LOCUS       Exported                8021 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pBbA5k-EPL123
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8021)
  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 8021)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8021
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             4358..4381
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      4417..4488
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4504..4531
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4559..4576)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4693..5238)
                     /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(4773..4792)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5352..5446
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5477..6271)
                     /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(5588..5607)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6198..6217
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6456..7538)
                     /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     7500..7519
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7539..7616)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7822..7841
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7927..7957
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    7965..7981
                     /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 atgaacaaaa acagagggtt tacgcctctg gcggtcgttc tgatgctctc aggcagctta
       61 gccctaacag gatgtgacga caaacaggcc caacaaggtg gccagcagat gcccgccgtt
      121 ggcgtagtaa cagtcaaaac tgaacctctg cagatcacaa ccgagcttcc gggtcgcacc
      181 agtgcctacc ggatcgcaga agttcgtcct caagttagcg ggattatcct gaagcgtaat
      241 ttcaaagaag gtagcgacat cgaagcaggt gtctctctct atcagattga tcctgcgacc
      301 tatcaggcga catacgacag tgcgaaaggt gatctggcga aagcccaggc tgcagccaat
      361 atcgcgcaat tgacggtgaa tcgttatcag aaactgctcg gtactcagta catcagtaag
      421 caagagtacg atcaggctct ggctgatgcg caacaggcga atgctgcggt aactgcggcg
      481 aaagctgccg ttgaaactgc gcggatcaat ctggcttaca ccaaagtcac ctctccgatt
      541 agcggtcgca ttggtaagtc gaacgtgacg gaaggcgcat tggtacagaa cggtcaggcg
      601 actgcgctgg caaccgtgca gcaacttgat ccgatctacg ttgatgtgac ccagtccagc
      661 aacgacttcc tgcgcctgaa acaggaactg gcgaatggca cgctgaaaca agagaacggc
      721 aaagccaaag tgtcactgat caccagtgac ggcattaagt tcccgcagga cggtacgctg
      781 gaattctctg acgttaccgt tgatcagacc actgggtcta tcaccctacg cgctatcttc
      841 ccgaacccgg atcacactct gctgccgggt atgttcgtgc gcgcacgtct ggaagaaggg
      901 cttaatccaa acgctatttt agtcccgcaa cagggcgtaa cccgtacgcc gcgtggcgat
      961 gccaccgtac tggtagttgg cgcggatgac aaagtggaaa cccgtccgat cgttgcaagc
     1021 caggctattg gcgataagtg gctggtgaca gaaggtctga aagcaggcga tcgcgtagta
     1081 ataagtgggc tgcagaaagt gcgtcctggt gtccaggtaa aagcacaaga agttaccgct
     1141 gataataacc agcaagccgc aagcggtgct cagcctgaac agtccaagtc ttaacttaaa
     1201 caggagccgt taagacatgc ctaatttctt tatcgatcgc ccgatttttg cgtgggtgat
     1261 cgccattatc atcatgttgg caggggggct ggcgatcctc aaactgccgg tggcgcaata
     1321 tcctacgatt gcaccgccgg cagtaacgat ctccgcctcc taccccggcg ctgatgcgaa
     1381 aacagtgcag gacacggtga cacaggttat cgaacagaat atgaacggta tcgataacct
     1441 gatgtacatg tcctctaaca gtgactccac gggtaccgtg cagatcaccc tgacctttga
     1501 gtctggtact gatgcggata tcgcgcaggt tcaggtgcag aacaaactgc agctggcgat
     1561 gccgttgctg ccgcaagaag ttcagcagca aggggtgagc gttgagaaat catccagcag
     1621 cttcctgatg gttgtcggcg ttatcaacac cgatggcacc atgacgcagg aggatatctc
     1681 cgactacgtg gcggcgaata tgaaagatgc catcagccgt acgtcgggcg tgggtgatgt
     1741 tcagttgttc ggttcacagt acgcgatgcg tatctggatg aacccgaatg agctgaacaa
     1801 attccagcta acgccggttg atgtcattac cgccatcaaa gcgcagaacg cccaggttgc
     1861 ggcgggtcag ctcggtggta cgccgccggt gaaaggccaa cagcttaacg cctctattat
     1921 tgctcagacg cgtctgacct ctactgaaga gttcggcaaa atcctgctga aagtgaatca
     1981 ggatggttcc cgcgtgctgc tgcgtgacgt cgcgaagatt gagctgggtg gtgagaacta
     2041 cgacatcatc gcagagttta acggccaacc ggcttccggt ctggggatca agctggcgac
     2101 cggtgcaaac gcgctggata ccgctgcggc aatccgtgct gaactggcga agatggaacc
     2161 gttcttcccg tcgggtctga aaattgttta cccatacgac accacgccgt tcgtgaaaat
     2221 ctctattcac gaagtggtta aaacgctggt cgaagcgatc atcctcgtgt tcctggttat
     2281 gtatctgttc ctgcagaact tccgcgcgac gttgattccg accattgccg taccggtggt
     2341 attgctcggg acctttgccg tccttgccgc ctttggcttc tcgataaaca cgctaacaat
     2401 gttcgggatg gtgctcgcca tcggcctgtt ggtggatgac gccatcgttg tggtagaaaa
     2461 cgttgagcgt gttatggcgg aagaaggttt gccgccaaaa gaagctaccc gtaagtcgat
     2521 ggggcagatt cagggcgctc tggtcggtat cgcgatggta ctgtcggcgg tattcgtacc
     2581 gatggccttc tttggcggtt ctactggtgc tatctatcgt cagttctcta ttaccattgt
     2641 ttcagcaatg gcgctgtcgg tactggtggc gttgatcctg actccagctc tttgtgccac
     2701 catgctgaaa ccgattgcca aaggcgatca cggggaaggt aaaaaaggct tcttcggctg
     2761 gtttaaccgc atgttcgaga agagcacgca ccactacacc gacagcgtag gcggtattct
     2821 gcgcagtacg gggcgttacc tggtgctgta tctgatcatc gtggtcggca tggcctatct
     2881 gttcgtgcgt ctgccaagct ccttcttgcc agatgaggac cagggcgtgt ttatgaccat
     2941 ggttcagctg ccagcaggtg caacgcagga acgtacacag aaagtgctca atgaggtaac
     3001 gcattactat ctgaccaaag aaaagaacaa cgttgagtcg gtgttcgccg ttaacggctt
     3061 cggctttgcg ggacgtggtc agaataccgg tattgcgttc gtttccttga aggactgggc
     3121 cgatcgtccg ggcgaagaaa acaaagttga agcgattacc atgcgtgcaa cacgcgcttt
     3181 ctcgcaaatc aaagatgcga tggttttcgc ctttaacctg cccgcaatcg tggaactggg
