LOCUS       sequence-63449-e        9417 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 9417)
  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 9417)
  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-EPL11
FEATURES             Location/Qualifiers
     source          1..9417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2250..2267
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5760..5777
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5813..5884
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5900..5927
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5955..5972)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6089..6634)
                     /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(6169..6188)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6748..6842)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6873..7667)
                     /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(6984..7003)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7594..7613
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7852..8934)
                     /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     8896..8915
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8935..9012)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9218..9237
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9323..9328,9329..9346,9347..9353)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9361..9377
                     /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 atgcaattca agccagccgt taccgctctg gtttccgccg tcgccctggc aaccctgctc
       61 agtggctgta agaaagaaga agcagcgcca gcggcgcagg ctcctcaggt cggcgtcgtg
      121 accatccagc cgcaagcctt caccctcacc tcggaattgc cggggcgaac cagtgcctac
      181 cgcgtcgccg aagtgcgccc gcaggtcaac ggcatcatcc tcaagcgcct gttcaaggaa
      241 ggcagtgagg tcaaggaagg ccagcagttg tatcagatcg accctgccgt gtacgaagcc
      301 accctggcca atgccaaggc caacctgctg gctacacgct cactggccga acgttacaag
      361 caactgattg acgaacaggc cgtctccaaa caggaatacg acgacgccaa tgccaaacga
      421 ttgcaggctg aggcttcgct caagagcgca cagatcgacc tgcgctacac caaggttctg
      481 gcgccaatca gcggccgtat cggtcgctct tcgttcaccg agggtgcact ggtgagcaat
      541 ggccagaccg acgccatggc caccatccag cagctcgatc ctatttatgt cgacgttacc
      601 cagtccaccg ccgagctgct caagctgcgc cgtgacctgg aaagcggcca gttgcagaag
      661 gccggcgaca acgccgcctc ggttcagctg gtactggaag acggcagcct gttcaagcag
      721 gaaggtcgcc tggaattctc cgaagtcgcg gtggacgaga ccaccggctc ggtgaccctg
      781 cgcgccctgt tccccaaccc cgatcacacc ctgctgccag gcatgttcgt gcatgcgcgg
      841 ctcaaggccg gggtcaacgc caacgccatc ctggcaccgc aacaaggcgt gacccgcgac
      901 ctcaagggcg cacccaccgc cctggtggtc aaccaggaga acaaggtcga actgcgccag
      961 ctcaaggcca gccgcacctt gggtagcgac tggctgatcg aggaaggcct caacccgggt
     1021 gaccgcctga tcaccgaagg gctgcagtac gtgcgcccag gcgtcgaggt gaaggtcagc
     1081 gatgccacca acgtcaagaa gccggccggt cctgatcagg ccaacgcggc gaaagcagac
     1141 gccaaagcgg agtaaaccat gtcgaagttc tttatcgatc gcccgatctt cgcctgggtg
     1201 atcgccttgg tgatcatgct ggtcggggcc ttgtccatcc tgaagctgcc gatcaaccag
     1261 taccccagta tcgcgccgcc agccatcgcc atcgccgtga cctacccggg cgcctcggcg
     1321 caaaccgtgc aggacaccgt ggtgcaggtg atcgagcagc agctcaacgg tatcgacaac
     1381 ctgcgttatg tgtcgtcgga aagtaactcc gacggcagca tgaccattac cgccaccttc
     1441 gaacagggca ccaaccccga caccgcgcag gtacaggtac agaacaagct gaacctggcc
     1501 accccgctgc tgccgcagga agtgcagcag caaggtatcc gcgtcaccaa agcagtgaag
     1561 aacttcctgc tggtgatcgg cctggtctcc gaagacggca gcatgaccaa ggacgacctg
     1621 gccaactata tcgtctcgaa catgcaggac ccgatctcgc gtaccgcggg tgtaggtgac
     1681 ttccaggtgt tcggtgcgca gtacgccatg cgtatctggc tcgatccggc caagctgaat
     1741 aagttccagc tgaccccggt cgacgtcaag accgctgtgg ccgcacagaa cgtgcaggtg
     1801 tcttccggcc agctcggcgg cctgccagcc ctgccgggca cccagctgaa cgccaccatc
     1861 atcggcaaga cccgcctgca aaccgccgag cagttcgaga gtatcctgct caaggtcaac
     1921 aaagacggtt cacaggtgcg cctgggcgat gtcgcccaag taggcctggg gggtgaaaac
     1981 tacgccgtca gcgcccagtt caacggcaag ccggcttccg gcctggcggt aaaactggca
     2041 accggcgcca acgcgctgga caccgccaag gcactgcgcg agaccatcaa aggcctggaa
     2101 ccgttcttcc cgccgggagt caaggcggta ttcccgtatg acaccacccc agtggtcacc
