Gibson Assembly of Highly Repetitive DNA

Highly repetitive DNA sequences remain difficult to synthesize, assemble, and verify, limiting their use in synthetic biology. Here, we report a modular plasmid framework for the scalable, sequence-defined assembly of repetitive DNA. As a model system, plasmids encoding repeats of the pentapeptide Gly–Val–Gly–Val–Pro (GVGVP) n were constructed to produce elastin-like polypeptides (ELPs) with temperature-dependent solubility. A synthetic DNA fragment encoding GVGVP 17 was incorporated into a plasmid architecture that enables iterative repeat amplification through a Gibson-based digest-and-assemble workflow. Sequential Hin dIII and Bam HI digestion followed by Gibson Assembly increased repeat number (2n–1 per cycle) while preserving plasmid architecture, yielding constructs up to GVGVP 1025, as verified by whole-plasmid sequencing. A superfolder GFP was added to the GVGVP library with expression of up to 513 repeats and functional characterization up to 257 repeats in E. coli NEB 5-alpha cells. These results establish a generalizable strategy for constructing large repetitive DNA sequences and encoding programmable protein polymers.