LOCUS       Exported                7807 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pBbA5k-EPL48
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7807)
  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 7807)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7807
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             4150..4167
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4203..4274
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4290..4317
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4345..4362)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4479..5024)
                     /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(4559..4578)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5138..5232
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5263..6057)
                     /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(5374..5393)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5984..6003
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6242..7324)
                     /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     7286..7305
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7325..7402)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7608..7627
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7713..7743
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    7751..7767
                     /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 atgttccgct atgccttgcc agtcagcctg gctttttttc tgtctgcttg tgggcacgaa
       61 gagcccgttc aaattggcat ccggccggcc atggtggtcc agccagagcc ttctgcaaga
      121 gcttcggaca gctacccggg cgaggtgcgc gcccgctatg agccggacct ggcattccgt
      181 atcggcggca aggtcagccg acggctggtg gaggagggcg agcgggtcaa ggccaaccag
      241 cccctggcgg agctcgaccc gcaggacgtg cgcctgcaac ttgaagccac ccgggcccag
      301 gtcactgcag ccagcgccaa cctcaacctg gtgcgcgccg agcgtgatcg ctacaagacc
      361 ctgctggagc ggcagatggt cagccattcc caatacgaca acgccgagaa cctctaccgt
      421 tccggcgaag cgcgcctgaa gcagatcaag gccgagttcg atgtggccaa caaccaggcc
      481 ggctacgcgg tgctgcgcgc gccgcaggac ggcgtggtgg ccaagcgtgc ggtggaagtg
      541 gggcaggtgg tggcggccgg gcagacggtc ttcaccctgg ccaccgatgg cgagcgtgag
      601 gtgctgatca gcttccccga gcagaactac ggccgcttca aggtcggcca accggtatcg
      661 gtggagctct ggacccagcc gggccagcgt ttcgacgggc gcatccgcga gctgtcgcct
      721 gctgccgatc ccaagtcccg gaccttcgcc gcgcgtattg ccttcactgc cggcaaggtt
      781 cccgccgagc tgggccagag tgcccgggtg ttcatccaga gcggcgatgc ggtgccgctg
      841 tcggtgccgc tgtcggcgtt gtccgcggaa ggcggggcga cttatgtctg gcgggtggat
      901 gccaacaata ccctgcacaa gaccccggtg cgggtcggcg ccttcggcga gaagagcgtg
      961 ccggtgctgg aagggctgag cgccagcgat tgggtggtgg ccgcgggggt gcatgtgctc
     1021 catgagggct tgcaggtgcg cccggtggat cgctccaacc gcgtggtcaa tctggcggcc
     1081 aaggagtaat ccccgatggg tttcaacctt tccgcctggg cgttgcgcaa tcgccagatc
     1141 gttctgttcc tgatgctgct gctggccatt gtcggtgcgc tgtcctacac caagttgggg
     1201 cagagcgagg atccgccttt caccttcaag gccatggtga tccgcaccaa ctggcccggg
     1261 gccacggccc aggaagtctc gcgccaggtc accgagcgca ttgaaaagaa gctgatggag
     1321 actggcgagt acgaccgcat cgtgtccttc tcgcggccgg gggagtccca ggtgaccttc
     1381 atcgcccggg actcgatgca ctccaacgag gttcccgagc tgtggtacca ggtacgcaag
     1441 aagatcagcg atattcggca gaccctgccg cccggtatcc aggggccgtt cttcaacgat
     1501 gagttcggca ccaccttcgg caatatctat gcgttgaccg ggcagggctt cgactacgcg
     1561 gtactcaagg actacgccga ccgcatccag atccagctgc aacgggtcaa ggacgtgggc
     1621 aaggtcgacc tgctgggcct gcaggacgag aaaatctgga tcgagctgtc caacgtcaag
     1681 ctggcgaccc tgggcctgcc cctggcggcg gtgcaacagg ccctggaaga acagaacgca
     1741 gtgtccaccg ccgggttctt cgagaccccg agcgagcggg tgcaactgcg ggtgtcgggc
     1801 aatttccaga ccgtggagca gatccgcaat ttcccgatcc gcgtcgccgg tcgtaccttc
     1861 cgcattggtg atgtggcgga cgtgcgtcgt ggtttcaacg atccacccgc gccgcgcatg
     1921 cgcttcatgg gtgaagacgc catcggcctg gctgtggcga tgaaggaggg gggtgacatc
     1981 ctggtgctgg gcaaggccct ggaaggcgag ttctcgcgcc tgcagaaaag cctgccggcg
     2041 gggatggaac tgcgcaaggt gtccgaccag ccggcggcgg tgaagactgg ggtgggcgag
     2101 ttcgtccggg tgctggccga ggccctgatc atcgtgctgc tggtgagttt cttctccctg
     2161 ggagtgcgga ccgggatggt ggtggccctg gcgattcccc tggtgctggc gatgaccttc
     2221 gccaccatgt actacctggg gatcggcctg cacaagattt ccctgggggc actggtgctg
     2281 gccctgggct tgctggtgga cgacgcgatc attgcggtag agatgatggc gatcaagatg
     2341 gagcagggct tcgaccggat caaggccgcc agcttcgcct ggaccagtac cgccttcccc
     2401 atgctcaccg gcaccctgat cactgcggcg ggcttcctgc cgatcgccac ggcccagtcc
     2461 ggcacgggcg aatacacccg ttcgatcttc caggtggtga ccattgccct ggtggcctcg
     2521 tggattgcag ccgtggtgtt cgtgccgtac ctgggggaaa aactgctgcc ggacctggcg
     2581 aagatccacg ccgccaagca cggcgccgaa tccaatcccc acggtacacc tttctaccag
     2641 cgggtccggc gggtagtggg ctggtgcgtg gagcggcgca agacggtgat cgtcctgacc
     2701 atcctgctgt tcgtcggctc ggtgatgctg ttccgtttcg ttccccagca gttcttcccg
     2761 gcctcgggtc gcctggaact gatggtcgac ctgaagctgg cggaaggcgc gtccctgagc
     2821 aacaccgccg atgaggtcaa gcgcctggaa gccctgctca aggagcatgc gggcatcgac
     2881 aactacgtgg cctatgtggg caccggttcg ccgcggttct acctgccgct ggaccagcaa
     2941 ctgccggcca ccagctttgc ccagttcgtg gtgctggcca agagcatcga ggagcgtgaa
     3001 gagctgcgcg gctggttgat cgaaaccctc aacgagcagt tcccggcgct gcgctcgcgg
