Plasmids constructed in this article and their assembly efficiencies.

<p>(<b>A</b>) The outer cycle is a plasmid map showing the main genetic elements; two inner green cycles show the overlapped SFs, along with the overlap lengths (bp); the center is plasmid name and size (bp). Two examples are provided to show how nicks or gaps form. In pOKCA construction, the antisense primer for SF1 (SF1Wa) and the sense primer for SF3 (SF3Ws) were designed to be back to back, such that a nick formed between SF1 and SF3. In pAcetone construction, primers SFTAWs and SFHBWa were designed to be apart, so a 110 bp gap formed between SFTA and SFHB. (B) ^aColony forming units per microgram DNA. ^bThe proportion of transformants caused by undigested PCR template, determined by colony PCR. ^cThe proportion of unwanted recombinants, determined by restriction mapping (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030267#pone.0030267.s002" target="_blank">Fig. S2</a></b>) and DNA sequencing. ^dThis plasmid was difficult to construct inherently (see text for details).