The green seaweed Ulva mutabilis is an ecologically important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed U. mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors, and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50%-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed.
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