Cloning and Sequencing of the Gene of Site-Specific Nickase N.BspD6I

T. A. Perevyazova¹, E. A. Rogulin¹, L. A. Zheleznaya¹, and N. I. Matvienko^2*

¹Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia; fax: (0967) 790-553

²Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia; fax: (095) 924-0493; E-mail: nikmatv@vega.protres.ru

^* To whom correspondence should be addressed.

A fragment of chromosomal DNA from Bacillus species D6 containing the gene of nickase N.BspD6I and the regions adjacent to its 5´- and 3´-ends was cloned and sequenced. The nucleotide sequence of the nickase gene, except of one neutral change, is homologous to the nicking endonuclease N.BstNBI gene sequenced by Higgens et al. (2001). After integration of a PCR-copy of the nickase gene into an expression vector pET28b under the control of the phage T7 promoter, specific nicking activity was detected in the lysates of transformed E. coli cells.

---

KEY WORDS: site-specific endonuclease, site-specific nickase, cloning, sequencing

---