pEG100s  
Indiana University, Bloomington, IN  
 

HOME     

   
 
pEarleyGate Vectors

PhD student Keith Earley in the Pikaard lab has engineered a set of plant transformation vectors that allow for simple recombinational cloning of cDNA or genomic sequences using Invitrogen's patented Gateway technology. We call these pEarleyGate vectors. pEarleyGate 100, 200 and 300 series vectors were built by insertion of a Gateway cloning cassette into plasmid pFGC5491 (a ChromDB plasmid; see www.chromdb.org), a binary vector suitable for Agrobacterium tumefaciens-mediated plant transformation that, in turn, was built using a pCAMBIA vector backbone.These vectors were designed with transformation of Arabidopsis and other dicots in mind and are described in the following publication:

Earley, Keith, Jeremy R. Haag, Olga Pontes, Kristen Opper, Tom Juehne, Keming Song, and Craig S. Pikaard (2006). Gateway-compatible vectors for plant functional genomics and proteomics. The Plant J. 45:616-629. Pubmed PDF

One set of pEarleyGate vectors (100 series) allows for rapid recombinational cloning of cDNAs (previously captured in Invitrogen pENTR vectors) to produce C-terminal fusions of encoded proteins in-frame with GFP, YFP or CFP or to produce N-terminal fusions to YFP. These vectors are useful for cell localization studies and use an enhanced CaMV 35S promoter to drive expression. Another set of pEarleyGate vectors (200 series) allows for encoded proteins to be epitope tagged with HA, Myc, AcV5, FLAG or a tandem affinity peptide (TAP) tag, the latter consisting of a calmodulin binding peptide and a 2X Protein A peptide (which will bind to IgG resin) separated by a TEV protease cleavage site (Rigaut et al, 1999, Nature Biotechnology 17: 1030-1032). 200-series vectors also make use of a CaMV 35S promoter to drive expression. A third set of vectors (300 series) allows for expression of genes from a promoter of choice rather than from the enhanced 35S promoter. We find the 300 series vectors to be particularly useful for adding C-terminal tags to cloned genomic sequences that include the natural promoter, exons and introns, with the tag being added to the final exon in lieu of the natural stop codon. All pEarleyGate vectors encode a kanamycin resistance cassette for plasmid selection in bacteria and a BAR gene cassette, encoding herbicide resistance, within the T-DNA for selection of transformed plants. For a table describing the features of all pEarleyGate vectors, with links to maps, complete sequences and ABRC ordering information, please click here.

For notes on cloning sequences of interest into pEarleyGate vectors, please click here.

Information concerning the use or intellectual property restrictions of Gateway technology or CAMBIA-derived plasmids can be found at the appropriate websites by following the links above. We have made the pEarleyGate vectors freely available through the ABRC (Arabidopsis Biological Resource Center) and these can be ordered online.

Construction of the pEarleyGate vectors has been made possible by grants from the United States National Science Foundation (grant numbers DBI-9975930 and DBI-0421619) and the National Institutes of Health (grant GM60380). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the National Institutes of Health.

 pEG 100 map  pEG 200 map  pEG 300 map

Numbers in parentheses represent the specific pEarleyGate plasmid vector carrying the indicated tag or fusion protein.

Kan= kanamycin resistance marker for bacterial selection; LB= T-DNA left border; RB=T-DNA right border;BaR= Basta resistance marker for transgenic plant selection driven by the mannopine synthase (mas) promoter and flanked by a mas 3' end ; 35S= enhanced 35S promoter; Gateway= Gateway recombination cassette; OCS 3'= 3' end of the octopine synthase gene

M400 series vectors

pEarleyGate M400 series vectors facilitate construction of N-terminal fusions of target proteins to YFP (M401), FLAG (M402) or HA (M403) tags and were built by insertion of a Gateway cloning cassette into plasmid pMCG1005, which was designed for maize transformation by Karen McGinnis and colleagues at the University of Arizona. Karen is now an assistant professor at the University of Florida. These plasmids do not use a pCAMBIA vector backbone but instead are based on a pBIN19 backbone. pMCG1005 and pEarleyGate M400 series vectors incorporate introns within key genes, which has been reported to improve expression in monocots.