pEGFP-F

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

Restriction Map of pEGFP-F. All restriction sites shown are unique.

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-F encodes farnesylated enhanced green fluorescent protein, a modified form of EGFP that remains bound to the plasma membrane in both living and fixed cells. The vector contains the 20-amino-acid farnesylation signal from c-Ha-Ras (1, 2) fused to the C-terminus of EGFP. This farnesylation signal directs EGFP-F to the inner face of the plasma membrane. pEGFP-F encodes a red-shifted variant of wild-type GFP (3每5) optimized for brighter fluorescence and higher expression in mammalian cells (excitation maximum = 488 nm; emission maximum = 507 nm). pEGFP-F encodes the GFPmut1 variant (6) 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 (7). Sequences flanking EGFP-F have been converted to a Kozak consensus translation initiation site (8) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-F is between the EGFP-F coding sequences and the SV40 poly A. SV40 polyadenylation signals downstream of the EGFP-F gene direct proper processing of the 3' end of the EGFP-F 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 virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-F backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

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)

Subcellular Localization Vectors


Use

EGFP-F is designed for use as a cotransfection marker. Because it remains attached to the plasma membrane, it can be detected by fluorescence microscopy in permeabilized cells after ethanol fixation (9). The vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (10). 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每 host and make fresh DNA.


Location of Features


Primer Locations


Propagation in E. coli


References

  1. Aronheim, A., et al. (1994) Cell 78:949每961.
  2. Hancock, J. F., et al. (1991) EMBO J. 10:4033每4039.
  3. Prasher, D. C., et al. (1992) Gene 111:229每233.
  4. Chalfie, M., et al. (1994) Science 263:802每805.
  5. Inouye, S. & Tsuji, F. I. (1994) FEBS Letters 341:277每280.
  6. Cormack, B., et al. (1996) Gene 173:33每38.
  7. Haas, J., et al. (1996) Curr. Biol. 6:315每324.
  8. Kozak, M. (1987) Nucleic Acids Res. 15:8125每8148.
  9. Jiang, W. & Hunter, T. (1998) BioTechniques 24:348每354.
  10. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK) pp. 143每190.