Construction of a large plasmid lacking linearizing single restriction sites by simultaneous in vivo recombination and plasmid shuffling in yeast

Creation of large (∼ 15 kb) recombinant plasmids can be done in a single step by co-transformation of yeast cells with a partial restriction digest of a plasmid vector and a linear insert whose ends overlap one of the vector restriction sites. This method is used to generate a plasmid expressing the Saccharomyces cerevisiae rRNA genes containing the Ca.LSU group 1 intron ribozyme from Candida albicans. This plasmid expresses functional rRNA and ribozyme.