Figure 6. Sub-cloning sub-combinations is the most time-efficient way to build any combination greater than four.

(A) The sub-cloning protocol can hasten the completion of large projects through parallel lines of construction in multiple vectors that are progressively joined together. For example, a combination of 11 could be made in 11 consecutive rounds of sequential PCR insertions, or more (time) efficiently in 5 rounds of sub-cloning sub-combinations. (B) The minimal number of rounds required to complete any given combination (n) was found by: ⌈log₂(n)⌉+1 (when starting from scratch). Calculations show that sub-cloning is the most time-efficient construction strategy for any combination of four or more insertions (solutions shown for all combinations up to 65).