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Abstract

Cover: In Papa and Shoulders (https://doi.org/10.1002/cpch.70), Schematic of DIRex recombineering. (A) In DIRex recombineering, intermediates containing the conditionally lethal gene (ccdB) are first selected for using kanamycin. The modification (Mod) is directly repeated in the homology arms. Thus, the direct and inverted repeats (IR) promote spontaneous excision to yield the final recombinant. Successful recombinants are selected for by removing arabinose. In the absence of arabinose, the ccdA antitoxin is no longer expressed, and ccdB then kills unmodified cells. (B) DIRex is hypothesized to promote spontaneous excision through hybridization between the two inverted repeats to form a hairpin during replication that brings the direct repeats into close proximity (Bzymek & Lovett, 2001; Näsvall, 2017). The direct repeats can then promote strand slippage during synthesis, which results in excision of one of the direct repeats and everything between the direct repeats. See e70.