Self-circularized Overhang-based Unrecombined Products (SOUP): a rapid in vitro method for generating backbone-free...

Plasmid DNA remains the standard tool for mammalian transfection but carries bacterial backbone sequences that can reduce expression efficiency and raise biosafety concerns. Minicircle DNA eliminates these elements and improves expression but requires bacterial recombination systems and multi-day protocols. Here we present Self-circularized Overhang-based Unrecombined Products (SOUP), a rapid in vitro workflow for generating backbone-free circular expression cassettes directly from polymerase chain reaction (PCR) products. The method involves cassette amplification with engineered overhangs, type IIS restriction digestion, self-ligation, and RecBCD exonuclease treatment, producing circular monomers alongside dimers and concatemers in less than 24 hours. We evaluated SOUP against its parental plasmid carrying an identical green fluorescent protein (GFP) cassette in Human embryonic kidney 293 (HEK293) cells. At 24 h post-transfection, SOUP yielded a larger proportion of GFP-positive cells, while the plasmid supported higher per-cell intensity. By 56 h, SOUP showed a clear advantage, with increased mean fluorescence, higher transfection efficiency, and a productivity index more than 2.5-fold greater than the plasmid. These results demonstrate that SOUP constructs, despite their heterogeneous composition, support strong gene expression. The workflow is fast and inexpensive, making it accessible as a practical complement to plasmid vectors and a rapid prototyping tool for backbone-free DNA constructs.