LOCUS       sequence-64019-e        9444 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 9444)
  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 9444)
  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-EPL59
FEATURES             Location/Qualifiers
     source          1..9444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1471..1482)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5787..5804
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5840..5911
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5927..5954
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5982..5999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6116..6661)
                     /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(6196..6215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6775..6869)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6900..7694)
                     /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(7011..7030)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7621..7640
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7879..8961)
                     /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     8923..8942
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8962..9039)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9245..9264
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9350..9355,9356..9373,9374..9380)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9388..9404
                     /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 ttgagacaac aggctcctgc cccgaggatc ttcatgcaat tcaagccagc tgttaccgtt
       61 ctggtcactg ccgtcgccct ggcaaccctg ctcagtggct gtagcaagaa ggaacaggcg
      121 gccccgcctg cgcaaacccc tcaggtcggc gtcgttaccc tcaaagcaca gccctatacc
      181 ctgaccaccg agctgccagg ccggaccacc gcattccgcg ttgccgaggt acgccctcag
      241 gtcaacggca tcattctcaa acgtctgttt accgaaggtg gcgacgtcaa agccggtcag
      301 cagctctatc agatcgaccc ggctgtctat gaggccaatg ccaacagcgc caaggcgacc
      361 cttcagtctg ccaagtcgat gtctgatcgc tacaagcaac tggtcagcga gcaagcggta
      421 agccgtcagg aatacgacac cgcccaggca tcgacgcagg aagctcaggc cgccttgcag
      481 accgcccaga tcaacctgcg ctacaccaag gtgctggcac ctatttcagg tcgtatcggc
      541 cgctccgcag tgaccgaagg cgcactggtc agcagcgccc agaccgaagc aatggcaacc
      601 atccagcagc tcgacccaat ctacgtggac gtggttcagt cgtctgccga catgctcaag
      661 ctgcgcaatg acctggaaag cggcaagctg cagaaagccg gtgacaacgc ggccaaggtc
      721 aagctgacgc tggaagacaa cagcgtctat ggccaggaag gcaagctgga gttctccgaa
      781 gtatcagttg atcagactac aggctcggtc accttgcgcg ccgtgttccc gaacccggaa
      841 catcgcttgt tgcctggcat gttcgtccac gcgcaattgc aggctggcgt cagtgcgaaa
      901 gccattctgg caccgcagca aggcgtgacc cgcgacctga aaggcacgcc gaccgccatg
      961 atcgtcaacc aggacaacaa ggttgaaatg cgtacgctgg tggccaaccg caccgtgggc
     1021 agcgactggc tgatcgaaga cggcctgaaa gaaggtgatc gggtcattac cgaaggcttg
     1081 cagtacgtca aaccaggcgc cgaagtgaaa gtcgccgaag cgaccaacgt caaaggagca
     1141 aacccggcac ctgccccggc cactgataaa gccgcaggca gcaaagggga gtaacccatg
     1201 tcgaaattct ttattgatcg acccattttt gcctgggtta ttgccctcgt gatcatgctg
     1261 gtcggtgcgc tgtcgatttc cagcctgccg atcaaccagt acccgagcat cgcaccgcca
     1321 gccatcggta tccaggtcac ctatccgggc gcctccgctc agacggtgca ggacactgtg
     1381 gtgcaggtga tcgagcaaca gctcaacggt atcgacaacc tgcgttatgt gagctcggaa
     1441 agtaactctg acggcagcat gaccattacc gcgaccttca atcagggtac caaccctgat
     1501 acggcgcagg ttcaggtgca gaacaagctc aacctcgcga ccccgctgtt gccacaggaa
     1561 gtgcagcaac agggtatccg cgtgaccaag gcagtgaaga acttcctgat ggttatcggc
     1621 ctggtgtccg aagacggcag catgggcaag gaagacctgg ccaactacat cgtgtcgaac
     1681 atgcaggacc cgatttcccg gacctccggt gtgggtgact tccaggtgtt cggttcgcag
     1741 tacgccatgc gtatctggct ggacccggca aagctgaaca acttccagct gactccggtg
     1801 gacgtgaaaa acgcgatcac cgcgcagaac gtccaggtat cgtccggcca gttgggtggc
     1861 ctgccgtcca tcagcggcca gcagttgaac gccacgatca tcggcaagac gcgtctgcag
     1921 accgccgagc agttcggcaa catcttcctc aaggtcaaca ccgacggttc gcaggttcgc
     1981 ctcaaggacg tcgcgactgt cggcctgggc gcagagaact acagcaccga ctcgcagttc
     2041 gacggtaaac cggcatcggg tctggcaatc aaactcgcga ccggtgccaa cgcactggac
     2101 accgccaagg ctattcgcgc gaccgtttcg tcgctggagc cgttcttccc gccaggcatg
     2161 aaagtcgttt acccgtatga caccacgcca gtggtcagcg aatcgatcaa tggtgtggtg
