LOCUS       Exported                5092 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Gene replacement vector pwFRT-PCS12-gat-sacB, complete sequence.
ACCESSION   GU450326
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5092)
  AUTHORS   Kang Y, Norris MH, Wilcox BA, Hoang T.
  TITLE     Knock-out and pull-out recombineering protocols for naturally 
            transformable Burkholderia thailandensis and Burkholderia 
            pseudomallei
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5092)
  AUTHORS   Kang Y, Norris MH, Wilcox BA, Hoang T.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-JAN-2010) Molecular Biosciences and Bioengineering, 
            University of Hawaii at Manoa, 2538 McCarthy Mall, Snyder Hall 207, 
            Honolulu, HI 96822, USA
REFERENCE   3  (bases 1 to 5092)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5092
                     /organism="Gene replacement vector pwFRT-PCS12-gat-sacB"
                     /mol_type="other DNA"
                     /db_xref="taxon:717933"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             201..1061
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /protein_id="ADD38966.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            201..1061
                     /gene="bla"
                     /label=bla
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      1266..1884
                     /label=ColE1ori
                     /note="ColE1ori"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2144..2174
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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)."
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_recomb     2289..2356
                     /note="FRT; flp recognition target"
     protein_bind    complement(2306..2353)
                     /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)."
     regulatory      2393..2434
                     /regulatory_class="promoter"
                     /note="PCS12; Burkholderia cenocepacia rpsL promoter"
     RBS             2445..2467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2503..2943
                     /codon_start=1
                     /gene="gat"
                     /product="glyphosate acetyl transferase"
                     /label=gat
                     /note="Gat; confers resistance to glyphosate"
                     /protein_id="ADD38967.1"
                     /translation="MIEVKPINAEDTYDLRHRVLRPNQPIEACMFESDLTRSAFHLGGF
                     YGGKLISVASFHQAEHSELQGKKQYQLRGVATLEGYREQKAGSSLVKHAEEILRKRGAD
                     MIWCNARTSASGYYRKLGFSEQGEVFDTPPVGPHILMYKRIT"
     gene            2503..2943
                     /gene="gat"
                     /label=gat
     gene            3000..4463
                     /gene="sacB"
                     /label=sacB
     CDS             3000..4460
                     /codon_start=1
                     /gene="sacB"
                     /product="levansucrase"
                     /label=sacB
                     /note="modified; counterselection marker; SacB"
                     /protein_id="ADD38968.1"
                     /translation="MTYSKQAVSRRHKRLALSAAALTAAACMSAHAQSDGATQAFAKET
                     NQKPYKETYGISHITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQ
                     NADGTVANYHGYHIVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFD
                     ANDSILKDQTQEWSGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNIN
                     GVEDYKSIFDGDGKTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTE
                     DGYQGEESLFNKAYYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKV
                     MKPLIASNTVTDEIERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTG
                     PYKPLNKTGLVLKMDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAP
                     SFLLNIKGKKTSVVKDSILEQGQLTVNK"
     terminator      4471..4516
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_recomb     4567..4640
                     /note="FRT; flp recognition target"
     protein_bind    complement(4584..4631)
                     /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)."
     primer_bind     complement(4698..4714)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"