LOCUS       Exported                8064 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pBbA5k-EPL3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8064)
  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 8064)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8064
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             4407..4424
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4460..4531
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4547..4574
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4602..4619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4736..5281)
                     /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(4816..4835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5395..5489
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5520..6314)
                     /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(5631..5650)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6241..6260
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6499..7581)
                     /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     7543..7562
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7582..7659)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7865..7884
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7970..8000
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    8008..8024
                     /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 atggaaccgt tcctatcccg gccccggcat ctcgctctgg ccatcaccgc ccttttgacc
       61 ctgaacgctt gccgcaaagc cccggaaacc gccccgtcgg cgccgccagt caaggtcagc
      121 gtcgcggaag tcatcgaaca gccgatcaac gaatgggacg aactctcggg acgcctcgag
      181 gcgccggagt cggtggaggt ccgcccgcgg gtgtccggct acatcgaccg ggtggccttc
      241 gtcgaaggcg gcctggtgaa gaaaggcgaa ctgctgttcc agatcgaccc gcgccccttc
      301 gaggccgagg tcaagcgcct ggaggccgaa ctgcagcagg cccgcaccgg ccttgcccgc
      361 gccgccaagg aggcccagcg cggcgagcgc ctgcgtgcca gcaaggccat ctccaccgaa
      421 ctggccgatg cccgggccag cgccgcccag gaggctcgcg cggcgctcgc cgcgatccag
      481 gcacagttgg acaatgcccg cctggacctc ggcttcaccc gtgtcgtctc gcccatcgac
      541 ggccgcgtcg gccgcgccga gatcaccgcc ggcaacctgg tcaacgccgg ccaggccctg
      601 ctgaccacgg tggtatccac cgatcgtgtc tacgcctatt tcgatgccga cgagcgcatc
      661 ttcctcaaat acgccgagcg ggcccggcgc aacggcggcg atacccgcgc cgccgcaccg
      721 gtgtacctcg gcctgaccgg cgagaatggc catccgtacc tgggccgact ggactttctc
      781 gacaaccagg tcaatccgcg caccggcacc atccgcggcc gcgcggtgct cgacaatgcc
      841 gatggccgct tcacgcccgg tctgtatgca cgcatccggc tggtcggtag cgacacctac
      901 gccgccaccc tgatccagga caccgcgatc ggcaccgacc tgggcaagaa attcgtcctg
      961 gtcctcggcg cggacaacac cctgagttac cgcagcgtcg agctgggacc gaagctggag
     1021 agcctgcgca tcgtccgcca gggcctggcc aagggcgagc ggatcgtggt caacggcctg
     1081 cagcgcgccc tgcccggcag cgccgtggaa gcgcaggacg tgcccatggc cgatgccgat
     1141 acccttgccg ccctggcccg ccagcgtcgg gcgctggaag gcgatgccgc atcccgcgtg
     1201 gcgagtcgcg cgagcgcggt cgaggacggg cgcccatgaa cttctcgcaa ttcttcatcc
     1261 accggccgat cttcgccgcc gtgctctcgc tgctgaccct gatcggcggc gccatcgcac
     1321 tgttccagtt gccgatcagc gaatacccgg aagtggtgcc gcccaccgtg gtggtgcgcg
     1381 ccaacttccc gggcgccaac cccaaggtga tcggtgaaac ggtcgcctcg ccgctggaac
     1441 aggccatcgt cggcgtcgag ggaatgctct acatgtcgtc ccaggccacc tccgacggca
     1501 agttgaccct gaccatcacc ttcgccctcg gcaccgatct ggacaacgcc caggtgcagg
     1561 tgcagaaccg cgtgacccgc accatgccga ccctgcccac cgaagtgcag cgcctcggcg
     1621 tgaccgtcga caaggcctcg ccggacctga ccatggtggt tcacctgacc tcgccggacg
     1681 aacgctacga catgctctac ctgtccaact acgccacgct caacgtcaag gacgagctgg
     1741 cgcgtctcga cggcgtgggc gacgtgcaac tgttcggcct cggcaactac tccctgcgcg
     1801 tctggctcga tccgcaccgg gtggcctcgc gcggcctcac cgccatggac gtggtcgacg
     1861 ccatccgcga gcagaaccgc caggtcgccg ccggctccct cggcgcaccg ccggcaccgg
     1921 gcgacaccgg cttccagctg tcgatcaaca cccagggccg cctggtcagc gaggaggagt
     1981 tcgaaaacat catcgtccgt gcaggtgagg acggtgagat cacccgcctg aaggacatcg
     2041 cccgcatcga actgggctcc agccagtacg ccctgcgctc gcaactgaac aaccggccgg
     2101 ccgtggccat gccggtcttc cagcgccccg gctccaacgc catcgatatc tccgacgcgg
     2161 tgcgcgcgcg catggccgag ctgaagcaga gctttccgca gggcgtcgac tactccatcg
     2221 tctacgaccc gaccgtgttc gtgcgcggct ccatcgaggc ggtggtgcac accctgttcg
     2281 aggcgatcgt cctggtggtg ctggtggtga tcctgttcct gcagacctgg cgcgcctcga
     2341 tcattcccct ggcggcggta ccggtgtcgc tgatcggtac cttcgcggtg atgcacctgt
     2401 tcggcttctc gctcaacgcc ctgtcgctgt tcggcctggt gctggccatc ggcatcgtgg
     2461 tggacgacgc gatcgtggtg gtggagaacg tcgagcgcaa catcgccctc ggcaagccgc
     2521 ccctggaggc gacccgccag gccatgcagg aggtgaccgg ccccatcgtc gccacggccc
     2581 tggtgctttg cgcggtgttc gtgccgacgg cgttcatctc cggcctgacc ggacagttct
     2641 accagcagtt cgccctgacc atcgccatct ccacggtgat ctcggcattc aattcgctga
     2701 ccctgtcgcc ggccctggcc gccgtgctgc tcaaggacca ccacgcgccc aaggaccgtt
     2761 tctcccgggt gctcgacagg ctgctcggcg gctggctgtt cgggcccttc aaccgcctgt
     2821 tcgagcgcgc cagccacggc tatgtcgcta ccgtgcgcca cgtgctgcgc ggcagtgcca
     2881 tcgccctgct ggtctacggc gggctgatgg gcctgacctg gctgggcttc tccagcaccc
     2941 cgacaggctt cgtaccgccg caggacaagc agtacctggt cgccttcgcc cagttgccgg
     3001 acgccgccac cctcgaccgc accgatgcgg tgatccagcg catgtcggag atcgccggcc
     3061 ggcatccggg ggtagccgac accgtggcct tccccggcct gtcgatcaac ggcttcacca
     3121 acagcccgaa cagcggcatc gtcttcaccc ccctcaagtc cttcgaggaa cgccaggacc
     3181 cgtcgctgtc ggcggatgcc atcgccgccg agctgaaccg ccggttcgcc gagatccagg
