Tn5 is a composite bacterial transposon that encodes a protein, transposase (Tnp), required for movement of the transposon. The initial step in the transposition pathway involves specific binding of Tnp to 19-bp end recognition sequences. Tn5 contains two different specific end sequences, termed outside end (OE) and inside end (IE). In Escherichia coli, IE is methylated by Dam methylase (IE(ME)). This methylation greatly inhibits recognition by Tnp and greatly reduces the ability of transposase to facilitate movement of IE defined transposons. Through use of a combinatorial random mutagenesis technique (DNA shuffling), we have isolated an IE(ME)-specific hyperactive form of Tnp, Tnp sC7v.2.0, that is able to promote high levels of transposition of IE(ME) defined transposons in vivo and in vitro while functioning at wild-type levels with OE transposons. This protein contains a critical glutamate-to-valine mutation at amino acid 58 that is responsible for this change in end specificity.