LOCUS       sequence-64014-e        9680 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 9680)
  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 9680)
  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-EPL52
FEATURES             Location/Qualifiers
     source          1..9680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             6023..6040
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      6076..6147
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6163..6190
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6218..6235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6352..6897)
                     /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(6432..6451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(7011..7105)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7136..7930)
                     /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(7247..7266)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7857..7876
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(8115..9197)
                     /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     9159..9178
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9198..9275)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9481..9500
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9586..9591,9592..9609,9610..9616)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9624..9640
                     /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 atgaatcaac agtctcgacg cacgctgatt gcgatcgcca tcgtggcgat cctgggcacc
       61 ggcgtggcaa cctcggttct gcgtccctgg cacggcgggg aagcacaggc caataccccg
      121 ccgccggcac cggcggtgga agtgtccgcg gcggtcggca agaccatcac cgaatgggat
      181 gaattctccg gccgcattga agccgtggac cgcgtggaga tccgtccgcg tgtttccggc
      241 acgatcgaaa ccgtgcattt ccgcgagggc tcgatcgtga agaagggcga tccgctgttc
      301 acgatcgatc cgcgtccgta tgcagccgaa gtggcacgcg ccgaagcggc tttggcggcg
      361 gcgcaggtac gcgcatcgct tgcgcagacc gagaaagccc gcgcccagcg cctgctggac
      421 gacaacgcga tctcgcgccg ggagttcgac gagcggatca acgccgccag cgagatgtcg
      481 gccgacgtgc gcggcgcgca ggcggcgctg gaagtggcga agctgaacct gagctacacg
      541 cgtatcgtcg cgccggtctc gggcaaggtg tcgcgtgccg agatcacggt gggcaacctg
      601 gtggccgccg gcgcggcgtc tccggcgctg accacggtgg tgtccgtgtc gccggtgtat
      661 gcgagcttcg acgtcgacga acagagctac ctgcgctaca cggcgccggg cgccagcggc
      721 ggcaagaatg atctgccggt gttcctgggg ctggccaacg aggacaacac gccgcacgaa
      781 ggcaagatcc actcggtgga caaccggctg gatacgcgca gcggcacgat ccgcgtgcga
      841 gcgatcttcg ccaatgaaga tggccgcctg ctgccgggcc tgtacgcgaa gatcaagctc
      901 ggtggcggca cgccgcatcc ggccgtgctg atcaatgatc gcgccatcgg cacggaccag
      961 ggcaagaagt tcgtgctcgt ggtggatcag gccaacaagc tcacgtatcg cgaagtcgaa
     1021 ctcgggccga cgtatgaagg gctgcgtgtg atccgcaagg gcttgaagcc gggcgagagc
     1081 atcgtcgtca atggcctgat gcgcgtgcgg ccaggcgaca ccgttgcgcc gaagccggta
     1141 tcgatggcct tccgcagcga gctcgagccg cgaggcaacg cgccggaccg caagcaggca
     1201 gcggaagaga aggccgagaa gcccaacgtg agctaagcaa cccagtctag ccagacacag
     1261 acaatttccc cccttcgtgg catcgaatgg cgtccgcttg cgagcggacc gccagggggg
     1321 ctgttttcgc cgagaccaag atgaatctct cgaaattctt tatcgaccgc cccattttcg
     1381 cgggtgtgct atcggtgctg atcttcctga tcggcgccat atcgatgttc aagttgccga
     1441 tctcggagta cccggaagtg gtgccgccgt cggtggtggt gcgcgcgcag ttcccgggcg
     1501 ccaacccgaa ggtgatcgcc gagaccgtgg ccacgccgct ggaagagcag atcaacggcg
     1561 tcgaggacat gctctacatg aactcgcagg ccaacagcga cggcacgctg acgctgacgg
     1621 tgaccttcaa gctgggcacg gacccggaca aggcccagca gctcgtgcag aaccgcgtct
     1681 cgcaggccga gccgcgtctg cccgaggacg tgcgtcgcct gggcattacc acggtcaaga
     1741 gctcgcctga tctgaccatg gtggtgcacc tggtctcgcc gaacaatcgc tatgacatga
     1801 cgtatctgcg caactacgcg ctgatcaacg tcaaggaccg cctggcgcgt atccagggcg
     1861 tcggtcaggt gcagatgttc ggctcgggcg actactcgat gcgtgtgtgg ctgaaccccg
     1921 acaagatcgc cgagcgtcat ctggcggcct cggacgtggt caaggccatc cgcgagcaga
     1981 acgtgcaggt ggcagccggc gtgatcggac aatcgccctc gctgccgggc accgatctgc
     2041 aactgtcggt caacgcgcag ggccgtctgc agacggtgga ggagtttggc gacatcatcg
     2101 tcaacacctc gccggacggc gcggtgacgt acctcaagga catcgcccgc gtcgagctgg
     2161 gtgcctccga gtacgcactg cgttcgctgc tcgacaacaa gtccgccgtg gcgctgccga
     2221 tcttccaggc gccgggttcc aacgcgatcc agatctcgga cgatgtgcgc aagacgatgg
