LOCUS       sequence-64004-e        9525 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 9525)
  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 9525)
  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-EPL36
FEATURES             Location/Qualifiers
     source          1..9525
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             5868..5885
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5921..5992
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6008..6035
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6063..6080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6197..6742)
                     /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(6277..6296)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6856..6950)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6981..7775)
                     /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(7092..7111)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7702..7721
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7960..9042)
                     /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     9004..9023
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9043..9120)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9326..9345
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9431..9436,9437..9454,9455..9461)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9469..9485
                     /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 atggaacagt catcccactt ctcctggcgc taccccctcg cactcgcggc cgtactggtc
       61 ctgagcgcct gcggcaaggc cccggaaacc acccaaggca tggcggcgcc caaggtcagc
      121 gtcgccgaag tcatcgaaca accgctgaac gagtgggacg aattcaccgg ccgcctggag
      181 gccccggagt cggtggagct gcgcccgcgg gtgtcgggct acatcgaccg cgtggccttc
      241 catgaaggcg cactggtgaa gaaaggcgac ctgctgttcc agatcgaccc gcgcccgttc
      301 gaggccgagg tcaagcgcct cgaagcccag ctgcaacagg cccgcgcggc ccaggcgcgg
      361 agcgtcaacg aagcccagcg cggcgaacgc ctgcgcgcca gcaacgcgat ctccgcggaa
      421 ctcgccgacg cccgcaccac cgccgcccag gaagccaagg cggcggtcgc cgcgacccag
      481 gcgcaactgg acgcggcgcg cctgaacctg agcttcaccc ggatcaccgc gccgatcgac
      541 ggtcgcgtca gccgcgccga ggtcaccgcc ggcaacctgg tcaactccgg ggagaccctg
      601 ctcaccaccc tggtcagcac cgacaaggtc tacgcctact tcgacgccga cgagcgcgtg
      661 ttcctcaagt acgtcgagct ggcccgccag gccggtcgcg acacgcgcag cgagagcccg
      721 gtgtacctcg gcctgagcag cgaggacggc aacccgcacc tgggccggct ggacttcctc
      781 gacaaccagg tcaacccgcg taccggcacc atccgcggcc gcgccgtgtt cgacaacgcc
      841 aagggcgagt tcaccccggg cctctacgtg cgcctgaagc tggtcggcag caagacctac
      901 gccgccaccc tgatcaagga cgaagcggtc ggcaccgacc tgggcaagaa gttcgtgctg
      961 gtcctggatg gcgacaacaa gaccgtctac cgcaccgtcg agatgggacc gaagctggag
     1021 ggcctgcgca tcgtccgcag cggcctgagc aagggcgacc ggatcgtcgt gaatggcctg
     1081 cagcgggtcc gcccgggcat gcaggtggat ccgcagaagg tcgagatggc cagcgccgac
     1141 accctggcca ccctcgcgcg cctgcggcag tcggtcggcg acagcgaacc accgaaggtg
     1201 gcggcgtcca aggacaacgc cactcgcaac gagccgcgcg gctgagcgcg gcgataggaa
     1261 gaaccgatga atttctccca attcttcatc cagcggccga tcttcgccgc ggtgctgtcg
     1321 ctgctgatcc tgattggcgg cgccatctcc ctgttccagc tacccatcag cgaatacccg
     1381 gaagtggtgc cgccgaccgt cgtggtccgc gccaacttcc ccggcgccaa cccgaaagtc
     1441 atcggcgaga ccgtcgcctc tccccttgaa caggcgatca ccggggtgga gaacatgctc
     1501 tacatgtcct cccagtcgac ctccgacggc aagctgaccc tgaccatcac cttcgccctc
     1561 ggcaccgacc tggacaacgc ccaggtgcag gtgcagaacc gcgtcacccg gaccgagccg
     1621 aagctcccgg aagaagtgac ccggctcggc atcaccgtcg acaaggcctc gcccgacctg
     1681 accatggtcg tgcacctgac ctcgccggat aaccgctacg acatgctcta cctgtcgaac
     1741 tacgcggtgc tcaacgtgaa ggacgaactg gcccgcctcg acggcgtcgg cgacgtccag
     1801 ttgttcggcc tcggcgacta ttcgctgcgc gtctggctgg acccgaacaa ggtcgcctcg
     1861 cgcaacctca ccgccaccga cgtggtcaac gccatccgcg agcagaaccg ccaggtcgcc
     1921 gccggcaccc tgggcgcgcc gccggcgccg agcgatacca gcttccagtt gtcgatcaac
     1981 acccagggtc gcctggtcac cgaggaagag ttcgagaaca tcatcatccg cgccggcgcc
     2041 aacggcgaga tcacccgtct gcgcgacatc gcccgggtcg agctgggctc caaccagtac
     2101 gccctgcgtt cgctgctgaa caacaagccg gcggtggcga tcccgatctt ccagcgtccc
     2161 ggctcgaacg ccatcgagat ctccaacctg gtgcgggaga agatggccga gctgaagcac
