To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C(30) carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C(40) synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies.