccg
      301 gacgaagccg tgcagcaagt acaaaaccag ctgcaatcag ccatgcgaaa gttaccgcag
      361 gcggtgcaaa atcagggcgt gacggtgcgt aaaaccggcg ataccaacat tctgaccatt
      421 gccttcgtct ctaccgatgg ttcgatggat aaacaggata ttgctgatta tgttgccagt
      481 aatattcagg acccgttaag ccgcgtgaat ggcgtcgggg atatcgatgc ctatggttcg
      541 caatattcca tgcgtatctg gctggacccg gcgaaactca acagtttcca gatgacggct
      601 aaagatgtca ctgatgccat tgagtcacag aacgcgcaga ttgcggttgg gcaacttggt
      661 ggtacacctt ccgtcgataa gcaggcgctc aacgccacca ttaacgccca gtcactgctg
      721 caaacaccag aacagttccg cgatatcacc ttgcgggtca atcaggacgg ctcagaggta
      781 aggctgggcg atgtcgccac cgtcgaaatg ggggcggaga aatacgatta tcttagccgc
      841 ttcaatggta agccagcctc cgggctgggg gtaaaactgg cctccggcgc taacgaaatg
      901 gcgacagcgg agctggtgct caatcgtctc gacgagctgg cgcagtattt cccgcatgga
      961 ctggaataca aggtggcgta tgaaaccacc tcgtttgtta aagcctccat tgaagacgtg
     1021 gtgaaaacgc tgctggaagc tatcgctctg gttttcctcg ttatgtatct gttcctgcaa
     1081 aacttccgcg ccacgctgat acccactatc gccgtgccgg tggtgttgat gggaaccttc
     1141 tccgtacttt acgccttcgg ttacagcgtc aacaccttaa ccatgttcgc gatggtgctg
     1201 gcgatcggtc tgctggtgga tgacgccatc gtggtggtgg aaaacgtcga acgtattatg
     1261 agtgaggaag gactcactcc tcgcgaagcc acacgtaaat cgatggggca gatccagggg
     1321 gcactggtcg ggattgcgat ggttctttcg gcggtatttg taccaatggc cttcttcggc
     1381 ggcaccaccg gtgccatcta tcgccagttc tctattacca ttgttgcggc gatggtgctg
     1441 tcagtactgg tagcgatgat cctcactccg gctctgtgtg ccacactact taagccactg
     1501 aaaaaaggtg agcatcatgg gcaaaaaggc ttttttgcct ggtttaacca gatgtttaac
     1561 cgcaacgccg aacgctacga aaaaggggtg gcgaaaattc tccaccgtag cctgcgctgg
     1621 attgtgattt atgtcctgct gcttggcggc atggtgttcc tgttcctgcg tttgccgacg
     1681 tcgttcttac cgctggaaga ccgtggcatg tttactacct cggtacagtt gcccagcggt
     1741 tcaacgcaac aacagaccct gaaagtcgtt gagcaaatcg agaaatacta cttcacccat
     1801 gaaaaagaca acatcatgtc ggtgtttgcc accgttggtt ctggccctgg gggtaacggg
     1861 caaaacgtgg cgcgaatgtt tatccgcctg aaagactgga gcgaacgcga cagtaagacc
     1921 ggcacctcgt ttgccattat cgagcgtgca acgaaggcgt ttaaccaaat taaagaagct
     1981 cgcgttatcg ccagcagccc gccagcaatt agcggtcttg gtagttctgc aggttttgat
     2041 atggagttgc aggaccacgc tggagcgggt cacgatgcgc tgatggcagc acgtaatcag
     2101 ttgctggcgc tggcggcgga aaacccggag ctaacccgtg tgcgccataa cggcctcgac
     2161 gacagtccgc agttgcagat tgatatcgac cagcgtaaag ctcaggcgct gggcgttgct
     2221 atcgacgata ttaacgacac actgcaaacc gcctggggtt cgagctatgt gaatgacttt
     2281 atggatcgcg gtcgcgtgaa gaaagtctat gtgcaggcag ctgcgccgta tcgcatgctg
     2341 ccagatgaca tcaatctctg gtatgtccga aataaagatg gcggcatggt gcccttctct
     2401 gctttcgcga cctcacgctg ggaaacaggc tcgccgcgtc tggaacgcta taacggttat
     2461 tctgcggttg agattgttgg ggaagccgca ccgggggtca gtaccggtac ggcgatggat
     2521 attatggaat cgttagtgaa gcagctgcca aacggctttg gtctggagtg gacggcgatg
     2581 tcgtatcagg agcggctttc cggcgcgcag gctccggcgc tgtacgccat ttccttgctg
     2641 gtggtattcc tgtgtctggc tgcgttgtat gaaagctggt cggtgccgtt ctcggtaatg
     2701 ctggtcgtgc cgctgggggt aatcggcgcg ctgctggcaa cctggatgcg cgggctggaa
     2761 aacgacgttt acttccaggt gggcctgtta acggtcattg gtttatcggc gaaaaacgcc
     2821 atcctgatcg tcgagtttgc taacgagatg aaccaaaaag gccacgacct gtttgaagcg
     2881 acgctccacg cctgccgtca gcgtttacgc ccgattctga tgacctcgct ggcatttatc
     2941 ttcggcgtat tgccaatggc aaccagcacg ggtgccggtt ccggtggtca gcatgcggtg
     3001 ggtactggcg taatgggcgg gatgatttcg gccactattc tggctattta cttcgtgccg
     3061 ctgttctttg tgctggtgcg ccgccgcttc ccgctgaagc cgcgcccgga acatcatcac
     3121 catcaccact gaggatccaa actcgagtaa ggatctccag gcatcaaata aaacgaaagg
     3181 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     3241 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatacctag ggatatattc
     3301 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     3361 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     3421 agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc
     3481 tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3541 cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
     3601 ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg
     3661 ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg
     3721 taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac
     3781 tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa
     3841 ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
     3901 ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga
     3961 ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct
     4021 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     4081 agttgttact agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc
     4141 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg
     4201 agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     4261 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     4321 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     4381 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     4441 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     4501 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     4561 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     4621 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     4681 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     4741 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     4801 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     4861 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     4921 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     4981 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     5041 ctcttgatca gatcatgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
     5101 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccgacgt
     5161 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5221 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5281 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5341 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5401 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5461 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     5521 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     5581 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     5641 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     5701 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     5761 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     5821 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5881 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     5941 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6001 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6061 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6121 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6181 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6241 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     6301 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     6361 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     6421 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     6481 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     6541 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     6601 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgtt
     6661 taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg aattgtgagc
     6721 ggataacaat ttcagaattc aaaagatctt ttaagaagga gatatacat
//
