LOCUS       40924_18821        6424 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gene replacement vector pEXKm4, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6424)
  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 6424)
  AUTHORS   Lopez CM, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-MAR-2009) Microbiology, Immunology and Patholofy, 
            Colorado State University, 3205 Rampart Rd., Fort Collins, CO 80523,
            USA
REFERENCE   3  (bases 1 to 6424)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6424)
  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 
            (02-MAR-2009) Microbiology, Immunology and Patholofy, Colorado State
            University, 3205 Rampart Rd., Fort Collins, CO 80523, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            complement(427..536)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(961..988)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(1080..1166)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     1516..1532
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1542..1559
                     /label=I-sceI site
                     /note="I-sceI site"
     misc_feature    1560..1583
                     /label=multiple cloning site
                     /note="multiple cloning site"
     misc_feature    1587..1604
                     /label=I-sceI site
                     /note="I-sceI site"
     primer_bind     complement(1623..1639)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1647..1663)
                     /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(1671..1701)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1716..1737)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2025..2613)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2759..2806
                     /note="FRT; Flp recombinase target site"
     protein_bind    complement(2759..2806)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3192..3983
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     promoter        4046..4074
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             4400..6208
                     /codon_start=1
                     /gene="gusA"
                     /product="GusA"
                     /label=gusA
                     /note="beta-D-glucuronidase"
                     /protein_id="ACO48891.1"
                     /translation="MLRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAI
                     AVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEV
                     MEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHD
                     FFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQ
                     VVATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGEQF
                     LINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDW
                     ADEHGIVVIDETAAVGFNLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARD
                     KNHPSVVMWSIANEPDTRPQVHGNISPLAEATRKLDPTRPITCVNVMFCDAHTDTISDL
                     FDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMYT
                     DMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRKP
                     KSAAFLLQKRWTGMNFGEKPQQGGKQ"
     gene            4400..6208
                     /gene="gusA"
                     /label=gusA
     protein_bind    complement(6350..6397)
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 aaagacgcgg ccagcgtttt tccagctgtc aatagaagtt tcgccgactt tttgatagaa
       61 catgtaaatc gatcctctag ctagagtcga tcttcgccag cagggcgagg atcgtggcat
      121 caccgaaccg cgccgtgcgc gggtcgtcgg tgagccagag tttcagcagg ccgcccaggc
      181 ggcccaggtc gccattgatg cgggccagct cgcggacgtg ctcatagtcc acgacgcccg
      241 tgattttgta gccctggccg acggccagca ggtaggccga caggctcatg ccggccgccg
      301 ccgccttttc ctcaatcgct cttcgttcgt ctggaaggca gtacaccttg ataggtgggc
      361 tgcccttcct ggttggcttg gtttcatcag ccatccgctt gccctcatct gttacgccgg
      421 cggtagccgg ccagcctcgc agagcaggat tcccgttgag caccgccagg tgcgaataag
      481 ggacagtgaa gaaggaacac ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct
      541 gacgccgttg gatacaccaa ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc
      601 gtgcgaaaaa ggatggatat accgaaaaaa tcgctataat gaccccgaag cagggttatg
      661 cagcggaaaa gcgctgcttc cctgctgttt tgtggaatat ctaccgactg gaaacaggca
      721 aatgcaggaa attactgaac tgaggggaca ggcgagagac gatgccaaag agctacaccg
      781 acgagctggc cgagtgggtt gaatcccgcg cggccaagaa gcgccggcgt gatgaggctg
      841 cggttgcgtt cctggcggtg agggcggatg tcgatatgcg taaggagaaa ataccgcatc
      901 aggcgcatgc atatttgaat gtatttagaa aaataaacaa aaagagtttg tagaaacgca
      961 aaaaggccat ccgtcaggat ggccttctgc ttaatttgat gcctggcagt ttatggcggg
     1021 cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga
     1081 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
     1141 ttcgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg catggggaga
     1201 ccccacacta ccatcggcgc tacggcgttt cacttctgag ttcggcatgg ggtcaggtgg
     1261 gaccaccgcg ctactgccgc caggcaaatt ctgttttatc agaccgcttc tgcgttctga
     1321 tttaatctgt atcaggctga aaatcttctc tcatccgcca aaacagccaa gctcgccatt
     1381 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     1441 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     1501 cccagtcacg acgttgtaaa acgacggcca gtgccaagct cattaccctg ttatccctac
     1561 ccgggcggcc gcctcgagaa ttcgggatta ccctgttatc cctagagctt ggcgtaatca
