LOCUS       sequence-64026-e        9708 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 9708)
  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 9708)
  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-EPL68
FEATURES             Location/Qualifiers
     source          1..9708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             6051..6068
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      6104..6175
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6191..6218
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6246..6263)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6380..6925)
                     /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(6460..6479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(7039..7133)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7164..7958)
                     /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(7275..7294)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7885..7904
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(8143..9225)
                     /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     9187..9206
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9226..9303)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9509..9528
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9614..9619,9620..9637,9638..9644)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9652..9668
                     /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 atgtttgttc aaagacgtac caccttcaac aaaagccggc gtcaatgcgt agtgctggca
       61 gccgtattca tcctctctgg ttgcatcaag gtggggccgg attttcaccg gccagatgtc
      121 gcggttccat ctgactggac ggaggcagga gaaaaccagg gggaaaccgg ccccgcggat
      181 tatcgcagtt ggtggcgggt attcaacgac ccgatcctcg accgccttat tgacctcgct
      241 taccgtgaga acctcaccct caggatcgcc ggagcgcgag tgctcgaagc cagggcgctg
      301 ctcggcatag tggtcggaga gtatttcccg cagacccagc agggctttgg ctcggttcac
      361 tacaacagga caagccgggg gtctgtgcag accggcaccc aacaatcttc cgccagccaa
      421 agcagtgcgt cgctcggagg tggctccggg ttatcgttct ggcaatcgca gatcgggttg
      481 acggccagtt gggaactcga tttctggggc aagtacaggc gggccatcga atcggccgat
      541 gcaagctggc ttgcctctgt ggccgactac gacaatgcac ttgtggctct tacggcggat
      601 gtcgccaatt cctacataac catcaggacc ctggaaacgc ggctcgacat tgcccgccgg
      661 aacacgcaaa cccagagaga gaacctgaac atcgccgaag cccggttcaa gtacggcaca
      721 gcaacgcagc tggatctgga tcaggcagaa acttcgctca ataccaccct tgccacgacc
      781 ccctccctgg aagcgcaact ccggcagaca aaacacggcc tcagcgtcct gttgggaatg
      841 gcgccgggcg atctgtccgg ctatctgaat ggtcctgcca ctatccccgt ctctcccccc
      901 caggttgtgt tcggcatccc ggccgacctg ctacgccgcc ggcccgatat ccgcgccgcg
      961 gaacaccgtg cagcggccca gagcgcccag atcggcgtgg caaaggctga cctctatccc
     1021 gctttctccc tctccggcac gtttggtttc ctctcaaccg atatcggcgg tgccagcctg
     1081 agcgacatgt tcaactggag cagccgtttc atacaggcag gcccttcgtt ccagtggaac
     1141 atcctcaatt acggccggat ttcgaacaat gtccggctcc aggatgcgcg gctccaggaa
     1201 ctgctcatca cctaccagaa catggtgctc accgcgcagc aggaggttga ggacaacctg
     1261 gtggccttcc tgcgggcccg cgaccgggcc gaattcctgg cgcggagcac agccgctgcg
     1321 gcgagatctc tcaatctcgc ggtcctccag tatcagcagg ggatcaagga cttcaccacg
     1381 gtacttatcg cgcagcagtc cctcttgaat gagcaggaca gcctggccac aaccatgggc
     1441 agcatcgcga gcagtctggt tggtgtgtac cgggcactgg ggggcggttg ggagatacgg
     1501 gaaggaaaag aactggttcc ccctgagatc agaaaagaaa tggaacagcg aaccgattgg
     1561 ggaaagctcc ttgccccggc gtcatacaat ccgcccgccc ccccggagca caagccagga
     1621 gtccgggggc cggactggtg accgttttgc cgcggcatca tgaatagatc agtactgaac
     1681 ggagacattc gatagtctga tgcggaccag ggaggcaatg cggctatgtt tcgcggaatg
     1741 aaaaccactt tccaaccctt acattgcctt gtgctctggg tcctgccact tctcgccctt
     1801 cttcaggccg gatgcaagaa gcccccgccg ccccctcccc cgcccccgaa ggtgaccgtt
     1861 tctcaccccc tgaaacgtga agttaccgat tacctggaac tgaccggcaa cacccaggcc
     1921 gtcaactctg tgcagttggt cgctcgtgtt cccgggtatc ttgagaaggt ctttttccgg
     1981 gacgggcaga tggtcaccaa tgggcaaccg ctctttcagc tacagcggaa cacctatgag
     2041 gccgccctcc gccaggccga aggccaggtc atggcgctga aatcgcagct tgagtacgcc
     2101 gaaggtcaac tgatccgcta ctccaacctg ttgccccata aggccgctac ccagactgac
     2161 gtggacaact ggagatacca gcgggactcc gccagggcta acctgaaagc cgcggaggcg
     2221 aatcgggacc tcgcaaggct caatcttggc tacaccctgg tctcggcccc ctttagcgga