     2221 agcttcccgc aaggcatgga ctactccatc gtctacgacc cgaccatctt cgtccgcggc
     2281 tccatcgagg cggtggtgca caccctgttc gaagccctgg tgctggtggt gctggtggtg
     2341 atcctgttcc tgcagacctg gcgcgcctcg atcatcccgc tggccgcggt gccggtgtcg
     2401 ctgatcggca ccttcgcggt gatgcacatg ctcggcttct cgctcaacgc gctgtcgctg
     2461 ttcggcctgg tgctggccat cggcatcgtg gtggacgacg ccatcgtggt ggtggagaac
     2521 gtcgagcgca acatcggcct cggcctcaag ccggtggaag ccaccaagcg tgccatgcgc
     2581 gaggtgaccg ggccgatcat cgccacggcg ctggtgctct gcgcggtgtt catcccgacc
     2641 gcgttcatct ccggcctcac cgggcagttc taccgccagt tcgccctgac catcgcgatc
     2701 tccacggtga tctcggcgtt caactcgctg accctgtcgc cagcgctggc ggcggtcctg
     2761 ctcaagggcc accacgagcc gaaggaccgc ttctcggtgt tcctcgacaa gctcctcggc
     2821 agttggctgt tccgtccgtt caaccgtttc ttcgaccgcg ccagccatgg ctacgtcggc
     2881 acggtgaacc gggtcctgcg cggcagctcg atcgccctgc tggtctacgg cggactgatg
     2941 gtgctgacct acttcggctt ctccagcacg ccgaccggtt tcgtcccgca gcaggacaag
     3001 cagtacctgg tggccttcgc ccagttgccc gacgcggcca gcctggaccg taccgaggcg
     3061 gtgatcaagc agatgtccga gatcgccctg gcgcagcccg gcgtggcgga ctcggtggcc
     3121 ttccccggcc tgtcgatcaa cggcttcacc aacagcccga acagcggcat cgtgttcacc
     3181 ccgctgaagc cgttcgacga gcgcaaggac ccgagccagt cggccggggc catcgccgcc
     3241 gcgctgaacg ccaagtacgc cgacattcag gacgcctaca tcgcgatctt cccgccgccg
     3301 ccggtacagg ggctggggac catcggcggc ttccgcctgc agatcgagga ccgtggcaac
     3361 cagggctacg aggagctgtt caagcagacc cagaacatca tcaccaaggc ccgtgcgctg
     3421 cctgagctgg aacccagctc ggtgttctcc agctaccagg tcaacgtgcc gcagatcgac
     3481 gccgacatcg accgcgagaa ggccaagacc cacggcgtgg cgatcagcga catcttcgac
     3541 accctgcagg tctacctcgg ctcgctgtac gcgaacgact tcaaccgctt cggccgtacc
     3601 tatcaggtca acgtccaggc cgagcagcag ttccgcctcg aacccgagca gatcggccag
     3661 ctgaaggtgc gcaacaacct cggcgagatg gtcccgctgg cgtccttcat caaggtcagc
     3721 gacacctccg gtccggaccg tgtgatgcac tacaacggct tcatcaccgc cgaactcaac
     3781 ggcgccccgg ccgcgggcta cagctccggc caggcgcagg cggcgatcga gaagctgctg
     3841 aaggaggaac tgcccaacgg catgacctac gagtggaccg agctgaccta ccagcagatc
     3901 ctcgccggca ataccgcgct gttcgtcttc ccgctctgcg tgctgctggc cttcctcgtg
     3961 ctggccgccc agtacgagag ctggagccta ccgctggcgg tgatcctgat cgtgccgatg
     4021 accctgctgt cggcgatcac cggggtgatc ctggccggca gcgacaacaa catctttacc
     4081 cagatcggcc tgatcgttct ggtggggctg gcgtgcaaga acgcgatcct gatcgtcgag
     4141 ttcgccaagg acaagcagga ggaaggcatg gaccgcgtcg ccgcggtgct ggaagcctgc
     4201 cgcctgcgcc tgcggccgat cctgatgacg tccatcgcct tcatcatggg tgtggtgccg
     4261 ctggtgatct ccaccggcgc cggcgccgag atgcgccatg cgatgggcgt ggcggtgttc
     4321 tccgggatga tcggggtgac cttcttcggc ctgctgctga cgccggtgtt ctacgtcctc
     4381 atccgccgct tcgtggagaa ccgcgaagcg cgccgcgccg ccaacgacaa aggcctgcca
     4441 gaggtgcatg catgattcac gcgcagtcga tccggagcgg gctcgcgtcc gccctgggtc
     4501 tgttcagtct gctggcgctc agcgcctgca cggtgggtcc ggactaccgg acccccgaca
     4561 ccgcggcggc gaagatcgac gccacggcga gcaagcccta cgaccgcagc cgcttcgaaa
     4621 gcctgtggtg gaaacagttc gacgatccga ccctgaacca gttggtcgaa cagtcgctga
     4681 gcggcaaccg cgacctgcgc gtggccttcg cccgcctgcg cgccgcccgc gccctgcgcg
     4741 acgacgtggc caacgatcgc ttcccggtgg tcaccagccg cgccagcgcc gacatcggca
     4801 agggccagca accgggagtg accgaggacc gggtcaacag cgagcgctac gaccttggcc
     4861 tggatagcgc ctgggagctt gacctgttcg ggcgcatccg ccgtcagctg gagtccagcg
     4921 acgccctcag cgaagcggcc gaggccgacc tgcagcaact gcaggtcagc ctgatcgccg
     4981 agctggtgga cgcctacggc caactgcgcg gcgcgcaact gcgcgagaag attgccctga
     5041 gcaacctgga gaaccagaag gagtcgcgcc agctcaccga gcaactgcgc gacgccgggg
     5101 tcggtgccga actcgacgta ctgcgcgccg atgcgcgcct ggcggccacc gccgccagcg
     5161 tgccgcaact gcaggcggaa gccgagcgcg ccaggcaccg tatcgccacc ctcctcggcc
     5221 aacggccgga agagttgaca gtggaccttt cgccgcgcga cctgccggcg atcaccaagg
     5281 ccctgccgat cggcgatccc ggcgaactgc tgcgccgccg gccggacatc cgcgccgccg
     5341 aacggcgcct ggccgccagc accgccgacg tcggcgtggc caccgccgac ctgttcccgc
     5401 gggtcagcct cagcggcttc ctcggcttca ccgccgggcg gggctcgcag atcggctcaa
     5461 gcgccgcccg cgcctggagc gtcggcccga gcatcagttg ggccgccttc gacctcggca
     5521 gcgtgcgtgc ccgcctgcgc ggcgccaagg ccgacgccga cgccgcgctg gccagctacg
     5581 aacagcaggt gctgctggcc ctggaagaat cggcgaatgc cttcagcgac tatggcaagc
