Francisella spp. is a bacterial pathogen that causes acute or chronic disease in fish species with high mortality associated with clinical signs such as exophthalmos, anorexia, anemia, erratic swimming and macroscopic changes such as nephro and splenomegaly, multifocal nodules disseminated in the organs. Therefore, vaccination is an effective strategy for preventing diseases in fish production systems, reducing the economic impact as well as the use of antibiotics, which in the long term can lead to antimicrobial resistance. The DNA vaccine consists of synthesizing a plasmid that encodes specific antigens of the target pathogen, inducing an immune response in the organism. The aim of this study was to produce a DNA vaccine to protect nile tilapia against infection caused by the bacterium Francisella orientalis. To this end, a synthetic plasmid was produced using GENEART tool (ThermoFisher®, Regensburg, Germany), which contains a sequence of a Francisella orientalis pathogenicity island gene (trade secret). The plasmid was inserted into Escherichia coli 10β by electroporation followed by sowing the transformed E. coli on Luria Bertani (LB) agar containing antibiotics for 12 hours at 37 ºC to select the strains that incorporated the plasmid. The plasmid was extracted using the commercial kit Wizard Plus SV Minipreps DNA purification System A1460 (Promega®, Madison, EUA). After extraction, an aliquot of the extracted plasmid was digested with the restriction enzyme pstI and the digested plasmid was subjected to electrophoresis on a 1% agarose gel, confirming the expected band pattern for the correct sequence of the plasmid, and then the entire extract was purified using the Purelink Endofree Giga Plasmid A31233 commercial kit (ThermoFisher®, Regensburg, Germany), following the manufacturer’s recommendations. The purified plasmid was added to a sterile saline solution (0.85%) and stored refrigerated at 4 ºC for later use. In this way, it was possible to construct a DNA vaccine with potential large-scale application in fish farms and its efficacy will be evaluated a posteriori in an in vivo trial; with the vaccination of fish and challenge with the Francisella orientalis bacterium.