CaLC2056: To generate a CAS5 homozygous deletion mutant, the NAT flipper cassette (pLC49)60 was PCR amplified as described for CaLC2034 and transformed into CaLC2034. NAT-resistant transformants were PCR tested for correct integration of the cassette as described for CaLC2034. The presence of deleted allele was verified by amplification with primer pairs oLC2034 in combination with oLC2035, and absence of wild-type allele was verified by amplification with primer pairs oLC2047 in combination with oLC2048. The SAP2 promoter was induced to drive expression of FLP recombinase to excise the NAT flipper cassette.