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Molecular Cloning is the process of producing recombinant DNA and transforming into host organisms to replicate and make more copies. Every cloning project is unique. Read More

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Vectors: Mammalian Reporter Change

Name Description Part Number
pSV-beta-Galactosidase Control Vector Open/Close Add
Co-transfect into cells as the control reporter in a dual-reporter assay
Assay in cell extracts using spectrophotometric, fluorescent or chemiluminescent methods
Can be used for in situ histochemical analysis using X-gal substrate

The pSV-Beta-Galactosidase Control Vector is a positive control vector for monitoring transfection efficiencies of mammalian cells. The SV40 early promoter and enhancer drive transcription of the lacZ gene, which encodes the beta-galactosidase enzyme. The pSV-Beta-Galactosidase Control Vector can be transfected individually or co-transfected with your DNA of interest. Beta-galactosidase is an excellent reporter enzyme that can be assayed quickly and directly in cell extracts using spectrophotometric, fluorescent or chemiluminescent assays. This reporter enzyme also is widely used for in situ histochemical analysis using the substrate X-Gal. The pSV-Beta-Galactosidase Control Vector can be co-transfected with your DNA of interest. For example, co-transfection with firefly luciferase gene vectors (pGL3 Vectors) provide cell extracts that can be assayed for both luciferase and beta-galactosidase activities. In this manner, the pSV-Beta-Galactosidase Vector acts as an internal control for transient expression assays. A negative control extract, prepared from mock-transfected cells, should also be assayed for the presence of endogenous beta-galactosidase activity in cultured cells. In addition, co-transfection with chloramphenicol acetyltransferase reporter gene vectors (pCAT3 Vectors) permits assaying for both CAT and beta-galactosidase activities. The pSV-Beta-Galactosidase Vector is a modification of pRSV-beta-Gal with SV40 and pUC18 sequences substituted for RSV and pBR322 sequences. The pSV-Beta-Galactosidase Vector will express beta-galactosidase in E. coli due to the presence of the E. coli gpt promoter located upstream of the lacZ gene. Colonies of E. coli containing the pSV-Beta-Galactosidase Vector will appear blue when plated on media containing X-Gal.

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pSP-luc+NF Fusion Vector Open/Close Add
Multiple cloning regions are positioned at the 5´ and 3´ ends of luc to provide maximum flexibility in cloning
Unique BstEII site located immediately downstream of the luciferase ATG translation codon for positioning N-terminal fusions
Not intended for expressing luciferase in eukaryotic cells

The pSP-luc+NF Fusion Vector is a luciferase cassette vector containing the engineered firefly luciferase gene, luc+NF. The luc+NF gene is related to the luc+ gene found in the pGL3 family of eukaryotic reporter vectors but has been further modified for maximum flexibility in constructing N-terminal fusions (NF) with luciferase. Subcloning luc+NF into expression vectors provides a useful genetic reporter with exceptional sensitivity. The pSP-luc+NF Fusion Vector is not itself intended for the expression of luciferase in eukaryotic cells, because it does not contain eukaryotic promoters, enhancers or polyadenylation signals. A unique BstEII site has been inserted immediately downstream of the luciferase ATG translation codon, allowing cloned inserts to be positioned immediately downstream of the luc+NF initiation codon. This vector is recommended specifically for applications where N-terminal fusion proteins do not contain an internal ATG codon at the luciferase junction. The luc+NF gene is positioned downstream of an SP6 promoter and a ribosome binding site. An opposing T7 promoter is located immediately downstream of luc+NF. Thus, the pSP-luc+NF Fusion Vector provides a convenient template for the in vitro synthesis of both sense and antisense luciferase transcripts for studies involving in situ hybridization, RNA processing, RNA transfection or coupled in vitro transcription/translation and protein folding. Multiple cloning regions containing recognition sequences for commonly used restriction enzymes are positioned at the 5' and 3' ends of luc+NF to provide maximum flexibility in cloning. Luciferase enzymatic activity can be assayed most efficiently using one of the Luciferase Assay Systems.

