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URL: http://www.addgene.org/160380
Proper Citation: RRID:Addgene_160380
Insert Name: FLP1
Organism: Saccharomyces cerevisiae
Bacterial Resistance: Ampicillin
Defining Citation: PMID:
Vector Backbone Description: Backbone Marker:Yeast, Vol 14: 847–852 (1998); Backbone Size:6861; Vector Backbone:pBIS-GALkFLP(URA3); Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Comments: NAT cassette amplified from pLND46 using primers GGT GAT GAC GGT GAA AAC CTC and CTG ATG CGG TAT TTT CTC CTT ACG . Vector flp(H305L)-URA3 (Yeast, Vol. 14: 847–852 (1998))amplified with oligos TGC GTA AGG AGA AAA TAC CGC & GTC AGA GGT TTT CAC CGT CAT outwards from the URA3 region to generate linear fragment with ends complementary to the NAT cassette. Both PCRs carried out using the Primestar MAX polymerase mix (Takara).The two products were then annealed using Gibson assembly, using 3X the amount of NAT PCR product compared to the flp PCR product. 2uL of the mix transformed into NEB 5-alpha competent cells, and transformants were miniprepped and checked for the correct marker replacement with BamH1-Nco1 restriction digest. New insert gives a smaller (1.2 kb) fragment than original vector (1.5kb fragment). Overexpression of flp(H305L) cures 2-micron plasmids.
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Source: Addgene
