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

Abstract

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 I intron ribozyme from Candida albicans. This plasmid expresses functional rRNA and ribozyme. Copyright © 2000 John Wiley & Sons, Ltd.