LOCUS       Exported                7778 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Gene replacement vector pEXKm5, complete sequence.
ACCESSION   GQ200735
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7778
                     /organism="Gene replacement vector pEXKm5"
                     /mol_type="other DNA"
                     /db_xref="taxon:655090"
     rep_origin      12..836
                     /label=oriT
                     /note="oriT"
     oriT            358..467
                     /note="incP origin of transfer"
     regulatory      complement(892..1217)
                     /regulatory_class="terminator"
                     /note="Escherichia coli T1 and T2 rrnB terminators"
     terminator      892..919
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      1011..1097
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1253..1558)
                     /codon_start=1
                     /product="LacZ alpha subunit"
                     /label=LacZ alpha subunit
                     /note="allows blue-white screening"
                     /protein_id="ACS73374.1"
                     /translation="MTMITPSSRDNRVIPNSRGGRPGRDNRVMSLALAVVLQRRDWENP
                     GVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRAWLFWRMREDFQPDTD"
     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 Reverse Primer
                     /note="M13 Reverse Primer"
     protein_bind    1578..1594
                     /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(1602..1632)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1647..1668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      1874..2547
                     /label=ori
                     /note="ori"
     rep_origin      complement(1956..2544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2690..2737
                     /note="FRT; Flp recombinase target site"
     protein_bind    complement(2690..2737)
                     /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             3123..3917
                     /codon_start=1
                     /gene="nptII"
                     /product="NptII"
                     /label=nptII
                     /note="neomycin phosphotransferase; kanamycin resistance 
                     protein"
                     /protein_id="ACS73371.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            3123..3917
                     /gene="nptII"
                     /label=nptII
     promoter        3977..4005
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /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"
     gene            complement(6249..7712)
                     /gene="sacB"
                     /label=sacB
     CDS             complement(6252..7712)
                     /codon_start=1
                     /gene="sacB"
                     /product="modified SacB"
                     /label=sacB
                     /note="modified levansucrase; counterselection marker"
                     /protein_id="ACS73373.1"
                     /translation="MTYSKQAVSRRHKRLALSAAALTAAACMSAHAQSDGATQAFAKET
                     NQKPYKETYGISHITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQ
                     NADGTVANYHGYHIVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFD
                     ANDSILKDQTQEWSGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNIN
                     GVEDYKSIFDGDGKTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTE
                     DGYQGEESLFNKAYYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKV
                     MKPLIASNTVTDEIERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTG
                     PYKPLNKTGLVLKMDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAP
                     SFLLNIKGKKTSVVKDSILEQGQLTVNK"