Assembly standard 28/Overview

The Lim standard is based on a multi-part/combinatorial cloning technique that is particularly well suited to shuffling protein domains. The key to this approach is the Type IIS restriction enzyme, AarI, a rare (7-cutter) that cuts 4bp offset from its binding site. Thus, AarI can generate four base overhangs of any sequence.
	Since the user can specify the overhangs, this method can be used to "stitch-together" fragments without a scar, which is sometimes necessary to preserve protein function. More importantly, these overhangs can be non-palindromic thereby avoiding a key problem of trying to do multipart ligations using standard restriction enzymes: the self ligation of a part (which blocks proper assembly of parts).
The Lim standard enables high efficiency ligations of up to 4 parts simultaneously (vector plus 3 inserts). While parts can be made with any 4 base overhangs, we chose a standard set, termed A, B, C, and D to facilitate exchange of parts between researchers. Note that most parts that adhere to the Lim standard are primarily intended for use when working with yeast.