Supplementary material for Chang and Gould (2000) Proc. Natl. Acad. Sci. USA 97 (10), 5249-5254.

To clone sid4⁺ into the pREP series of vectors, the internal NdeI site was removed and an NdeI site was introduced at the initiating methionine by site-directed mutagenesis using pKG1098 as template. The NdeI/SmaI fragment was cloned into similarly cut pREP vectors. To clone sid4DN into the pREP series of vector, pKG1098 was digested with NdeI and SmaI and the fragment ligated into similarly cut pREP vectors.

The intron of sid4⁺ was removed by site-directed mutagenesis of pKG1098 to generate pKG1353. PCR amplification of pKG1353 was performed to introduce a NotI site one codon before the stop codon and a NotI fragment encoding ~13 copies of the myc epitope (from Dan McCollum, pKG1452) was subcloned into it to generate pKG1935. PCR amplification of genomic DNA using PfuTURBO (Stratagene) from KGY1576 using primers just upstream of the internal NdeI site and within the Kan^R gene was used to obtain a sid4DN-HA DNA fragment. This fragment was cloned into pCRblunt. An EcoRI fragment including the entire sid4DN-HA DNA fragment was subcloned into pSK to generate pKG1936.