LOCUS       sequence-64027-e        9297 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 9297)
  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 9297)
  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-EPL69
FEATURES             Location/Qualifiers
     source          1..9297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(568..579)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5640..5657
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5693..5764
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5780..5807
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5835..5852)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5969..6514)
                     /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(6049..6068)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6628..6722)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6753..7547)
                     /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(6864..6883)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7474..7493
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(7732..8814)
                     /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     8776..8795
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8815..8892)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9098..9117
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(9203..9208,9209..9226,9227..9233)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    9241..9257
                     /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 atgaacgtat caaaaatatg catgcatgcc ttgcttcttt tcgcggttcc ccttgccggt
       61 tgcaagggag acgaagcgaa gaagtccccg gcggtgaaac cgccggtggc ggtggaggtc
      121 attgccgttg ccccccggga cctggtggaa ggggtcgagg tgaccggcag cctggaaccg
      181 aagttctatg ccgatgtgaa gacccagatt ccggggctcg tgaagcaggt ctatgtgact
      241 gaatggctgc gggtccgcaa ggggaccccc cttgcccgaa tcgacgtggc cgaggcggag
      301 gcaatggtga agcgggctga ggcgtcggtg gagtcggccc gggccggctt ggcccaggcc
      361 caggtggggg caaaccgggc ggagcgggag ctgacccgga ttctcaaact gaaggaatcg
      421 gggctcgcga ctcagcaggg ggtagacgac gcccgcaccg agtcggccgc ggctcaggcc
      481 aggatagagg cggcccaggc ccagatccgg gtggcccagg aagaggtgcg ccaggcccgg
      541 gcacggcagg caaaggggct tgtggtctct cccctcgatg gagtagtggc cctgcgggat
      601 gtgaacgtgg gggatctggc aagcgacgcc gccgccagca agcccatatt ccggatagtg
      661 gacaaccgtg tccttaacct gacggtcacc gttccctcgg ccgattcggc ccgggtcaag
      721 gtgggacagc cgctggagtt caccgtggac gccctccccg ggaaaacctt catgggcagg
      781 gtgatgtaca tcaatccgga actctcctct gccgaccggt cgctgcgggt gatcgccgaa
      841 gtgagaaatg acggagaccg cctcaaggga gggctcttcg ccaagggacg gattatcact
      901 ggccagcggc ccaaggtggt tcaggtcccc cgtggggcct tgaccacgtg ggatacggcg
      961 gcccggaagg ggaacctgtt cattgtcgcc ggcaccaccg cgaagctcag gcaggtggag
     1021 accggagccg tcagcggcga ttcagtggag atcgtgaaag gggtaggggc cggcgagcag
     1081 atcgtggccc gcggaggctt caacctcaag gacggggata cggtttccgt tgccggtgga
     1141 aggaagtagc ggatgatcct ctccgatctt tccatcaagc gtcccatctt tgccgcagtg
     1201 atgatgctgg tgcttgtgac cctcggcatc ttctcctacc ggcggctctc ggtggagatg
     1261 tatcccaatg tggagtttcc ggtcatctcc atcgtcacca agtatcccgg cgccagcccc
     1321 gagaccgtgg aacgggaggt ctccaagcgg atcgaggagg cggtgaacca gattgccggg
     1381 gtgaagcacg tcttctccta ttctcgggag agcgtctcca cggtggtggt ccagttccgg
     1441 ctggaggagc ggatcaatga ggtgtcccag gaggcccggg ccaagatcaa ttccgtgcgg
     1501 ggagagcttc ccaccggcat agaagacccc gttatccaaa agatcgattt caacgccctg
     1561 ccggtagtgt ccctggcgct ccagtccgga agcctctcac agcgggagct gacaacgctc
     1621 atggacaagc ggatcagaaa gcgtttcgag ggtgtggccg gggtcggtaa ggtggaggta
     1681 gtgggagggg ccaagcgcga ggtgagtgtc cacgtggacc cggtgcggct ggagtccctg
     1741 gggctcggag taaacgacgt ggtggcgggg cttcagagtg agaacgtgaa cactcccctt
     1801 ggccgcatca cccggggagg agtcgagtat cccctccgca tcgagggaaa gccggaccgc
     1861 gtggccgact accgcgccat ggtcatcgcc cagaaggggg ggcgccccgt aaccctgggg
     1921 gaagtggcca ccgtcaccga cggggtggag gagcggcgga ccctggccct ggtgaacgga
     1981 gcgccggcca tcggtatcga catctacaag cagtcggggg ccaaccaggt ggaggtggtg
     2041 gataacgtca ggaagaccat ggagaaggtg cggcgggagc ttccgccggg ggtcgaactc
     2101 tccctggtgc gcgatggctc catcatgacc cgccaatctt tggccgacgt ggaggaaacc
