Plant-based expression systems are promising platforms for the rapid and scalable production of medical recombinant proteins. In this study, we optimized regulatory elements for recombinant protein production in Nicotiana benthamiana using a synthetic biology approach. Three novel 5′ untranslated regions (5′UTRs) derived from N. benthamiana were selected and evaluated using turboGFP as a reporter. All three enhanced protein expression to levels comparable to those achieved with the Tobacco mosaic virus omega sequence, suggesting that host-derived 5′UTRs can serve as effective alternatives to viral elements. We also developed compact terminators to reduce expression cassette size. Although pinII, act3, and extensin terminators provide strong expression, their large sizes may hinder vector construction and multigene assembly. Truncation analysis showed that a 500-bp pinII terminator retained activity, whereas further truncation and shortening of the other terminators reduced expression. Because double terminators outperformed single terminators, a compact pinII 500–rbcS double terminator was selected. The optimized 5′UTRs and double terminator were applied to express griffithsin, a well-known antiviral protein. Western blot analysis confirmed successful accumulation of griffithsin in N. benthamiana. These elements may improve the efficiency and design flexibility of plant-based platforms for medical recombinant protein production.