A hemagglutinin (HA) tag was introduced at the 59 end of muSOX by PCR using the primers 59-CGGAATTCATGGCT-TACCCATACGATGTACCTGACTATGCGATGGAAG GG-TCGATTATTC-39 and 59-ATAGTTTAGCGGCCGCTTA-GGGGGTTATGGGTTTTCT-39. HA-muSOX was then cloned into the EcoRI/NotI sites of pCDEF3 containing a T7 promoter to generate pCDEF3-T7-HA-muSOX. MuSOX was randomly mutagenized by PCR with the Genemorph kit (Strategene) according to the manufacturer's protocol using 35 ng of pCDEF3-T7-HA-muSOX as template, the above primers, and 30 PCR cycles to generate a pool of random mutants. The mutants were then cloned into the EcoRI/NotI sites of pCDEF3-T7. R443I was generated by QuickChange (Stratagene) using the primers 59-GCTCATCATCACTCCTGT TATAATTCCATC-TACTGTGCTGC-39 and 59-GCAGCACAGTAGATGGAAT-TATAACAG GAGTGATGATGAGC-39.