Chimeric Antigen Receptors (CARs) have shown extraordinary efficacy in numerous clinical trials as an adoptive cell therapy to treat hematological malignancies. Still, CAR T therapy faces significant challenges, ranging from long lead times and expensive manufacturing to complicated vector engineering. Here we describe nano-S/MARt (nS/MARt), a novel DNA vector platform for stable CAR expression with minimal disruption of T cell activity. This antibiotic- free, nanovector technology uses scaffold/matrix attachment regions (S/MARs) for DNA vector maintenance and replication, and transfects primary human T cells efficiently without toxicity. When combined with GMP-compliant MaxCyte Flow Electroporation® and CliniMACS Prodigy® automated cell processing, nS/MARt enabled the production of recombinant T cells with stable CAR expression and enhanced anti-tumor activity in only five days. The result was a shortened manufacturing protocol, producing safer cell therapeutics for thousands of patients from a single batch.