1. Cloning of Partner plasmids and amplification of the ume6Δ::r1HIS1r1 cassette.
Cloning of Partner plasmids and amplification of the ume6Δ::r1HIS1r1 cassette. (A) pMH01 and pMH02 are derived from pRS424, which contains KpnI and SapI restriction sites. The vector sequence between these restriction sites becomes the repeat sequence for the ume6Δ::r1HIS1r1 cassette. This repeat sequence can be lengthened or shortened through use of different restriction enzymes. (B) Amplification from pMH01 using primers His1 CRIME/F and UME6-SapI/R; the latter primer contained an 80-bp region of homology to the downstream region of UME6 and generated one of the two halves of the ume6Δ::r1HIS1r1 cassette. Amplification from pMH02 using primers His1 CRIME/R and UME6-KpnI/F, the latter primer containing an 80-bp region of homology to the upstream region of UME6, generated the other half of the ume6Δ::r1HIS1r1 cassette. Following transformation, split-marker recombination (16) reconstituted the whole ume6Δ::r1HIS1r1 cassette, revealing the direct repeat.
Manning Y. Huang, and Aaron P. Mitchell mSphere 2017; doi: /mSphere