Gateway® TagRFP-AS-C entry clone is a vector containing red (orange) fluorescent protein TagRFP gene variant with codon usage optimized for high expression in Arabidopsis and Saccharomyces. TagRFP coding sequence is flanked by attL1 and attL2 sites allowing easy site-specific recombination. The Invitrogen Gateway® Technology provides a rapid and highly efficient way to transfer the TagRFP gene into a number of Gateway® destination vectors for expression in different experimental systems.
|Application Notes:||Generation of TagRFP-tagged fusions|
A localization signal or a gene of interest can be cloned into MCS of the vector both before and after site-specific recombination with a destination vector. It will be expressed as a fusion to the TagRFP C-terminus when inserted in the same reading frame as TagRFP and no in-frame stop codons are present. Alternatively, TagRFP gene can be fused to the 3’-end of a gene of interest by LR recombination of the Gateway® TagRFP-AS-C with a destination vector containing this gene in a correct reading frame. In this case, the protein of interest will be expressed as a fusion to the TagRFP N-terminus. TagRFP-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo.
Note: The plasmid DNA was isolated from dam+-methylated E.coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam- host and make fresh DNA.
LR site-specific recombination
Please refer to Invitrogen Gateway® Technology description for detailed instructions regarding to perform LR site-specific recombination reaction. In general, to transfer TagRFP gene or TagRFP-fusion construct into the destination vector you will need:
- Purified plasmid DNA of Gateway® TagRFP-AS-N
- A destination vector of choice
- Invitrogen LR ClonaseTM II enzyme mix (Invitrogen Cat# 11791-020)
- Proteinase K solution (supplied with the LR ClonaseTM II enzyme mix)
- TE Buffer, pH 8.0 (10mM Tris-HCl, pH 8.0, 1mM EDTA)
- Appropriate chemically competent E. coli host and growth media for expression
- Appropriate selective plates
Propagation in E. coli
Suitable host strains: DH5alpha, HB101, XL1-Blue and other general purpose strains.
Selectable marker: plasmid confers resistance to kanamycin (30μg/ml) to E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Plasmid incompatibility group: pMB1/ColE1
|Long Term Storage:||-20°C|
|Background / Technical Information:||For the Original Manufacturer’s Data Sheet please click here.|
Our product description may differ slightly from the Original Manufacturer’s Data Sheet.
Product Literature References
An analysis of 5’-noncoding sequences from 699 vertebrate messenger RNAs
: M. Kozak; Nucleic Acids Res. 15
, 8125 (1987), Abstract