pEGFP-C2

Annotated Map/MCS (pdf)
Sequence (text)
Sequence (pdf)
Restriction Digest (pdf)

 

Click here to view the MCS for this vector.

 

Restriction Map and Multiple Cloning Site of pEGFP-C2. (Unique restriction sites are in color or bold.) Note that the Eag I site is not unique.) The Xba I site cannot be used for fusions since it contains an in-frame stop codon. The Xba I and Bcl I sites (*) are methylated in the DNA provided by CLONTECH. If you wish to digest the vector with these enzymes, you will need to transform the vector into a dam– host and make fresh DNA.

Note: The vector sequence file has been compiled from information in the sequence database, published literature, and other sources, together with partial sequences obtained by CLONTECH. This vector has not been completely sequenced.


Description

pEGFP-C2 encodes a red-shifted variant of wild-type GFP (1-3) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-C2 encodes the GFPmut1 variant (4) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-C2 is between the EGFP coding sequences and the SV40 poly A. Genes cloned into the MCS will be expressed as fusions to the C-terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin-resistance cassette (neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex thymidine kinase gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette (Pamp) expresses kanamycin resistance in E. coli. The pGFP-C1 backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

C-Terminal Fluorescent Protein Vectors

Each vector is provided with a Vector Information Packet and either the BD Living Colors™ User Manual (PT2040-1) or the BD Living Colors User Manual, Volume II (PT3404-1).

Reef Coral Fluorescent Protein Vectors (RCFPs)

GenBank Accession # U57606


Use

Fusions to the C-terminus of EGFP retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo. The target gene should be cloned into pEGFP-C2 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7). pEGFP-C2 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).


Location of Features


Primer Locations


Propagation in E. coli


References

  1. Prasher, D. C., et al. (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111:229-233.
  2. Chalfie, M., et al. (1994) Green fluorescent protein as a marker for gene expression. Science 263:802-805.
  3. Inouye, S. & Tsuji, F. I. (1994) Aequorea green fluorescent protein: Expression of the gene and fluorescent characteristics of the recombinant protein. FEBS Letters 341:277-280.
  4. Cormack, B., et al. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38.
  5. Haas, J., et al. (1996) Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6:315-324.
  6. Kozak, M. (1987) An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15:8125-8148.
  7. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK), pp. 143-190.