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Description: Purchased from Clontech
Notes for Use FROM CLONTECH WEBSITE:
pDsRed2-ER is a mammalian expression vector that encodes a fusion consisting of Discosoma sp. red fluorescent protein (DsRed2; 1, 2); the endoplasmic reticulum (ER) targeting sequence of calreticulin (3), fused to the 5' end of DsRed2; and the ER retention sequence, KDEL (4, 5), fused to the 3' end of DsRed2a human codon-optimized (6) DsRed variant engineered for faster maturation and lower non-specific aggregation.
To drive expression of DsRed2, this vector contains the immediate early promoter of cytomegalovirus (PCMV IE). SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3'-end of the DsRed2 mRNA transcript. The vector also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 T-antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassetteconsisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK poly A) geneallows stably transfected eukaryotic cells to be selected using G418 (7). A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.
Vector
Size
Cat. #
pDsRed2-ER
20 µg
6982-1
Use
pDsRed2-ER is designed for fluorescent labeling of the endoplasmic reticulum in living cells (8, 9). The vector can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7). DsRed2 (excitation/emission maxima: 558 nm/583 nm) can be detected by fluorescence microscopy and by flow cytometry. Filter sets optimized for detecting DsRed by microscopy are available from Chroma Technology Corporation and Omega Optical Inc. Please see their websites (www.chroma.com and www.omegafilters.com) and the Living Colors® Vol. II User Manual, provided with this vector, for more information. To detect DsRed2-expressing cells by flow cytometry, use the instrument's argon-ion laser to excite the fluorophore at 488 nm and the FL-2 channel to detect the fluorophore's emission at 583 nm.
Location of Features
• Human cytomegalovirus (CMV) immediate early promoter: 1589
Enhancer region: 59465; TATA box: 554560
Transcription start point: 583
• Calreticulin signal sequence: 597647
• Discosoma sp. red fluorescent protein (DsRed2) gene: 6631337
KDEL coding sequence (in frame with DsRed2): 13501361
Stop codon: 13621364
• SV40 early mRNA polyadenylation signal
Polyadenylation signals: 15761581 & 16051610; mRNA 3' ends: 1614 & 1626
• f1 single-strand DNA origin: 16732128 (packages the noncoding strand of DsRed2-ER)
• Bacterial promoter for expression of Kanr gene
35 region: 21902195; 10 region: 22132218
Transcription start point: 2225
• SV40 origin of replication: 24692604
• SV40 early promoter
Enhancer (72-bp tandem repeats): 23022373 & 23742445
21-bp repeats: 24492469, 24702490 & 24922512
Early promoter element: 25252531
Major transcription start points: 2521, 2559, 2565 & 2570
• Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 26532655; stop codon: 34453447
• Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 36833688 & 36963701
• pUC plasmid replication origin: 40324675
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 (50 µg/ml) to E. coli hosts.
• E. coli replication origin: pUC; copy number: ~500
• Plasmid incompatibility group: pMB1/ColE1
References
1. Living Colors DsRed2 (July 2001) CLONTECHniques XVI(3):23.
2. Matz, M. V., et al. (1999) Nature Biotech. 17:969973.
3. Fliegel, L., et al. (1989) J. Biol. Chem. 264:2152221528.
4. Munro, S. & Pelham, H. R. (1987) Cell 48:899907.
5. Pelham, H. R. (1996) Cell Struct. Funct. 21:413419.
6. Haas, J., et al. (1996) Curr. Biol. 6:315324.
7. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK), pp. 143190.
8. Roderick, H. L., et al. (1997) FEBS Letters 405:181185.
9. Kendall, J. M., et al. (1994) Anal. Biochem. 221:173181.
Reference: Published reference: A. Kaykas and R. T. Moon, 2004, "A plasmid-based system for expressing small interfering RNA libraries in mammalian cells." BMC Cell Biology 2004, 5:16.