Table 2.
Correct integration event |
Precise excision event |
|||
---|---|---|---|---|
Parental allele | Resulting allele | Success rate | Resulting allele | Success rate |
GAL7wt | Δgal7::ura3-IR | 50% (3/6) | Δgal7 | 100% |
trp1-1 | Δtrp1::ura3-IR | 66% (4/6) | Δtrp1 | 100% |
ADH4wt | adh4 T451::ura3-IR | 60% (3/5) | adh4 T451T | 100% |
ADH7wt | adh7 Q149::ura3-IR | 50% (1/2) | adh7 Q149Q | 100% |
GPD1wt | gpd1 D391::ura3-IR | 100% (1/1) | gpd1D391Lfs*27 | 100% |
trp1-1 | trp1::ura3-IR | 83% (5/6) | TRP1wt | 100% |
Initial integration is not perfect due to the presence of a DR leading to improper integration (Supplementary Figure 1). Excision events were always observed to have precise removal of the IR within the DR. Mutations introduced in adh4 and adh7 were synonymous codon changes (ACC to ACT and CAA to CAG, respectively). In gpd1, codon 391 (GAT) was replaced with C resulting in a substitution and frameshift of the reading frame). The trp1-1 allele is an amber mutation at codon 83. Exact sequences of all sequenced mutant loci are in Supplementary Figure 3.