LOCUS       sequence-64031-e        8238 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 8238)
  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 8238)
  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-EPL90
FEATURES             Location/Qualifiers
     source          1..8238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4581..4598
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4634..4705
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4721..4748
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4776..4793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4910..5455)
                     /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(4990..5009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5569..5663)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5694..6488)
                     /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(5805..5824)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6415..6434
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6673..7755)
                     /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     7717..7736
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7756..7833)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8039..8058
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(8144..8149,8150..8167,8168..8174)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    8182..8198
                     /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 atgaacggat tatcatgctg tttccgggca gccgaaggct tccggacggt tcaaactgca
       61 acgaacggaa ccaccatgaa cgaccattct gccatgcgcg ccgtctcctc cttctcgaca
      121 atggcctcgg tgcgtcgtgt ggcatgcact ctgctggcgt gttgcctcgt ggtcgcgggc
      181 gcgtgctcac gcgatgaggg caaggcgaag ggggcgcgcg ccaagggccc ggcacctgtg
      241 acggcggctg ttgccgcccg gcaggacatc ccggtgcgtc tcaaggccgt gggcaatgtg
      301 gaggcatccg cgacggtgca ggtgcggtct cgcatcacgg gtgaactcac gggcatccat
      361 ttccgtgagg gcgacgacgt gcgcaagggg cagaaactct tcaccatcga cccgcggccc
      421 atggaggcgg ccctgcgcga ggcgcgtgca cggcaagagc gcacccgtgc ccagctgacc
      481 aaggcggagg acgacctcag gcgcttcgcc agccttgtgg agggcggttt cgtcagccgt
      541 gagcagtatg agcagatacg caccaccgcc gaggggttgc gcgcgtcgct gcgcgaggct
      601 gaggccgccg tggagagtgc ggccctgcag cgcgactact gcatcatcac cgcaccggcc
      661 gatgcccggg caggggccgt gggcgcgcac atcggcaaca tgatcaaggc caatgccgac
      721 gactggctgg tgacgctgga caccgtggaa cccgtgtacg tgagtttctc gattcccgag
      781 gtgcacctcc cggcggtgct tgccttgagg caatcgggca ctggcagggt gcttgcgcgc
      841 cctgacggcg gggacgaggt ggagggccga ctggacttcg tcgacaacag cgtcgacacg
      901 gcgacgggca ccatccgtct gcgggccacc ttcgacaacc ggacgcgcag tctgtggccg
      961 gggcgtttcg tggatgtccg ccttacgctg gccacgcaca aggatgccgt ggtcgttccc
     1021 tcgcgggccg tacaggcggg tgtggacggc ccctatgcct atgtggtggc tgacggacgc
     1081 gccgagttgc gcaacctcgc cgtgggcgtg caggcggata cgatgacgga ggtgctgcag
     1141 ggcatcgcac ccggtgagaa ggtggtcgtt gaaggcctga tgcggctcac gcccggggcc
     1201 gaggtcgtca tccgggcagc ggatgcgggc cggaatgcga cagcatctcc agcagacacc
     1261 acggcgacgc caggcgggcc atccggggac actccgccag gtggcgcgtc gaagagtgct
     1321 ggggctgcgg ctgaaaaggg agcggcagat acggcaaccg acagcaagca ggcaggcggc
     1381 aacgccgcac agacgtccgt gccgacgcag gggccgacgc aggggccgac gggcacgaag
     1441 gctgcggagg cccgccagtg aacgtcacgg agttcttcat ccgcaggccg gtgacgacca
     1501 cgctcgtcat gctggggctg ctcttcttcg gcgtgatggg ctaccgccag cttccggtga
     1561 gcgacctgcc caacgtcgac ttccccacca tacaggtgcg ggcgcaactg ccgggggcgg
     1621 acccggagac catggcctcg tcggtggctg cccctctgga acggcaattc gccaccatcg
     1681 cgggcatcga ctccatgagt tccaccaaca cgcagggcaa cacgcgcatc accatccagt
     1741 tcaacctttc gcgtgacatc gacgcggcgg cgcaggacgt gcaagccgcc atatcgacag
     1801 cgcagcgcaa gctgccgtca gacctgacca cgccacccag ctaccagaag gtgaacccgg
     1861 ccgaccagcc catcctcttt ctcgcgctca catcgcccac gcttcccctc tccaaggtca
     1921 acgaatacgc cgagacgcgc atcgggcaga ccatctccat ggtctcgggg gtggcgcagg
     1981 tcaacgtcta cggtgccaag aagtacgcgg tgcgggtgca ggtcgacccg aggcggctga
     2041 aggcgctggg cctcggtatc gacgaggtgt ccgccgccgt ggacaaggcc aacagcaacc
     2101 tgcccacggg caccctgcaa ggcgaacaca cggcgagcat catcaaggca tccgggcagt
     2161 tgtatgacgc tgcggcctac aggcccgtgg tggtcgccta ccgcaacgga gcgcccgtgc
     2221 gtctcgaaga gttgggccgg gtcgtggata gcgttgaaca ggaccgcatc ctcaactggt
     2281 tcaacgacga acggggcatc gtgctggccg tgcaacgcca gcccggcacc aacacggtgg
     2341 aggtggtcga atccatcaag aggctgctgc ccgaattcga gcggcagttg cccgccgccg
     2401 tgaagctgaa catcctgttc gaccggtccg agtccatcaa ggcgtcggtg gccgaggtta
     2461 agttcacgct ggtgctcacg gtgtgcctcg tggtgctggt catcttcctg tttctgcgca
     2521 gcctgtcggc caccgtcatt cccagcctcg ccctgcccat gtcggtggtg gtgaccttcg
     2581 ccgccatgca catgatgggc ttcagcctcg acaacctctc tctcatggcc ctgacgctgg
     2641 cggtgggctt cgtcgtggac gatgccatcg tcatgctgga gaacatcgtg cggcacatgg
     2701 agatgggcaa gactccgcta cgggccgccc tcgacggtgc gcgcgagata ggcttcacca
     2761 tcatatccat gacggtgtcc cttgcggcgg tgttcatccc cgtactgttc atggggggca
     2821 tcgtgggacg gttgttccac gaattcgccg tggtcatcac cgtcgccatc ctcctttcgg
     2881 gcttcgtctc cctgtcgctc acgcccatgc tgtgcgcgct gttcctcaag ccgcatgtgg
     2941 ggcagagacg gcacgggcgg gtatacaacg cgctggagac cttcttcgag acgctgcacc
     3001 gggggtatga acgttcgttg cggttcacga tgcggcacca ccgcatgacg ttcggcctgt
     3061 cgatggtggt gcttggtgtc acggtgtggc ttttcgccgc catgcccaag ggctttctgc
     3121 ccagtgagga taccgggcag atttccggct tcaccgaggc cgaccagtcc gtctccttcg
     3181 ggcagatggt gaagctgcag aagaccctgc accccatcat cgcggctgac cccggcgtgg
     3241 actcgttctc gtccaccgtg ggcgcgggcg gtcccaacgt gggcggcaac tccgggcgtt
