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MCS for this Vector.
|Restriction Map and
Multiple Cloning Site of pECFP-C1. The Xba
I and Bcl I sites (*) are methylated in the DNA provided by
CLONTECH. If you wish to digest the vectors 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.
pECFP-C1 encodes an enhanced cyan fluorescent variant of
the Aequorea victoria green fluorescent protein gene (GFP). The ECFP gene
contains six amino acid substitutions. The Tyr-66 to Trp substitution gives ECFP
fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and
emission (major peak at 475 nm and a minor peak at 501 nm) similar to other cyan
emission variants (1-3). The other five substitutions (Phe-64 to Leu; Ser-65 to
Thr; Asn-146 to Ile; Met-153 to Thr; and Val-163 to Ala) enhance the brightness
and solubility of the protein, primarily due to improved protein-folding
properties and efficiency of chromophore formation (2, 4, 5). In addition to the
chromophore mutations, ECFP contains >190 silent mutations that create an
open reading frame comprised almost entirely of preferred human codons (6).
Furthermore, upstream sequences flanking ECFP have been converted to a Kozak
consensus translation initiation site (7). These changes increase the
translational efficiency of the ECFP mRNA and consequently the expression of
ECFP in mammalian and plant cells.
The MCS in pECFP-C1 is between the ECFP coding sequence
and the stop codon. Genes cloned into the MCS will be expressed as fusions to
the C-terminus of ECFP if they are in the same reading frame as ECFP and there
are no intervening in-frame stop codons. ECFP with a C-terminal fusion moiety
retains the fluorescent properties of the native protein and thus can be used to
localize fusion proteins in vivo.
The vector contains an SV40 origin for replication and a
neomycin resistance (Neor) gene for selection (using G418) in
eukaryotic cells. A bacterial promoter (P) upstream of Neor
expresses kanamycin resistance in E. coli. The vector backbone also
provides a pUC19 origin of replication for propagation in E. coli and an
f1 origin for single-stranded DNA production. The recombinant ECFP vector can be
transfected into mammalian cells using any standard transfection method. If
required, stable transfectants can be selected using G418 (8). pECFP-C1 can also
be used simply to express ECFP in a cell line of interest (e.g., as a
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)
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
- Enhanced cyan fluorescent protein gene
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615; stop codon: 1408-1410
Insertion of Val at position 2: 616-618
ECFP mutations (Phe-64 to Leu; Ser-65 to Thr; and Tyr-66 to Trp): 805-813;
Asn-146 to Ile: 1051-1053; Met-153 to Thr: 1072-1074; Val-163 to Ala:
1102-1104 His-231 to Leu mutation (AÆT): 1307
Last amino acid in ECFP coding region: 1327-1329
- MCS: 1330-1417
- SV40 early mRNA polyadenylation signal
>Polyadenylation signals: 1550-1555 & 1579-1584;
mRNA 3' ends: 1588 & 1600
- f1 single-strand DNA origin: 1647-2102 (Packages the
noncoding strand of ECFP)
- Bacterial promoter for expression of Kanr
-35 region: 2164-2169; -10 region: 2187-2192
Transcription start point: 2199
- SV40 origin of replication: 2443-2578
- SV40 early promoter
Enhancer (72-bp tandem repeats): 2276-2347 &
21-bp repeats: 2423-2443, 2444-2464 & 2466-2486
Early promoter element: 2499-2505
Major transcription start points: 2495, 2533, 2539 & 2544
- Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2627-2629; stop codon: 3419-3421
G->A mutation to remove Pst I site: 2809
C->A (Arg to Ser) mutation to remove BssH II site: 3155
- Herpes simplex virus (HSV) thymidine kinase (TK)
- Polyadenylation signals: 3657-3662 & 3670-3675
- pUC plasmid replication origin: 4006-4649
- EGFP-N Sequencing Primer (#6479-1): 679-658
- EGFP-C Sequencing Primer (#6478-1): 1266-1287
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 number: ~500
- Plasmid incompatibility group: pMB1/ColE1
- Heim, R., et al. (1994) Wavelength mutations and
posttranslational autoxidation of green fluorescent protein. Proc. Natl.
Acad. Sci. USA 91:12501-12504.
- Heim, R. & Tsien, R. Y. (1996) Engineering green
fluorescent protein for improved brightness, longer wavelengths and
fluorescence resonance energy transfer. Curr. Biol. 6:178-182.
- Miyawaki, A., et al. (1997) Fluorescent
indicators for Ca2+ based on green fluorescent proteins and
calmodulin. Nature 388:882-887.
- Cormack, B. P., et al. (1996) FACS-optimized
mutants of the green fluorescent protein (GFP). Gene 173:33-38.
- Yang, T. T., et al. (1996) Optimized codon usage
and chromophore mutations provide enhanced sensitivity with the green
fluorescent protein. Nucleic Acids Res. 24:4592-4593.
- Haas, J., et al. (1996) Codon usage limitation
in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6(3):315-324.
- Kozak, M. (1987) An analysis of 5'-noncoding sequences
from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15:8125-8148.
- Gorman, C. (1985) In DNA Cloning: A Practical
Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK), pp.