© 2019 Many strategies have been developed to manipulate DNA molecules and investigate protein–DNA interactions with single-molecule resolution. Often, these require long DNA molecules with a length of several 10s of kb that are chemically modified at specific regions. This need has traditionally been met by commercially available DNA from bacteriophage λ. However, λ DNA does not allow for the generation of highly customizable substrates in a straightforward manner, an important factor when developing assays to study complex biochemical reactions. Here we present a generalizable method for the design and production of very long chemically modified DNA substrates derived from a single plasmid. We show the versatility of this design by demonstrating its application in studying DNA replication in vitro. We anticipate this strategy will be broadly useful in producing a range of long chemically modified DNA molecules required for a diverse range of single-molecule approaches.