Fig. 2.

TR construction strategy to control the length of synthetic SRT proteins. (A) DNA and protein sequence of the TR unit (n = 1). Restriction sites introduced for DNA manipulation are indicated. (B) The TR procedure. (B, I) The TR unit is removed from its vector by digestion and gel purification. (B, II) The TR unit is circularized by intramolecular ligation. (B, III) The circular unit is nicked to create a priming site for RCA. (B, IV) RCA in the presence of standard dNTPs plus 5-methyl-dCTP causes 5mC to be incorporated into the RCA product at random cytosine positions. (B, V) Digestion of the RCA product with restriction enzymes that are blocked by 5mC yields TR products with a distribution of different lengths. (B, VI) The mixture of TR products is separated on a gel; the size range of interest is gel-purified and cloned into an expression vector.