|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.
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).
Coral Fluorescent Protein Vectors (RCFPs)
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
- Human cytomegalovirus (CMV) immediate early promoter:
Enhancer region: 59-465; TATA box: 554-560
Transcription start point: 583
C->G mutation to remove Sac I site:569
- Farnesylated enhanced green flourescent protein (EGFP-F)
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615; Stop codon: 1408-1410
Insertion of Val at position 2: 616-618
GFPmut1 chromophore mutations (Phe-64 to Leu; Ser-65 to Thr): 805-810
His-231 to Leu mutation (AÆT): 1307
Last amino acid in wild-type GFP: 1327-1329
c-Ha-Ras farnesylation signal: 1345-1406
- MCS: 1414-1492
- SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1625-1630 & 1654-1659;
mRNA 3' ends: 1663 & 1675
- f1 single-strand DNA origin: 1722-2177 (packages the
noncoding strand of EGFP)
- Bacterial promoter for expression of Kanr
-35 region: 2239-2244; -10 region: 2262-2267
Transcription start point: 2274
- SV40 origin of replication: 2518-2653
- SV40 early promoter
Enhancer (72-bp tandem repeats): 2351-2422 &
21-bp repeats: 2498-2518, 2519-2539 & 2541-2561
Early promoter element: 2574-2580
Major transcription start points: 2570, 2608, 2614 & 2619
- Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2702-2704; stop codon: 3494-3496
G->A mutation to remove Pst I site: 2884
C->A (Arg to Ser) mutation to remove BssH II site: 3230
- Herpes simplex virus (HSV) thymidine kinase (TK)
Polyadenylation signals: 3732-3737 & 3745-3750
- pUC plasmid replication origin: 4081-4724
- EGFP-N Sequencing Primer (#6479-1): 679每658
- EGFP-C Sequencing Primer (#6478-1): 1266每1287
Propagation in E. coli
- Suitable host strains: DH5a, HB101, and other general
purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM109 or XL1-Blue.
- Selectable marker: plasmid confers resistance to
kanamycin (30 µg/ml) to E. coli hosts.
- E. coli replication origin: pUC; copy
- Plasmid incompatibility group: pMB1/ColE1
- Aronheim, A., et al. (1994) Cell 78:949每961.
- Hancock, J. F., et al. (1991) EMBO J.
- Prasher, D. C., et al. (1992) Gene 111:229每233.
- Chalfie, M., et al. (1994) Science 263:802每805.
- Inouye, S. & Tsuji, F. I. (1994) FEBS Letters 341:277每280.
- Cormack, B., et al. (1996) Gene 173:33每38.
- Haas, J., et al. (1996) Curr. Biol. 6:315每324.
- Kozak, M. (1987) Nucleic Acids Res. 15:8125每8148.
- Jiang, W. & Hunter, T. (1998) BioTechniques 24:348每354.
- Gorman, C. (1985) In DNA Cloning: A Practical
Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK) pp.