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. The pNL1.3[secNluc], pNL3.3[secNluc/minP], pNL2.3[secNluc/Hygro] and pNL1.3.CMV[secNluc/CMV] Vectors are used for cloning putative regulatory elements for secreted expresson in mammalian cells. Select for stable expression in cells using hygromycin resistance. 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, NanoLuc-PEST (NlucP) closely couples protein expression to changes in transcriptional activity and increased signal-to background ratios, and NanoLuc luciferase fused to an N-terminal secretion signal (secNluc) is suitable when a secreted reporter is preferred. 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: 1) very small, monomeric enzyme (171 amino acids; 513bp); 2) high thermal stability (Tm = 60°C); 3) active over a broad pH range (pH 6-8); 4) no post-translational modifications or disulfide bonds; 5) uniform distribution in cells; 6) emission spectrum well suited for bioluminescence resonance energy transfer (BRET; lambdamax = 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: 1) pNL1: cloning of a known or putative promoter region; 2) pNL2: cloning of a known or putative promoter region and establishment of a stable cell line through Hygromycin selection; 3) 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-kB-RE vector); 4) 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).