LOCUS       sequence-64365-e        9375 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 9375)
  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 9375)
  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-EPL98
FEATURES             Location/Qualifiers
     source          1..9375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             5718..5735
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5771..5842
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5858..5885
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5913..5930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6047..6592)
                     /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(6127..6146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6706..6800)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6831..7625)
                     /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(6942..6961)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7552..7571
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7810..8892)
                     /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     8854..8873
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8893..8970)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9176..9195
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9281..9286,9287..9304,9305..9311)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9319..9335
                     /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 atgcagcgtt cccgcatcgc gctattggta ttttcggccc tcgttggttc tgtcgtttta
       61 actggttgtg atcaggctgc tgagcaacct aaagctactg cttctcaagg cactccagta
      121 ggtgtagtta cgataaaaag ccaaacactt acacttaaac aagaattacc agggcgtgta
      181 agtgcatatc aaatagcaga aattcgtcct caagttagcg gtattgtgca atctcgtttg
      241 tttacagaag gtacgcaagt agaaaaaggc caagcgctat atcaaataaa tccagccatt
      301 tttgaagctg agcttgcagc aagtgaagca gctgtagccc gtgctgaggc gagtattgcc
      361 agcagtaaat caaaagctgc gcgttataaa gagttgttgg caattaaagc tgtaagtcag
      421 caagactttg acgaagcaga tgcaactgct aaacaggcaa aggctgagtt acttactgcg
      481 aaagcgcaat tacaggctgc acaaattaat ttagattaca gccatgtggc atcgcctatt
      541 agtggtcaaa ttagtaaatc gagtgtgact gttggtgctt tggtaagtgc taatcaagca
      601 atggcattgg caacagtaac gcaacttgat ccaatttatg ttgatttaac acaatcgagt
      661 aatgagttaa ccaagcttaa aaaagcgttg tcgtcgggct tgttaagtgt tgatgcagat
      721 attcaaaccg aggttgagct ttatatggaa gacggttcgg tttatccgca taagggcacc
      781 ttacagtttt cagaagtaac ggttaatcca agtacgggtt cggtaacatt gcgagctaaa
      841 ttttcaaacc ctgagaaact acttttaccg ggtatgtatg cccgtgcatc tattgttgaa
      901 ggggttcaag aaaatgctgt tttggttccg cagcgtggtg taacgcgtaa taccaaaggc
      961 cagccaacgg ctatggtggt aagtgaaaat aatacagtag agagccgagt actagaagtt
     1021 gatagaacaa taggttcgaa ctggttagtg actaaaggtt tatctgctgg tgataagtta
     1081 attgttgaag gcttacaaaa aattcgtcct ggcgcgccag tgattgtcac tgaagcaaca
     1141 cctttagcca ctcaagcgca ataacggaga gtttaaatgt cacgatattt tatcgataaa
     1201 ccgatatttg cttgggtact cgctattatt gttatgttag cgggtatttt agcagttaaa
     1261 agtttaccta ttgcacagta tccttctatt gcaccgcctg cgattagtat tactgcaaat
     1321 tacccaggtg catcagcgca aaccttagaa gactctgtaa cgcaagttat tgagcaaaaa
     1381 atgaaagggc tagacggctt gttatatatg tcgtcaacgt cagagtcgaa tggctcagca
     1441 acgcttacat taacgtttaa tgctgataca gaccctgata ttgctcaggt tcaagtacaa
     1501 aacaaattag caaccgcaac gcccttgtta ccagaggaag tgcaacgcca aggtgttgta
     1561 gtggccaaag cggcgcgtaa ctttttatta gtattagctt ttgtgtctaa agatggcagc
     1621 atgactaata ttgatattgg tgattatgta gcctcaaacg tgcaagatat tgtttctcgt
     1681 gttgatggtg taggtgaagc gcagttattt ggctcgcagt acgctatgcg tatttggtta
     1741 gatccagcaa aattacaaaa ctttaagtta acgccaaacg atattggcgc ggcaattact
     1801 gcacaaaatg cgcaggtttc tgccggccaa ctcggtggta tgcctgcgat tgcaggacag
     1861 cagttaaatg cgacaataac ggcacagagc cgtttgcaaa cacctgagca gtttgaaaac
     1921 attttagtta aaaccaatag tgatggctca gtagtgcgtc taaaagatgt agctagagta
     1981 gaactgggcg gtgaaaatta tggtgtagtt gcacgttaca atggtgatcc ggctgcgggt
     2041 attggtataa aacttgccag tggtgctaac gcactagata ccgcagaggg cgttaaaaaa
     2101 gcattagagg acttaaaacc gttttttcct gaaggtttag atgttgctat tccttacgac
     2161 accacaccat ttgtatcttt gtcgatagaa aaagtagtgc acacgctaat tgaagctgtg
