CaLC3908/CaLC3909: To generate a CAS5 heterozygous deletion mutant in a strain carrying the FKS1 F641S mutation, the NAT flipper cassette (pLC49)60 was PCR amplified as described for CaLC2034 and transformed into CaLC2087 (SN95 FKS1 F641S /FKS1). Correct upstream and downstream integration at the CAS5 locus was verified as described for CaLC2034. The SAP2 promoter was induced to drive expression of FLP recombinase to excise the NAT flipper cassette. To generate a CAS5 homozygous deletion mutant, the NAT flipper cassette (pLC49)60 was again PCR amplified and transformed into the SN95 FKS1 F641S /FKS1 CAS5/cas5::FRT strain. Correct upstream and downstream integration at the CAS5 locus was verified as described for CaLC2034. The absence of a wild-type allele was confirmed with oLC2034 in combination with oLC2164. The SAP2 promoter was induced to drive expression of FLP recombinase to excise the NAT flipper cassette.