LOCUS       Exported                7999 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pBbA5k-EPL33
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7999)
  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 7999)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7999
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             4342..4359
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4395..4466
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4482..4509
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4537..4554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4671..5216)
                     /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(4751..4770)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5330..5424
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5455..6249)
                     /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(5566..5585)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6176..6195
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6434..7516)
                     /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     7478..7497
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7517..7594)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7800..7819
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7905..7935
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    7943..7959
                     /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 atgcacatcc aatggaccgg ctcgctgcgc gggctgctgg cggccctggt cgccctattc
       61 ctgctgggct gcgaagaagc agcggacgcc gggaagactg cggaggcccc cgccgaggtc
      121 ggcgtgatcg tcgccaggcc ggcgcctatc ggcatcacca gcgagctgcc cggacgcctg
      181 gaagcgtacc gccaggctga agtgcgggcg cgcgtcgccg gcatcgtcac ccgtcgcctg
      241 tacgaggaag gccaggacgt ccgcgccggc accgtgctgt tccagatcga ccctgcgccc
      301 ttgaaggcgg ccctggacat cagccgcggc gccctggccc gggccgaggc cagccacgcg
      361 gcggcggccg acaagctcaa gcgctacgcc gacctgatca aggaccgcgc catcagcgaa
      421 cgcgagtaca ccgaagcgca gaccgacgcg cgccaggccc tggcgcagat cgcctcggcc
      481 aaggccgaac tggagcaggc ccgcctgcgc ctgggctacg ccacggtcac cgcgccgatc
      541 gacggccgcg cgcggcgtgc gctggtcacc gaaggcgcgc tggtcggcga ggactcgccg
      601 acaccgctga cccgcgtcga gcagatcgat ccgatctacg tgaacttctc ccagccggcc
      661 ggcgaagtcg ccgccatgca gcgggcgatc cgcgaaggcc aggtgaaggg tgtcgccgac
      721 aaggacatcg ccgtgcgcct ggtcctggcc gacggcagcg agtacccgct ggccggcgag
      781 ctgctgttct ccgacctggc ggtcgacccc ggcaccgaca ccatcgccat gcgtgccctg
      841 ttccgcaatc cgcatcgcga attgctgccc ggcggctacg tgcaggtgcg cctgcagcgc
      901 gcggtgaacc cgcaggcgat caccgtcccg cgcgacgcgc tgatccgtac cgcccagtcc
      961 gccgtggtca aggtggtcaa cccaaagggc ttggtggaag acgtggaggt ccgcgccgac
     1021 accctgcagg gccgcgactg gatcatcagc cgcgggctca agggcggcga gtgggtgatc
     1081 gtcgagaacg ccgcccagca tgccgccggc tccagcgtcc aggcggtggt ccgccagccg
     1141 gccagcgccg acgccccctc accgctggcc gcctcgccgg cgggccagtg acggagaacg
     1201 ctacgaatgg ctcgtttctt cattgaccgg ccggtcttcg cctgggtgat ctccctgctg
     1261 atcgtgctcg ccggggtcct ggcgatccgc ttcctgccgg tcgcccagta cccggacatc
     1321 gcgccgccgg tggtcaacgt cagcgccagc tatcccggcg cctcggccaa ggtggtcgag
     1381 gaagcggtga ccgcgatcat cgagcgcgag atgaacggcg cgcccggcct gctctacacc
     1441 aaggccacca gcagcaccgg ccaggcctcg ctgaccctga ccttccgcca gggcgtgaac
     1501 gccgacctcg ccgcggtgga agtgcagaac cgcctgaaga tcgtcgagtc gcgcctgccc
     1561 gaatcggtgc ggcgcgacgg catctacgtg gagaaggcgg cggacagcat ccagctgatc
     1621 gttaccctta cctcctccag cggccgctac gacgccatgg agctgggcga gatcgcctcg
     1681 tccaacgtgt tgcaggcgct gcgccgggtg gagggcgtgg gcaaggtcga gacctggggc
     1741 gccgagtacg ccatgcgcat ctggcccgac ccggccaagc tgacctcgat gaacctcagc
     1801 gccagcgacc tggtcaacgc cgtgcgccgg cacaacgccc gcctcaccgt gggcgacatc
     1861 ggcaacctcg gggtccccga ctcggcgccg atcagcgcca cggtgaaggt cgacgacacc
     1921 ctggtgacgc ccgagcagtt cggcgaaatt ccgctgcgca tccgcgccga cggcggcgcg
     1981 atccgcctgc gcgacgtggc ccgcgtcgag ttcggccaga gcgagtacgg cttcgtctcg
     2041 cgggtcaacc aaatgaccgc caccggcctg gcggtgaaga tggcgcccgg ctccaacgcg
     2101 gtggccaccg ccaagcgcat ccgcgccacc ctcgacgagc tgtcgcgcta cttcccggag
     2161 ggcgtgagct acaacatccc ctatgacacc tcggcgttcg tcgagatctc gatcaggaag
     2221 gtggtcagca ccctgctcga ggcgatgctg ctggtgttcg ccgtgatgta cctgttcatg
     2281 cagaacttcc gcgccaccct gatcccgaca ctggtggtgc cggtggccct gctgggcacc
     2341 ttcacggtga tgctcggcct gggcttctcg atcaacgtgc tgaccatgtt cggcatggtc
     2401 ctggcgatcg gcatcctggt ggacgacgcg atcatcgtgg tggagaacgt cgagcggctg
     2461 atggccgagg aaggcctgtc gccgcacgac gccacggtca aggcgatgcg ccagatcagc
     2521 ggggccatcg tcggcatcac cgtagtgctg gtctcggtgt tcgtgccgat ggcgttcttc
     2581 agcggcgcgg tgggcaacat ctaccgccag ttcgcggtga ccctggcggt ctccatcggc
     2641 ttctcggcgt tcctcgcgct gtcgctgacc ccggccctgt gcgccaccct gctgcgcccg
     2701 atcgacgccg accaccacga gaagcgcggc ttcttcggct ggttcaaccg cgccttcctg
     2761 cgcctgaccg gacgctaccg caacgcggtg gccggcatcc tcgcccggcc gatccgctgg
     2821 atgctggtct acaccctggt catcggcgtg gtcgccctgc tcttcgtgcg cctgccgcag
     2881 gcgttcctgc cggaagagga ccagggcgac ttcatgatca tggtgatgca gcccgaaggc
     2941 acgccgatgg cggagaccat ggccaacgtc ggcgacgtcg agcgctacct ggcggagcac
     3001 gaaccggtgg cctacgccta tgcggtcggc ggcttcagcc tgtacggcga cggcaccagc
     3061 tcggcgatga tcttcgccac cctgaaggac tggtcggaac gccgggaggc cagccagcac
     3121 gtcggcgcca tcgtcgagcg catcaaccag cgcttcgccg gcctgcccaa ccgtacggtg
