LOCUS       sequence-64000-e        7922 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 7922)
  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 7922)
  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-EPL15
FEATURES             Location/Qualifiers
     source          1..7922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      823..832
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4265..4282
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4318..4389
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4405..4432
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4460..4477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4594..5139)
                     /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(4674..4693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5253..5347)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5378..6172)
                     /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(5489..5508)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6099..6118
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6357..7439)
                     /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     7401..7420
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7440..7517)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7723..7742
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7828..7833,7834..7851,7852..7858)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    7866..7882
                     /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 gtgggcagga tcgcccgttg tcgtgtagaa gcgaataaca actgcatcca aggaaccgga
       61 cctatgctgc gccgccgcat gctcatcatg ttggccgtgg ttctgctgat cgtgctggcc
      121 ctggggggtt acaaagcctt ctcgatctac cagcagatcc agatgttcac tgcccccaag
      181 ccccccatca ccgtggcggc tgccatggcc gaacagcgcc cgtggcaaga gcgcctgccc
      241 gctgtcggca gcctcaaggc gctgcagggc gtggatctga gcctggaggt ggaaggcatc
      301 gtcaaatccc tgcacttcga ctcaggccaa caggtcaagg ccgggcaact gctcttgcag
      361 ctcaacgacg atcaggaaac agccctgctc ggtaccgccc tggccgacct gggcctggcc
      421 aaggtcgact tcggccgtgg cagccagctg gtgggcgatt cggccatctc ccgcggcgag
      481 ttcgaccgcc tttcctccca gtaccggcgc aaccaggcgg tggtcgagca gcttaaggcg
      541 cagaagatca agaaacgcat caatgcacct ttcagcggca ccatcggcat ccgccaggtg
      601 gacatcggcc agtacctggc cgccggcact gtgatcgcca ccctgcaaga cacctccagc
      661 ctgtacgtag acttcaacgt gccggaacag gcgctgccac acctgagcct gggccagcag
      721 gtgctggtgc aagtggccgc ctacccgggt cagcgcttcc ccggcagcct cagcgccatc
      781 aaccccaagg tcgaggaaac cacgcgcaac ctgctggtac gcgccaccat ggccaacccc
      841 gacggcaaac tgctgccagg catgttcgcc agcctgttga ttctgctgcc caacccgcag
      901 ccgcaagtgg tggtgccgga aagcgccatt acctacaccc tgtatggcaa ctcggtgtat
      961 gtcgcctcac cgagaaagga caaggatggc aaaccggtgt acgacgacaa gggccaaccc
     1021 cagctggtcg ccgaacagcg cacggtaaag actggcgagc gccgagacgg cgtggttgta
     1081 gtcagcgaag gcctgcaggc cggggaacag gtggtcaccg ccgggcagct gaaactcaca
     1141 cccggtgccg caatacgcat aggccaggac aacgccctca agcccgaacc gggcgcagcg
     1201 gccaaggact caggaggctg acatggcgtt caccgacacg ttcatccaac gcccggtgct
     1261 ggccagcgtg gtcagcctgc tgattctgct gctcggcttc caggcctgga acaagctgca
     1321 gatccgccag tacccgcaaa tggaaaacgc cctgatcacg gtcaccaccg cctaccccgg
     1381 ggccaacgcc gaaaccattc agggctatat cacccagcca ttgcagcaaa gcctggccag
     1441 cgccgaaggc atcgactaca tgacctcggt gagccggcag aatttctcga tcatttccat
     1501 ttacgcgcgc attggcgccg acagcgaccg gctgttcacc gagctgctgg ccaaggccaa
     1561 tgaggtgcga aacaagctac cccaggactc cgaagacccg gtactgagca aggaagctgc
     1621 cgacgcctcg gcgctgatgt acgtcagctt ctacagcaag gaaatgagca acccgcagat
     1681 caccgactac ctgtcacggg ttatccagcc caagctggcc actctgccgg gcatggccga
     1741 ggcggagatc ctcggcaacc aggtgttcgc catgcgcctg tggctggacc cggtgaagct
     1801 ggccggtttc ggcctttcgg ctaccgacgt gaccaatgcc gtacgccgct acaacttcct
     1861 ttccgccgct ggcgaggtga aaggcgaata cgtggtcacc agcgtcaacg ccagcaccga
     1921 gctgaagtcg gccgaggcgt tcgccgccct gccgctcaag accagcggtg acagccgcgt
     1981 gctgctgggc gatgtggcgc gggtcgagat gggcgcagaa aactacgaca cggtcagctc
     2041 gttcgacggc accccgtcgg tctatatcgg catcaaggcc accccggccg ccaacccgct
     2101 ggatgtcatc aaggaagtgc ggcgcatcat gcctgagctg gaaagccagc tgccctcggc
     2161 actgaacgta tcgattgcct acgacgccac gcggttcatc caggcctcga tcgacgaagt
     2221 gatcaagacc ttgggcgaag cggttctgat cgtcatcgtc gtggtgttcc tgttcctcgg
     2281 tgccctgcgc tcggtactga ttccggtggt gaccataccg ctgtcgatga tcggtgtact
     2341 gttcttcatg cagatgatgg gctactcgct caacctgctg accctgctcg ccatggtgct
     2401 ggccatcggc ctggtggtgg acgatgccat tgtggtggtg gagaacattc accggcacat
     2461 ggaagaaggc aagtcgccgt tcgatgcagc cctggaaggc gcgcgcgaaa ttgccatgcc
     2521 agtggtgtcc atgaccatca cccttgccgc cgtgtatgca ccgatcggct tcctcaccgg
     2581 gctcaccggc gcgctgttca aggagtttgc cctgacgctg gccggggcgg tgatcatttc
     2641 cggaatcgtt gccctgaccc tgtcaccgat gatgtgcgcc ctgctgctgc gccgggagca
     2701 gaacccgagc ggcctggccc accgcctcga tcagctgttc gaacgcctga aagggcgcta
     2761 ccagcgtttg ctgcacgcca ccctcgacag ccggccggtg gtgctggtgt ttgccgtgat
     2821 catcctgtgc ctgatcccgg tgctgctcaa attcacccag aacgaactgg cgcccaacga
     2881 ggaccagggg gtgatcttca tgatgagcag ctcgcctcaa ccggccaacc tcgactacct
     2941 caacgcctac accgaccagt tcacaccgct gttcaaggcc ttccccgagt attactcgtc
     3001 gtttcagatc aacggcttca acggcgtgca gaccggtatc ggtggttttt tgctcaagcc
     3061 ctggaacgag cgcgaacgca cgcagatgga gctactgccg ctggtgcagg ccaaactcga
