Extended Data Fig. 8: Strain construction using the HiT vector strategy.

a, Schematic of C-terminal tagging HiT vectors (top), as described previously³⁸. Targeted integration of BsaI-linearized constructs (bottom) is facilitated by a gRNA specific to the 3′ end of the coding sequence and 40-bp homology regions (H1 and H2), both encoded in the gene-specific cutting unit. Transcription of the gRNA is driven by a type III promoter (pU6). A heterologous 3′ untranslated region (3′_CDPK3) allows expression of the gene product. DHFR denotes a pyrimethamine-resistance cassette to enable mutant selection. b, N-terminal HiT vector configuration for the generation of conditional overexpression strains. Construct integration endogenously tags the targeted gene with the Shield-1-stabilized DD domain and replaces the native promoter with that of α-tubulin (pTUB1). Cutting units were designed similarly to those in a, with a gRNA that targets the 5′ end of the coding sequence encoded in the reverse orientation. For the inducible BFD2 strain in particular, HA was also designed into the cutting unit to enable detection of the protein by the same epitope used for the examination of endogenously regulated BFD2.