     5641 gccaggagcg cctggtctcg ctggtccgcc agtcggaagc cagccgcgcc gccgcgcaac
     5701 aggcggcgat ccgctaccgc gaaggcacca ccgatttcct ggtgctgctg gacgccgaac
     5761 gcgagcaact ctccgccgaa gatgcccagg cccaggccga ggtcgagctg taccgcggca
     5821 tcgtggcgat ctaccgctcc ctcggcggtg gctggcaacc cagcgcccat catcaccatc
     5881 accactgagg atccaaactc gagtaaggat ctccaggcat caaataaaac gaaaggctca
     5941 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag
     6001 tcacactggc tcaccttcgg gtgggccttt ctgcgtttat acctagggat atattccgct
     6061 tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac
     6121 gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg
     6181 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac
     6241 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6301 gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt
     6361 tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc
     6421 aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac
     6481 tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt
     6541 aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac
     6601 aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca
     6661 gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac
     6721 gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat
     6781 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
     6841 gttactagtg cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa
     6901 caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct
     6961 ctcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     7021 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     7081 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     7141 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     7201 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
     7261 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     7321 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
     7381 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     7441 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
     7501 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
     7561 gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
     7621 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     7681 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     7741 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     7801 tgatcagatc atgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt
     7861 actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cgacgtcggt
     7921 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     7981 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     8041 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     8101 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     8161 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     8221 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     8281 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     8341 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     8401 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     8461 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     8521 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     8581 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     8641 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     8701 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     8761 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     8821 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     8881 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     8941 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     9001 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     9061 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     9121 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     9181 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     9241 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     9301 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     9361 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgtttagg
     9421 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggaatt gtgagcggat
     9481 aacaatttca gaattcaaaa gatcttttaa gaaggagata tacat
//
