pGAL-flp(H305L)-NAT
(Plasmid #160380)

• Purpose: overexpression of Flp recombinase step-arrest mutant in yeast
• Depositing Lab: John McCusker
• Publication: Yeast viruses (unpublished)

Backbone

• Vector backbone: pBIS-GALkFLP(URA3)
• Backbone manufacturer: Yeast, Vol 14: 847–852 (1998)
• Backbone size w/o insert (bp): 6861
• Total vector size (bp): 7002
• Vector type: Yeast Expression
• Selectable markers: Nourseothricin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: FLP1
• Alt name: R0010W
• Alt name: Flippase
• Species: S. cerevisiae (budding yeast)
• Insert Size (bp): 1610
• Mutation: H305L
• Promoter: GAL

Cloning Information

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: NA
• 3′ sequencing primer: NA

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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.