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RAS Pathway Clone Collection

By Dominic Esposito

Frederick National Laboratory



Published on


The R3 KRAS Pathway Clone Collection #1 is a set of vectors for use with the Gateway cloning platform (Life Technologies, Carlsbad, CA) to permit construction of expression vectors for in vitro and in vivo research. While these clones can be used for standard Gateway cloning, their utility is greatly enhanced with the use of the FNLCR Combinatorial Cloning Platform (CCP) which allows simplified construction of vectors with different elements. Some uses of this system might be:
- Construction of protein expression constructs with various fusion tags
- Generation of expression constructs with different promoters for in vivo expression
- Production of clones with fluorescent tags for localization experiments
- Generation of constructs for making mutant cell lines or transgenic animals
- Production of vectors for shRNA or miRNA delivery

The advantage of the CCP is in the exquisite specificity of the Multisite Gateway reactions, which permit linkage of multiple elements in a directional fashion, and which involve no additional DNA amplification, thus ensuring the accuracy of the DNA sequence of the final products without the need for additional sequencing. There is also no need for restriction-based cloning processes which have a high rate of failure and may require optimization depending on the sites available in a given clone.

The KRAS Pathway clone collection contains a series of RAS pathway genes in two separate formats. The "closed" format contains a Kozak translational initiation sequence and ATG start site at the 5' end, and a stop codon at the 3' end. These constructs can be used to make native or amino-terminally tagged constructs using Gateway vectors or the CCP. The "open" format has the same 5' sequence, but lacks a stop codon and is in frame with the Gateway attB2 site at the 3' end. This allows production of C-terminal fusions using Gateway vectors or the CCP. These clones have been completely sequence validated and functionally tested in Gateway reactions to ensure proper performance.




Hartley JL, Temple GF, and Brasch MA. (2000) "DNA cloning using in vitro site-specific recombination." Genome Res. 10, 1788-1795.

Wall VA, et. al. (2014) "Combinatorial assembly of clone libraries using site-specific recombination." Meth. Mol. Biol. 1116, 193-208.

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Researchers should cite this work as follows:

  • Dominic Esposito (2014), "RAS Pathway Clone Collection,"

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