     3181 tatgcgatga actcgccgcc gctgccggac ctgggttcca ccagcggctt cgacttccgc
     3241 ctgcaggacc gtggcggggt tggctacgag gccctggtca aggcccgcga ccagttgctg
     3301 gcgcgcgccg ccgaggaccc gcgcctggcc aacgtgatgt tcgccggcca gggcgaggcg
     3361 ccgcagatcc gcctggacat cgaccggcgc aaggcggaga cccttggcgt gagcatggac
     3421 gagatcaaca ccaccctggc ggtgatgttc ggctcggact acatcggcga cttcatgcac
     3481 ggcagccagg tgcgcaaggt ggtggtccag gccgacggcg ccaagcgcct gggcatcgac
     3541 gacatcggcc ggcttcacgt gcgcaacgag cagggcgaga tggtgccgct ggcgacgttc
     3601 gccaaggccg cctggaccct cggcccgccg caactgaccc gctacaacgg ctatccctcg
     3661 ttcaacctcg agggccaggc cgcgccgggc tacagcagcg gcgaagccat gcaggcgatg
     3721 gagcaattga tgcagggact gcccgagggc atcgcccacg agtggtccgg ccagtccttc
     3781 gaagaacgcc tgtccggcgc ccaggcgccg gcgctgttcg ccctctcggt gttgatcgtg
     3841 ttcctcgccc tggccgccct ctacgaaagc tggtcgatcc cgctggcggt gatcctggtg
     3901 gtgccgctgg gcgtactcgg cgcactgctc ggggtgagcc tgcgcggtct gcccaacgac
     3961 atctacttca aggtcggcct gatcaccatc atcggcctct cggcgaagaa cgccatcctc
     4021 atcatcgagg tggccaagga ccattaccag gaaggcatga gcctgctgca ggcgaccctg
     4081 gaggccgcgc gcctgcgcct gcgaccgatc gtcatgacct cgctggcgtt cggtttcggc
     4141 gtggtcccgc tggcgctctc cagcggcgcc ggttccggcg cccaggtcgc catcggcacc
     4201 ggggtgctcg gcgggatcgt caccgccacg gtactcgcgg tgttcctggt accgctgttc
     4261 ttcctggtgg tcgggcgcct gttccggttg cgcaaggcgc cgcgcaccgg caactcgccc
     4321 cagatcccca cggagcaagc ccatcatcac catcaccact gaggatccaa actcgagtaa
     4381 ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     4441 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     4501 ctttctgcgt ttatacctag ggatatattc cgcttcctcg ctcactgact cgctacgctc
     4561 ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga
     4621 tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg
     4681 ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg
     4741 acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt
     4801 cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg
     4861 cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc
     4921 cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag
     4981 acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga
     5041 agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa
     5101 gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca
     5161 aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag
     5221 atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg
     5281 tagcacctga agtcagcccc atacgatata agttgttact agtgcttgga ttctcaccaa
     5341 taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca
     5401 ttactggatc tatcaacagg agtccaagcg agctctcgaa ccccagagtc ccgctcagaa
     5461 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     5521 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     5581 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     5641 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     5701 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
     5761 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     5821 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     5881 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
     5941 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
     6001 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
     6061 ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
     6121 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     6181 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     6241 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatcatgatc ccctgcgcca
     6301 tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc
     6361 agagggcgcc ccagctggca attccgacgt cggtgcctaa tgagtgagct aacttacatt
     6421 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6481 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     6541 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     6601 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     6661 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     6721 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     6781 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     6841 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     6901 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     6961 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     7021 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     7081 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     7141 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     7201 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     7261 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     7321 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     7381 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     7441 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     7501 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     7561 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     7621 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     7681 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     7741 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     7801 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     7861 atgcgtccgg cgtagaggat cgagatcgtt taggcacccc aggctttaca ctttatgctt
     7921 ccggctcgta taatgtgtgg aattgtgagc ggataacaat ttcagaattc aaaagatctt
     7981 ttaagaagga gatatacat
//
