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  • pSV40-CLuc Control Plasmid

    Description

    The pSV40-CLuc Control Plasmid is a mammalian expression vector that encodes the secreted luciferase from the Ostracod Cypridina noctiluca (1) as a reporter, under the control of the constitutive SV40 promoter. Cypridina Luciferase (CLuc) is a 62 kDa protein with a native signal peptide at the N-terminus that allows it to be secreted from mammalian cells (1) so that CLuc activity can be detected in the culture medium. There is a multiple cloning site (MCS) upstream of the SV40 promoter. 

    Recommended sequencing primers for pSV40-CLuc:

    Forward primer (23-mer) (not available from NEB)
    5´-GAGTTCAAGAAAGAATGCTACAT-3´ (1888–1910)

    Reverse primer (24-mer) (not available from NEB)
    5´-GTAAGGACAGTCCTGGCAATGAAC-3´ (360–337)

    Figure 1: Activity of Cypridina Luciferase in supernatants and lysates from a stable CLuc-expressing cell line. CLuc activity was measured from 20 µl of cell culture supernatant (500 µl total culture volume) and from 20 µl of cell lysate (100 µl total lysate volume).
    Figure 2: The high sensitivity of both the CLuc and GLuc assays allows detection of very small numbers of cells expressing each protein. 20 µl of culture supernatant from the indicated number of cells expressing each reporter were assayed.
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    Figure 3: pSV40-CLuc multiple cloning site (MCS). The Cypridina Luciferase sequence is located downstream of the SV40 promoter (not shown); unique restriction sites are shown.

    DNASU is a central repository for plasmid clones and collections that may also be helpful.

    Highlights

    • Polylinker MCS: 1–51
    • SV40 promoter: 51-246
    • CLuc ORF: 291–1952
    • Start codon of CLuc: 291–293   Stop codon: 1950–1952
    • Signal peptide: 291–344
    • SV40 poly-A site: 1967–2188
    • SV40 enhancer: 2195–2441
    • Bacterial replication ori (pMB1): 3347–2759
    • Amp resistance: 4378–3518

    Advantages and Features

    Features

    • Multiple samples can be obtained from the same transfected cells (i.e., before and after experimental treatments or at multiple time points).
    • 90–95% of CLuc activity is found in the cell culture medium, with the remaining 5-10% detectable in cell lysates (Figure 1). This allows flexibility when assaying CLuc along with other co-transfected reporters.
    • The activity of CLuc is high and the CLuc assay is sensitive enough to detect very small amounts of CLuc enzyme activity (Figure 2).
    • CLuc does not use the same substrate as other marine luciferases (e.g. Renilla, Gaussia). Therefore, it is possible to assay both CLuc and GLuc independently in cell culture medium from cells expressing both reporters.
    • The pSV40-CLuc Control Plasmid can be transfected into cells using any standard transfection protocol.

    Applications

    The pSV40-CLuc Control Plasmid can be used as a control for assessing the efficiency of transfection in mammalian cells. Plasmids containing other constitutive promoter elements are also available (see Related Products). CLuc can be used as a stand alone reporter or in conjunction with other compatible reporters such as Gaussia luciferase (GLuc)(2). CLuc and GLuc are ideally suited for co-expression as both are secreted and highly active enzymes providing ease of use and sensitivity.

    Properties and Usage

    Storage Temperature

    -20°C

    References

    1. Nakajima, Y. et al. (2004). Biosci. Biotechnol. Biochem.. 63, 565-570.
    2. Wu, C., Suzuki-Ogoh, C. and Ohmiya, Y. (2007). BioTechniques. 42, 290-292.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.
    1. Where can I find the sequence of this plasmid?
    2. Can I make a stable cell line with pSV40-CLuc?
    3. Can I transfect this plasmid into mammalian cells?
    4. How do I assay for CLuc expression?
    5. Can I use assay kits designed for other reporters (Gaussia, Renilla & Firefly luciferases) to assay CLuc activity?
    6. Is there another secreted reporter that can be used with CLuc?

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