     3121 ggaaatcagc gggctgcaga tcttcggctt caacctgcct tcgttacccg gcactggcga
     3181 gggcttgcca ttccagttcg tgatcaatac cgctggcgac tacccggcac tgctggacgt
     3241 ggcccaacgc atcaagacgc gcgctcagga atcgggcaag ttcgccttcc tcgatatcga
     3301 tcttgccttc gacaaacccg aagtggtggt ggacatcgac cgggcaaagg cggcgcagat
     3361 gggggtgtcg atggacaccc tcggcggcac cttggcgacc ctgctgggcg aggcggaaat
     3421 caaccgtttc acccttgaag gccgcagtta caaggtgatc gcccaggttg aacggcctta
     3481 ccgcgacaac cccggctggc tgaacaacta ctacgtgaag aacgaccagg gccagttgct
     3541 accgctgtcg accctgatca ccctcaccga ccgcgctcgc ccacgccagc tcaaccagtt
     3601 ccagcaactg aacgctgcga tcattcaggg cgtgcccatg gtcagcctgg gcgaggcgct
     3661 caagaccgtg caggacatcg ctcgcgaaga ggccccggaa ggctttgcct tcgactacgc
     3721 cggcgcggcc cgccagtatg tacaggaagg cagcgcattg tgggtcacct tcggcctggc
     3781 gctggcgatc attttcctcg tactggctgc acagttcgaa agcttccgcg acccgttggt
     3841 gattctggtg acagtgccgt tgtccatctg tggcgcgctt ttgccgctgt tcctgggtgt
     3901 gtcgagcatg aacatctaca cccaggtcgg gctggtgaca ttgatcgggc ttatcagcaa
     3961 gcacggtatc ctgatcgtcg agttcgccaa tcagctacgc gatgagcgtg gcctgagtgt
     4021 gcgcgaagcg atcgaggaag cagcggccat tcgcctgcgg ccggtgctga tgaccactgc
     4081 ggctatggtg ttcggcatgg tgccgctgat tctggcaagc ggggcaggag cggtcagccg
     4141 gtttgacatc ggtatggtga ttgccaccgg gatgtcgatt ggcacgttgt ttaccttgtt
     4201 tgtgttgccg tgcatctaca ccctgctggc gcacaaggcg agcgccaagg cacctgcaat
     4261 cgctcatcat caccatcacc actgaggatc caaactcgag taaggatctc caggcatcaa
     4321 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     4381 aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatacc
     4441 tagggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc
     4501 gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta
     4561 acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa
     4621 gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata
     4681 ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac
     4741 cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg
     4801 ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct
     4861 gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac
     4921 tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa
     4981 ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc
     5041 aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt
     5101 tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag
     5161 ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     5221 cccatacgat ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg
     5281 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     5341 aggagtccaa gcgagctctc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc
     5401 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
     5461 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
     5521 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     5581 ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca
     5641 tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     5701 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     5761 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     5821 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     5881 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     5941 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat
     6001 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     6061 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     6121 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
     6181 atcctcatcc tgtctcttga tcagatcatg atcccctgcg ccatcagatc cttggcggca
     6241 agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg
     6301 gcaattccga cgtcggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     6361 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6421 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     6481 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     6541 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     6601 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     6661 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     6721 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     6781 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     6841 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     6901 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     6961 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     7021 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     7081 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     7141 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     7201 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     7261 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     7321 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     7381 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     7441 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     7501 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     7561 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     7621 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     7681 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     7741 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     7801 gatcgagatc gtttaggcac cccaggcttt acactttatg cttccggctc gtataatgtg
     7861 tggaattgtg agcggataac aatttcagaa ttcaaaagat cttttaagaa ggagatatac
     7921 at
//
