Provided herein are methods and compositions useful in the screening and production of recombinant AAV (rAAV) that have structural fitness and potency in insect cells and mammalian cells. The disclosure provides a directed evolution system for generating and selecting rAAV variants based on their Kozak sequences. In some embodiments, methods disclosed herein comprise the use of modified Kozak sequences to express AAV2 VP1 proteins in amounts that are useful for producing rAAV particles having high infectivities and/or transduction efficiencies. In some embodiments, methods of producing, and methods of screening, rAAV are provided that comprise the steps of infecting insect cells by administering rAAV to these cells, recovering rAAV-integrated DNA from these insect cells, and transfecting recovered rAAV into mammalian cells. Also provided herein are nucleic acids comprising nucleotide sequences encoding novel Kozak sequences. Further provided herein are mammalian cells, baculovirus particles and insect cells comprising these nucleic acids.