For teams developing RNA and DNA therapeutics, viral vectors, cell-free protein synthesis workflows or CRISPR gene editing applications, shortening the design-to-test cycle can be critical for success. The EnClose Cell-free dbDNA Synthesis Kit (E9301) is designed to provide a faster alternative to conventional plasmid DNA manufacturing. The EnClose Cell-free dbDNA Synthesis workflow combines phi29-XT DNA Polymerase for high-yield rolling circle amplification and TelN Protelomerase for deconcatenation and covalent closure of linear dsDNA ends in a streamlined one-day process. For RNA therapeutic development, an IVT template can be generated in approximately 24 hours with only 1.5 hours of hands-on time. Applications include RNA therapeutics, DNA therapeutics, AAV and lentiviral payload production and other workflows where rapid iteration and clean DNA starting material are important.