LOCUS       sequence-64016-e        9539 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 9539)
  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 9539)
  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-EPL54
FEATURES             Location/Qualifiers
     source          1..9539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             5882..5899
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5935..6006
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6022..6049
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6077..6094)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6211..6756)
                     /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(6291..6310)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6870..6964)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6995..7789)
                     /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(7106..7125)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7716..7735
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7974..9056)
                     /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     9018..9037
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9057..9134)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9340..9359
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9445..9450,9451..9468,9469..9475)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9483..9499
                     /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 atggcgagtg gactggagtc caagggcagg cagtatgcgt tcatgttcct caccgcactg
       61 gcagtagcgg gatgcggaaa gcaggaggcg aaggggccgg ccgcgccgcc cgtgaccgag
      121 gttggggtcg tcaaggtcga cctccacgac gtacccgtcg cgttcgagtt cgtcgggcag
      181 accgagagct cgcagcaggt tgaaatccgg gcgcgggtca acggtttcct ggagcaacgg
      241 ctctataccg aaggcacgat ggtcaaggcg gggcaggtgc tgttccgcat ggaccagaag
      301 ccattcaagg ccgcgctgga cgcggccatg gccgaactgg cgcagcaaca ggcgcgcctc
      361 accaccgcgc gccagaacct tgcccgcgtg cgcccgctgg cggccaagaa tgcgttgagc
      421 cagaaggatc tcgacgattc cgttgggcag gagcagggcg ccgccgccgc ggtggagcag
      481 gccaaggcca acgtgacaaa cgcccgtctg aatctggact acacgacgat caccgcgccg
      541 gtctccgggt tatccagcta cgccaagaag caggtcggtt cgtatatcga cgcgagcaac
      601 agcttgctga cttatgtcgc caagctcgac ccgatctggg tcaatttcag tttgtcggag
      661 aacgaggtcc tccagtacca gtcccagcaa gcctccgggc agttgaagat gccgccgcgc
      721 ggctcgatgg aggtggagat cgtcttggcg gatggcgcga cctatggcaa gcgtggccac
      781 atcacgtttg ccgatgcatc gttcagctcc gagactggca cctatctgct gcgcgcggaa
      841 ttcccgaata ccgaaggcgc gctgcgtccg gggcaattcg tgcgcgttcg cgtgcttggc
      901 gcggagcgca cgcgcgcgat cacggtgccg caggcggccg tggtgcaagg gcaacgtgga
      961 cagtttgtct ggaccgtcgg caaggacaac aaggcagaga ccagagtggt ggatgtcggt
     1021 gaatgggcgc agggcaactg gattatccgc tccggcctgc gcgaaggcga taccgtggtg
     1081 gtcgatggca cggtgcgcct cgctcccggc gcgcccctca agccagtggc cgcgcctccc
     1141 cagccggtgt ccaccgaacg gccgcctggc tcccccggcg ccgcgcccac gccagcctcg
     1201 gccccaaccg ccgcccctga gaagggggcg acgtcatgaa gctttcacac ttcttcatcg
     1261 accgtccgat cttcgcatcg gtcctgtcga tcatcattgt cgttggcggt ctggttgcca
     1321 tggtcaacct gccgatcgcg cagttccctg acatcacgcc cccgaccatc acagtgacca
     1381 cgacctatcc gggcgccagc tcggacgtgg tggcacagaa cgtcgcggcc ccgatcgagc
     1441 agcaggtcaa cggcgcggac aacatgctgt atatgtcgtc caccagttcg tccaccggca
     1501 acatgacgct gaccgtctac ttcgagatcg gcacggaccc gtcactggca caggtggacg
     1561 tgcagaaccg cgtgaacctg gcgctgccga cgttgccgga cgcggtgacc aagcagggcg
     1621 tgagcgtgca gaagcgttca tcggccttca tgatggtgat cgcggtctat tcgccggata
     1681 acagctatcc gcagaccttc gtcgataact tcacgaacat ctatattctc gatgcgctca
     1741 agcgcatccc cggtgccaac caggcgtcga tcttcggcac gcccgactac gccatgcgca
     1801 tctggctgaa gccggaccgg atggcccagc taggcatcac cgtcgaggac atcaagaacg
     1861 cggtggccaa ccagaacgag cagttctccg ctggccgaat aggacagtca cccaccgacg
     1921 ggccggtcac actgaccttc ccggtgacga cgaaggggcg gatgaccgag ccggccgagt
     1981 tcgagaacat catcctgcgt gccgcctcgg gtgacgcggc gctggtgcgc ctcaaggacg
     2041 tcggccgtgc cgaactgggc tccaaggatt attcgttgcg cagccgctac aagggcaaga
     2101 ccgccacgct gctggcggtc tatcaacagc cgggcgccaa cgcgctggcc gtggccgagc
     2161 aggtccggaa gacgctggag gaagtaaaga agagcttccc gccgggcatg gagtatgaag
