DNA vaccines have the potential to provide a safe route for protective immunity to neoplasms and infectious agents. However, current DNA vaccine plasmids are not optimal with additional non-essential DNA, nor do they facilitate controlled or flexible targeting of antigens to various intracellular destinations. A family of DNA vaccine vectors, optimized and minimized to comply with FDA guidelines regarding content and elimination of extraneous materials, was constructed. The resulting vectors are much smaller than existing vectors, drive higher levels of target gene expression, facilitate high throughput cloning applications, and allow simultaneous cloning into multiple vectors that feature various intracellular targeting destinations for the protein product. The ability to control expression and trafficking is intended to provide a rapid, rational approach to cancer therapy and emerging infectious diseases.