Ace2^{em1(ACE2)Synbl}

Enhanced specificity eSpCas9 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 inserted for enhanced expression, 872 bp human ACE2 3' UTR, 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 followed by a Flp Recombinase Target (FRT) site and 3-frame ATG short open reading frames. This cassette is designed to trap and truncate any mouse Ace2 isoforms that may splice past the hACE2 cassette. The FRT site allows Flp-mediated collapse of targeted tandem integration events if required. (J:101977)