     2161 cttgtcgtcg gcggcattct caccatcatc atcgtcttcc tgttcatcaa ctcctggcgc
     2221 tccacggtca tcaccggggt gacgctcccc atctcggtca tctccgcctt cattgtcatg
     2281 aacgccatgg ggatgaccct caatgtcatg accctcatgg cgctctccct tgccatcggg
     2341 ctcctcatcg acgatgccat cgtggtgcgg gagaatattg tccggcacct ggagatgggg
     2401 aaggaccaca tggaggcctc gcggttcggc accagcgaga tcggtctggc ggttttcgcc
     2461 acgactatgt cgattcttgc ggtttttgtg cccgtggctt tcatgaaggg gatcgtgggg
     2521 cgcttctttt tccccttcgg catcacggtt tccttcgcag tgctggtgtc gctcttcgtc
     2581 tccttcaccc tggatccgat gctctcttcc cgctggcacg acccggcaat ccatgcccac
     2641 ggcaaacgga aggggcttgc ccgctggctc gaaaccttca acgactggtt cgaccggagc
     2701 gccgaccgct accgcctcgc catcgcctgg gcactagacc accgcaaaac ggtggtcgcc
     2761 acggctggca tcaccttttt cctggggctt gccctcatgg ggaccctgga atcatcattc
     2821 atggtcgacg aggatacgag cgagttccag gtttcgttca agtccgcccc cgaggccagc
     2881 atctccgaaa ccgaggatcg gcttaaccgg gtgctggcgg cactgaaggg cattcccgag
     2941 atcgaccatt cctatgccac catcggggcc ggcgacagcg gcacggtgcg cgacgggctt
     3001 ctctacatga agctgaagga aaaatcggcc cgcaatcgga gccagttcga gatagagcgc
     3061 ctggtgcgcg agcggctgcg gcaggtggcc ggcatcacct tttccatcga aaaggtgggg
     3121 gtcatcgcga gcacggcgaa acccctgaac gttgacctga aaggggacga tatcgccctc
     3181 ctcaagaagt acgcggccca gctcaaggag ctgatgtata ccatcccggg gatcgtcgac
     3241 atctccgcca ccctggagca cgacatcccc gagtaccggc tccgggtcga ccgggagaag
     3301 gccctctctg cgggcgtctc cagtaacgac gttgtcatgt ccctttcccg cctcgtgggg
     3361 ggggaggcgg tcactaccta cgaggacgag gacggcgatg ccgtggacgt tcgggtccgg
     3421 cttcccgagg ggctgcgcga gaatccggcc caagtgcggg atttgaagat ctcggtgcct
     3481 gacggcgcgg gcggcaccaa gctgatacct ctggcgagcg tcagcaccga aaccgtcgcc
     3541 gccaccccca ccgaggtgaa ccgccgcgac cagtcgcgtc ttgtgacggt gtcggcgaac
     3601 ctggaccggc tccccatcgg cacggcggtg aagaaggtgg aaggggtttc gaagaagatc
     3661 accatggagc cggggtacag catcaacttc tccggcgagg ccgaggatat ggccgaatcc
     3721 ttcggctaca tgggagaatc gctcatgctg gccgtggtat tcgtcttcct tattctggcc
     3781 gcccagttcg agtcgttctt cgagcccctg gcaatcatgc tgtcgctgcc gctctccatc
     3841 gtcggcatgg ccggcatgct gaaactgacc ggcgacacca tcaatatcat ggtgctcatc
     3901 ggcctcatca tgctcatggg acttgtcacc aagaacgcca ttctgttggt ggactacgcg
     3961 aaggttctcc ggcggcggga tggcctcccg cgccgggagg cgattatcga ggcggggcgg
     4021 acgagactcc gccccatcgt catgaccacc ctggccatga tcttcggcat gctgccgctc
     4081 tttcttggta tcggcgctgg cggcgagggg cgtgctccca tggcccgtgc cgttatcggc
     4141 gggctcatca cctcgtcgct cctcaccctc atcgtcgtgc cggtcatgta cacctacatg
     4201 gacgacctgg cgctgtggct caggcgaaag tggcggggtg aggagcaacg gagtaacgga
     4261 gggacaggca tttcatgact gaccaacgct ttgatgatac cgggaagagt ggcatttaca
     4321 gggcgctcga gaataaattg aggattttct ccgttgctcc gttgctctgt gttatttggc
     4381 ttttgcatgg tatcagtatg gccgccgagc atcgtgtcct caccctcgat caggccctgg
     4441 aggtggctgc ggagcgtaac cgcgacatcc tcaaggcaaa ggaattccac aaccaggtga
     4501 gggggcgcta tgtggaggaa cggtctgctg cccttcccca tctgacgatc accggacagg
     4561 ctgtcaccca gcacgacgag agcctcaatg cctatgcgga cggattcatg ccggtgcgcc
     4621 aggatatcta tggcgccgaa ctcggtctct ctcaggctct ctacacctgg gggaaggtgg
     4681 gggctgcaat ccgggcggca gagaaggggt ttgccaccgc cgacgagcag tttcgcaggg
     4741 cgcggcagga tgcctggcgc gatgtgtcgg tggcctttta caatatcctt cttgccaggg
     4801 agcagcacgc cattgcttcc cagaatcttg cgcagaaggt gcggcaccag gatgaggccc
     4861 agcgcaggta tgcggccggc gtggccaccg attacgacgt cctggcagcg gatgttgcgg
     4921 tgcaaaatgc catgcccgac gtgatccggg ccggcaacag tgtccgcgtg gcccgggacc
     4981 ggctctcctt cctcctggcc ctggagggtg aggtggatgt gaccggctcg ctggagactg
     5041 tgctgactac ctatccctcc tacgacaaag ccctcgcagt ggcccgggat aaacgtcccg
     5101 agctgaagga gatcaagcat cgcctcgcca ttgccggtga actggtgaag gtggctgacg
     5161 ccgacgacaa gccgcgtctg gacctgacgg ggaaatacgg atggcggcac ctggaggtgg
     5221 gggacggttc cgggagcggc cagatatgga ccgtgggcct tcagttgagt tttcccctgt
     5281 tcgatggcct caagacccgg ggcaaggtgg cccaggccga gagcgaacgg cgaagcatgc
     5341 agatcgacga ggctaaactc ctggagtcgg ttgccctgga aatccgcgat gcggtcaatg
     5401 ctgttcgcga gtccgaggag attgtcaagg ccctgtccgg tacggtggtc caggcggagc
     5461 gccttctgca gatggccgaa aagggattcg agctgggggt caagattcgg ctggaggtgg
