Schematic representation of integration of a reporter construct after a double crossover event using the
targeting system. A) The truncated pyrG gene (-112) + 3’ UTR fragment (1255 bp) was amplified by PCR using primers ABpyrGP12f and ABpyrGP10r. The 3’ pyrG fragment (684 bp) was amplified by PCR using primers ABpyrGP11f and ABpyrGP13r. Both PCR products were digested (EcoRI-NotI for pyrG-3’ UTR and NotI-SstII for 3’ pyrG) and ligated in EcoRI-SstII opened pBluescriptSK, yielding plasmid pMA334. The mluc reporter cassette was obtained by PCR using SL1 and TtrpCP2rev-NotI as primers and pMA313 (containing PgpdA-mluc-TtrpC-AOpyrG, unpublished vector) as template. pMA334 was opened with NotI and the NotI digested Pgpd-mluc-TtrpC fragment was inserted, giving plasmid pMA349. Both plasmids have been deposited at Fungal Genetic Stock Centre. pMA349 was digested with AscI to release the complete pyrG** targeting transformation DNA. B) Integration of the pyrG** targeting construct via a double cross over at the pyrG locus.