     3241 acgcctacat cgccatcttc ccgccgccgc cggtgcaggg cctcggcacc atcggcggct
     3301 tccgcgtgca ggtccaggac cgtggcggcc tcggctacga ggagctgtat cggcaggtgc
     3361 agaacgtcat cgccaagagc cgcacggtgc cggagctggc cggcctgttc accagctacc
     3421 aggtgaacgt gccgcaggtc gacgccgaca tcgaccggga aaaggccaag acccacggcg
     3481 tggccatcag cgacattttc gacaccctgc aggtgtatct gggatcgctg tacgccaacg
     3541 acttcaaccg cttcggccgt acctatcaag tcaatgtcca ggccgagcag cggtttcgcc
     3601 tggaaccggc acagatcggc cagctcaagg tgcgcaacaa ccgtggcgag atggtgccgc
     3661 tgtcgacctt cgtcaggatc gaaggcagcg ccggtcccga tcgagtgatg cactacaacg
     3721 gcttcctcac cgccgagatc aacggcgccg ccgcccccgg ctacagctcc ggccaggccg
     3781 aggcggcgat ggagcggctg ctgcgcgagg aactgccggg cggcatgacc ttcgagtgga
     3841 ccgagctgac ctaccagcag atcctcgccg gcaacaccgc catcttcgtg ttcccgctct
     3901 gcgtgctgct ggcgttcctg gtcctggccg cccagtacga gagctggggc ctgccgctgg
     3961 cggtgatcct gatcgtgccc atgaccctgc tgtcggccat cgtcggcgtc aggatcgcgg
     4021 gcggagacaa caacgtcttc acccagatcg gcctgatcgt gctggtgggg ctggcctgca
     4081 agaacgccat cctcatcgtc gagttcgcca aggatcggca ggcggagggc ctggaccgca
     4141 tcgccgccgt gctggaggcc tgccgcctgc gcctgcggcc gatcctgatg acctccatcg
     4201 ccttcatcat gggcgtggtg ccgctggtgc tgtcctccgg ggccggcgcc gagatgcgcc
     4261 atgccatggg ggtggcagtg ttctccggga tgatcggcgt gacgctcttc ggcctgctgt
     4321 tgaccccggt attcttcgtg ctggtacggg ctttcgtcga acgacgcgaa gcccgtcgga
     4381 accggcaggc actcgaagcg cagggacatc atcaccatca ccactgagga tccaaactcg
     4441 agtaaggatc tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     4501 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     4561 tgggcctttc tgcgtttata cctagggata tattccgctt cctcgctcac tgactcgcta
     4621 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg
     4681 gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc
     4741 ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa
     4801 acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc
     4861 ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt
     4921 ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa
     4981 ccccccgttc agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5041 gaaagacatg caaaagcacc actggcagca gccactggta attgatttag aggagttagt
     5101 cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc
     5161 tccaagccag ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc
     5221 ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca
     5281 agaagatcat cttattaatc agataaaata tttctagatt tcagtgcaat ttatctcttc
     5341 aaatgtagca cctgaagtca gccccatacg atataagttg ttactagtgc ttggattctc
     5401 accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
     5461 ggtcattact ggatctatca acaggagtcc aagcgagctc tcgaacccca gagtcccgct
     5521 cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata
     5581 ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg
     5641 gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat
     5701 ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg
     5761 acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg
     5821 agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta
     5881 cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc
     5941 gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga
     6001 gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca
     6061 gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc
     6121 gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc
     6181 gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt
     6241 gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca
     6301 tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagatca tgatcccctg
     6361 cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc
     6421 ttaccagagg gcgccccagc tggcaattcc gacgtcggtg cctaatgagt gagctaactt
     6481 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6541 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     6601 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     6661 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     6721 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     6781 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6841 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     6901 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6961 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     7021 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     7081 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     7141 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     7201 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     7261 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     7321 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     7381 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     7441 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     7501 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     7561 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     7621 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     7681 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     7741 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     7801 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     7861 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     7921 ccacgatgcg tccggcgtag aggatcgaga tcgtttaggc accccaggct ttacacttta
     7981 tgcttccggc tcgtataatg tgtggaattg tgagcggata acaatttcag aattcaaaag
     8041 atcttttaag aaggagatat acat
//
