LOCUS       40924_18826        7778 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gene replacement vector pEXKm5, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  AUTHORS   Lopez CM, Rholl DA, Trunck LA, Schweizer HP.
  TITLE     Versatile dual-technology system for markerless allele replacement 
            in Burkholderia pseudomallei
  JOURNAL   Appl. Environ. Microbiol. 75 (20), 6496-6503 (2009)
  PUBMED    19700544
REFERENCE   2  (bases 1 to 7778)
  AUTHORS   Rholl DA, Lopez CM, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-MAY-2009) Microbiology, Immunology and Pathology, 
            Colorado State University, 1692 Campus Delivery, Fort Collins, CO 
            80523, USA
REFERENCE   3  (bases 1 to 7778)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7778)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl. 
            Environ. Microbiol."; date: "2009"; volume: "75"; issue: "20"; 
            pages: "6496-6503"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (22-MAY-2009) Microbiology, Immunology and Pathology, Colorado State
            University, 1692 Campus Delivery, Fort Collins, CO 80523, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            complement(358..467)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(892..919)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(1011..1097)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     1420..1443
                     /label=M13 Forward Primer
                     /note="M13 Forward Primer"
     primer_bind     1447..1463
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_binding    1473..1490
                     /label=I-SceI homing endonuclease binding site
                     /bound_moiety="I-SceI homing endonuclease"
     misc_feature    1491..1514
                     /label=MCS
                     /note="MCS"
     misc_binding    1518..1535
                     /label=I-SceI homing endonuclease binding site
                     /bound_moiety="I-SceI homing endonuclease"
     primer_bind     complement(1554..1570)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1578..1594)
                     /label=lac operator
                     /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)."
     promoter        complement(1602..1632)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1647..1668)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(1956..2544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    complement(2690..2737)
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3123..3914
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        3977..4005
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             4331..6139
                     /codon_start=1
                     /gene="gusA"
                     /product="GusA"
                     /label=gusA
                     /note="beta-D-glucuronidase; blue phenotype in presence of 
                     X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid)"
                     /protein_id="ACS73372.1"
                     /translation="MLRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAI
                     AVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEV
                     MEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHD
                     FFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQ
                     VVATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGEQF
                     LINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDW
                     ADEHGIVVIDETAAVGFNLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARD
                     KNHPSVVMWSIANEPDTRPQVHGNISPLAEATRKLDPTRPITCVNVMFCDAHTDTISDL
                     FDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMYT
                     DMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRKP
                     KSAAFLLQKRWTGMNFGEKPQQGGKQ"
     gene            4331..6139
                     /gene="gusA"
                     /label=gusA
     terminator      6196..6241
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(6255..7607)
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="GATQAFAKETNQKPYKETYGISHITRHDMLQIPEQQKNEKYQVSE
                     FDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYHIVFALAGDPKNADDTSIYMFYQ
                     KVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEWSGSATFTSDGKIRLFYTDFSGK
                     HYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDGKTYQNVQQFIDEGNYSSGDNHT
                     LRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKAYYGKSTSFFRQESQKLLQSDKK
                     RTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDEIERANVFKMNGKWYLFTDSRGS