     2221 tcgcgctgga taccaccgag tttgtgcatg agtcgatcaa cgaggtggtg catacgttgc
     2281 gcgacgcagt gatcctggtg atcctggtcg tgttcctgtt cctgcagagc ttccgcgcca
     2341 cgctggtccc gattctggcc gtgccggtgt cgatcatcgg cacgttcatg gccatgtcgg
     2401 cgttcggctt ctcggtcaat atgctgacgt tattcgggat ggtgctggcc atcggcattg
     2461 tggtggacga cgcgatcgtc gtgatcgaga acgtcgaacg gaacatggtc gagttcaaga
     2521 tgccgcccaa agaggcggcc aagcacgcca tggacgaggt gggcggcccg gtgatcgcga
     2581 tcgtgctggt gctgctggcc gtgttcatcc cggtggcgtt cctgtccggc atcacgggcc
     2641 agttgtacaa gcagttcgcc attacgctgg cggtgtcggt gctgctgtcc ggtgtcgtcg
     2701 cgctgacctt gtcgccggca ctggcggcca tcctgcttaa accgggctcc cacgagaaga
     2761 atcgcttctt ccgctggttc aaccgcagtt tcgatcggct cgtggaaggg tacggcaggg
     2821 cgatccagct catcatccgg cgctcggtga tatcgctggc aatcgtggcc gcgctgattg
     2881 cagtcagcgt gctgatgttc atgcgcatcc cgtcgtcgtt cctgccggtg gaagaccagg
     2941 gttatctgct gggcgcggtg atcatgcccg atgcggccag cctggaccgc acgtccgcgt
     3001 tgtcctcgcg cgccgcggac tggttctcca agcagccggg cgtcaaggcg gtggcggtac
     3061 cggacggcta cagcctgatc gactcgcaga acaagtccaa tgccggcacg ctgtttgtca
     3121 cactgaaggg ctttggcgag cgcggcaagg gcgaaagcgc ggcggacctg atcgcgaagg
     3181 ctaacaagga gttctccacc taccgcgagg gagtcgtggt gccggtcaat ccgccatcga
     3241 tcccgggcct gggtacgacc ggcggcttcg agttctggct gcaaagcacc ggggatggca
     3301 cgtatccgca actcgaggaa cgcgtcaggg cgtttatcgc caaggcacat cagcggcccg
     3361 aactgcgcgg cgtgaccagc acgatcaata cgcgctccag acagttgctg gtggacctcg
     3421 accgtgagcg cgcggaaaca cagggcgtgg cggtggagtc ggtctactcg gccttgcaga
     3481 cgatgttcgg ctcgctctac gtcagccagt tcccgaagaa cagtcgcctg ttccaggtga
     3541 tcctgcaggc ggagcccaag taccgttcgc ggcccgacga cctcgacaat gtctacgtgc
     3601 gtaatgccaa gggagatatg gtcccgatca aggccgtgac gaattctcgc tgggtctctg
     3661 gcgcggatct ggtcacgcgg ttcaacaact tccccgcagc gaagattact ggcgacgccg
     3721 caccgggcta tagctccggg caggcgctgg cagcaatgga agaggtagcc cacgaagtcc
     3781 ttggcgaagg ctacagcttt gcgtggagcg gacaggcgta cgaagagaag aaggcagggt
     3841 ccaccgccgc gctggttttc gccttcgcat tgctgatggt gttcctgatc ctggccgcgc
     3901 agtacgagaa atggtcgttg ccgctgggcg tgctgctggc ggtgccattc gcgttgtttg
     3961 gcgcgctaat gggcatcttg ctgcggggta tggaaaacga cgtgtacttc cagatcgggt
     4021 tgaccgtgct gatcgcgctg gcggccaaga atgccatcct gatcttcgag ttcgccgtgg
     4081 aaatgcaaga gaaggaaggg ctgagcccgt ttgaggccgc ggtcaaggca gccaagatgc
     4141 gtctgcgtcc gatcatcatg acttcgctgg ccttcatcct gggttgcgtg ccgctggcca
     4201 tcgccagcgg ggcatccgct gccagccgcc agtcgctcgg taccggcgtg attggcggga
     4261 tgcttggcgc gaccgtcatc gccatcttct tcatcccgat gttcttctgg ggcctggagc
     4321 agcgcagcga gaagaggaag ggcaagcagc cggcttcagg ggctgggacc gggccagggg
     4381 cggggccggc gagtgacacc gaggctactc cgggaggcac gtcatgaagc gccgcacggt
     4441 gtcccgattg atgcttctcg ccacgctggc cgccgccggg tgcgcggtcg ggccggacta
     4501 cgttcgtccg cctgtcgatg ttccgcaggc ctaccgcttc gcggacgaag gtatcagcgt
     4561 gccgcagaca ccggccacgt attggtggtc gcagatgaac gatccggtgc tggacgggct
     4621 gattgccaca gcgcgggcca acaacaagga cttgctgatt gccgcggcac gggtcgagga
     4681 gtactacggc cgtgtcatga ccacccggtc tggcctgttc cctcaggtgg gaatcgaggc
     4741 tggcggtggg cgccagcgca tcagcaccac caatggcacg gcgctgccga tcaacaaccc
     4801 gttcaaccag acgcacgtcg ccgccaccgc atcctgggaa atcgacctgt ttggccggat
     4861 acgccgcctg accgaggcgg ccaaggctga ctggcaggcc agcgaactgg cgcgtcggag
     4921 cacggaaatc tcggtggtcg ccgcgactgc cagcgcctat atcacgctgc gggatctcga
     4981 taaccgcctg cgtatctcgc aagaaaccct ggaaagccgt cggggcgcgc ttgagttgtt
     5041 ccgggagcgc ttccagggcg gcgtggtctc cgaggtcgag ttcgcgcaag cgcggtcgga
     5101 atacgcatcg gcactgacct cggtcgagca gttccgccag caagtcgcgc tgcaggagga
     5161 tctgctggcc ggtctgctgg gcgagaatcc gcgtgaaatt ccgcgcggca agtcgatctc
     5221 cgagttgacc ttgccgggcg tgccggtcgg cctgccttcg acgttactgg agcagcggcc
     5281 ggacgtgctc ctggccgagc agaatctcgt ggcggccaat gcccgcatcg gcgccgcgcg
     5341 tgccgaatac ttcccgacga tttcgctgac cggcgcgttc ggcgctgcca gcacggcact
     5401 gtcggggctg tggaccggtc cggcacgggc ctggtcgttc gcagggctca tcagtatgcc
     5461 gatcttcacc ggtggcgcga tcgccggcga cgtcaagcag gcggaggcga ttcagacgca
     5521 gtcgctgcag gcctaccagc aatccatcca gacagcgttc cgggaagtgg cggatgcgct
     5581 ggtcagcgtg cagcgtacgc gcgatcagct caatgccaag accgaccagg tcgatgcgct
