pSV40-CLuc Control Plasmid

  • This product was discontinued on August 02, 2018

The pSV40-CLuc Control Plasmid is a mammalian expression plasmid that constitutively expresses the secreted Cypridina luciferase (CLuc) under the  SV40 early promoter.

  • Moderate constitutive expression of CLuc
  • CLuc does not use the same substrate as other marine luciferases, e.g., Renilla and Gaussia.  Therefore, it is possible to assay both CLuc and GLuc independently in cell culture medium from cells expressing both reporters.
  • CLuc can be assayed in the culture media or cell lysate

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N0318
  • Discontinued
    Discontinued
  • Product Information
    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: Cypridina Luciferase (CLuc) activity obtained from different CLuc plasmidsCypridina Luciferase (CLuc) activity obtained from different CLuc plasmids
    HeLa cell supernatants (20 μl) and lysates (5 μl) were assayed with the BioLux CLuc Assay Kit (NEB #E3309). HeLa cells were seeded in 12-well plates and transfected with 50 ng of CLuc-expressing plasmid per well. At 24 hr post-transfection, supernatants were collected and cell lysed in 100 μl per well using Luciferase Cell Lysis Buffer (NEB #B3321). The CLuc activity was measured in a Mithras LB940 (Berthold Technologies) luminometer set to: 50 μl of injection, 2 seconds of delay and 2 seconds of integration.
    Figure 2: pSV40-CLuc multiple cloning site (MCS)Figure 2: 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 and Addgene are central repositories 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
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