Figure 4

A versatile integration vector to facilitate gene expression in the ade2 locus. A) The pMJ-023 vector contains gene-targeting sequences (FLa 1: upstream region of ade2 and FLa 2: downstream region of ade2) for integration into ade2 locus (in pink); a pyr2 selectable/counter selectable marker (in light blue) flanked by direct repeats (in striped blue); and a USER cassette (in green) flanked by the PgpdA promoter (in purple) and the TrpC terminator (in turquois). The NotI cut sites that are used for excising the gene-targeting substrate are indicated. The E. coli vector backbone is marked in black; see Materials and methods for more information. B) The gene of interest (GOI) (orange) is inserted into the vector by USER cloning. C) The liberated gene-targeting substrate containing the GOI expression cassette integrates into ade2 by homologous recombination, in a process that results in ade2 deletion. Primer targets for diagnostic PCRs are indicated.