From Gateway to MultiSite Gateway in one recombination event

We created a construct, pDONR-R4-R3, to easily convert any available Gateway destination vector to a MultiSite Gateway vector in a single recombination reaction. In addition, we designed pDONR-R4-R3 so that DNA fragments already cloned upstream or downstream of the Gateway cassette in the original destination vectors can still be utilized for promoter-gene or translational fusions after the conversion.Our tool makes MultiSite Gateway a more widely accessible technology and expands its applications by exploiting all the features of the Gateway vectors already available.Recombinase-based cloning technologies are becoming increasingly popular because of their easy use and high efficiency. These tools exploit bacterial or viral site-specific recombinases like the bacteriophage P1 Cre, the Saccharomyces cerevisiae FLP or the bacteriophage lambda integrase [1-3]. These enzymes catalyze a reciprocal double-stranded DNA exchange between two specific DNA sites [4].Gateway (Invitrogen) is one of the most popular recombination cloning technologies [3]. It is based on the Escherichia coli bacteriophage lambda integrase/att system [5]. Two sets of reactions are employed in this technology: LR and BP recombinations (Fig 1). The BP reaction is catalyzed by the BP Clonase enzyme mix (Invitrogen), which recombines attB sites with attP sites. The LR Clonase mix (Invitrogen) is responsible for recombination of attL sites with attR sites. Any DNA fragment of interest can be PCR amplified with primers containing attB sites and cloned into a donor vector carrying attP sites in the presence of the BP Clonase mix. The recombination of attB with attP sites leads to the formation of attL and attR sites. The reaction creates an entry clone bearing the insert of interest flanked by attL sites and a byproduct flanked by attR sites (Fig 1A). The insert can then be mobilized into any destination vector having attR sites via an LR reaction (Fig 1B). The resulting construct can be easily selected u