     2281 cggatagatc ggagacagca ggatccggga aatctggtgg gctctgaaac gaacaacacc
     2341 gttcttgccc aactcatcca gatcgacccc atttacgtgt atttcacggt cagcgacctg
     2401 gacctggccc ggttgctcaa gacaaaccgt cttcccggcg ttgccaaaac agaaaaacgc
     2461 ccggttttta tcgcccttgc acaggaagag ggttattcgc acaagggtta tgtcgatttt
     2521 gccgccacaa gcctcacccc taccaccgga acgctgctgt tacggggcgt cttccggaat
     2581 ccgtcaggaa ggatactccc cggattgtac gcgaaggtcc gtctcccgct ggagagaagg
     2641 gtcgctctcc tgactcccga cacggcaata agaagcgacc agcaggggtc gtacgtgctg
     2701 accgtgaacg gcaaaaatac ggtggagcgg cgcgccgtca agacaggcca ccaggaggat
     2761 tccctgcgcg tgatcgaaca aggccttgac ggaagcgaat gggtcatcgt caaaggacag
     2821 ctgaaagccc cctctggaag ccgtgtaacc cccgtacggg aggatgcgga gcctgcgtct
     2881 gccccttccc gcaccaccgc gggaccgaag gtcaagccgt gatttcccag ttcttcatcg
     2941 aacgtcccat cttcgccaac gtcatcgcgc tggtcacggt catcatcggg ctgatcttcc
     3001 tgaatcgcct gccagtggcc cagtaccccc agatcgtgcc cccgaccata caggtgaccg
     3061 cgcgctaccc cggcgcaagc gcggaggttg tcgcggcgac catcggagta cctatcgaac
     3121 aggcggtgaa cggcgtggaa ggctccatct acatgtcgtc caccagcggg agtgacggct
     3181 cctataacct gaccatcacc ttcgatgtcg gaaccgacct caatacctcc ctggctcttg
     3241 tccagaacct ggtaaacacc tccctggccc aacttccggg cggggcgcag cagcagggag
     3301 tcaccgtaaa aaaagtctcg cccaacatcc tgctggtggc cagcctctat gcggacgatg
     3361 agagattcga ggagatcttc ctctccaact atgccgtcat aaacctgcaa aacccgctgg
     3421 ccaggctccc cggcgtcggc caggtgcggg tattcggcgc tggtccctac agcatgaggg
     3481 tctggctcga cgccaagaga ctccagagct tcgacctcac cacaagcgac attctgaccg
     3541 ccatccagag acagaacgtc caggtggtgg cgggacagct cggggccccg ccggttccgg
     3601 cgagccagag tttccagttc accgttacca cccttggccg gctctctgat gtggggcagt
     3661 tcgaagatat cgtcgtcaag accgcaaccg gcccggctcc gcaggtggtg cggctgcgag
     3721 acgtggcacg ggtggatctg agccagcaga acttcagcaa ttacgcccgg ttcaccggcc
     3781 acaaatcagc ccagatcgtg gttttcgccc ttccggacgc caacgccatc gatgtggccg
     3841 acagcgttta caaggccctg gacgggatga gcaagaaatt ccccgacggg atgaaatacg
     3901 ccatccgcta cgacaccacc aagtttgtca gggaggcgat ctccagcgtc tacgacacac
     3961 tcttcatagc cgcattcctg gtgctcttcg tcatcctcct gttcctgcag aacatcaggg
     4021 cgatgctggt tccggccacc actgtgccgg tcaccatcat tggcgccttt atcgccatgg
     4081 ccggactcgg tttcacgatc aacctgatga ccctgttcgc gctggtgctg gtcatcggga
     4141 tcgtggtgga cgacgccatc gttatcgtgg agagcagttc atactatatc gaaaaggggc
     4201 ttgccccgaa ggaggccacc atcaaggcga tgcgggaact gaccgggccg gtgatgggga
     4261 ttacccttgc cctggtcgcg gtcttccttc cggcggcctt cttcccggga atcaccgggc
     4321 agatattccg ccagttcgcc ctggtaatcg cctcaacggc cgtgatcagc gccatcaacg
     4381 cacttaccct gaagccggtc cagtgcgcct tgtggctgag gccgcgcggg gaaaagcggc
     4441 ctaactggtt ctaccgggga ttcaaccggg cgttccagtc cttcaccaat gtctacattg
     4501 ggatcgtcgc ccggatggtg aaacggccgg tcctgttcgt tgtcattttc gccatcatcg
     4561 tcacaacctc tttcctggta ttcataaatc gccccctcgg ctttctcccc accgaggacc
     4621 agggttacgg cgtcctggtg tcccgcatcc ccgaaggggc ctcacagccc aggtcggaga
     4681 aggtggcggt gcagatcaac gagattctca aaaagacctc cggtgtcgac ttctgggtcc
     4741 acatcggcgg gttgtcgatc ctggatggtg ccaacgtctc aaacatgtcc accaccttcg
     4801 tcgtttacaa agactggagc gagcggggct ccgccctgaa ccaggacagg atcatagcga
     4861 gcatcaatcg ccagcttgcc gagatccagg aggcacaggc gtttgtggtc atccctccgc
     4921 cgatccgggg cctcggtcag actggcggtt tccagatgat ggtggaggat cgggggagcg
     4981 tcggtcttga acggctcaag gatgccgctg acgagctgat gcggagcggc aactcacagc
     5041 ctaacctgaa gggcctcgcc tccaccttca gcctaagcag cccccaattg tacctggata
     5101 tcgacaggac caaggcggag gccctcctgg tgcccctgtc gaatgtcttc gagaccctcc
     5161 aggcatacct cggttcctcg ttcgtgaacc tcttcaacaa atacaaccag gtcttccagg
     5221 tctatattca ggccgacagc ccttaccgca tcaggcccga ggacatagga aaactctatg
     5281 tgcgcaacca acggggggag atggttcccc tcggcgccct gattaatgtc aggcagaccc
     5341 agggccccga actgataacc cggtacaacc tctacccggc cgccgccatt ttcggctctg
     5401 ccgcccccgg cttcagctcc ggtcaggccc tgactctgat ggagaggatg gccgccgaaa
     5461 agctccctga ggggattgac ttcgattgga cctccaccag cttccaggaa aagaaggtgg
     5521 gcaaacaagc ctacttcatc tacgccctct ccgtcatcct ggtctacatg gtcctggcgg
     5581 ccctgtacga aagctggact tcgcccgcag cgatcattct cgtggtcccg atagcgctgg
     5641 tcggagtcct tctggccctc atcatccggg gctacgacaa caacctctat acccaggtgg
     5701 ggctggtgct gatgattgca ctggcgagca aaaacgccat ccttatcgtg gaattcgccc
     5761 gcgacctcca ccacggaggg atgtccatag ccgaggccgc catcgaggca acccgccgcc
