pTubHA4C, a new versatile vector for constitutive expression in Drosophila. Stephanie M. Arcia^1,2, Yan Zhang¹, Pedro Fernandez-Funez^1,3,4, Diego E. Rincon Limas^1,4. 1) Department of Neurology; 2) HHMI Science for Life Undergraduate Program; 3) Department of Neurosciences; 4) Genetics Institute and Center for Translational Research on Neurodegenerative Diseases; McKnight Brain Institute, University of Florida, Gainesville, FL.

   Several vectors for gene expression are available in Drosophila, a hub for genetics and genomics innovation. However, the vectors for ubiquitous expression have a complex structure, including coding exons, that makes in-frame cloning of cDNAs very complicated. We describe a new Drosophila expression vector (pTubHA4C) for ubiquitous expression of coding sequences under the control of a minimal, 0.9 kb promoter of 1tubulin (1t). This plasmid was designed to include optimized multiple cloning sites (polylinker) to provide flexibility in cloning strategies. We also added the option of double labeling the expressed proteins with two C-terminal tags, the viral epitope hemagglutinin (HA) and a synthetic tetracysteine (4C) tag that binds small fluorescent compounds. This dual tag allows both in situ and biochemical detection of the desired protein. In particular, the new 4C tag technology combines easy fluorescent labeling with small arsenical compounds in live or fixed cells and tissues, while producing minimal alterations to the tagged protein due to its small size. To demonstrate the potent and ubiquitous expression under the control of the Tub promoter, bacterial lacZ was expressed and monitored in cell culture and transgenic flies. We found that the modified 0.9 kb Tub promoter induced similar expression levels to the intact 2.6 kb 1t promoter, supporting the inclusion of all critical regulatory elements in the new and flexible TubHA4C vector.