Basic Vector Information
pwFRT-PCS12-gat-sacB vector Vector Map
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
LOCUS 40924_46528 5092 bp DNA circular SYN 18-DEC-2018 DEFINITION Gene replacement vector pwFRT-PCS12-gat-sacB, complete sequence. ACCESSION . 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recombineering protocols for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei JOURNAL Unpublished REFERENCE 2 (bases 1 to 5092) AUTHORS Kang Y, Norris MH, Wilcox BA, Hoang T. TITLE Direct Submission JOURNAL Submitted (09-JAN-2010) Molecular Biosciences and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) TITLE Direct Submission REFERENCE 4 (bases 1 to 5092) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Unpublished" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (09-JAN-2010) Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 2538 McCarthy Mall, Snyder Hall 207, Honolulu, HI 96822, USA" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..5092 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 96..200 /label=AmpR promoter CDS 201..1058 /label=AmpR /note="beta-lactamase" rep_origin 1232..1820 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 2108..2129 /label=CAP binding site /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 /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 /note="PCS12; Burkholderia cenocepacia rpsL promoter" /regulatory_class="promoter" RBS 2445..2467 /label=RBS /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 CDS 3105..4457 /label=SacB /note="secreted levansucrase that renders bacterial growth sensitive to sucrose" terminator complement(4474..4517) /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" protein_bind complement(4584..4631) /label=FRT /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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