This is a modified protocol for URA3 counter-selection in S. cerevisiae and similar yeasts. It is identical to the standard counter-selection with two transformations, with the exception of the replica-plating step. This protocol is appropriate for genomic loci where URA3 is over-expressed and the Ura3 protein is likely to be present in the colonies and cells at higher toxic levels. Because this method required five times as many plates for the replica-plating, it is more wasteful and should only be used when necessary.