To create the Kog1 variants Kog1S491A/S494A, Kog1S491D/S494D, and Kog1S491E/S494E we cloned Kog1 (with Ser 491 and 494 mutated to Ala, Asp, or Glu) into a plasmid carrying URA3 under an ADH1 promoter (pRS306). The plasmids were then cut using PflF1, and transformed into a wild type train (ACY044). In all cases this led to insertion of the plasmid at the Kog1 locus, creating a wild-type copy of Kog1, followed by the URA3 gene, followed by a mutant copy of Kog1 (AA, DD, or EE; as confirmed by sequencing). We then isolated colonies that excised the plasmid by selection on 5FOA. In this final step, we expected to find colonies that retain the wild-type copy of Kog1 as well as colonies that retain the mutated copy of Kog1 (depending on the site of recombination during the loop out event). In line with our expectations, we found 7/10 of the colonies created by looping in, and looping out, Kog1S491A/S494A had the AA mutation (the other 3 were wild-type). In contrast, we found 0/20 colonies had the DD mutation and 0/12 strains had the EE mutation. Specifically, we can calculate the probability of finding 0/20 DD and 0/12 EE mutations by chance, given that we found 7/10 AA mutations, at p<0.0001 and p<0.001, respectively, using Fisher’s exact test. The probability of finding 0/32 DD and EE mutants by chance, given that we found 7/10 AA mutants, is less than 1 x 10-–5. This strongly suggests that strains carrying Kog1 with phosphomimetic substitutions at Ser 491 and 494 are inviable.