     3061 gtcacgcgcc tggagaacgg gccgcccgtg ggctacccgg tgcagttccg ggtcaccggc
     3121 gagcacatcg aggaagtccg cgccctggcg cgcaaggtag cggccaaggt gcgagagaac
     3181 ccccatgtgg tcaacgtgca cctggactgg gaagagccca gcaaggtggt ctacctgaac
     3241 atcgaccagg accgggcccg ggccttggga gtgagcaccg ccaacctgtc gaaattcctc
     3301 cagagttcgc tgatcggctc caccgtcagc cagtaccggg aagacaacga gctgatcgag
     3361 atcctgctgc ggggcacccg cgaggaacgc accgagctgt cgttgctgcc gagcctggcg
     3421 gtgcctaccg agaatggcca gagcgtgtcc ctgtcccagg tggcgaccct ggagtacggc
     3481 ttcgaagaag gggtgatctg gcaccgcaac cgcctgccca acgtcaccgt gcgcgccgac
     3541 atctacggca aggagcagcc ggcgaccctg gtgcagcaga tcctgccgac cctggagccg
     3601 gttcgcgccg agctgcccga cggttacctg ctggaagtgg gtggcacggt cgaggactcg
     3661 acccggggcc agaactcggt gaaggccggc gtgccgctgt tcatcgtggt ggtgctgacc
     3721 ttgctgatga tccagctgcg cagtttctcg cgcacggcca tggtgttcct cacggcgccc
     3781 ctgggattga tcggggtcac cttgttcctg ctggtgttcc gccagccctt cggcttcgtg
     3841 gccatgctcg ggaccattgc cctgtcgggg atgatcatgc gcaactcggt gatcctggtg
     3901 gaccagatcg agcaggacat caaggcgggg cacaagccct gggatgcgat catcgaagcc
     3961 acggtgcggc gtttccggcc catcgtgctg accgccctgg cggcggtgct ggcgatgatc
     4021 ccgctgtcgc gcagtgtgtt cttcgggccg atggcagtgg ccatcatggg tgggttgatc
     4081 gtggcgacgg cgctgaccct gttgttcctg ccggcgctgt atgcggcctg gttcagggtc
     4141 aagaaaaccc atcatcacca tcaccactga ggatccaaac tcgagtaagg atctccaggc
     4201 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     4261 cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt
     4321 atacctaggg atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact
     4381 gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat
     4441 acttaacagg gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct
     4501 gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa
     4561 agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg
     4621 tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag
     4681 ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga
     4741 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc
     4801 accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg
     4861 gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc
     4921 ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt
     4981 cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta
     5041 atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     5101 tcagccccat acgatataag ttgttactag tgcttggatt ctcaccaata aaaaacgccc
     5161 ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta
     5221 tcaacaggag tccaagcgag ctctcgaacc ccagagtccc gctcagaaga actcgtcaag
     5281 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     5341 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     5401 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     5461 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     5521 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     5581 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     5641 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     5701 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     5761 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     5821 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag
     5881 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     5941 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     6001 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     6061 aaacgatcct catcctgtct cttgatcaga tcatgatccc ctgcgccatc agatccttgg
     6121 cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc
     6181 agctggcaat tccgacgtcg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     6241 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6301 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6361 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     6421 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6481 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     6541 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6601 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6661 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6721 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6781 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6841 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6901 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6961 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     7021 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     7081 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     7141 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     7201 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     7261 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     7321 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     7381 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     7441 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     7501 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     7561 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     7621 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     7681 tagaggatcg agatcgttta ggcaccccag gctttacact ttatgcttcc ggctcgtata
     7741 atgtgtggaa ttgtgagcgg ataacaattt cagaattcaa aagatctttt aagaaggaga
     7801 tatacat
//
