Figure 3.

Construction of gene-targeting substrates by in vivo DNA assembly. (A) General strategy to perform a one-step cloning-free gene deletion of a gene of interest, GOI, mediated by in vivo DNA assembly. pyrG is used as an example of a selectable marker. Three PCR fragments are combined by in vivo DNA assembly to produce a classical gene-targeting substrate for one-step gene deletion. (B) NID1 was transformed with plasmid pAC572 (plate to the top left) or three PCR fragments: one that contains the pyrG marker and two that contain 1000 bp of up- and downstream sequences of yA (plate in the top right) or up- and downstream of wA (plate to the bottom).