Oligomeric forms of tau are the primary source of neurotoxicity in a wide range of neurodegenerative diseases. Structural studies of pathological tau aggregates isolated from tauopathy patient brains have uncovered a diversity of disease-specific tau conformations and structural polymorphs, but the mechanism for how they misfold into pathogenic species remains unknown. Here we aim to test a long-standing hypothesis that post- translational modifications (PTMs), such as phosphorylation and acetylation at disease-associated sites, mediate tau oligomerization, structural polymorph-dependent propagation, and neurotoxicity. We propose a bottom-up approach for directly assessing the impact of pre-defined, disease-associated phosphorylation and acetylation of full-length human tau. For the first time, we demonstrate that a state-of-the-art synthetic biology approach enables the production of recombinant site-specifically phosphorylated tau (rp-tau), and that rp-tau with a single modification is sufficient to adopt pathological conformations. This new capability will be expanded to include site-specific lysine acetylation of tau to produce full-length tau with defined PTMs (including simultaneous phospho- and acetyl-) implicated in Alzheimer’s disease (AD) and AD-related dementias (ADRD). The new reagents developed here will be used to determine the impact of site-specific tau PTMs on tau conformation using cryo-EM, microtubule binding, oligomerization, neurotoxicity, and tau seeding. We will generate monoclonal antibodies against tau with defined PTMs and AD/ADRD relevant conformations validated through cryo-EM. The impact of the project will be to provide transferrable resources to test tau PTMs with unprecedented resolution and uncover the molecular signature and mechanisms that mediate tau oligomerization, conformational change, and seeding.