     2161 gaatcgatca gcggcgtgat ccacaccctg atcgaagccg tggtgctggt gttcctggtg
     2221 atgtacctgt tcctgcagaa cttccgcgcc accatcatca ccaccatgac cgtaccggtg
     2281 gtattgctgg gtaccttcgg catccttgcc gccgcgggct tcagcatcaa caccctgacc
     2341 atgttcgcca tggttctggc catcggcttg ctggtggacg acgccatcgt cgtggtggag
     2401 aacgtcgagc gggtcatgtc cgaggaaggc ttgccgccca aggaagcgac caaacgctcg
     2461 atggaacaga tccagggcgc cctggtgggt atcgccctgg tgctctcggc cgtactgctg
     2521 cccatggcgt tctttggcgg ctccacgggt gtgatctacc ggcagttctc catcaccatc
     2581 gtctcggcca tgggcctgtc ggtgctggtt gcgctgatct tcaccccggc gctctgcgca
     2641 accatgctca aaccgctgaa gaagggcgag caccacaccg ccaaaggtgg cttcttcggc
     2701 tggttcaacc gcaacttcga ccgcagcgtc aacggctacg agcgcagtgt aggcgccatc
     2761 ttgcgcaaca aggtgccatt cctgctggcc tatgcgctga tcgtggtcgg catgatctgg
     2821 ctgttcgccc gcatccctac cgcgttcctg ccagaagaag accagggcgt actgttcgct
     2881 caggtgcaga ccccggccgg ctccagtgcc gaacgcacgc aggtcgtggt cgaccagatg
     2941 cgtgaatacc tgctcaagga cgaagccgat accgtatcgt cggtgttcac ggtcaacggt
     3001 ttcaacttcg caggccgcgg ccagagctcg ggcatggcct tcatcatgct caaaccctgg
     3061 gatgaacgct cgaaggaaaa cagcgtgttc gccctggccc agcgtgccca gcagcacttc
     3121 tttaccttcc gtgatgcgat ggtgttcgcc ttcgccccgc cagcggtgct tgaactgggt
     3181 aacgccaccg gcttcgacgt gttccttcag gaccgcggcg gtgtcggcca cgagaagttg
     3241 atggaggcgc gcaaccagtt cctggccaaa gctgcgcaga gcaagatcct cagcgccgtg
     3301 cgcccgaacg gcctgaacga tgaaccgcag taccagctga ccattgatga cgaacgtgcc
     3361 agcgccctgg gcgtgaccat tgccgacatc aacaacaccc tgtcgattgc cttgggtgcc
     3421 agctacgtca acgacttcat cgaccgtggc cgggtcaaga aggtgtacat ccagggcgaa
     3481 cccagcgcgc ggatgagccc ggaagacctg caaaaatggt acgtgcgcaa cggcgcaggg
     3541 gagatggtgc cgttctcctc cttcgccaaa ggcgaatgga cctacggttc gccgaagctg
     3601 tcgcgttaca acggtgtcga agcgatggaa atcctcggtg caccggcgcc tggctacagt
     3661 accggtgaag ccatggccga ggtcgagcgc attgcaggcg agctgccaag cggtatcggc
     3721 ttctcctgga ccggcatgtc ctacgaggaa aaactctccg gctcgcagat gccggcgctg
     3781 ttcgccctct cggtgctgtt cgtgttcctg tgcctggcgg ccctgtacga aagctggtcg
     3841 atcccgatcg ctgtggtgct ggtagtaccg ctgggtatca tcggtgcgct gatcgccacc
     3901 agcctgcgcg gcctgtccaa cgacgtgtac ttcctggttg gcctgttgac caccatcggt
     3961 cttgcggcga aaaacgccat tctgatcgtg gaattcgcca aggagctgca cgaacaaggc
     4021 cgcagcctgt acgacgcagc gatcgaggcg tgccgcatgc gtctgcgccc gatcatcatg
     4081 acctcgctgg cgttcatcct gggcgtggta ccgttgacca tcgccagcgg cgccggcgcc
     4141 ggcagccagc acgccatcgg tactggcgtg atcggcggca tgatcagtgc gaccgtgctg
     4201 gctatcttct gggtaccact gttcttcgtc gcagtgtcgt cgctgttcgg cagcaaagag
     4261 ccggaaaaag acgtcacccc tgaaaatcca cgttatgagg ctgggcaatg accaagtctt
     4321 tgttgtccct ggcggtaacc gctttcattc ttggcggctg ctcgctgatc cctgactacc
     4381 agaccccgga ggcgccggtg gctgcgcagt ggccgcaagg ccctgcttac tcgccgacgc
     4441 aatcggcgga tgttgccgct gctgaacagg gctggcgcca gttcttccac gacccggcgc
     4501 tgcaacagct gatccagacc tcgctggtca ataaccgcga cctgcgcgtc gcggcgttga
     4561 acctcgacgc ctaccgtgcg caataccgca ttcagcgcgc cgacctgttc ccggcggttt
     4621 cggccaccgg cagcggcagc cgccagcgtg tcccggcgaa catgtcgcaa acgggcgaat
     4681 ctggcatcac cagccagtac tcggccacct tgggcgtcag cgcctacgag ctggacctgt
     4741 tcggccgcgt gcgcagcctg accgagcagg ccctggaaac ctacctctcc agcgagcagg
     4801 cgcgtcgttc cacgcaaatc gccctggtag ccagcgtggc caacgcctac tacacctggc
     4861 aggccgacca ggccctgttc aagctgaccg aagaaacgct gaagacctac gaggaaagct
     4921 acaacctcac ccgtcgcagc aacgaagtcg gcgtggcctc ggccctggac gtcagccagg
     4981 cgcgtaccgc cgtggaaggc gccagggtca agtactcgca gtaccagcgc ctggtcgccc
     5041 aggacgtcaa cagcctgacc gtgctgctgg gcaccggcat tcctgccgac ctggccaagc
     5101 cgctggagct tgatgccgac caactggccg aagtaccggc cggcctgcca tcggatattc
     5161 ttcagcgtcg cccggacatt caggaagccg agcacctgct caaggctgcc aacgccaaca
     5221 ttggtgcagc ccgcgcagcg ttcttcccga gcatcagcct gaccgcgaac gccggcagcc
     5281 tgagccccga catgggccac ctgttctcgg gcggccaggg cacctggctg ttccagccgc
     5341 agatcaacct gccgatcttc aacgccggca gcctcaaagc cagcctggac tactcgaaaa
     5401 tccagaagga catcaacgtc gccaagtacg aaaaaaccat ccagacggcc ttccaggaag
     5461 tctccgatgg cctggcggca cgcaagacct tcgaagagca gttgcaggcc cagcgcgacc
     5521 tggtgcaggc gaaccaggac tactaccgct tggccgaacg ccgttaccgc atcgggattg
