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EcoRV (pronounced "eco R five") is a type II restriction endonuclease isolated from certain strains of Escherichia coli. It has the alternative name Eco32I.

In molecular biology, it is a commonly used restriction enzyme. It creates blunt ends. The enzyme recognizes the palindromic 6-base DNA sequence 5'-GAT|ATC-3' and makes a cut at the vertical line. The complementary sequence is then 3'-CTA|TAG-5'. The ends are blunt and can be ligated into a blunt cloning site easily but with lower efficiency than sticky ends.