Using Multisite LR Cloning to Generate a Destination Clone

UMMS Affiliation

Program in Systems Biology; Program in Molecular Medicine

Publication Date


Document Type



Genetic Phenomena | Genetics and Genomics | Genetic Structures | Investigative Techniques | Laboratory and Basic Science Research | Molecular Biology | Systems Biology


This protocol describes using the Gateway recombinatorial cloning system to simultaneously transfer a promoter and an open reading frame (ORF) from two different Entry clones into the same Destination vector using LR enzymes. A multisite cloning reaction transfers the inserts from multiple Entry clones into a single Destination vector. This type of recombination is much less efficient than transferring a single DNA fragment; however, the variety of Destination clones that can be generated in this manner is vast. In this example protocol, we describe using pDEST-MB14 to make a Destination clone that features a promoter fragment fused upstream to an ORF that is cloned in-frame with a carboxy-terminal green fluorescent protein (GFP) moiety encoded by the plasmid backbone. This method can be used as a guide for other multisite cloning reactions.

DOI of Published Version



Cold Spring Harb Protoc. 2018 Jan 2;2018(1):pdb.prot094946. doi: 10.1101/pdb.prot094946. Link to article on publisher's site

Journal/Book/Conference Title

Cold Spring Harbor protocols

Related Resources

Link to Article in PubMed

PubMed ID