     2221 atacttgtgt ttgttgtaat gtatttgttt ttgcaaaact ttcgtgcaac gttaattcct
     2281 actattgcag tacctgttgt actactgggt acgtttgcaa ttttgtatac ctttggttat
     2341 tcaatcaata cgttaactat gtttgctatg gtacttgcca ttggcctgtt ggttgatgat
     2401 gcgattgttg tagtagaaaa cgttgagcgc ttaatgactg aagaaaaact ctcggcatta
     2461 gatgcaacac gtaaatcaat ggacgaaatt aaaggcgcat tggtaggtat tgctatggtg
     2521 ctgtcggcag tatttattcc gatggcgttt tttagtggct ctaccggtgt tatttaccgt
     2581 cagttctcta ttactattgt gtcggcaatg ggcttgtcgg tattagtggc acttatttta
     2641 acgcctgcat tatgtgcaac cttacttaag ccaagccatg tacacgataa aacatcattt
     2701 attggacgtt ttttctctgg ctttaaccgt ggttttgata aaacaaaccg cggtgcacaa
     2761 ggtattgttg ggcgtatgat cagccaatca aagcgctact tacttattta tggtgtgctt
     2821 gtgggtggta tggtatatgt gttttcaagc ttaccaactg cttttttacc agacgaagac
     2881 caaggtatat tatttaacca agtaatgttg cctgcaggct ctaccattga gcaaacgtta
     2941 ggtgtagttg aaaaagtaga aaaccattat ttgaatgatc aagccgaagc agtaggttct
     3001 atctttacgg taacaggctt tagttttgcc ggttctgggc aaaactcagc aattggtttt
     3061 gtgaacttaa agcattggga tgagcgccag cgtgacgact tatcggtaaa tgcagtagca
     3121 ggtaaaggga tggggtattt ttcaaccata aaagaagcgt ttgtatttgc tttcccacca
     3181 ccggcaattg tagagcttgg tacagctaat ggctttaata tgtttttaca agaccgcgta
     3241 ggcttaggcc atgacgagct attaaatgca cgtaatatgc tattgggtat ggcaagtaaa
     3301 agccctgttt tagcgggtgt gcgtccaaat ggccaagaag acatgccaga gctgcaactt
     3361 gatattgacc ttgcaaaagc ggaagcattg ggcgttactc aaaccgatat aaatagtact
     3421 ttgtctaccg catggggtag ccgctatgtt aatgacttta ttgaccgcgg ccgcgtgaaa
     3481 aaagtataca tgcaaggcga agctgattcg cgcatggtgc cagaggactt aaataagtgg
     3541 tacgtgcgta ataataatgg cgatatggtg ccgtttgcag catttgctag ttcgcactgg
     3601 tcttacggct caccacgctt agagcgctac aatggttttt ctgcgatgga aattcaaggt
     3661 agtgcagcac ctggctatag tacagggcag gctatggatg aaatggagcg tttagttaag
     3721 caattgccaa acggtattgc ctctgagtgg tcgggtatat cttatcaaga acgctcaagt
     3781 ggtgggcaag cgcctttact gtatggattg tcgctgttgt ttgtattttt atgtttagct
     3841 gcgttatacg aaagttggtc agtaccattt gctgtaatga ttattgtgcc actgggtata
     3901 tttggtgcga ttatggcggc attaataggt aacttatcaa acgatattta cttacaagta
     3961 ggtttgttaa ccaccatagg tttggcttct aaaaacgcga tattaatagt tgaatttgct
     4021 attcataaaa tggaagaagg cctgagctta gtggatgctg caatagctgc tgttaaactg
     4081 cgtttacgcc cagtattaat gacctcgctg gcctttattt gtggtgttgt acctttggct
     4141 attgcgtcga gcgcagggtc gggtgcacaa aatgcgctgg gtatctctat tattggtggt
     4201 actttagctg cctctacctt agtggtgtta tttgtgcctt tgttttttgt tttagtacgt
     4261 aaaacattct caagtaaatc ggcagtagca acgaaggaga gcgaataatg aacttaaaaa
     4321 cattaggttt aaacgcactt actttagctg tattaacggg gtgccaactt gccccagagc
     4381 aacaagaaat agctttacct gtgcctgatg cgtatgcatc gggtgcagag caagcgcaag
     4441 ctacgcagct aaattggcaa cagtttttta acgatgaaaa actgcaagcg ttaattgcac
     4501 agagccttga gcataataaa gacttacaaa ttgctgcatt aaatgtgcag cgcgtgcgtg
     4561 gcttatatca aatagaagac tcggcattat ttccatcgct tgatataaat ggatctggat
     4621 cgcgtcagcg cttgccgggt gatttatcga gtactggtga gccgcgtatt aattcgcagt
     4681 acagtgctac cgtaggtatt acctcgtatg agcttgatat atggggcaag gttcgcaatc
     4741 agtcagcgca agcactgcaa accttatact ctacgcagct gagccaatac agtatgcaag
     4801 tgtcgcttat tgctgagctt gctaatgcct ggcttaacta tgcaaccgac cagcagttat
     4861 tagcactggc cgaggaaacc ctaacgtcgc aacagtcgtc gttatcactt actcaacaaa
     4921 gttttgattt aggtgctgca tcagcaatta ccctagagca acttaaaagt acggttgcta
     4981 cagctaaagt tgatatagca aagtataagc gtttattaaa acgcgataaa aatgcgcttg
     5041 atttactagt aggtaatagc gttgccagtg atttactgcc aaatgcgtca cttaatcatt
     5101 tactcgactt accagaagta ccagttggtt taccgtctga tttactcacg caacgccctg
     5161 atattaaagc ggttgagcat cagttgttag cggctaatgc aaatattggt attgcgcgtg
     5221 ctgcgtttta cccaagtatt agtttaaccg ctaatgcggg tactgcatca agcgacttaa
     5281 gtggtttatt tgatggtggc tcgggtacgt ggagttttgt acctactatt aatttgccta
     5341 tttttaatat gggccgtaac caagctaatt tagatgttgc taaagcggcg caacaaatag
     5401 cgcttaccac ctatcagcaa aaaattcagc aagcgtttcg tgaagttgct gacgtacttg
     5461 cagatagaga aggttataac gcccagctaa gcgcacttga gcagttagtg caaagccgtc
     5521 aagcaacctt tgatatttcg caagcgcgtt acgacaaagg tgccgacagt tacttgcaag
