CoS-MAGE was carried out in three strains (EcNR2, EcNR2.*xseA*^{−}, and nuc5^{−}), using sets of ten oligos encoding TAGTAA mutations, and a co-selection oligo designed to revert a mutated selectable marker located within 500 kb of the targeted loci. Set 1 (**A**) was co-selected with chloramphenicol acetyltransferase (*cat,* inserted at the *mutS* locus). In comparison with EcNR2 (n = 319), both EcNR2.*xseA*^{−} (**p = 0.0001, n = 135) and nuc5^{−} (***p<0.0001, n = 257) show statistically significant increases in mean oligo conversion, a decreased proportion of clones exhibiting no allele conversions, and more clones with 5+ conversions. Set 2 (**B**) was co-selected with beta lactamase (*bla,* inserted with the λ prophage). Here, nuc5^{−} (n = 142) shows a statistically significant increase in recombineering performance compared to both EcNR2 (***p<0.0001, n = 268) and EcNR2.*xseA*^{−} (***p<0.0001, n = 184). Set 3 (**C**) was co-selected with *tolC*. Here, nuc5^{−} (n = 139) shows a statistically significant increase in mean allele conversion compared to EcNR2 (*p = 0.002, n = 327). EcNR2.*xseA*^{−} (n = 92) shows an intermediate phenotype between EcNR2 (p = 0.2) and nuc5- (p = 0.3). All oligos used in this experiment had two PT bonds on both ends. Data shown in the right panels are presented as the mean with the standard error of the mean. Statistical significance is denoted using a starred system where ns denotes a non-significant variation, * denotes p<0.003, ** denotes p<0.001, and *** denotes p<0.0001.

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