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pwFRT-PCS12-gat-sacB vector (V002139#)

Basic Vector Information

Basic Vector Information
Vector Name pwFRT-PCS12-gat-sacB Antibiotic Resistance Ampicillin
Length 5092 bp Type Gene replacement vector
Source Kang Y, Norris MH, Wilcox BA, Hoang T.

pwFRT-PCS12-gat-sacB vector Vector Map

pwFRT-PCS12-gat-sacB5092 bp6001200180024003000360042004800AmpR promoterblaoriCAP binding sitelac promoterlac operatorM13 revFRTPCS12RBSgatsacBrrnB T1 terminatorFRTM13 fwd

Plasmid Resuspension protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

pwFRT-PCS12-gat-sacB vector Sequence

Copy Sequence

Download GeneBank File(.gb)

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  JOURNAL   Unpublished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"

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