LOCUS       sequence-64012-e        7866 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 7866)
  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 7866)
  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-EPL45
FEATURES             Location/Qualifiers
     source          1..7866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      4008..4017
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4209..4226
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4262..4333
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4349..4376
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4404..4421)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4538..5083)
                     /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(4618..4637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5197..5291)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5322..6116)
                     /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(5433..5452)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6043..6062
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6301..7383)
                     /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     7345..7364
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7384..7461)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7667..7686
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7772..7777,7778..7795,7796..7802)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    7810..7826
                     /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 atggtgtccg tcggtgtaac caagcccccc tccctgcgcg catggctggc gctgatcgcg
       61 gcgctgctga ccgtcggcct gctggccggg tggctgtggc acggggcccg gcccggcccc
      121 cagacctatg cacaggcacc ggtcaaggtc gccgtggcca cggccagtac cctgcccttc
      181 cccaactacc tggaagccat cggcgaactg gaggcgatcc agcaagtcag cgtgccggcg
      241 gaaatcggcg ggcgcatcgt caacctgccg atccagtcag ggcagcgggt cgaacgcggc
      301 caggtcctgg tccagctcaa cgacgcgccc cagcgcggca acctggtgcg cctgcaaggg
      361 gaaatggaaa acgcccggat ccgcctcgaa cgctcgcggc gcctggtgtc ggtcaacgcc
      421 acctccaggg aagccttcga caatgcccag accgagctgt ccaccgccaa gggcgccttg
      481 caggcgctga ccgcggaaat cgagcagcgc accatccgcg cgccctttgc cggcaccctg
      541 ggcattcgca aggtgcacct ggggcaatac gtgaaccccg gggacgtact gatcaacctg
      601 attggcgaca agggcctgta cgtcaacttc agcgtgcccg agcaggcgct ggcccaggtc
      661 acgccagggg cgaccctgga agtgcttctc gacgccctgc ccgggcaggt gctcagcgcc
      721 atagtgagcg ctgtcgatcc gtttctcgat cgcacccgca ccctgagcat ccaggcacgc
      781 ctgcaagacc cgccagccag cgccctgccc catatgttcg ctcgtgtgcg gctggcccag
      841 ccgctgccca gcggcacctt gagcgtgccg gaaaccgcgg tgacctataa cgcctacggc
      901 gaagagttgt tcgtggtcca gccggccaag gatgcccagg gcgcgccgac ggtgcagcgg
      961 gtatcggtga aaaccggcgc gcggcatgcc ggccgggtgg taatcgacag cggcctggcg
     1021 ccgggcgcgc aagtggtgac gtccgggcag atcaagctca gtgacggcgc cccgatcgaa
     1081 ccgcagcagc acagcgtgct cagcgatgcc ggcggcagtg cgacgaccct ggccgcggga
     1141 gaagagccgt gaagttcacc gatctgtttg tccgccgccc ggtgctggcg ctggtggtca
     1201 gcctgctgat cctgctggcg gggctcaagg cctacagcaa cctgccggtg cgccagtacc
     1261 cgttgctgga aagctcggtg atcagcgtca ccaccgacta ccccggggcg ccggctgagc
     1321 tgatgcaggg ctttgtcacc cagccgatca accagtccct ggcctcggtg gaaggggtgg
     1381 actacgtcag ctcgtcctcg gtccaggggc gcagcctgat caccctgcgc atggccctca
     1441 accaggattc gtcgaagacc ctgagcgagg tgatcgccaa ggtcaaccag gtcaagtacc
     1501 gccttcccga gcaggcctac gacccggtga tcgagcgttc cgccggggac gccaccgccg
     1561 tggcctatgt cggtttcgcc agccgctcgc tgtccgcctc ggcgctgagc gactacctca
     1621 gccgggtggt acagccgctg ttttccagca tcgagggcgt ggccaaggtc gagaccttcg
     1681 gcggccagcg cctggccatg cgcctgtggc tggacccgca gcgcatggcc ggccacggcg
     1741 tcagcgcggc ccaggtggaa caggcgatcc gcgccaacaa cttccaggcc gcccccgggc
     1801 agatcaaggg ccagtacgtg atctcgtcga tccaggtcaa caccgacctg agcaacctcg
     1861 acgagtttcg cgacatggtc atcagcaacc gtggcgacca cctgctgcgc ctgcgggaca
     1921 tcgcccgggt cgagctgggc tcggcgtcct atgaaaccag tgcgctgatg gacggcaagc
     1981 cggcggtgca cctgggcctg tatgccacgc ccaagggcaa cccgctgctg atcgtcgccg
     2041 acatcaagcg cctgctgccg gagatccaga agaccctgcc gccgggagtg gaggtggccc
     2101 tggcgttcga gaccgcccac ttcatccagg cctcgatcat cgaggtcatc gaaaccctga
     2161 tcgaagccat cctgatcgtg gtggcgatca tctacctgtg cctgggctcg gcccgcagcg
     2221 tgctgatccc ggtggtcagc attccgctgt cgatggtcgg cgcactggcg ctgatgcacc
     2281 tgttcggctt cagcatcaac ctcctgactc tgctggccat ggtcctggcc atcggcctgg
     2341 tggtggacga tgccatcgtg gtcatggaga acatccaccg ccacctcaag gagggtcgca
     2401 cgccgttgca ggcggccttt gtcggggccc gcgaaatcgc ctcgccggtg atcgccatga
     2461 ccatcaccct ggctgcggtg tacatgccca tcggcctgat gagcgggctg accggcgcgc
     2521 tgttcaagga gttcgccctg accctggccg gcgcggtgct gatctccggg gtagtggccc
     2581 tgaccctgac cccggtgatg tgcgccacgc tgctgccggc ggggcagcag gaaggccgca
     2641 tgagccgctg ggccgagcag ttcttccact ggctggcacg tctctaccag cacggcctgg
     2701 tgcgttccct gcgctggcgc ggcgcggtcc tggcgtttgc cgccctggtg ctgctcagcc
     2761 tgccgtggct ctacggccac gccagcaagg agctggcgcc caacgaagac cagggcaacc
     2821 tgctgctggc gatcaagtcg ccgcagcacg ccaacctgga ctatgtcgag cgctatgcct
     2881 accagctgga cgcgatcctc cagggcctgc cggaaaccgt cagcacctgg atcatcaatg
     2941 gcagcgacgg cgtggctgcc agtttcgccg gggtcaactt cagcgagtgg tcgcaacgcc
     3001 agcgcagcgc ggccagcatc cagcagcagt tgcagcagga ggtcgatgca gtcgagggca
     3061 actcgatctt cgtcttccag ttgccgccgc tgccggcctc caccggcggc ctgccggtgc
