A lox2272 flanked polyadenylation signal was further enclosed in "Sleeping Beauty" inverted terminal repeats. This structure was inserted in intron 17 and followed by an FRT flanked puromycin resistance cassette. In addition, a T1910A point mutation modified codon 637 in exon 18 so that it encoded glutamic acid rather than valine (p.V637E), recapitulating the human p.V600E mutation. Properly targeted males were mated to females ubiquitously expressing flp recombinase in order to remove the selection cassette. The remaining STOP sequence prevents allele expression but can be removed by cre expression.