Fig. 1

Overview. a Partially overlapping DNA fragments are transformed into yeast cells together with a linearized plasmid backbone, or fused together by PCR, Seamless or Gibson assembly prior to the transformation. Homologous recombination (HR) enables the fusion of the DNA fragments without transformation. b The assembled plasmid insert contains 4.5 kb DNA encoding two expression units, the TEF1 promoter driving KanR expression, and the TDH3 promoter driving a fusion of the TRP1 and GFP genes. The insert has DNA sequences at the ends that are homologous to the ends of the linearized plasmid backbone (not shown). Assembly success was tested when the insert was broken into two, three, four or five fragments with short or long regions of homology to neighboring fragments or the linearized RS416 plasmid. The transformation used 2 ng or 20 ng of total DNA, including the linearized plasmid. The linearized plasmid DNA was added at the pre-transformation step for Seamless and Gibson assembly