     2281 ccgagctgaa agcgaacatg ccggagggcg tggactacag catcgtctat gacccgaccc
     2341 agttcgtgcg ccacagcatc gaggccgtga cgcacacgct gttcgaggcc atcgcgctgg
     2401 tggtgctggt ggtgatcctg ttcctgcaga cctggcgcgc gtcgatcatt ccgctgctgg
     2461 ccgtgccggt gtcgatcgtc ggcacgttcg ggctgatgca cgcgttcggg ttctcgatca
     2521 acgcgctgag cctgttcggt ctggtgttgg cgatcggtat cgtggtggac gatgcgatcg
     2581 tggtagtgga gaacgtcgag cgcaatatcg aggaagggtt gtcgccgaag gaagccacgt
     2641 acaaggccat gcgcgaggta agtggtccga tcatcgccat cgcgctgacg ctgatcgccg
     2701 tgttcgtgcc gctggcattc atgactggcc tgaccgggca gttctacaag cagttcgcgc
     2761 tgacgatctc gatctcgacg atcatctcgg cgttcaactc gctgacgctg tcgcccgcgc
     2821 tgtccgcgct gctgctcaag agccacgacg cgccgaagga ttggctgacg cgcgccatgg
     2881 acaaggtgct cggcaagttc ttcgtccgct tcaaccagtt cttccgccgc agctcggaca
     2941 actatggccg gggcgtgaag ggcgtgattc gccgcaaggg ctcggtgttc ggcgtgtacg
     3001 cgctgatgct ggtcgcgacc tggggcgtgt tccagatggt gccgaagggc ttcgtaccgg
     3061 cgcaggacaa gcagtacctg gtgagcttcg ccaagctgcc tgacggcgcg acgctggatc
     3121 gcacggaaga cgtgatccgc cgcatgtcgg atatcgcgct caagcatccg ggcgtggagt
     3181 cggcggtggc attcccgggc ctgtcgatca acggcttcac caatagcccg agcgccggca
     3241 tcgtgttcgt cacgctcgat ccgttcgaca agcgcaagag caaggaactg tctggcaatg
     3301 cgatcgcggc tgatctgaac aagcagtacg gctcgatcca ggacgccttc attgcggtgt
     3361 tcccgccgcc gccggtgcag ggcctcggta cgatcggcgg tttcaagatg atgatcgagg
     3421 accgcgccgc gctgggctat gacgaactgt ttaacgcgac caacgcgttc atggccaagg
     3481 cgcgcgccac gcccgaactg gctggcatct tcagcaacta tcaggtgaac gtgccgcagc
     3541 tagacgtgaa gctggaccgc accaaggcca agcaactcgg cgtgcccgtg accgatgtgt
     3601 tcgacacgct ccagacctac ctcggctctg cgtacgtcaa cgacttcaac aagtttggtc
     3661 gcacgtacca ggtcaaggtg caggccgatg cgcagttccg ccagcatgcg gaggacatcc
     3721 tgcagctgaa gacgcgcaac gtgagtggtg agatggtgcc gctgtcgtcg ctggtgcagg
     3781 tcaagcagag cttcggcccc gacagcgtga tccgctacaa cggcttcacg gctgccgata
     3841 tgaatggtgg tccggctcct ggcttctcgt cgggccaggc gcaggccgcc gccgagcgga
     3901 tcgccgcgga gacgctgccc aagggtatcg ccttcgagtg gaccgagctg acgtatcagg
     3961 acatcctggc cggcaatgcg ggcgtctgga tcttcccgct gtgcgtgttg ctggtgttcc
     4021 tggtgctggc cgcccagtac gaaagcctga cgctgccgct ggcggtgatc ctgatcgtgc
     4081 cgctgagcct gctggccgcc atgactggcg tgtggctgac ccgtggcgat aacaacatct
     4141 tcacgcagat cggtttcatc gtgctggtgg ggttatccgc gaagaacgcg attctgatcg
     4201 tcgagttcgc gcgtgaactg gaacatcacg gccgtacggt ggtgcaggcc gcgatcgagg
     4261 ccagccgcct gcgtctgcgc ccgatcctga tgacgtcgtt cgctttcatc atgggtgtgg
     4321 tgccgctggt ggtgtccacc ggcgccggcg cggaaatgcg ccatgcgatg ggcgtggccg
     4381 tgttcgcggg gatgctgggg gtgacgttct tcggcctgtt cctgacgccg gtcttctatg
     4441 tggcgctgcg tttgctggcc acgcgaggcc agcgccagaa ggtggctgag cagcctgcgt
     4501 cggcgtcgca ggcggtggca tcggcagaat aagggtgaac aacatgaata acgcaatgaa
     4561 gctggtcctg ccgaagctgg gtatgctggc cgccgcgctg gtgctggcgg gctgctcgct
     4621 ggcgccgact tacaaggtgc ccgagacgcc caccgcgtcg acgttcaagg aagccgaggc
     4681 cgctgctgcg gaaggcgcgc aatggaagac cgcgacgccg gccgaaggcc agcatcgcgg
     4741 tgaatggtgg aaggtcttcg gcgacgccga tctcgatcgt ctgatcgacg cggccaacgc
     4801 ctacaaccag gacctcgctg ccgccgcggc gcgtctgaag caggcacgtg ccttcacggg
     4861 cgcgaccgag gcggacctgt atccgcaatt gagcgcgggg ttcagcccga cgcgcgcgca
     4921 gccctcggca gcctcgcaag gcttgcccga tggcacgcgg gtgtcgccgc agaccgtgta
     4981 ccaggcgcgt ctgatcgcca actacgagct tgatctcttc ggccgcgtgg ccaatagcgt
     5041 caatgcagca cgcgccgacg agcagggcgc cgaagacctg tatcgttcgg tgcagctcgc
     5101 gctgcaggcc gatacggcgc aggcgtactt cgcgctgcgc acgcttgata gcgaccgcga
     5161 tctgctcaac gcgacgatca agctgcgcga ggacgcgctg acgctgctgc gcaagcgcta
     5221 cgaggctggc gaaaccaccg atctcgatcc ggcccgcgcc gaggccgagc ttggcaccgc
     5281 ccgcgccgat ctggctggca tcgagcggcg ccgtgccaac caggagcacg cgctggccgt
     5341 actgaccggg gtgtcgccgt cgcagttcgc gctggcggcg cgaccgtttg acacgccgcc
     5401 ggttgcggtt cccgccggct tgccgtcgga actgctcgaa cgtcgccccg atatcgctgc
     5461 ggcggaacgc cagatggcgg ccgccaacgc acggatcggt gtggcaaagg cggcgttctt
     5521 cccacgcatc tcgctgacag cgctgttcgg cttcgagtcg tcggatatct cgaacctgtt
     5581 caagtggtcg tcgcgcgcat ggttgcttgg gccgctggtg ggttccactg ttgcccagac
     5641 cgtgtttgac ggtggccgca acagttcgaa cctggccggc gcgcgcgcgg cgcacgagga
     5701 aagcgtggcg cgatatcgcc agaccgtgct ggtggcgttc cgcgaggtcg aggacagtct