     2221 cacaccctga tcgaagcgat cgtgctggtg ttcctggtga tgtacctgtt cctgcaaaac
     2281 ttccgcgcca cggtcatcac gaccatgacc gtaccggtag tattgctggg tacgttcggc
     2341 atcctcgcgg cattcggctt caccatcaac accctgacca tgttcggtat ggtgctggcc
     2401 atcggcttgc tggtggacga tgcgatcgtg gtggtggaaa acgtcgagcg ggtcatggag
     2461 gaggaaaagc tctccccgcg cgacgccacg atcaaatcca tgacccagat ccagggtgcg
     2521 ctggtcggta ttgcgctggt gctgtcggcg gttctgctgc ccatggcgtt cttcggcggc
     2581 tctaccgggg tgatctataa acagttctcg atcaccatcg tctcggccat ggccctgtcg
     2641 gtggttgtcg ccctgatctt caccccggcg ctgtgcgcaa ccatgctcaa gcctatcgat
     2701 cacgagaagc acggccagcc aaaacgtggt ttcttcggct ggttcaaccg cacctttgac
     2761 cgcagcgttc tgagctacga acgtggcgtc ggcaacatgc tcaagcacaa atggccggcg
     2821 tatctgggct atatcctgat ctgcgcaggc atggtctgga tgttcatgcg tatcccggcc
     2881 gcgttccttc cggaagaaga tcagggcgtc atctttgccc agatccagac cccggcaggc
     2941 tcctctaccg agcgtacgca ggaagtcatc gacaacatgc gtgaatacct actgaccaag
     3001 gaaagcggtg cagtgaagtc ggtgttctcg gtaaacggct tcaactttgc gggccgcggt
     3061 caaagctcgg cgattgcgtt cgtcatgctc aagccgtggg aagaacgtga ttccaacaac
     3121 agcgtcttcg aactggccaa gcgcgctcag ggctatttct tcagcctgcg cgacgcaatg
     3181 gtattcgcgg tcgttccacc gtctgttctc gagctgggta acgccacagg tttcgacgtt
     3241 tacctgcagg atcagggcgg cgtgggtcac gacaaactga tggaggcgcg taaccagttc
     3301 ctcggcatgg cagcgcagag caagattctc gcaggcgtgc gtccgaacgg gctgaatgac
     3361 gaaccgcaat accagttgat catcgacgac gaacgcgcca gtgcgctggg cattaccctg
     3421 tcggacatca acaacacact gtccaccgcg ttgggcggca gctacgtcaa tgacttcatc
     3481 gaccgtggcc gtgtgaagaa ggtgtacatc cagggcgatg cgggtgcccg tatgacgcct
     3541 gaagacctca agaagtggta cgtgcgcaac agctcgggtg aaatggtgcc gttctcggcc
     3601 ttcgccagcg gcaaatggac ctacggctca ccgaaactgt cgcgctacaa cggcgtggca
     3661 gcagaggaag tgctcggtac tcctgccccg ggttacagca gcggcgaggc gatggccgag
     3721 gtagaagccc tggccaagaa actgccgcaa ggtatcggca tttcgtggac cggtctgtcc
     3781 tacgaggaac gtttgtccgg ctcgcaggcc cctgccctct acgcgctgtc gttgctggtg
     3841 gtgttcctgt gtctggcggc gctgtatgaa agctggtcga ttccgatcgc ggtcatcctc
     3901 gtggtgccgc tgggggttat cggtgcactg atggcaacca gcctgcgtgg cctgtccaac
     3961 gacgtgttct tccaggtggg cttgctggtg acggtgggtc tggcggcgaa gaacgccatt
     4021 ctgatcgtcg agttcgccaa agagctgcac gaacagggca agagcctggt cgactcggca
     4081 atcgaggcgt gccgcatgcg tctgcgcccg attatcatga cgtccatggc gtttatcctc
     4141 ggcgttgtgc cgctggcaat ctccacgggc gcaggttcag gtagtcaaca ttcgatcggt
     4201 actggtgtaa ttggcggtat gatcaccgcc gttatcctgg cgatcttctg ggtgcccctg
     4261 ttctttgtct cggtttcagg gctcttcaag agcaagaaga aacatatttc acacgatgag
     4321 gctggccaat gagcaagtca ttgatctctc ttgcagttac cgcgttcatt ctcggcggct
     4381 gctctttgat ccccgagtac aagcagccgg aagcaccggt cgccgctcaa tacccgcaag
     4441 ggccggcgta ctcgccggtc gaaggtgcca aaatggctac cgcggagcaa ggctggcggc
     4501 acttcttcaa tgatccggcg ctgcaacagc tgattcagac tgcactgctc aacaaccgcg
     4561 acctgcgggt cgcggccctg aacattgacg cttaccgtgc ccagtaccgt attcagcgct
     4621 cggacctgtt ccctgctgtt tcggcgacag gtagcggtac tcgtcaacgc gtgccagccg
     4681 accagtcgca aaccggtcaa gcaggcatca ccagcagcta ttcggcaacg ctgggtgtca
     4741 gctcgtacga gctggacctc tttggccggg tgcgcagcct cagcgagcag gcgctggaga
     4801 cttacttctc cagcgaagaa gcgcgtcgca gcacccagat cagtctggtg gccagcgtcg
     4861 ccaatgccta catgacctgg caggccgaca aggaactgct caagctgacg caggacaccc
     4921 tcaagaccta cgaagaaagc tatcgcctga cctcgcgcag caatgaagtc ggtgtttcct
     4981 cggccctgga cctgagccag tcacgcaccg ccgtcgaaag cgcgcgtgcc aaactggctc
     5041 agtacgagcg tctggttgca caggatcaga acagtctggt tctgctgctc ggcaccagcc
     5101 tgcctgacaa cctgccggcc agccacttgc tggattccga catgctgacc gaaatgccgg
     5161 ctggcctgcc gtccgacctg ctgcagcgtc gtccggacat tctggaggcc gagcatacgc
     5221 tcaaggcagc caacgccaac attggcgcag cacgcgcagc gttcttcccc agcatcagcc
     5281 tgacggccag tgccggaacc agcagcagcg agctgtccgg cctgttcaag ggcggatcgg
     5341 gtgcatggct gttccagcca agcatcaaca tcccgatctt caacgccggc aacctgcgcg
     5401 ccagcctgga ctactcgaaa atccagaaag aaatcagcgt cgccaactac gagaaggcga
     5461 ttcaaaccgc cttccaggaa gtgtctgacg gcctggcttc acgcaagacc tacgacgagc
     5521 agctccaggc gcaacgtgac ttcgtcgcgg ccaaccagga ctactaccgt ctggccgagc
