Hi there,

There is no direct counterselection but if your strain is ura3- (or trp1-), you can consider exchanging the G418 cassette with the equivalent cassette bearing the URA3 (or TRP1) gene then check transformants for ura+ (or trp+) and G418 sensitivity then transform this strain with whatever gene containing cassette and counterselect URA3 (or TRP1) on 5FOA or (5FAA) containing medium.

Thank you Dominique  and Kariona... am planning to take a wild type strain replace the gene with zeo-phes casette (positive selection for zeocin) and then replace this by my gene of interest and negatively select by growing on chloro phenylalanine...(similar to what Dominique suggested).... there is no one direct step i guess....

Hi,

you can try the to replace the G418 cassette by a cassette containing another selectable marker, NatR, for example, and the negative selectable marker GALp-GIN11 cassette. You check insertion of the whole cassette by selecting in CloNAT plates and then you can replace the whole cassette with any DNA of interest selecting for colonies that grow on galactose plates. GIN11 is a gene that inhibits growth so if the whole cassette is present colonies will not grow on galactose plates.

The original work is described in:

Methods Enzymol. 2006;409:329-45.

The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.

Storici F1, Resnick MA.

the description of the KanMX GALp-GIN11 is described in:

Genetic engineering of industrial Saccharomyces cerevisiae strains using a selection/counter-selection approach. Kutyna, D., Cordente, A.G. & Varela, C. In Methods in Molecular Biology. (2014) 1152: 157-68

Thank you Cristian