     5581 acagcaacct gaccttcctc gatgcccaac gcaacctgtt cagtgcccag caagcgctga
     5641 tcggagaccg cctgtcgcag ctgaccagcg aggtcaacct gtacaaggcg cttggcggtg
     5701 gctggtacga gcagaccggg caggccaacc agcaggcatc ggtggaaaca ccgaaaggcc
     5761 atcatcacca tcaccactga ggatccaaac tcgagtaagg atctccaggc atcaaataaa
     5821 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     5881 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt atacctaggg
     5941 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
     6001 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg
     6061 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc
     6121 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg
     6181 cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg
     6241 tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag
     6301 gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc
     6361 ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca
     6421 gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta
     6481 aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga
     6541 gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga
     6601 gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa
     6661 atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat
     6721 acgatataag ttgttactag tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc
     6781 gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag
     6841 tccaagcgag ctctcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag
     6901 aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
     6961 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg
     7021 tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg
     7081 atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc
     7141 gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca
     7201 tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
     7261 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc
     7321 atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact
     7381 tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa
     7441 ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg
     7501 gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac
     7561 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc
     7621 acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct
     7681 catcctgtct cttgatcaga tcatgatccc ctgcgccatc agatccttgg cggcaagaaa
     7741 gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc agctggcaat
     7801 tccgacgtcg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     7861 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     7921 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     7981 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     8041 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     8101 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     8161 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     8221 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     8281 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     8341 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     8401 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     8461 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     8521 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     8581 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     8641 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     8701 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     8761 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     8821 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     8881 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     8941 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     9001 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     9061 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     9121 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     9181 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     9241 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     9301 agatcgttta ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggaa
     9361 ttgtgagcgg ataacaattt cagaattcaa aagatctttt aagaaggaga tatacat
//
