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pNL2.2[NlucP/Hygro] Vector

by Promega
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  • The pNL2.1[Nluc/Hygro] and pNL2.2[NlucP/Hygro] Vectors are used to clone a putative promoter to drive expression of the bright NanoLuc® reporter protein. Select for stable expression in mammalian cells using hygromycin.

    NanoLuc® (Nluc) luciferase is a small enzyme (19.1kDa) engineered for optimal performance as a luminescent reporter. The enzyme is about 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase using a novel substrate, furimazine, to produce high intensity, glow-type luminescence. The luminescent reaction is ATP-independent and designed to suppress background luminescence for maximal assay sensitivity.

    • Transcription regulation.
    • Virus-cell interactions.
    • Compound screening.
    • Post-translational modifications.
    • GPCR signaling.
    • Cell signaling.
    • Promoter analysis.

    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).

    A comparison of light output and response dynamics between NanoLuc® and firefly luciferase constructs.
    A comparison of light output and response dynamics between NanoLuc® and firefly luciferase constructs.
    10327MA-W

    pNL2.1[Nluc/Hygro] Vector GenBank® Accession Number JQ513372 and vector sequence text file.

    10328MA-W

    pNL2.2[NlucP/Hygro] Vector GenBank® Accession Number JQ513373 and vector sequence text file.

    NanoLuc® Technology performs similarly to firefly luciferase but gives higher light output.
    NanoLuc® Technology performs similarly to firefly luciferase but gives higher light output.
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    Storage Conditions

    -30C TO -10C

    Use Restrictions

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

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