                     KMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLKMDLDPNDVTFTYSHFAVPQAKG
                     NNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSVVKDSILEQGQLTVNK"
ORIGIN
        1 cgatcctcta gctagagtcg atcttcgcca gcagggcgag gatcgtggca tcaccgaacc
       61 gcgccgtgcg cgggtcgtcg gtgagccaga gtttcagcag gccgcccagg cggcccaggt
      121 cgccattgat gcgggccagc tcgcggacgt gctcatagtc cacgacgccc gtgattttgt
      181 agccctggcc gacggccagc aggtaggccg acaggctcat gccggccgcc gccgcctttt
      241 cctcaatcgc tcttcgttcg tctggaaggc agtacacctt gataggtggg ctgcccttcc
      301 tggttggctt ggtttcatca gccatccgct tgccctcatc tgttacgccg gcggtagccg
      361 gccagcctcg cagagcagga ttcccgttga gcaccgccag gtgcgaataa gggacagtga
      421 agaaggaaca cccgctcgcg ggtgggccta cttcacctat cctgcccggc tgacgccgtt
      481 ggatacacca aggaaagtct acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa
      541 aggatggata taccgaaaaa atcgctataa tgaccccgaa gcagggttat gcagcggaaa
      601 agcgctgctt ccctgctgtt ttgtggaata tctaccgact ggaaacaggc aaatgcagga
      661 aattactgaa ctgaggggac aggcgagaga cgatgccaaa gagctacacc gacgagctgg
      721 ccgagtgggt tgaatcccgc gcggccaaga agcgccggcg tgatgaggct gcggttgcgt
      781 tcctggcggt gagggcggat gtcgatatgc gtaaggagaa aataccgcat caggcgcatg
      841 catatttgaa tgtatttaga aaaataaaca aaaagagttt gtagaaacgc aaaaaggcca
      901 tccgtcagga tggccttctg cttaatttga tgcctggcag tttatggcgg gcgtcctgcc
      961 cgccaccctc cgggccgttg cttcgcaacg ttcaaatccg ctcccggcgg atttgtccta
     1021 ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga
     1081 gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcatggggag accccacact
     1141 accatcggcg ctacggcgtt tcacttctga gttcggcatg gggtcaggtg ggaccaccgc
     1201 gctactgccg ccaggcaaat tctgttttat cagaccgctt ctgcgttctg atttaatctg
     1261 tatcaggctg aaaatcttct ctcatccgcc aaaacagcca agctcgccat tcgccattca
     1321 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     1381 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     1441 gacgttgtaa aacgacggcc agtgccaagc tcattaccct gttatcccta cccgggcggc
     1501 cgcctcgaga attcgggatt accctgttat ccctagagct tggcgtaatc atggtcatag
     1561 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1621 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1681 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1741 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1801 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1861 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1921 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1981 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2041 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2101 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2161 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2221 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2281 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2341 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     2401 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2461 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2521 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2581 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2641 acctagatcc ttttgggtac cgagctcgaa ttagcttcaa aagcgctctg aagttcctat
     2701 actttctaga gaataggaac ttcggaatag gaacttcaag atcccctgat tccctttgtc
     2761 aacagcaatg gataattcga ttaacaaatg catggcgcaa gggctgctaa aggaagcgga
     2821 acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg
     2881 ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta
     2941 catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg
     3001 gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc
     3061 caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc
     3121 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3181 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3241 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3301 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3361 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3421 aggatctcct gtcatcccac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3481 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3541 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3601 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3661 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3721 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3781 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3841 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3901 ttgacgagtt cttctgaatt gaaaaaggaa gagtatgagg atccaacatt tccatcacta
     3961 gtgaacacga acccagttga cataagcctg ttcggttcgt aaactgtaat gcaagtagcg
     4021 tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt