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Promoterless Renilla Luciferase Vectors with Puromycin Selection Open/Close Add
Designed for cloning a putative promoter element for investigating gene transcription control regions
Available with three varieties of engineered Renilla luciferase genes: hRluc, hRlucP or hRlucCP
Select for stably transfected cells using puromycin

Promoterless Renilla luciferase vectors are designed primarily to accept a putative promoter element for investigation of important regions controlling gene transcription. Alternatively, they may be used as promoterless control vectors in a dual-reporter system with a firefly luciferase vector serving as the experimental vector. The promoterless vectors are available with three varieties of engineered Renilla luciferase genes: hRluc, hRlucP or hRlucCP. The hRluc gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The hRlucP and hRlucCP and RapidResponse genes are hRluc genes appended with degradation sequences to influence the cellular half-life of the hRluc gene. The RapidResponse genes respond more rapidly to stimuli but at the expense of signal intensity. The hRlucP gene responds more rapidly than hRluc2 with moderate signal intensity, and the hRlucCP responds more quickly with the lowest signal intensity. The promoterless pGL4.82[hRluc/Puro], pGL4.83[hRlucP/Puro] and pGL4.84[hRlucCP/Puro] Vectors are available with the selectable marker puromycin.

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Promoterless Renilla Luciferase Vectors with Neomycin Selection Open/Close Add
Designed for cloning a putative promoter element for investigating gene transcription control regions
Available with three varieties of engineered Renilla luciferase genes: hRluc, hRlucP or hRlucCP
Select for stably transfected cells using neomycin

Promoterless Renilla luciferase vectors are designed primarily to accept a putative promoter element for investigation of important regions controlling gene transcription. Alternatively, they may be used as promoterless control vectors in a dual-reporter system with a firefly luciferase vector serving as the experimental vector. The promoterless vectors are available with three varieties of engineered Renilla luciferase genes: hRluc, hRlucP or hRlucCP. The hRluc gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The hRlucP and hRlucCP and RapidResponse genes are hRluc genes appended with degradation sequences to influence the cellular half-life of the hRluc gene. The RapidResponse genes respond more rapidly to stimuli but at the expense of signal intensity. The hRlucP gene responds more rapidly than hRluc2 with moderate signal intensity, and the hRlucCP responds more quickly with the lowest signal intensity. The promoterless pGL4.79[hRluc/Neo], pGL4.80[hRlucP/Neo] and pGL4.81[hRlucCP/Neo] Vectors are available with the selectable marker neomycin.

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Promoterless Renilla Luciferase Vectors with Hygromycin Selection Open/Close Add
Designed for cloning a putative promoter element to investigate gene transcription control regions
Available with three varieties of engineered Renilla luciferase genes: hRluc, hRlucP or hRlucCP
Select for stably transfected cells using hygromycin

Promoterless Renilla luciferase vectors are designed primarily to accept a putative promoter element for investigation of important regions controlling gene transcription. Alternatively, they may be used as promoterless control vectors in a dual-reporter system with a firefly luciferase vector serving as the experimental vector. The promoterless vectors are available with three varieties of engineered Renilla luciferase genes: hRluc, hRlucP or hRlucCP. The hRluc gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The hRlucP and hRlucCP and RapidResponse genes are hRluc genes appended with degradation sequences to influence the cellular half-life of the hRluc gene. The RapidResponse genes respond more rapidly to stimuli but at the expense of signal intensity. The hRlucP gene responds more rapidly than hRluc2 with moderate signal intensity, and the hRlucCP responds more quickly with the lowest signal intensity. The promoterless pGL4.76[hRluc/Hygro], pGL4.77[hRlucP/Hygro] and pGL4.78[hRlucCP/Hygro] Vectors are available with the selectable marker, hygromycin.

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Promoterless Renilla Luciferase Basic Vectors Open/Close Add
Designed for cloning a putative promoter element for investigating gene transcription control regions
Available with three varieties of engineered Renilla luciferase genes: hRluc, hRlucP or hRlucCP
Use as a promoterless control vector in a dual reporter assay

Promoterless Renilla luciferase vectors are designed primarily to accept a putative promoter element for investigation of important regions controlling gene transcription. Alternatively, they may be used as promoterless control vectors in a dual-reporter system with a firefly luciferase vector serving as the experimental vector. The promoterless vectors are available with three varieties of engineered Renilla luciferase genes: hRluc, hRlucP or hRlucCP. The hRluc gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The hRlucP and hRlucCP and RapidResponse genes are hRluc genes appended with degradation sequences to influence the cellular half-life of the hRluc gene. The RapidResponse genes respond more rapidly to stimuli but at the expense of signal intensity. The hRlucP gene responds more rapidly than hRluc2 with moderate signal intensity, and the hRlucCP responds more quickly with the lowest signal intensity. The promoterless vectors are available with or without selectable markers (hygromycin, neomycin or puromycin).

