A loxP site was inserted into intron 3 and the following elements into intron 4: an FRT site, a second loxP site, engrailed homeobox 2 (en2) intron and splice acceptor sequences, IRES sequence, the tdTomato red fluorescent protein gene with sequence for a C-terminal HA tag, poly(A) signal sequence, a neomycin resistance gene cassette, a second FRT site and a third loxP site. The sequence between the FRT sites (RFP and neo cassettes) were removed through subsequent flp-mediated recombination. Exon 4 was then deleted through cre-mediated recombination.