Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. Finally, we successfully generated a potent rAAV vector based on serotype 5 with a balanced VP1/VP2/VP3 stoichiometry.