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Promoterless NanoLuc Genetic Reporter Basic Vectors Open/Close Add
Multiple cloning site offers an array of restriction sites for cloning
Offers two engineered genes for your reporter assay: Nluc and NlucP
NanoLuc® enzyme and its substrate are about 100-fold brighter than other luciferases, producing a high intensity, glow-type luminescence

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 pNL1.1[Nluc] and pNL1.2[NlucP] Vectors are used for cloning putative promoter regions and used for transient transfection experiments. 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).

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Promoterless Firefly Luciferase Vectors with Puromycin Selection Open/Close Add
Designed for cloning a putative promoter element for investigating gene transcription control regions
Available with three varieties of engineered firefly luciferase genes: luc2, luc2P or luc2CP
Select for stably transfected cells using puromycin

Promoterless firefly luciferase vectors are designed primarily to accept a putative promoter element for investigation of important regions controlling gene transcription. The promoterless vectors are available with three varieties of engineered firefly luciferase genes: luc2, luc2P or luc2CP. The luc2 gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The luc2P and luc2CP and RapidResponse genes are luc2 genes appended with degradation sequences to influence the cellular half-life of the luc2 gene. The RapidResponse genes respond more rapidly to stimuli but at the expense of signal intensity. The luc2P (1-hour half-life) gene responds more rapidly than luc2 (3-hour half-life) with moderate signal intensity, and the luc2CP (0.4-hour half-life) responds more quickly with the lowest signal intensity. The promoterless pGL4.20[luc2/Puro], pGL4.21[luc2P/Puro] and pGL4.22[luc2CP/Puro] vectors are available with a selectable marker, puromycin.

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Promoterless Firefly Luciferase Vectors with Neomycin Selection Open/Close Add
Designed for cloning a putative promoter element for investigation of gene transcription control regions
Available with three varieties of engineered firefly luciferase genes: luc2, luc2P or luc2CP
Select for stably transfected cells using neomycin (G418)

Promoterless firefly luciferase vectors are designed primarily to accept a putative promoter element for investigation of important regions controlling gene transcription. The promoterless vectors are available with three varieties of engineered firefly luciferase genes: luc2, luc2P or luc2CP. The luc2 gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The luc2P and luc2CP and RapidResponse genes are luc2 genes appended with degradation sequences to influence the cellular half-life of the luc2 gene. The RapidResponse genes respond more rapidly to stimuli but at the expense of signal intensity. The luc2P (1-hour half-life) gene responds more rapidly than luc2 (3-hour half-life) with moderate signal intensity, and the luc2CP (0.4-hour half-life) responds more quickly with the lowest signal intensity. The promoterlesspGL4.17[luc2/Neo] Vector, pGL4.18[luc2P/Neo] and pGL4.19[luc2CP/Neo] Vectors are available with a selectable marker, neomycin.

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Promoterless Firefly Luciferase Vectors with Hygromycin Selection Open/Close Add
Designed for cloning a putative promoter element for investigation of gene transcription control regions
Available with three varieties of engineered firefly luciferase genes: luc2, luc2P or luc2CP
Select for stably transfected cells using hygromycin

Promoterless firefly luciferase vectors are designed primarily to accept a putative promoter element for investigation of important regions controlling gene transcription. The promoterless vectors are available with three varieties of engineered firefly luciferase genes: luc2, luc2P or luc2CP. The luc2 gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The luc2P and luc2CP and RapidResponse genes are luc2 genes appended with degradation sequences to influence the cellular half-life of the luc2 gene. The RapidResponse genes respond more rapidly to stimuli but at the expense of signal intensity. The luc2P (1-hour half-life) gene responds more rapidly than luc2 (3-hour half-life) with moderate signal intensity, and the luc2CP (0.4-hour half-life) responds more quickly with the lowest signal intensity. The promoterless pGL4.14[luc2/Hygro], pGL4.15[luc2P/Hygro] and pGL4.16[luc2CP/Hygro] Vectors are available with the selectable marker hygromycin.

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Promoterless Firefly Luciferase Basic Vectors Open/Close Add
Designed for cloning a putative promoter element for investigation of gene transcription control regions
Available with three varieties of engineered firefly luciferase genes: luc2, luc2P or luc2CP

Promoterless firefly luciferase vectors are designed primarily to accept a putative promoter element for investigation of important regions controlling gene transcription. The promoterless vectors are available with three varieties of engineered firefly luciferase genes: luc2, luc2P or luc2CP. The luc2 gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The luc2P and luc2CP and RapidResponse genes are luc2 genes appended with degradation sequences to influence the cellular half-life of the luc2 gene. The RapidResponse genes respond more rapidly to stimuli but at the expense of signal intensity. The luc2P (1-hour half-life) gene responds more rapidly than luc2 (3-hour half-life) with moderate signal intensity, and the luc2CP (0.4-hour half-life) responds more quickly with the lowest signal intensity. The promoterless pGL4.10[luc2], pGL4.11[luc2P] and pGL4.12[luc2CP] Vectors do not have a selectable marker.

