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Nano-Glo® HiBiT Dual-Luciferase® Reporter System

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Multiplexed Detection of HiBiT-Tagged Proteins and Firefly Luciferase in the Same Sample

  • Rapidly identify specific and non-specific effects of compounds
  • Compatible with batch processing and automation
  • Separate and bicistronic systems for co-expression of HiBiT and Fluc available

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Nano-Glo® HiBiT Dual-Luciferase® Reporter System
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Protein Quantitation & Detection Hero Art Floating

Sequential Measurement of Firefly Luciferase and HiBiT-Tagged Proteins from the Same Sample

The Nano-Glo® HiBiT Dual-Luciferase® Reporter System (HiBiT NanoDLR™) allows sequential measurement of firefly luciferase (Fluc) and HiBiT-tagged proteins from the same sample in an “add-read-add-read” assay format. Fluc is used as a constitutively expressed control to indicate nonspecific compound effects on cell viability or protein expression, rather than specific changes in the levels of the HiBiT-tagged protein of interest, aiding in the interpretation of protein modulation screens and studies.

The 11-amino acid HiBiT peptide tag can be added to a protein of interest using CRISPR/Cas9 gene editing coupled with ectopic expression of Fluc. Alternatively, use a bicistronic HiBiT entry vector to express both the HiBiT-protein fusion and constitutive Fluc on the same mRNA.

HiBiT NanoDLR™ Workflow

18324ma - HiBiT NanoDLR™ workflow

Firefly luciferase (Fluc) is first detected after addition of the ONE-Glo™ EX reagent supplemented with LgBiT (1). The NanoDLR™ Stop & Glo® Reagent is added to the cells to both quench the firefly signal and detect the now-complemented NanoBiT® (HiBiT + LgBiT) signal (2).

Use the Fluc Signal to Distinguish Specific and Nonspecific Compound Effects on Protein Levels

18325ma-a - Selective HiBiT-BRD4 degradation with MZ1
18325ma-b - Selective HiBiT-BRD4 degradation with dBET1
18325ma-c - General cellular toxicity with staurosporine

Selective HiBiT-BRD4 degradation with two PROTACs compared to general cellular toxicity. HEK293 cells were transiently transfected with a construct expressing the HiBiT-BRD4-IRES-luc2 cassette from the weak TK promoter. Cells were treated with titrating concentrations of MZ1 (Panel A), dBET1 (Panel B) or staurosporine (Panel C) before the Fluc (blue) and HiBiT (red) signals were measured using the lytic HiBiT NanoDLR™ Assay. MZ1 and dBET1 selectively link BRD4 to two different E3 ligase complexes for targeted degradation, and staurosporine causes general toxicity, resulting in loss of both HiBiT-BRD4 and firefly luciferase (Fluc).

Quantifiable Over Seven Orders of Magnitude

18326ma - Background-subtracted RLU from firefly luciferase in ONE-Glo EX/LgBiT reagent
18327ma - Background-subtracted RLU from HiBiT in NanoDLR Stop & Glo Reagent

The broad linear dynamic range accurately quantifies tagged proteins regardless of expression level to measure changes in protein abundance. The HiBiT NanoDLR™ Assay can quantify even low-abundance proteins at endogenous levels of expression.

Express HiBiT and Fluc on the Same mRNA with Bicistronic Vectors

18328ma - Create N- or C-terminal HiBiT fusion vectors and express from different promoter strengths (CMV, PGK or TK)

A variety of bicistronic HiBiT entry vectors with constitutive expression of firefly luciferase are available to create N- or C-terminal HiBiT fusion vectors and express from different promoter strengths (CMV, PGK or TK). All are compatible with stable cell line generation with a blasticidin selection marker.

Patents and Disclaimers

Materials may be covered by pending or issued patents or may have certain limitations.
Information is available upon request and will be included on applicable quotes.

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Nano-Glo® Luciferase Assay Limited Use Label License BY USE OF THIS MATERIAL, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. Researcher may use this material for research use only; no commercial use is allowed. Commercial use means any and all uses of this material by a party in exchange for consideration, including, but not limited to, (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the material, whether or not such material is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the material. No other use or transfer of this material is authorized without the prior express written consent of Promega. For uses of Nano-Glo®-branded reagents intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researcher must: (a) use NanoBRET™-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material; or (b) contact Promega to obtain a license for use of the material for energy transfer assays to energy acceptors not manufactured by Promega. With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA. \

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Nano-Glo® Luciferase Assay Limited Use Label License BY USE OF THIS MATERIAL, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. Researcher may use this material for research use only; no commercial use is allowed. Commercial use means any and all uses of this material by a party in exchange for consideration, including, but not limited to, (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the material, whether or not such material is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the material. No other use or transfer of this material is authorized without the prior express written consent of Promega. For uses of Nano-Glo®-branded reagents intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researcher must: (a) use NanoBRET™-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material; or (b) contact Promega to obtain a license for use of the material for energy transfer assays to energy acceptors not manufactured by Promega. With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA. \