     3121 aactggtgat ccgcagcacc gaggactacc gctcgctgtt ccaggccatg gaggagatca
     3181 agcagcaggc ccgggccagc ggcctgttcg cggtggtgga cagcgatctg gagttcaaca
     3241 acccggtgac cgaggtgcgc atcgatcgca gcaaggccaa cagcctgggc atccgcatgc
     3301 aggacatcgg caccaccctg gcggtgctga tcggcgagaa ctacaccaac cgcttcgccc
     3361 tgcagggccg ctcctacgac gtgattcccc agagcgccgc ggcccagcgc ctgacacccc
     3421 aggccctcaa cggcttctac gtggagaccg acagcggcac ccaggtgccg ttgtccaccg
     3481 tggtcagcct gcatgacagc atccagccca acaagctcaa gcagttcaac cagcagaatg
     3541 ccgcgacctt ccagggcatc cccgccccgg gggtcagcct gggccaggcg gtggccttcc
     3601 tgcagcagca aagccaggcc ctgcccgcca gtttcagcca tgactggcag gccgattcgc
     3661 ggcagttcat ccacgaaggc aatgccctgc tgctgacttt cctgttcgcc ctcatcgtga
     3721 tctacatggt gctggccgcg cagtacgaaa gcctgagcga cccgctgatc atcctgatca
     3781 ccgtgccgct gtcgatctgt ggcgcgctga ttcccctggc gctggggctg gcgaccatca
     3841 atatctacac gcagatcggc ctggtgaccc tgatcggcct catcagcaag cacggcatcc
     3901 tgatggtgga gttcgccaat gaactgcgcg ccgccctggg ctgcactgcg gcccaggcga
     3961 ttatccgcgc ggcacggatc cgcctgcggc cgatcctcat gaccaccgcc gccatggtgg
     4021 tggggctggt gccgctgctg ttttcttccg gcgccggcgc cagcagccgt ttcggcctgg
     4081 ggctggtgat cgtcatcggc atgctggtgg gcaccctgtt caccctgttc gtgctgccgt
     4141 cgatctacag cctgatggca cgcgcccaca gtcgctccag cgagctggag gaactgcagc
     4201 aactggccca tcatcaccat caccactgag gatccaaact cgagtaagga tctccaggca
     4261 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     4321 ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta
     4381 tacctaggga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
     4441 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
     4501 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg
     4561 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
     4621 gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt
     4681 ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt
     4741 tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac
     4801 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca
     4861 ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg
     4921 ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg
     4981 gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc
     5041 gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa
     5101 tcagataaaa tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt
     5161 cagccccata cgatataagt tgttactagt gcttggattc tcaccaataa aaaacgcccg
     5221 gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat
     5281 caacaggagt ccaagcgagc tctcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga
     5341 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     5401 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     5461 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     5521 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg
     5581 ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg
     5641 tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga
     5701 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt
     5761 gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc
     5821 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca
     5881 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt
     5941 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac
     6001 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat
     6061 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga
     6121 aacgatcctc atcctgtctc ttgatcagat catgatcccc tgcgccatca gatccttggc
     6181 ggcaagaaag ccatccagtt tactttgcag ggcttcccaa ccttaccaga gggcgcccca
     6241 gctggcaatt ccgacgtcgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     6301 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6361 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     6421 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     6481 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     6541 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     6601 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     6661 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     6721 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     6781 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     6841 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     6901 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     6961 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     7021 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     7081 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     7141 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     7201 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     7261 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     7321 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     7381 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     7441 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     7501 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     7561 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     7621 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     7681 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     7741 agaggatcga gatcgtttag gcaccccagg ctttacactt tatgcttccg gctcgtataa
     7801 tgtgtggaat tgtgagcgga taacaatttc agaattcaaa agatctttta agaaggagat
     7861 atacat
//