     5521 acgacgccga actgaacctg cgtcaggccc gggggaacct ggcccgggca cggcgcgact
     5581 atctcgtggc ccgggttaat ctggagcggg tcatgggggt gctgggggag ggaagtctcc
     5641 atcatcacca tcaccactga ggatccaaac tcgagtaagg atctccaggc atcaaataaa
     5701 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     5761 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt atacctaggg
     5821 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
     5881 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg
     5941 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc
     6001 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg
     6061 cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg
     6121 tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag
     6181 gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc
     6241 ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca
     6301 gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta
     6361 aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga
     6421 gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga
     6481 gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa
     6541 atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat
     6601 acgatataag ttgttactag tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc
     6661 gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag
     6721 tccaagcgag ctctcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag
     6781 aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
     6841 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg
     6901 tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg
     6961 atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc
     7021 gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca
     7081 tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
     7141 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc
     7201 atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact
     7261 tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa
     7321 ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg
     7381 gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac
     7441 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc
     7501 acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct
     7561 catcctgtct cttgatcaga tcatgatccc ctgcgccatc agatccttgg cggcaagaaa
     7621 gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc agctggcaat
     7681 tccgacgtcg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     7741 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     7801 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     7861 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     7921 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     7981 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     8041 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     8101 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     8161 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     8221 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     8281 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     8341 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     8401 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     8461 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     8521 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     8581 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     8641 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     8701 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     8761 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     8821 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     8881 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     8941 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     9001 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     9061 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     9121 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     9181 agatcgttta ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggaa
     9241 ttgtgagcgg ataacaattt cagaattcaa aagatctttt aagaaggaga tatacat
//
