Saccharomyces cerevisiae has proven a valuable system for the construction of plasmids via gap repair or in vivo cloning. The method allows cloning with superior accuracy and without the need to use restriction enzymes. However, despite its remarkable efficiency, the process may occasionally require the screening of large number of candidates. We have previously reported that by simply using shuttle plasmids that allow blue/white selection in Escherichia coli, it is possible to pre-select for positive clones. Here, we demonstrate that the same strategy can be used to assemble plasmids from several ectopic DNA fragments, which are all introduced in yeast cells by a simple transformation step. Further, to facilitate the subcloning of the fragment cloned into other targeting or expression vectors, the multi-cloning sites of three shuttle plasmids have been extended to include fifteen new restriction enzyme recognition sites. © 2011 Elsevier B.V.