ptdTomato-N1載體描述 ptdTomato-N1 is a mammalian expression vector designed to express a protein of interest fused to the N-terminus of tdTomato. td Tomato is 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). Expression of td Tomato as a tandem dimer prevents the fused protein of interest from being forced into a dimeric complex. Fusions that retain the fluorescence properties of tdTomato can be monitored by flow cytometry and localized by fluorescence microscopy. The multiple cloning site (MCS) in ptdTomato-N1 is positioned upstream of the tdTomato coding sequence. A Kozak consensus sequence, located between the MCS and the tdTomato coding sequence, enhances translational efficiency of the unfused tdTomato protein in eukaryotic cells (3). SV40 polyadenylation signals downstream of the tdTomato coding sequence direct proper processing of the 3' end of the tdTomato (or fusion gene) 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. The vector also has a neomycin resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells. This cassette consists of the SV40 early promoter, the Tn5 neomycin/kanamycin resistance gene, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette confers kanamycin resistance in E. Coli.
The gene of interest must be cloned into ptdTomato-N1 so that it is in-frame with the tdTomato coding sequence. The gene must include an initiation codon (ATG), and lack in-frame stop codons.
ptdTomato-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). ptdTomato-N1 can also be used as a cotransfection marker, as the unmodified vector will express tdTomato in mammalian cells.
For Western analysis, either the Living Colors DsRed Polyclonal Antibody (Cat. No. 632496) or the DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393) can be used to detect the tdTomato protein.
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) in E. coli hosts.
E. coli replication origin: pUC
Copy number: high
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 Biotechnol. 22(12):1567-72.
2. Matz, M. V., et al. (1999) Nature Biotechnol. 17(10):969–973.
3. Kozak, M. (1987) Nucleic Acids Res. 15(20):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.
