We report the construction of pRAUW122, a plasmid capable of directing the integration of gene-constructs by gene replacement at the his-3locus of Neurospora crassa. Gene targeting at his-3 is selected by growing the transformants in histidine-free medium. The elimination of a second selectable marker (Bmlr), flanking the gene construct, is detected by screening for benomyl sensitivity of the his-3+ transformants. Therefore, his-3+ Bmls transformants have a high probability of having arisen by the desired gene replacement event.

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