We study regulatory grammar rules in yeast S. cerevisiae by exploring a simplified transcriptional architecture composed of a minimal core promoter and a synthetic upstream regulatory sequence (sURS) encoded with 3 motifs from a list of 41 conserved motifs.  We identify at least 13 (of 41) motifs which displayed a significant regulatory function. Motif function was validated in S. cerevisiae using unseen sURS designs. The validation sURS experiments indicated that a modular set of grammar rules for up- and down-regulation may exist in yeast. Using synthetic TFs, we further demonstrated that this regulatory grammar is encoded within the effector domains of the TF rather than in the binding sites themselves. Finally, we demonstrate the universality of these rules by characterizing the regulatory effect of the same sURSs in CHO-K1 cells. Our work provides a resource the design of functional promoters in eukaryotic organisms.