     5761 ggccgacgta cgctggttta gccagcaggg cgcggcgctc gacagtgcat tggccggcgc
     5821 gaagcgcgcg cagcgcactt cgcgcagccg ctatgacgcg ggcgcggtgg actacctcac
     5881 cgtgatcgat gcggaccgta cggtgctgca atcgcaacgc gatgccaatg cggtggccgg
     5941 gctgcgggcg gcggcaacgg tgtcgctcgt gcgtagcctt ggtggcggtt ggggccctct
     6001 gccggagaat gtggcgaaga accatcatca ccatcaccac tgaggatcca aactcgagta
     6061 aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     6121 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     6181 cctttctgcg tttataccta gggatatatt ccgcttcctc gctcactgac tcgctacgct
     6241 cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag
     6301 atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag
     6361 gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc
     6421 gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt
     6481 tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac
     6541 gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc
     6601 ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa
     6661 gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg
     6721 aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca
     6781 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc
     6841 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa
     6901 gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat ctcttcaaat
     6961 gtagcacctg aagtcagccc catacgatat aagttgttac tagtgcttgg attctcacca
     7021 ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc
     7081 attactggat ctatcaacag gagtccaagc gagctctcga accccagagt cccgctcaga
     7141 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     7201 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     7261 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     7321 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     7381 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     7441 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     7501 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     7561 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     7621 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     7681 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     7741 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     7801 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     7861 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     7921 gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatcatgat cccctgcgcc
     7981 atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc ccaaccttac
     8041 cagagggcgc cccagctggc aattccgacg tcggtgccta atgagtgagc taacttacat
     8101 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     8161 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     8221 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     8281 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     8341 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     8401 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     8461 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     8521 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     8581 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     8641 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     8701 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     8761 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     8821 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     8881 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     8941 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     9001 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     9061 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     9121 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     9181 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     9241 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     9301 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     9361 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     9421 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     9481 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     9541 gatgcgtccg gcgtagagga tcgagatcgt ttaggcaccc caggctttac actttatgct
     9601 tccggctcgt ataatgtgtg gaattgtgag cggataacaa tttcagaatt caaaagatct
     9661 tttaagaagg agatatacat
//
