Our project’s aim is to create a centromeric plasmid for Pichia Pastoris, a species of yeast commonly used in the biotechnology industry. Building upon our work from last year, we created a yeast shuttle vector using the pSB1CRmut plasmid that contained a Hpa1 restriction site. This restriction site allowed us to linearize and thus Gibson Assembly our backbone with the ArgArs sequence necessary to facilitate proper centromeric activity in our test plasmid. Then, we repeated the restriction and Gibson process to insert our possible centromeric sequences along with an RFP test gene into our plasmid. Sectoring assays of the centromeric test plasmid versus a regular replicating plasmid with RFP allowed us to determine the effectiveness of our plasmid–which, if reliable enough over generations, could be an effective product for use in the biotechnology and biomanufacturing industries.