Figure 1.

Pairwise selection assembly. A target assembly sequence is broken down into sub-fragments (I–IV) that are synthesized with flanking tags (grey arrows). At PSA level 0, sub-fragments are inserted into one of two PSA vectors where tags activate two, divergently oriented selectable markers (A and B). Level 0 sub-fragment pairs are excised so that only one activation tag is retained for each sub-fragment. Subsequent pair ligation occurs in a second PSA vector where tags activate a second set of selectable markers (C and D), producing a PSA level 1 product. This hierarchical process is repeated, switching between two vectors with different selectable markers, until the full-length product is assembled.