Ace2^{em2.1(ACE2)Synbl}

SpCas9 and gRNA Ace2-cRNA_32 (protospacer sequence 5-AAGGCTGAGAAGGAGCCAGG-3) are used to introduce the following sequence 13 bp downstream of the mouse Ace2 start codon in exon 2: signal peptide coding sequence (translation produces amino acid sequence Ace2 WLLLSLVAVTTA) in-frame with first 5 amino acids of mouse Ace2, human ACE2 cDNA coding sequence with SV40 intron, 872 bp human ACE2 3UTR, 100 bp human intergenic sequence immediately 3 of ACE2 polyadenylation region, cassette comprised of a mouse U6 promoter-driven Ace2-gRNA32 guide RNA with 2xPolIII terminator, all in reverse orientation relative to Ace2, splice acceptor and SV40 polyadenylation sequence designed to trap and truncate any mouse Ace2 isoforms that may splice past the hACE2 cassette, and a FRT-flanked PGK neomycin cassette with bGHpA. The FRT sites allow Flp-mediated removal of the neomycin cassette from targeted embryonic stem cell clones as well as collapse of targeted tandem integration events if required. Flp-mediated recombination removed the FRT-flanked neo cassette. (J:101977)