An amber codon-scanned library of nisA with all sense codons of the core peptide individually replaced by TAG was generated via multiple parallel overlap extension PCR reactions (Ho et al., 1989). A synthetic construct pET-21a_P_His6_leader_nisA served as a template. This construct contains the WT sequence of nisA (GenBank: HM219853.1) together with an N-terminal His-tag fused by a small linker to the coding sequence (complete encoded amino acid sequence: MGSSHHHHHHSQDP). Primers used for the generation of the library are listed in Supplementary Table S2. The individual nisA(amber) variants were cloned via NdeI and XhoI restriction sites into the expression vector pET-21a (Merck).