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Promega DNA Vector Limited Use Label License (LULL) BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. 1. Usage Restrictions: 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation. 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including product manufacture, providing a service, information or data to unaffiliated third parties, resale of the material for any use and sale of derivatives for any use. 2. Modification Restrictions: 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors. 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes. 3. For determinations of luminescence activity of this material and its derivatives, recipient must either: 3.1. Use Promega-branded luminescent assay reagents (LARs); or 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either: 4.1. Use Nano-Glo®-branded luminescent assay reagents; or 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either: 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must: 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein. 7. For uses of HaloTag® Technology in this material, recipient must either: 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used. 8. Transfer of Materials: 8.1. Unmodified material cannot be transferred. 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license. 8.3. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party. 9. Other Uses: 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information. 10. Disclaimer and Governing Law: 10.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

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Promega DNA Vector Limited Use Label License (LULL) BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. 1. Usage Restrictions: 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation. 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including product manufacture, providing a service, information or data to unaffiliated third parties, resale of the material for any use and sale of derivatives for any use. 2. Modification Restrictions: 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors. 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes. 3. For determinations of luminescence activity of this material and its derivatives, recipient must either: 3.1. Use Promega-branded luminescent assay reagents (LARs); or 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either: 4.1. Use Nano-Glo®-branded luminescent assay reagents; or 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either: 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must: 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein. 7. For uses of HaloTag® Technology in this material, recipient must either: 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used. 8. Transfer of Materials: 8.1. Unmodified material cannot be transferred. 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license. 8.3. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party. 9. Other Uses: 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information. 10. Disclaimer and Governing Law: 10.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

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Promega DNA Vector Limited Use Label License (LULL) BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. 1. Usage Restrictions: 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation. 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including product manufacture, providing a service, information or data to unaffiliated third parties, resale of the material for any use and sale of derivatives for any use. 2. Modification Restrictions: 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors. 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes. 3. For determinations of luminescence activity of this material and its derivatives, recipient must either: 3.1. Use Promega-branded luminescent assay reagents (LARs); or 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either: 4.1. Use Nano-Glo®-branded luminescent assay reagents; or 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either: 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must: 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein. 7. For uses of HaloTag® Technology in this material, recipient must either: 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used. 8. Transfer of Materials: 8.1. Unmodified material cannot be transferred. 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license. 8.3. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party. 9. Other Uses: 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information. 10. Disclaimer and Governing Law: 10.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

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Promega DNA Vector Limited Use Label License (LULL) BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. 1. Usage Restrictions: 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation. 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including product manufacture, providing a service, information or data to unaffiliated third parties, resale of the material for any use and sale of derivatives for any use. 2. Modification Restrictions: 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors. 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes. 3. For determinations of luminescence activity of this material and its derivatives, recipient must either: 3.1. Use Promega-branded luminescent assay reagents (LARs); or 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either: 4.1. Use Nano-Glo®-branded luminescent assay reagents; or 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either: 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must: 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein. 7. For uses of HaloTag® Technology in this material, recipient must either: 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used. 8. Transfer of Materials: 8.1. Unmodified material cannot be transferred. 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license. 8.3. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party. 9. Other Uses: 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information. 10. Disclaimer and Governing Law: 10.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

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Promega DNA Vector Limited Use Label License (LULL) BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. 1. Usage Restrictions: 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation. 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including product manufacture, providing a service, information or data to unaffiliated third parties, resale of the material for any use and sale of derivatives for any use. 2. Modification Restrictions: 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors. 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes. 3. For determinations of luminescence activity of this material and its derivatives, recipient must either: 3.1. Use Promega-branded luminescent assay reagents (LARs); or 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either: 4.1. Use Nano-Glo®-branded luminescent assay reagents; or 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either: 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must: 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein. 7. For uses of HaloTag® Technology in this material, recipient must either: 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used. 8. Transfer of Materials: 8.1. Unmodified material cannot be transferred. 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license. 8.3. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party. 9. Other Uses: 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information. 10. Disclaimer and Governing Law: 10.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

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Promega DNA Vector Limited Use Label License (LULL) BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. 1. Usage Restrictions: 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation. 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including product manufacture, providing a service, information or data to unaffiliated third parties, resale of the material for any use and sale of derivatives for any use. 2. Modification Restrictions: 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors. 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes. 3. For determinations of luminescence activity of this material and its derivatives, recipient must either: 3.1. Use Promega-branded luminescent assay reagents (LARs); or 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either: 4.1. Use Nano-Glo®-branded luminescent assay reagents; or 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either: 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega. 6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must: 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein. 7. For uses of HaloTag® Technology in this material, recipient must either: 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used. 8. Transfer of Materials: 8.1. Unmodified material cannot be transferred. 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license. 8.3. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party. 9. Other Uses: 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information. 10. Disclaimer and Governing Law: 10.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

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