     3241 tactgcaacc ggctttgact ttgagctgat tgaccaggct ggccttggtc acgaaaaact
     3301 gactcaggcg cgtaaccagt tgcttgcaga agcagcgaag caccctgata tgttgaccag
     3361 cgtacgtcca aacggtctgg aagatacccc gcagtttaag attgatatcg accaggaaaa
     3421 agcgcaggcg ctgggtgttt ctatcaacga cattaacacc actctgggcg ctgcatgggg
     3481 cggcagctat gtgaacgact ttatcgaccg cggtcgtgtg aagaaagttt atgtcatgtc
     3541 agaagcgaaa taccgtatgc tgccggatga tatcggcgac tggtatgttc gtgctgctga
     3601 tggtcagatg gtgccattct cggcgttctc ctcttctcgt tgggagtacg gttcgccgcg
     3661 tctggaacgt tacaacggcc tgccatccat ggaaatctta ggccaggcgg caccgggtaa
     3721 aagtaccggt gaagcaatgg agctgatgga acaactggcg agcaaactgc ctaccggtgt
     3781 tggctatgac tggacgggga tgtcctatca ggaacgtctc tccggcaacc aggcaccttc
     3841 actgtacgcg atttcgttga ttgtcgtgtt cctgtgtctg gcggcgctgt acgagagctg
     3901 gtcgattccg ttctccgtta tgctggtcgt tccgctgggg gttatcggtg cgttgctggc
     3961 tgccaccttc cgtggcctga ccaatgacgt ttacttccag gtaggcctgc tcacaaccat
     4021 tgggttgtcg gcgaagaacg cgatccttat cgtcgaattc gccaaagact tgatggataa
     4081 agaaggtaaa ggtctgattg aagcgacgct tgatgcggtg cggatgcgtt tacgtccgat
     4141 cctgatgacc tcgctggcgt ttatcctcgg cgttatgccg ctggttatca gtactggtgc
     4201 tggttccggc gcgcagaacg cagtaggtac cggtgtaatg ggcgggatgg tgaccgcaac
     4261 ggtactggca atcttcttcg ttccggtatt ctttgtggtg gttcgccgcc gctttagccg
     4321 caagaatgaa gatatcgagc acagccatac tgtcgatcat catcatcatc accatcacca
     4381 ctgaggatcc aaactcgagt aaggatctcc aggcatcaaa taaaacgaaa ggctcagtcg
     4441 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac
     4501 actggctcac cttcgggtgg gcctttctgc gtttatacct agggatatat tccgcttcct
     4561 cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg gcttacgaac
     4621 ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg agagggccgc
     4681 ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa tctgacgctc
     4741 aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggcgg
     4801 ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg
     4861 gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt cgctccaagc
     4921 tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc ggtaactatc
     4981 gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc actggtaatt
     5041 gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga aaggacaagt
     5101 tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt agctcagaga
     5161 accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga gattacgcgc
     5221 agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt ctagatttca
     5281 gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata taagttgtta
     5341 ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa
     5401 tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctctcg
     5461 aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     5521 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     5581 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     5641 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     5701 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
     5761 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     5821 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     5881 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     5941 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     6001 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     6061 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
     6121 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     6181 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     6241 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     6301 cagatcatga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt
     6361 tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccgac gtcggtgcct
     6421 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6481 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6541 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     6601 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     6661 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     6721 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     6781 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     6841 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     6901 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     6961 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     7021 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     7081 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     7141 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     7201 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     7261 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     7321 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     7381 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     7441 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     7501 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     7561 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     7621 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     7681 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     7741 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     7801 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     7861 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg tttaggcacc
     7921 ccaggcttta cactttatgc ttccggctcg tataatgtgt ggaattgtga gcggataaca
     7981 atttcagaat tcaaaagatc ttttaagaag gagatataca t
//
