pECFP-C1

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 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.

Description

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 (Neo^r) gene for selection (using G418) in eukaryotic cells. A bacterial promoter (P) upstream of Neo^r 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 transfection marker).

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)

Location of Features

• Human cytomegalovirus (CMV) immediate early promoter: 1-589
  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 Kan^r gene.
  -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 & 2348-2419
  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 signal
• Polyadenylation signals: 3657-3662 & 3670-3675
• pUC plasmid replication origin: 4006-4649

Primer Locations

• 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 礸/ml) to E. coli hosts.
• E. coli replication origin: pUC
• Copy number: ~500
• Plasmid incompatibility group: pMB1/ColE1

References

1. Heim, R., et al. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91:12501-12504.
2. 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.
3. Miyawaki, A., et al. (1997) Fluorescent indicators for Ca²⁺ based on green fluorescent proteins and calmodulin. Nature 388:882-887.
4. Cormack, B. P., et al. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38.
5. 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.
6. Haas, J., et al. (1996) Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6(3):315-324.
7. Kozak, M. (1987) An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15:8125-8148.
8. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK), pp. 143-190.