id	author	title	date	pages	extension	mime	words	sentences	flesch	summary	cache	txt
cord-103540-x18pz2uz	Yellman, Christopher M.	Precise replacement of Saccharomyces cerevisiae proteasome genes with human orthologs by an integrative targeting method	2020-03-26		.txt	text/plain	6426	335	49	title: Precise replacement of Saccharomyces cerevisiae proteasome genes with human orthologs by an integrative targeting method We used the IT method to introduce phosphorylation site mutations into the gene SPO12 from oligonucleotide repair templates, and to precisely replace essential yeast proteasome genes with their human orthologs. The cassettes, amplified by PCR with flanking target identity, can be integrated into a yeast chromosomal target locus by high-efficiency transformation 36 and selected for by complementation of a ura3 mutation in the host strain. We have previously shown, in plasmid-based complementation tests, that many human genes encoding subunits of the proteasome can functionally replace their yeast orthologs under the control of a strong constitutive yeast promoter and terminator 30 . The IT4 cassettes were then replaced by inducing I-SceI and transforming with PCR-amplified human ORFs, flanked by homology to the promoter and terminator of the yeast gene.	./cache/cord-103540-x18pz2uz.txt	./txt/cord-103540-x18pz2uz.txt