LOCUS       sequence-64020-e        7993 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 7993)
  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 7993)
  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-EPL61
FEATURES             Location/Qualifiers
     source          1..7993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4336..4353
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4389..4460
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4476..4503
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4531..4548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4665..5210)
                     /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(4745..4764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5324..5418)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5449..6243)
                     /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(5560..5579)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6170..6189
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6428..7510)
                     /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     7472..7491
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7511..7588)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7794..7813
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7899..7904,7905..7922,7923..7929)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    7937..7953
                     /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 atgagtggtt tgagcgattg gcgtgtcatg cctgcggtag cgctgctggc agttttgggt
       61 ctggccgggt gcgagcagaa acagcagcag gatcacgctg catcgacagc gcccaaagag
      121 gtggaggcca ttgcggtcaa agccgaatcg ttcacggtcg ttgatgaact gcccggccgt
      181 atcgaaccgg tccgtgtggc gcaggtacgc gcccgggtgg ccggtatcgt gctcacgcgc
      241 aacttcgagg aaggtgccga tgtgaaagcg ggcgcggtgc tgttccagat cgacccggca
      301 ccgttcaagg ccgcgctgtc caaagctcag ggcgacctgg cccgcacaga agccacactg
      361 ttcgaaactc aggctacggt caaacgctac gattcgctgg tcgaaatcga agcggtcagc
      421 cgccagacct tcgacaccgc ccgctctgcg ctgcaaaacg ccatcgccgc taaaaagtcg
      481 gctcaggcag atgtcgaaac ggcccgactg aacctgggat acgccacagt caaggcaccg
      541 atttcagggc gcatcggccg cgagatggtc actgaaggcg cgctggtcgg tcagggcgaa
      601 accaccctgc tggcaaccat ccagcaactc gacccggtct acgccgactt cacccagccg
      661 gttgccgacg ccttgcaaat gcgcgaggcc attcaggaag gtcgcctgcc caagggtggc
      721 gaagacgcgc tgtcgctgag cgtcgacggt actgactatc aaagcactgg caccctgttg
      781 tttaccgacg ttgcagtaga ccggaccacc ggccaggttt cgttacgcgc ccgcttcgcc
      841 aaccccaaag gcgtgttgct gccgggcatg tatgtgcggg tgcgcacacc gcagcgggtc
      901 gatgccaatg ccatcctcgt cccgcaacgt gccgttcagc gctccagtga tggcgcagcc
      961 cgcgtacttg tggttggcca ggacggtacg gtcgaagcgc gtccggtcaa aaccggcgca
     1021 atgcaggatt cgcgctggca ggtcaccgag gggctgaagg caggtgaaca gattgtcgtc
     1081 ggcaacatga ccgggctcaa ttccggtgac aaggtcgtcg ccaaattgcc ctccgcccag
     1141 ccgcaatcca agtcgcaata aagccgcctt cagcgcgatc gaggatcacc gttcatgtct
     1201 ctgttcttta tcaagcgacc caactttgcc tgggtgctgg ccctgttcat tctgctggca
     1261 ggcttgatgg ccctgccctc cttgccagtg gcgcagtacc ctgatgtggc accgccgcag
     1321 atcaccatta ccgctactta ccccggcgca tccgcgaaag tgctggtcga ctcggtgacc
     1381 agcgtcatcg aggaagagct gaacggtgcc aagggcatgc tgtattacga atcgaccagc
     1441 aactcgaccg gcagcgccga aatcaacgtg acattcgtac cgggcacgaa cccggacatg
     1501 gcacaggttg aagttcagaa ccgtatcaag aaggctgaag cgcgcctgcc gcagacggtg
     1561 ctcagccagg gcctgcaagt cgaacaggcc agctccggct tcctgttgat ctttaccctg
     1621 aactacaagg acggctcggc caccaaggat acggtcgcgc tggccgacta cgcggcccgc
     1681 aacgtcaata acgaaatcag tcgggtgaac ggtgtcggca ggttgcagtt tttcgccgcc
     1741 gaagccgcaa tgcgggtctg gatcgacccg cagaagctgg tgggctatgg gctgtccatc
     1801 gacgacgtga atgcggccat tcgtgcgcag aacgtgcagg ttcccgcagg cagcttcggc
     1861 agcagcccgg gctcatcact gcaggaattg accgcaacac tggcggtcaa gggcacgctg
     1921 gacaacccgg aggagttcgg ccgcatcgta ttgcatgcca acgaagacgg ctccacggtg
     1981 catttgtccg atgtggcacg cgtggccatc ggcagtcagg attacagttt tgaatcgcgc
     2041 ctgaacgggc aacgtgcagt ggctggtgcc gtgcaactgt cgccaggtgc caatgcgatc
     2101 cagactgccc gggcagtcaa tcagcgcctc accgagctgt cggtcaattt cccggaaggc
     2161 gtcgagtttt cgataccgta cgacacctcg cgcttcgtcg acgtggccat cgacaaggtg
     2221 atctacaccc tgatcgaggc catggtgctg gtgtttctgg tgatgtttct gtttctgcag
     2281 aacattcgct acacgcttat cccgaccatc gtggtgccgg tgtgcctggc cgggaccctg
     2341 gcgatcatgt acctgatggg cttctcggtg aacatgatga ccatgttcgg catggtgctg
     2401 gccatcggca ttctggtcga cgacgccatt gtggtggtgg aaaacgtcga gcggatcatg
     2461 gccgaagaag gcctttctcc ggcagccgca acagtcaagg cgatgcagca ggtctccggc
     2521 gcgattttcg gtatcacgct ggtactggcc gcagtgtttc tgccgctggc gttcatgggg
     2581 ggttcggtcg gggtcatcta ccagcagttc tcgctgtcgc tggccgtgtc gattctgttc
     2641 tcgggtttcc tggccctgac ctttaccccg gcactgtgtg ccacgttgct caagccgatt
     2701 ccggccgggc acagtgagaa gcgcgggttc ttcggcggct tcaatcggct gttcggtaga
     2761 ttcaccgacc gttacgagcg ggtcagttcc agcatgatca aacgcgctgg ccgctacatg
     2821 ctgttgtacg tcggtatcgt cggtctgctg ggttttttct acctgcgtct gccggaatcc
     2881 tttgtgcccg tggaagatca gggctacctg atcatcgacg tgcaactgcc gcccggcgcg
     2941 acccggtcgc gtaccgacct caccgcgcaa ttgctcgaga actacatgtt gtcccgcgaa
     3001 gccacaggtg cggtgaccat gctgctgggc ttcagcttct cgggcatggg tgaaaacgcc
     3061 ggtcttgcgt tcccgaccct gaaggactgg tcggagcgcg gcaagggcca gtcggcagct
     3121 gaagaagcgg cggcgttcaa ccagcatttt tccgggctcg ccgacggtac ggtgatggcg
