CaLC3859: To regulate the expression of CAS5, the tetracycline-repressible transactivator, the tetO promoter, and the NAT flipper cassette were PCR amplified from pLC60561 using primers oLC2088 in combination with oLC2089. The PCR amplified product was transformed into CaLC2034. Correct upstream and downstream integration at the CAS5 locus was verified by amplifying across both junctions using primer pairs oLC2034 in combination with oLC534 and oLC300 in combination with oLC2145. The absence of a wildtype CAS5 promoter was verified with oLC2034 in combination with oLC2035. To C terminally HA tag CAS5, the CAS5-HA-HIS1 cassette was released from pLC818 with SacII and transformed into the tetO-CAS5/cas5Δ. Correct upstream integration at the C terminus of CAS5 was verified by amplifying across the junction using primer pair oLC2163 in combination with oLC2164. Expression of HA-tagged Cas5 was verified by western blot.