Integration of fusion fragments and subsequent pop-out event for allele replacement. Fusions L and R (Fig. 2) are cotransformed into the appropriate yeast strain. Recombination between the two fusion fragments generates a functional, intact K. lactis URA3 gene. Recombination between each fragment and the homologous chromosomal locus results in a duplication of the gene of interest where both copies contain the altered site (*). During the integration, the tags are deleted from the ends of the fragments. The left copy of the duplication lies adjacent to the endogenous promoter (purple box labeled Pro). After the subsequent pop-out event, the altered site is always preserved in the genome.
