pCLuc-Basic 2 Vector

The pCLuc-Basic 2 Vector is a cloning vector for expression of Cypridina Luciferase in mammalian cell. It contains the secreted CLuc reporter gene but lacks promoter elements.  It can be used to express CLuc under the control of sequences cloned in the MCS.

  • CLuc can be assayed in the culture medium of mammalian cells expressing Cypridina luciferase
  • 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.
  • Can be used to generate Neomycin-resistant stable cell lines

Ordering Information

$name
  • 500 μg/ml
    20 μg
    $293.00
  • Product Information
    pCLuc-Basic 2 is a cloning vector for expression in mammalian cells, containing a reporter gene but lacking promoter elements. The reporter gene is the secreted luciferase from the Ostracod Cypridina noctiluca. 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. The pCLuc-Basic 2 Vector contains a multiple cloning site (MCS) upstream of the CLuc coding sequence. A neomycin resistance gene under the control of the SV40 promoter allows selection for stable integration of the plasmid into the mammalian cell genome using G418.

    Recommended sequencing primers for pCLuc-Basic 2:
    Upstream of MCS:
    pBasic Forward Sequencing Primer (23-mer) (not available from NEB)
    5´-GGGGTTCCGCGCACATTTCCCCG-3´(6020–6042)

    pBasic Reverse Primer (25-mer) (not available from NEB)
    5´-TCAGAAGCCATAGAGCCCACCGCAT-3´(1888–1864)

    CLuc 3´ End Forward Primer (23-mer) (not available from NEB)
    5´-GAGTTCAAGAAAGAATGCTACAT-3´(1671–1693)

    CLuc 5´ End Reverse Primer (24-mer) (not available from NEB)
    5´-GTAAGGACAGTCCTGGCAATGAAC-3´(143–120)

    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:  pCLuc-Basic 2 multiple cloning site (MCS)  Figure 2:  pCLuc-Basic 2 multiple cloning site (MCS)
    Figure 2:  pCLuc-Basic 2 multiple cloning site (MCS)
    The Cypridina Luciferase sequence is shown with a blue background. Only unique restriction sites are shown.

    DNASU and Addgene are central repositories for plasmid clones and collections that may also be helpful.

    Highlights

    • Polylinker MCS: 20–68
    • Start codon of CLuc: 75–77 Stop codon: 1734–1736
    • Signal peptide: 75–128
    • Synthetic poly-A site: 1745–1793
    • Neo promoter (SV 40): 2379–2714
    • Neomycin resistance gene: 2766–3560
    • Bacterial replication ori (pMB1): 4894–4306
    • Amp resistance: 5925–5065
    Product Categories:
    Cypridina Luciferase Products,
    DNA Plasmid Products
    Applications:
    Gaussia Luciferase,
    Yeast Protein Expression,
    Protein Labeling
    ,
    Proteomics
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