Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker.

Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects, and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome.