Abstract: A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or glutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

Abstract: A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or gutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

Abstract: A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or glutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

Abstract: A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or gutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

Abstract: RsaI, a restriction enzyme from the bacterium Rhodopseudomonas sphaeroides, recognizes the DNA sequence 5′-GTAC-3′. Because RsaI is commercially valuable, we sought to overproduce it by cloning the genes for RsaI and its accompanying, modification, enzyme. The ‘methylase-selection’ method, the customary procedure for cloning restriction and modification genes, was applied to RsaI. The method yielded clones containing the methylase gene (rsaIM), but none containing both the methylase gene and the restriction gene (rsaIR). Inverse-PCR was then used to recover sections of the DNA downstream of rsaIM. These sections were sequenced, and the sequences were joined in silico to reveal the gene organization of the RsaI R-M system.