     5581 gtcgctaccg gatcggtatc gacagcaacc tgaccttcct cgacgcccag cgctcgttgt
     5641 tcagcgcgca acaaacgctg atcaccgatc gtctgtcgca gctgaccagc gaggtcaacc
     5701 tgtacaaggc cctcggcggc ggctggtacg aaagaaccgg gcagaccgaa accatctcca
     5761 aagagaagcc ttcggttcag ctgttccatc atcaccatca ccactgagga tccaaactcg
     5821 agtaaggatc tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     5881 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     5941 tgggcctttc tgcgtttata cctagggata tattccgctt cctcgctcac tgactcgcta
     6001 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg
     6061 gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc
     6121 ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa
     6181 acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc
     6241 ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt
     6301 ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa
     6361 ccccccgttc agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6421 gaaagacatg caaaagcacc actggcagca gccactggta attgatttag aggagttagt
     6481 cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc
     6541 tccaagccag ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc
     6601 ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca
     6661 agaagatcat cttattaatc agataaaata tttctagatt tcagtgcaat ttatctcttc
     6721 aaatgtagca cctgaagtca gccccatacg atataagttg ttactagtgc ttggattctc
     6781 accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
     6841 ggtcattact ggatctatca acaggagtcc aagcgagctc tcgaacccca gagtcccgct
     6901 cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata
     6961 ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg
     7021 gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat
     7081 ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg
     7141 acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg
     7201 agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta
     7261 cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc
     7321 gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga
     7381 gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca
     7441 gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc
     7501 gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc
     7561 gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt
     7621 gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca
     7681 tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagatca tgatcccctg
     7741 cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc
     7801 ttaccagagg gcgccccagc tggcaattcc gacgtcggtg cctaatgagt gagctaactt
     7861 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     7921 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     7981 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     8041 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     8101 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     8161 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     8221 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     8281 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     8341 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     8401 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     8461 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     8521 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     8581 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     8641 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     8701 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     8761 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     8821 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     8881 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     8941 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     9001 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     9061 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     9121 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     9181 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     9241 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     9301 ccacgatgcg tccggcgtag aggatcgaga tcgtttaggc accccaggct ttacacttta
     9361 tgcttccggc tcgtataatg tgtggaattg tgagcggata acaatttcag aattcaaaag
     9421 atcttttaag aaggagatat acat
//
