pCLuc Mini-TK 2 is a cloning vector for mammalian cells, containing a minimal promoter fragment from the HSV thymidine kinase (TK) promoter adjacent to a reporter gene, 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 of mammalian cells expressing the reporter gene. The pCLuc Mini-TK 2 Vector contains a MCS upstream of the minimal TK promoter for cloning promoter or enhancer elements. A neomycin resistance gene under the control of an SV40 promoter allows selection for stable integration of the plasmid into the mammalian cell genome using G418.Recommended sequencing primers for pCLuc Mini-TK 2 Vector (not available from NEB)
Upstream of MCS:
pBasic Forward Primer (23-mer)
pBasic Reverse Primer (25-mer)
CLuc 3´ end Forward Primer (23-mer)
CLuc 5´ End Reverse Primer (24-mer)
- 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 pCLuc Mini-TK 2 Vector can be transfected into cells using any standard transfection protocol.
Product SourceIsolated from E. coli strain ER2272 by a standard DNA purification procedure.
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