     5821 ggttccgtcc gatcgtcatg acctcgttcg cgttcatcct gggagtcgtt ccgcttgtgg
     5881 tcgcgttcgg agcgggttcg gcgggccaac gtgccatcgg ctctgtcgtc ttcgggggca
     5941 tgatctcctc cacccttctc gccatcccgt tcgttccggt gttctatgtg gtcctggaac
     6001 gtatgagtga atgggtgggc aattggcgtt cgaagaggac cctgcgtcga catcatcacc
     6061 atcaccactg aggatccaaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc
     6121 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     6181 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacctagg gatatattcc
     6241 gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct
     6301 tacgaacggg gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga
     6361 gggccgcggc aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct
     6421 gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6481 ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc
     6541 tgttatggcc gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc
     6601 tccaagctgg actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt
     6661 aactatcgtc ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact
     6721 ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag
     6781 gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc
     6841 tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat
     6901 tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta
     6961 gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa
     7021 gttgttacta gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct
     7081 gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagcga
     7141 gctctcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg
     7201 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
     7261 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
     7321 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
     7381 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc
     7441 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
     7501 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
     7561 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
     7621 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat
     7681 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
     7741 gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac
     7801 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     7861 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     7921 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc
     7981 tcttgatcag atcatgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag
     8041 tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa ttccgacgtc
     8101 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     8161 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     8221 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     8281 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     8341 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     8401 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     8461 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     8521 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     8581 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     8641 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     8701 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     8761 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     8821 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     8881 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     8941 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     9001 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     9061 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     9121 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     9181 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     9241 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     9301 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     9361 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     9421 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     9481 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     9541 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatcgttt
     9601 aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga attgtgagcg
     9661 gataacaatt tcagaattca aaagatcttt taagaaggag atatacat
//
