pEXKm5 vector (V006426)

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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"