A loxP site flanked neomycin resistance gene cassette was inserted into intron 10 and cystine codon 462 (TGT) in exon 12 was changed to alanine (GCC) (p.C462A). This amino acid substitution abolishes E3 ubiquitin ligase activity without affecting DNA binding ability. The neo cassette was subsequently removed by Cre-mediated recombination.