Strategy for pCM433-based "in-out" allelic exchange. Scheme depicted for the example of introducing a mutant allele with a point mutation (orfmut) in place of the wt allele (orfwt). PCR amplification of orfmutwith primers complementary to the boundaries of the coding sequence, followed by cloning of the resulting fragment into pCM433, results in the desired orfmutdonor plasmid. This donor can then be conjugated into the orfwtrecipient strain, for which selection of one of the three encoded antibiotic resistances (Ap, Cm, Tc) will result in strains that have experienced recombination at the desired allele, and incorporation of the donor plasmid into the chromosomal locus. Although the recombination is drawn here to have occurred to the right of the point mutation, resulting in the orfwtupstream, recombination to the left of the point mutation would situate orfmutupstream. Selection for sucrose resistance, and screening for antibiotic sensitivity results in clean exchange of the orfmutallele or reversion to orfwt. Finally, isolates bearing clean, single copies of the locus can be screened to identify a strain bearing the desired allele.