Replisomes are the multi-protein complexes that coordinate the enzymatic activities required for DNA replication. How replisomes bypass lesions on the template DNA during replication has been studied using conventional ensemble biochemical methods. The proposed models of lesion bypass by replisomes in bacteria do not account for the inherit plasticity of the replisome during replication. Where various components may exchange into the replisome in a manner dependent on their concentration. Currently there are no suitable DNA substrates containing site-specific lesions suitable to study E. coli DNA replication using single-molecule fluorescence microscopy. To determine the molecular details of replisome bypass of template DNA lesions at the single-molecule level, a linear DNA template containing site specific lesions is required. To date we have designed and constructed a modular linear DNA substrate that is readily visualized with single-molecule resolution. Moreover, using E. coli replisomes reconstituted from purified proteins we have observed rates of replication consistent with previous studies. Continued development of this single-molecule assay will allow for investigation of the interplay between leading-strand lesion skipping and polymerase exchange dynamics.