AbstractWe have constructed and tested a dominant resistance module, for selection ofS. cerevisiaetransformants, which entirely consists of heterologous DNA. ThiskanMXmodule contains the knownkanropen reading‐frame of theE. colitransposonTn903 fused to transcriptional and translational control sequences of theTEFgene of the filamentous fungusAshbya gossypii.This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed alacZMTreporter module in which the open reading‐frame of theE. coli lacZgene (lacking the first 9 codons) is fused at its 3′ end to theS. cerevisiae ADH1terminator.KanMXand thelacZMTmodule, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked byNotI sites. Using the double module for constructions of in‐frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection somekanMXmodules are flanked by 470 bp direct repeats, promotingin vivoexcision with frequencies of 10–3–10–4. The 1·4 kbkanMXmodule was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR‐added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin‐resistant colonies carried the correctly int
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