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pNL1.1.TK[Nluc/TK] Vector

by Promega
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  • Overview
  • Protocols
  • Specifications
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  • The pNL1.1.CMV[Nluc/CMV], pNL1.1.PGK[Nluc/PGK] and pNL1.1.TK[Nluc/TK] Vectors are promoter-driven NanoLuc® (Nluc) control vectors that can be co-transfected with experimental firefly luciferase vectors when using the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System.

    NanoLuc® luciferase is a small (19.1kDa), stable reporter enzyme that can be up to 100-fold more sensitive than the flash-type Renilla signal in the DLR™ Assay and more than 3,000-fold more sensitive than the Renilla signal in the Dual-Glo® Assay. The increased brightness of the NanoLuc® Luciferase means you use less control DNA, minimizing assay artifacts and providing a stable control signal for normalization of the experimental Fluc reporter. Firefly luciferase, which is derived from Photinus pyralis, can be used be used as the control when NanoLuc® Luciferase is the experimental reporter. The vectors are engineered with minimal consensus transcription factor-binding sites to reduce anomalous expression.

    The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System uses the same protocol as the popular Dual-Glo® Luciferase Assay, with improved sensitivity, performance and convenience. Control vectors are ready to substitute into your assay. In addition, the NanoDLR™ Assay is compatible with multiple experimental configurations. This flexibility means you can use either Fluc or Nluc as the experimental reporter and normalize with either the Nluc or Fluc control, respectively.

    About NanoLuc® Luciferase and the pNL Vectors

    For use as a genetic reporter, multiple forms of NanoLuc® luciferase have been configured to meet differing experimental objectives. Unfused Nluc offers maximal light output and sensitivity, and NanoLuc®-PEST (NlucP) closely couples protein expression to changes in transcriptional activity and increased signal-to background ratios. When a secreted reporter is preferred, there is NanoLuc® luciferase fused to an N-terminal secretion signal (secNluc). Luminescence is linearly proportional to the amount of NanoLuc® protein over a 1,000,000-fold concentration range, with a signal half-life ?2 hours when detected with Nano-Glo® Luciferase Assay Reagent.

    NanoLuc® luciferase possesses a number of physical properties that make it an excellent reporter protein:

    • very small, monomeric enzyme (171 amino acids; 513bp)
    • high thermal stability (Tm = 60°C)
    • active over a broad pH range (pH 6–8)
    • no post-translational modifications or disulfide bonds
    • uniform distribution in cells
    • emission spectrum well suited for bioluminescence resonance energy transfer (BRET; ?max = 465nM).

    NanoLuc® luciferase is made available in a variety of plasmids designed for use in reporter gene assays of transcriptional control and with each of the NanoLuc® forms (unfused Nluc, PEST destabilized NlucP, and secreted secNluc). The different pNL variations are designed for the following:

    • pNL1: cloning of a known or putative promoter region
    • pNL2: cloning of a known or putative promoter region and establishment of a stable cell line through Hygromycin selection
    • pNL3: cloning of a binding site or response element not in need of a basic promoter (such as are present in the pNL3.2.NF-?B-RE vector)
    • Control plasmids for the unfused, PEST-destabilized and secreted Nluc forms also are available.

    The pNL vector series uses a pGL4-based backbone for easy sequence transfer from existing plasmids. This backbone design also reduces anomalous results by removing many transcription factor binding sites and other potential regulatory elements. The Nluc gene variations are codon optimized and have had many potential regulatory elements or other undesirable features removed (such as common restriction enzyme sites).

    Relative luminescence for firefly and NanoLuc® luciferases expressed from constitutive promoters in multiple cell types.
    Relative luminescence for firefly and NanoLuc® luciferases expressed from constitutive promoters in multiple cell types.

    Sequence Information

    pNL1.1.CMV[Nluc/CMV] Vector GenBank® Accession Number JQ513379 and vector sequence text file.

    pNL1.1.PGK[Nluc/PGK] Vector GenBank® Accession Number KM359770 and vector sequence text file.

    pNL1.1.TK[Nluc/TK] Vector GenBank® Accession Number KM359774 and vector sequence text file.

  • Certificate of Analysis

    Lookup Certificate of Analysis

    Storage Conditions

    -30C TO -10C

    Use Restrictions

    For Research Use Only. Not for Use in Diagnostic Procedures.

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