pCMV-tdTomato载体描述 pCMV-tdTomato encodes tdTomato, a member of the family of fruit fluorescent proteins (1) derived from the Discosoma sp. red fluorescent protein, DsRed (2). Because the Tomato protein has a tendency to dimerize, the vector was designed with two copies of the Tomato coding region linked together to allow intramolecular dimerization. As a result, each tdTomato RNA transcript encodes a tandem dimer of the Tomato protein (excitation and emission maxima equal 554nm and 581nm, respectively). The tdTomato coding sequence is positioned just downstream of the constitutively active cytomegalovirus immediate early promoter (PCMV IE). As a result, mammalian cells transfected with this vector will constitutively express tdTomato. A Kozak consensus sequence is located immediately upstream of the tdTomato coding sequence to enhance translational efficiency in eukaryotic cells (3). SV40 polyadenylation signals downstream of the tdTomato coding sequence direct proper processing of the 3' end of the tdTomato mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists 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. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.
pCMV-tdTomato is designed for use as a marker for cotransfection or cell tracking. The red fluorescence of tdTomato can be detected by fluorescence microscopy, allowing direct visual imaging. In addition, flow cytometry can be used to enrich transfected cell populations. pCMV-tdTomato can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4).
Propagation in E. coli
Suitable host strains: DH5α, 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 (50 μg/ml) to E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Plasmid incompatibility group: pMB1/Col E1
Excitation and emission maxima of tdTomato
Excitation maximum = 554 nm
Emission maximum = 581 nm References 1. Shaner, N. C., et al. (2004) Nature Biotech. 22(12):1567-1572.
2. Bevis, B. J. & Glick, B. S. (2002) Nature Biotech. 20:83–87.
3. Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.
4. Gorman, C. (1985) In DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.
