Multiple amino acid sequence alignment (ClustalW) in the catalytic domains of Nt.BstNBI, MlyI, AlwI, and BceSIII. Nt.BstNBI, the large subunit of BstNBI, a top-strand nicking enzyme (GAGTCN4↓). The recognition sequences of MlyI, AlwI, and BceSIII are GAGTC 5/5, GGATC 4/5, and ACGGC 12/14, respectively. The putative catalytic residues of BceSIII (E—PD-X12-E—Q-X8-9-E-X6-H) are shown in bold and underlined. Asterisk, identical amino acid residues; colon or period, amino acid residues with similar properties.