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 GVGVP17 was incorporated into a plasmid architecture that enables iterative repeat amplification through a Gibson-based digest-and-assemble workflow. Sequential HindIII and BamHI digestion followed by Gibson Assembly increased repeat number (2n-1 per cycle) while preserving plasmid architecture, yielding constructs up to GVGVP1025, 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.