     3181 gtgacgcctc cgcccattga cggcctgggc acctcgggcg gcttctcgct gcgcctgcag
     3241 gaccgtgcag gcctgggccg cgaagccctg ctggcggcac gcgacaagct gctcggcgag
     3301 gccaacggca acccgaagat tctctacgcg atgatggaag gtctggccga agcgccgcaa
     3361 ttgcgcctga atatcgaccg cgaaaaagcc cgtgcgcttg gcgtgagttt cgaatcgatc
     3421 agcaatgcgc tgtccaccgc cttcggctcc tcggtgatca gcgatttcgc caatgccgga
     3481 cgtcagcagc gtgtggtcgt gcaggcagaa caaagttcgc gaatgactcc ggaaagcgta
     3541 ctcaagctct acgtgcccaa cagcagcggc acgctggtgc cgctgggcgc attcgtttcg
     3601 acgcaatggg aacaaggccc ggtgcagatc gcccgctaca acggctaccc cgcgttcagg
     3661 atttccggcg atgcagcacc gggtgtgagt acaggtgaag cgatggcgga aatcgaacgg
     3721 atcgtttccg agctgccgca aggcatcggc tatgaatgga ccggcctctc ctatcaggaa
     3781 agagtcgcca gcggccaggc agccagcctg ttcggcctgg cgctgctggt ggtgttcctg
     3841 ctgctggttg cgctgtatga aagctgggcg attccgctgg tggtcatgct gatcgtaccc
     3901 gtcggtgcgc tgggctccgt gctggcagtg actgcggtcg gcatgcctaa cgatgtgtat
     3961 ttcaaggtcg ggttgatcac catcatcggg ctggcagcaa aaaacgcgat tctcatcgtc
     4021 gaattcgcca aggagctgtg ggatcaaggc cattcgctgc gtgacgcggc acttcaggct
     4081 gcgcgactgc gcttgcggcc gattgtcatg acatcgctgg ccttcatcct tggcgtcgtg
     4141 cccctcacgc tggcgaccgg cgcaggcgct gcgagccagc gcgcgatcgg caccggcgtc
     4201 ataggcggca tgctcagcgc tacactgctg ggcgtgattc tggtgccgat attttttgtc
     4261 tgggtgctgt cagtgttgcg ccgtaagccg catgtacagc aggttgatga cgataaacac
     4321 ctgcccgaga aacaacatca tcaccatcac cactgaggat ccaaactcga gtaaggatct
     4381 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     4441 gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct
     4501 gcgtttatac ctagggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt
     4561 tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag
     4621 gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc
     4681 ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga
     4741 ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc
     4801 tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac
     4861 actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca
     4921 gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc
     4981 aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat
     5041 gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt
     5101 tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg
     5161 ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc
     5221 ttattaatca gataaaatat ttctagattt cagtgcaatt tatctcttca aatgtagcac
     5281 ctgaagtcag ccccatacga tataagttgt tactagtgct tggattctca ccaataaaaa
     5341 acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg
     5401 gatctatcaa caggagtcca agcgagctct cgaaccccag agtcccgctc agaagaactc
     5461 gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac
     5521 gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc
     5581 tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg
     5641 gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc
     5701 gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg
     5761 ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc
     5821 gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg
     5881 ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag
     5941 atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc
     6001 gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc
     6061 ctgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg
     6121 cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata
     6181 gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat
     6241 catgcgaaac gatcctcatc ctgtctcttg atcagatcat gatcccctgc gccatcagat
     6301 ccttggcggc aagaaagcca tccagtttac tttgcagggc ttcccaacct taccagaggg
     6361 cgccccagct ggcaattccg acgtcggtgc ctaatgagtg agctaactta cattaattgc
     6421 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     6481 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     6541 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     6601 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     6661 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
     6721 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     6781 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     6841 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     6901 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     6961 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     7021 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     7081 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     7141 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     7201 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     7261 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     7321 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     7381 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     7441 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     7501 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     7561 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     7621 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     7681 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     7741 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     7801 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     7861 ccggcgtaga ggatcgagat cgtttaggca ccccaggctt tacactttat gcttccggct
     7921 cgtataatgt gtggaattgt gagcggataa caatttcaga attcaaaaga tcttttaaga
     7981 aggagatata cat
//
