Cloning methods are fundamental to synthetic biology research. The capability to generate custom DNA constructs exhibiting predictable protein expression levels is crucial to the engineering of biology. Golden Gate cloning, a modular cloning (MoClo) technique, enables rapid and reliable one-pot assembly of genetic parts. In this study, we expand on the existing MoClo toolkits by constructing and characterizing compatible low- (p15A) and medium-copy (pBR322) destination vectors. Together with existing high-copy vectors, these backbones enable a protein expression range covering a 500-fold difference in normalized fluorescence output. We further characterize the expression- and burden profiles of each vector and demonstrate their use for the optimization of growth-coupled enzyme expression. The optimal expression of