     5581 ttcttgattc gcaaagaact tggtacagtg cccaacagca gttaattacg ggtaagcaag
     5641 cattacttgc cagtaaaatt aacttataca aagcagttgg cggcggttgg caagttacct
     5701 caccagcggt gcaagatcat catcaccatc accactgagg atccaaactc gagtaaggat
     5761 ctccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     5821 ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt
     5881 ctgcgtttat acctagggat atattccgct tcctcgctca ctgactcgct acgctcggtc
     5941 gttcgactgc ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc
     6001 aggaagatac ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc
     6061 gcccccctga caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag
     6121 gactataaag ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg
     6181 cctttcggtt taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg
     6241 acactcagtt ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt
     6301 cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat
     6361 gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc
     6421 atgcgccggt taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca
     6481 gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc
     6541 ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca
     6601 tcttattaat cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc
     6661 acctgaagtc agccccatac gatataagtt gttactagtg cttggattct caccaataaa
     6721 aaacgcccgg cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac
     6781 tggatctatc aacaggagtc caagcgagct ctcgaacccc agagtcccgc tcagaagaac
     6841 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     6901 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     6961 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     7021 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc
     7081 tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     7141 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc
     7201 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     7261 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     7321 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     7381 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     7441 tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     7501 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     7561 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     7621 atcatgcgaa acgatcctca tcctgtctct tgatcagatc atgatcccct gcgccatcag
     7681 atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac cttaccagag
     7741 ggcgccccag ctggcaattc cgacgtcggt gcctaatgag tgagctaact tacattaatt
     7801 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     7861 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     7921 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     7981 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     8041 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     8101 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     8161 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     8221 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     8281 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     8341 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     8401 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     8461 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     8521 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     8581 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     8641 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     8701 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     8761 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     8821 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     8881 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     8941 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     9001 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     9061 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     9121 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     9181 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     9241 gtccggcgta gaggatcgag atcgtttagg caccccaggc tttacacttt atgcttccgg
     9301 ctcgtataat gtgtggaatt gtgagcggat aacaatttca gaattcaaaa gatcttttaa
     9361 gaaggagata tacat
//
