Principals of RCME and plasmids created.

(A). Schematic showing the process of RCME (Watson 2008): (i) starting with a base strain in which the urg1 ORF is replaced by an antibiotic marker (each of hphMX6, natMX6 and kanMX6 are available) that is flanked by (incompatible) loxP and loxM3 sites, a plasmid (ii) is introduced. This plasmid contains the cloned gene of interest and any tagging sequences positioned between loxP and loxM3 sites. It also expresses Cre recombinase. Site-directed recombination next exchanges the sequences between the plasmid and the chromosome (iii). Successful exchange can easily be identified by loss of the antibiotic marker, typically seen in greater than 50% of cells. Plasmid loss in these colonies is then confirmed by replica plating to verify colonies are leu⁻. In our experience, all of these are successful integrants. (B). Plasmid for expression of untagged sequences (NO DSR) as previously published [7]. Shown is a schematic of the sequence between loxP (P) and loxM3 (M3) for pAW8ENdeI. A start codon is formed from an NdeI site. (C) Equivalent schematic of pAW8ENdeI containing various DSR sequences. (D) Schematic of plasmid used to express proteins with either a yEGFP tag, a 3xHA tag or an HA combined with an IAA17 degron tag (HAIAA17) (all with NO DSR). L  =  poly-tyrosine–glycine–serine (TGS) linker: TAG  =  yEGFP, 3xHA or HAIAA17 protein tag. (E) Equivalent schematic of pAW8ENdeI C-terminal tagging plasmids that also contain various DSR sequences. HA  =  human influenza hemagglutinin protein tag, yEGFP  =  yeast codon optimised green fluorescent protein, HAIAA17  =  Degron from Arabidopsis thaliana transcription repressor.