     5641 gggccgttac gcttcgctgg cacgagatcg ctacgaaggc ggttatacga gctatcttga
     5701 agtgctggat tccgagcgcg cgctgttcgc cgcgcaactc gaccagtcca ccctcattgg
     5761 ttcggagttt ggccaacttg tccagctcta caaggcgctg ggtggtggct ggcccgtgga
     5821 gtcggaagca gcacaggcgc ctcaggcaag cctgagtcaa ccggaaaccc gtaacaagga
     5881 gcatcatcac catcaccact gaggatccaa actcgagtaa ggatctccag gcatcaaata
     5941 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     6001 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatacctag
     6061 ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag
     6121 cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca
     6181 gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca
     6241 tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca
     6301 ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg
     6361 tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt
     6421 aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg
     6481 ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg
     6541 cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc
     6601 taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa
     6661 gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca
     6721 gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata
     6781 aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc
     6841 atacgatata agttgttact agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa
     6901 ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg
     6961 agtccaagcg agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat
     7021 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     7081 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     7141 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     7201 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     7261 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
     7321 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
     7381 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     7441 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     7501 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     7561 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca
     7621 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     7681 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     7741 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     7801 ctcatcctgt ctcttgatca gatcatgatc ccctgcgcca tcagatcctt ggcggcaaga
     7861 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     7921 attccgacgt cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     7981 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     8041 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     8101 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     8161 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     8221 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     8281 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     8341 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     8401 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     8461 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     8521 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     8581 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     8641 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     8701 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     8761 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     8821 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     8881 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     8941 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     9001 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     9061 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     9121 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     9181 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     9241 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     9301 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     9361 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     9421 cgagatcgtt taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg
     9481 aattgtgagc ggataacaat ttcagaattc aaaagatctt ttaagaagga gatatacat
//
