Figure 3.

Overview of the cloning steps to append an ODD to a CAR

(1) Identify locus on the vector (SFG-HRE) to be modified along with proximal unique restriction sites (in this case NcoI/XhoI). (2) Synthesize fragment containing the ODD flanked by sequence identical to the CAR sequence including the proximal restriction sites identified (NcoI/XhoI) and at least 6 additional random b.p. overhangs (to allow efficient restriction endonuclease cleavage) to either side. (3) Double digest the SFG-HRE CAR and the ODD-containing synthetic fragment and recover DNA from gel (fragments to be recovered from gel are indicated). (4) Ligate DNA fragments (A) and (B).