     3301 tcttcatcag gctgaaggat ttcgacgagc gcgacgagca tgtcgacacc atcatcaacc
     3361 gcctgcgggc caaactctcc ggctttccgg gcatcaacgt ctttctcgtc aacccgccca
     3421 gtatcaacgt gggcggacgc gcttccaaga gcctctacca gtacacactg caagggcctg
     3481 acaccgccga actgtacaag gcgggaacgg aacttgaggc ggccctgcgc gagttgcccc
     3541 aactgcgcga cgtgaccagc gacctccaga tacgcaaccc cgaagtgcgg gtggacatcg
     3601 accgtgacaa ggccgccgca ctgggcctca gcgtgcatca ggtggaggac gccctgcaat
     3661 cggcctacgg tacgcggcag gtctcgacca tcctcgcacc ggacaacgac tatcaggtca
     3721 tcctcgaact gttgcccgaa taccagcgcg acgcgtcgtc gatgtcgttg ctcaatgtgc
     3781 gttccgccag cgggcgtctg gtgccgctgg ataccatcgc caccctgcgc ccctcggtgg
     3841 ggccgctggc cgtgaaccac tccgggcaat tcccgtccgt gaccctgtcc ttcaacctgc
     3901 gccccggcgt gtccctcagt gaggcggtgc aggctgtgga gggcatcgcg gggcagtatc
     3961 tcccgcccac cgccaccgga accttccagg gcacggcgca ggccttccag tcgtccatgc
     4021 agggcatggc gatgctgctg ttcatggcag tggtggtcat ctacatcgtg cttggcgtgc
     4081 tgtacgaaag cttcatccat ccgctgacca tcctttccgg tctgccctcg gccgggctgg
     4141 gggcactggt gacgctgttc atcttcggca tcgacctcaa cctgtacgcc ttcgtgggca
     4201 tcatcatgct catcggcatc gtgaagaaga acgccatcat gatgatcgac ttcgcggtgg
     4261 aggccgaacg caagaacggt gcaagccctt atgaggccat cctgcaggga tgcctcatac
     4321 gattccggcc catcatgatg acgaccatgg ccgcactcat gggcacgctg cccatcgccg
     4381 tgggctgggg gccgggggcc gaggcccgcc agcctcttgg cctcgcggtg gtgggcgggc
     4441 tgctggtctc gcagttgctc acgctgtaca tcacccccgt gtactacacc tacctcgatg
     4501 ccctgagtcg ccgcatcaaa cggcgcatgg ggcgggtggc gcaggacgtc gaggccgtgg
     4561 cctccggtgg tgacggcgca catcatcacc atcaccactg aggatccaaa ctcgagtaag
     4621 gatctccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     4681 ctgttgtttg tcggtgaacg ctctctacta gagtcacact ggctcacctt cgggtgggcc
     4741 tttctgcgtt tatacctagg gatatattcc gcttcctcgc tcactgactc gctacgctcg
     4801 gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat
     4861 gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc
     4921 tccgcccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga
     4981 caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc
     5041 ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct cattccacgc
     5101 ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc
     5161 gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga
     5221 catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa
     5281 gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag
     5341 ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa
     5401 ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga
     5461 tcatcttatt aatcagataa aatatttcta gatttcagtg caatttatct cttcaaatgt
     5521 agcacctgaa gtcagcccca tacgatataa gttgttacta gtgcttggat tctcaccaat
     5581 aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc agatggagtt ctgaggtcat
     5641 tactggatct atcaacagga gtccaagcga gctctcgaac cccagagtcc cgctcagaag
     5701 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     5761 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     5821 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     5881 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
     5941 tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     6001 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
     6061 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     6121 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     6181 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     6241 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     6301 tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     6361 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     6421 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     6481 tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag atcatgatcc cctgcgccat
     6541 cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca
     6601 gagggcgccc cagctggcaa ttccgacgtc ggtgcctaat gagtgagcta acttacatta
     6661 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6721 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     6781 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     6841 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     6901 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     6961 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     7021 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     7081 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     7141 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     7201 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     7261 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     7321 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     7381 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     7441 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     7501 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     7561 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     7621 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     7681 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     7741 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     7801 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     7861 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     7921 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     7981 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     8041 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     8101 tgcgtccggc gtagaggatc gagatcgttt aggcacccca ggctttacac tttatgcttc
     8161 cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcagaattca aaagatcttt
     8221 taagaaggag atatacat
//
