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Open Access Highly Accessed Methodology article

From Gateway to MultiSite Gateway in one recombination event

Enrico Magnani12*, Linnea Bartling2 and Sarah Hake12

Author Affiliations

1 Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA

2 Plant Gene Expression Center, United States Department of Agriculture-Agriculture Research Service, Albany, California 94710, USA

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BMC Molecular Biology 2006, 7:46  doi:10.1186/1471-2199-7-46

Published: 6 December 2006



Invitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors. One or three DNA segments can be cloned using Gateway or MultiSite Gateway respectively. A vast number of single-site Gateway destination vectors have been created while MultiSite Gateway is limited to few destination vectors and therefore to few applications. The aim of this work was to make the MultiSite Gateway technology available for multiple biological purposes.


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.