pPIC3.5K vector carries the kanamycin resistance gene, thereby allowing Resistant to Geneticin™ reagent. The natural occurrence of multiple insertion events can be identified by the development of resistance to elevated levels of Geneticin™ reagent. Pichia pastoris transformants were selected on histidine-deficient medium and screened for resistance levels to Geneticin™ reagent. The ability to grow in high concentrations of Geneticin™ indicates that multiple copies of the kanamycin resistance gene and gene of interest have been integrated into the genome. The pPIC3.5K vector directly addresses the secretion of proteins that are not expressed but remain within the cell. This vector is included in the Multi-copy Pichia pastoris Expression Kit.