FIG 1.
PRBT5 expression system. (A) DNA segments containing the NAT1 marker and RBT5 promoter, assembled as PCR products in plasmid pTH10. (B) Sequence [i], with homology to the upstream region of YFG, is appended to the cassette with a long primer containing an 80-bp region of homology. Sequence [ii], with homology to the start of the YFG open reading frame, is also appended to the cassette with long primer containing an 80-bp region of homology. The cassette is transformed into a recipient strain along with DNA cassettes expressing CAS9 and a single guide RNA targeting upstream of the YFG locus. The double-strand break introduced by Cas9 complexed with an sgRNA targeting the YFG upstream region is indicated by two black triangles. Expected homologous recombination events are depicted as single crosses, and together they should yield a locus containing NatR and PRBT5 fused to the YFG open reading frame.