For promoter analysis, the pMLuc-1 vector contains a multiple cloning site upstream of the Renilla luciferase cDNA, followed by the rabbit β-globin polyadenylation sequence upstream from transcription termination. A second synthetic terminator sequence upstream of the cloning site was introduced to decrease read-through transcription. The Renilla luciferase cDNA contained in the pMLuc vectors has been codon-optimized to maximize expression in mamalian cells. Promoter activity of fragments cloned into the pMLuc-1 vector can be monitored with the MightyLight™ Rluc Assay Kit.
The pMLuc-2 vector contains a minimal thymidine kinase (TK) promoter driving the expression of the optimized Renilla luciferase reporter (see vector map below). To assess enhancer activity, the cloning site has been placed downstream of the rabbit β-globin polyadenylation signal. Placement at this distal position separates the enhancer from the minimal promoter and provides a more accurate measure of enhancer function, since activity should be independent of orientation and location relative to the promoter. The vector contains an artificial polyadenylation site upstream of the minimal TK promoter to suppress read-through transcription. The minimal TK promoter is derived from herpes simplex virus and retains low activity in the vast majority of cell lines used (see data below). Enhancer activity of fragments cloned into the pMLuc-2 vector can be monitored with the MightyLight™ Rluc Assay Kit.
For promoter analysis, the pMLuc-3 vector contains a multiple cloning site upstream of a secreted form of Renilla luciferase cDNA, followed by the rabbit β-globin polyadenylation sequence upstream from transcription termination (see vector map below). A second synthetic terminator sequence upstream of the cloning site was introduced to decrease read-through transcription. Secretion of Renilla luciferase is made possible by a fusion of DNA coding the signal peptide of the human interleukin-2 (IL-2) to the DNA encoding the normally cytoplasmic Renilla luciferase (1). Promoter activity of fragments cloned into the pMLuc-3 vector is monitored using the MightyLight™ Rluc Assay Kit to detect secreted Renilla luciferase in the medium. Additionally, sampling of time points can be performed without cell disruption.
Two pMLuc CMV Postive Control vectors are available:
The vector sequences are provided in GenBank format, with significant features indicated. To convert the sequence into other formats, first select the sequence and copy (Command-C on Mac or Ctrl-C on Windows). Next, click here to open the NIH ReadSeq web page in a new window. Paste the sequence into the input field (Command-V on Mac or Ctrl-V on PC), choose your favorite format from the dropdown menu, and hit the "submit" button.You can access sequences, maps, ordering information, newsletter articles and technical bulletins through the following links: |
