LOCUS       Exported                6424 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Gene replacement vector pEXKm4, complete sequence.
ACCESSION   FJ797516
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6424
                     /organism="Gene replacement vector pEXKm4"
                     /mol_type="other DNA"
                     /db_xref="taxon:629244"
     rep_origin      81..905
                     /label=oriT
                     /note="oriT"
     oriT            427..536
                     /note="incP origin of transfer"
     regulatory      961..1286
                     /regulatory_class="terminator"
                     /note="Escherichia coli T1 and t2 rrnB terminators"
     terminator      961..988
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      1080..1166
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1322..1627)
                     /codon_start=1
                     /gene="lacZ alpha"
                     /product="beta-galactosidase alpha"
                     /label=lacZ alpha
                     /protein_id="ACO48889.1"
                     /translation="MTMITPSSRDNRVIPNSRGGRPGRDNRVMSLALAVVLQRRDWENP
                     GVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRAWLFWRMREDFQPDTD"
     gene            complement(1322..1627)
                     /gene="lacZ alpha"
                     /label=lacZ alpha
     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"
     regulatory      1641..1746
                     /regulatory_class="promoter"
                     /note="lacZ promoter/operator region"
     protein_bind    1647..1663
                     /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)."
     promoter        complement(1671..1701)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1716..1737
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      1943..2616
     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..3986
                     /codon_start=1
                     /gene="nptII"
                     /product="neomycin phosphotransferase"
                     /label=nptII
                     /note="NptII; kanamycin resistance protein"
                     /protein_id="ACO48890.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            3192..3986
                     /gene="nptII"
                     /label=nptII
     promoter        4046..4074
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /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
     misc_feature    complement(6350..6397)
                     /note="FRT; Flp recombinase target site"
     protein_bind    complement(6350..6397)
                     /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)."