     4081 ggcggttttc atggcttgtt atgactgttt ttttgtacag tctatgcctc gggcatccaa
     4141 gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac
     4201 gcagcagggc agtcgcccta aaacaaagtt aggcagccgt tgtgctggtg ctttctgata
     4261 gttgttgtgg ggtaggcagt cagagctcga tttgcttgtc gccataatag attcacaaga
     4321 aggattcgac atgttacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg
     4381 cctgtgggca ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag
     4441 cgcgttacaa gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga
     4501 tgcagatatt cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa
     4561 aggttgggca ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt
     4621 gtgggtcaat aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga
     4681 tgtcacgccg tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga
     4741 actgaactgg cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa
     4801 gcagtcttac ttccatgatt tctttaacta tgccgggatc catcgcagcg taatgctcta
     4861 caccacgccg aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg
     4921 taaccacgcg tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg
     4981 tgatgcggat caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt
     5041 gaatccgcac ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa
     5101 aagccagaca gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa
     5161 gggcgaacag ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga
     5221 agatgcggac ttacgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt
     5281 aatggactgg attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat
     5341 gctcgactgg gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt
     5401 taacctctct ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga
     5461 agaggcagtc aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc
     5521 gcgtgacaaa aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg
     5581 tccgcaagtg cacgggaata tttcgccact ggcggaagca acgcgtaaac tcgacccgac
     5641 gcgtccgatc acctgcgtca atgtaatgtt ctgcgacgct cacaccgata ccatcagcga
     5701 tctctttgat gtgctgtgcc tgaaccgtta ttacggatgg tatgtccaaa gcggcgattt
     5761 ggaaacggca gagaaggtac tggaaaaaga acttctggcc tggcaggaga aactgcatca
     5821 gccgattatc atcaccgaat acggcgtgga tacgttagcc gggctgcact caatgtacac
     5881 cgacatgtgg agtgaagagt atcagtgtgc atggctggat atgtatcacc gcgtctttga
     5941 tcgcgtcagc gccgtcgtcg gtgaacaggt atggaatttc gccgattttg cgacctcgca
     6001 aggcatattg cgcgttggcg gtaacaagaa agggatcttc actcgcgacc gcaaaccgaa
     6061 gtcggcggct tttctgctgc aaaaacgctg gactggcatg aacttcggtg aaaaaccgca
     6121 gcagggaggc aaacaatgaa tcaacaactc tcctggcgca ccatcgtcgg ctacagcctc
     6181 ggtgacgtct cagattaaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt
     6241 gaaagctact attatttgtt aactgttaat tgtccttgtt caaggatgct gtctttgaca
     6301 acagatgttt tcttgccttt gatgttcagc aggaagctcg gcgcaaacgt tgattgtttg
     6361 tctgcgtaga atcctctgtt tgtcatatag cttgtaatca cgacattgtt tcctttcgct
     6421 tgaggtacag cgaagtgtga gtaagtaaag gttacatcgt taggatcaag atccattttt
     6481 aacacaaggc cagttttgtt cagcggcttg tatgggccag ttaaagaatt agaaacataa
     6541 ccaagcatgt aaatatcgtt agacgtaatg ccgtcaatcg tcatttttga tccgcgggag
     6601 tcagtgaaca ggtaccattt gccgttcatt ttaaagacgt tcgcgcgttc aatttcatct
     6661 gttactgtgt tagatgcaat cagcggtttc atcacttttt tcagtgtgta atcatcgttt
     6721 agctcaatca taccgagagc gccgtttgct aactcagccg tgcgtttttt atcgctttgc
     6781 agaagttttt gactttcttg acggaagaat gatgtgcttt tgccatagta tgctttgtta
     6841 aataaagatt cttcgccttg gtagccatct tcagttccag tgtttgcttc aaatactaag
     6901 tatttgtggc ctttatcttc tacgtagtga ggatctctca gcgtatggtt gtcgcctgag
     6961 ctgtagttgc cttcatcgat gaactgctgt acattttgat acgtttttcc gtcaccgtca
     7021 aagattgatt tataatcctc tacaccgttg atgttcaaag agctgtctga tgctgatacg
     7081 ttaacttgtg cagttgtcag tgtttgtttg ccgtaatgtt taccggagaa atcagtgtag
     7141 aataaacgga tttttccgtc agatgtaaat gtggctgaac ctgaccattc ttgtgtttgg
     7201 tcttttagga tagaatcatt tgcatcgaat ttgtcgctgt ctttaaagac gcggccagcg
     7261 tttttccagc tgtcaataga agtttcgccg actttttgat agaacatgta aatcgatgtg
     7321 tcatccgcat ttttaggatc tccggctaat gcaaagacga tgtggtagcc gtgatagttt
     7381 gcgacagtgc cgtcagcgtt ttgtaatggc cagctgtccc aaacgtccag gccttttgca
     7441 gaagagatat ttttaattgt ggacgaatca aattcagaaa cttgatattt ttcatttttt
     7501 tgctgttcag ggatttgcag catatcatgg cgtgtaatat gggaaatgcc gtatgtttcc
     7561 ttatatggct tttggttcgt ttctttcgca aacgcttgag ttgcgccgtc gctctgcgcg
     7621 tgcgccgaca tgcaagcggc tgcggtgagc gcggcggcgc tcagggccaa acgtttgtgt
     7681 ctacgggaaa cggcttgttt ggagtacgtc atcgtttcct cctgtgcagc gagtgctaat
     7741 tatatggacg ggttttgaca aattcaagaa gtgactag
//
