ssOR requires only one strand and expression of a phage annealing protein. A. Outline of the ssOR assay in E. coli. The Tn5 gene for kanamycin resistance (neo) in pGKneo was cut at its Nco1 site and either end filled or treated with exonuclease to create pGKneo* or pGKneoΔ respectively. pGKneo plasmids also encode for ampicillin resistance (bla). The direction of leading strand synthesis initiated from the pUC-derived origin is indicated. A pGKneo plasmid was mixed with either single or double stranded oligonucleotides encoding the unmutated sequence across the Nco1 site and co-electroporated into an E. coli host containing or not various phage proteins. Relative oligo repair efficiencies were scored by counting kanamycin resistant colonies adjusted for transformation efficiency by scoring ampicillin resistant colonies for each data point. B. Two 48 mer oligos encompassing 22 nts of homology either side of the inner 4 nts of the Nco1 site were synthesized and used either alone (lagging, leading) or after annealing together (double). They were co-electroporated with pGKneoΔ into E. coli hosts that contained the expressed phage proteins indicated; Vector; the pBAD24 expression plasmid without any cloned phage protein. "Lagging" denotes the oligo that hybridizes to the lagging strand template, hence could prime Okazaki fragment synthesis. Numbers denote the relative ssOR efficiency.