     1621 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1681 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1741 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1801 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     1861 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1921 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1981 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2041 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2101 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2161 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2221 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2281 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2341 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2401 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2461 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2521 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2581 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2641 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2701 aggatcttca cctagatcct tttgggtacc gagctcgaat tagcttcaaa agcgctctga
     2761 agttcctata ctttctagag aataggaact tcggaatagg aacttcaaga tcccctgatt
     2821 ccctttgtca acagcaatgg ataattcgat taacaaatgc atggcgcaag ggctgctaaa
     2881 ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca
     2941 gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca
     3001 gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat
     3061 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
     3121 tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag
     3181 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3241 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3301 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3361 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3421 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3481 tgccggggca ggatctcctg tcatcccacc ttgctcctgc cgagaaagta tccatcatgg
     3541 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3601 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3661 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     3721 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     3781 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     3841 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3901 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     3961 atcgccttct tgacgagttc ttctgaattg aaaaaggaag agtatgagga tccaacattt
     4021 ccatcactag tgaacacgaa cccagttgac ataagcctgt tcggttcgta aactgtaatg
     4081 caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa
     4141 cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttgtacagt ctatgcctcg
     4201 ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa
     4261 cgatgttacg cagcagggca gtcgccctaa aacaaagtta ggcagccgtt gtgctggtgc
     4321 tttctgatag ttgttgtggg gtaggcagtc agagctcgat ttgcttgtcg ccataataga
     4381 ttcacaagaa ggattcgaca tgttacgtcc tgtagaaacc ccaacccgtg aaatcaaaaa
     4441 actcgacggc ctgtgggcat tcagtctgga tcgcgaaaac tgtggaattg atcagcgttg
     4501 gtgggaaagc gcgttacaag aaagccgggc aattgctgtg ccaggcagtt ttaacgatca
     4561 gttcgccgat gcagatattc gtaattatgc gggcaacgtc tggtatcagc gcgaagtctt
     4621 tataccgaaa ggttgggcag gccagcgtat cgtgctgcgt ttcgatgcgg tcactcatta
     4681 cggcaaagtg tgggtcaata atcaggaagt gatggagcat cagggcggct atacgccatt
     4741 tgaagccgat gtcacgccgt atgttattgc cgggaaaagt gtacgtatca ccgtttgtgt
     4801 gaacaacgaa ctgaactggc agactatccc gccgggaatg gtgattaccg acgaaaacgg
     4861 caagaaaaag cagtcttact tccatgattt ctttaactat gccgggatcc atcgcagcgt
     4921 aatgctctac accacgccga acacctgggt ggacgatatc accgtggtga cgcatgtcgc
     4981 gcaagactgt aaccacgcgt ctgttgactg gcaggtggtg gccaatggtg atgtcagcgt
     5041 tgaactgcgt gatgcggatc aacaggtggt tgcaactgga caaggcacta gcgggacttt
     5101 gcaagtggtg aatccgcacc tctggcaacc gggtgaaggt tatctctatg aactgtgcgt
     5161 cacagccaaa agccagacag agtgtgatat ctacccgctt cgcgtcggca tccggtcagt
     5221 ggcagtgaag ggcgaacagt tcctgattaa ccacaaaccg ttctacttta ctggctttgg
     5281 tcgtcatgaa gatgcggact tacgtggcaa aggattcgat aacgtgctga tggtgcacga
     5341 ccacgcatta atggactgga ttggggccaa ctcctaccgt acctcgcatt acccttacgc
     5401 tgaagagatg ctcgactggg cagatgaaca tggcatcgtg gtgattgatg aaactgctgc
     5461 tgtcggcttt aacctctctt taggcattgg tttcgaagcg ggcaacaagc cgaaagaact
     5521 gtacagcgaa gaggcagtca acggggaaac tcagcaagcg cacttacagg cgattaaaga
     5581 gctgatagcg cgtgacaaaa accacccaag cgtggtgatg tggagtattg ccaacgaacc
     5641 ggatacccgt ccgcaagtgc acgggaatat ttcgccactg gcggaagcaa cgcgtaaact
     5701 cgacccgacg cgtccgatca cctgcgtcaa tgtaatgttc tgcgacgctc acaccgatac
     5761 catcagcgat ctctttgatg tgctgtgcct gaaccgttat tacggatggt atgtccaaag
     5821 cggcgatttg gaaacggcag agaaggtact ggaaaaagaa cttctggcct ggcaggagaa
     5881 actgcatcag ccgattatca tcaccgaata cggcgtggat acgttagccg ggctgcactc
     5941 aatgtacacc gacatgtgga gtgaagagta tcagtgtgca tggctggata tgtatcaccg
     6001 cgtctttgat cgcgtcagcg ccgtcgtcgg tgaacaggta tggaatttcg ccgattttgc
     6061 gacctcgcaa ggcatattgc gcgttggcgg taacaagaaa gggatcttca ctcgcgaccg
     6121 caaaccgaag tcggcggctt ttctgctgca aaaacgctgg actggcatga acttcggtga
     6181 aaaaccgcag cagggaggca aacaatgaat caacaactct cctggcgcac catcgtcggc
     6241 tacagcctcg gtgacgtcgc caataacttc gccttcgcaa tgactagtga attatctaga
     6301 attattccat tgagtaagtt tttaagcaca tcagcttcaa aagcgctctg aagttcctat
     6361 actttctaga gaataggaac ttcggaatag gtacttcaag atccccaatt cgagctcggt
     6421 accc
//