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Promoter-Driven Control Renilla Luciferase Vectors Open/Close Add
Co-transfect into cells where firefly luciferase is the experimental reporter gene
CMV, SV40 and TK promoter options offer different expression levels suitable for most dual-reporter assays

The Promoter-driven Renilla control vectors are commonly co-transfected with experimental firefly luciferase vectors for use in the Dual-Luciferase or Dual-Glo Reporter Assay Systems. The control Renilla vectors should give an almost invariant level of activity, while the experimental firefly vector varies with treatment.

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Promoter-Driven Control NanoLuc Luciferase Vectors Open/Close Add
Multiple promoter options (CMV, TK and PGK) are available to obtain appropriate levels of the control reporter in your experimental system
Co-transfect with experimental firefly luciferase vectors as normalization control
Measure signal using the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System

The promoter-driven NanoLuc (Nluc) control vectors can be used to co-transfect 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. The vectors are engineered with minimal consensus transcription factor-binding sites to reduce anomalous expression.

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Promoter-Driven Control Firefly Luciferase Vectors Open/Close Add
Co-transfect into cells where Renilla or NanoLuc® luciferase is the experimental reporter gene
SV40, PGK and TK promoter options offer different expression levels suitable for most dual-reporter assays

The Promoter-driven firefly control vectors are commonly co-transfected with experimental Renilla or NanoLuc luciferase vectors for use in the Dual-Luciferase or Dual-Glo Reporter Assay Systems. The control firefly vectors should give an almost invariant level of activity, while the experimental Renilla or NanoLuc vector varies with treatment. The pGL4.13[luc2/SV40] ,pGL4.53[luc2/PGK] and pGL4.54[luc2/TK] Vectors offer three different promoter options for your dual-reporter assay.

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pRL Renilla Luciferase Control Reporter Vectors Open/Close Add
A T7 promoter is located immediately upstream of Rluc for in vitro synthesis of Renilla luciferase
The SV40 late poly(A) signal sequence is positioned downstream of Rluc to provide efficient transcription termination and mRNA polyadenylation
A prokaryotic origin of replication and β-lactamase gene allow selected propagation of the pRL vectors in E. coli host strains

The pRL Vectors are wildtype Renilla luciferase (Rluc) control reporter vectors. The pRL Vectors, which provide constitutive expression of Renilla luciferase, can be used in combination with a firefly luciferase vector to cotransfect mammalian cells. Expression of Renilla luciferase provides an internal control value to which expression of the experimental firefly luciferase reporter gene may be normalized. The pRL Vectors contain the cDNA encoding Renilla luciferase (Rluc) cloned from the anthozoan coelenterate Renilla reniformis (sea pansy). Four different promoter configurations are available. The HSV-thymidine kinase promoter (pRL-TK) is relatively weak and may be particularly useful in providing neutral constitutive expression of the Renilla luciferase control reporter. The early SV40 enhancer/promoter region (pRL-SV40) and the CMV immediate early enhancer/promoter region (pRL-CMV) typically provide high-level transcription and, therefore, may be less suitable for co-reporter applications involving experimental vectors with robust regulatory elements. In general, we recommend validating the performance of specific co-reporter combinations in the desired target cells. In addition to the modified Rluc reporter gene, all pRL Vectors are isolated from a dam-/dcm- E. coli K host strain, allowing digestion with restriction enzymes that are sensitive to dam and dcm methylation.

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pNLF1 NanoLuc Protein Fusion Vectors Open/Close Add
Use traditional cloning to insert protein of interest into the multiple cloning site (MCS)
Bright NanoLuc® reporter enables endogenous expression levels of fusion proteins for biologically relevant results
Choose from transient transfection or select for stable expression using hygromycin

The small size (19.1kDa) and extreme brightness (about 100-fold brighter than either firefly [Photinus pyralis] or Renilla reniformis) of NanoLuc luciferase (Nluc) make it an ideal protein fusion partner. NanoLuc fusion proteins can be used in a variety of applications including: reporters of protein stability, probes for bioluminescent cell imaging (BLI) or as the donor signal in bioluminescent resonance energy transfer (BRET) applications for protein:protein or protein:small-molecule interaction studies. The NanoLuc protein fusion vectors enable simple generation of N- or C-terminal fusions of NanoLuc luciferase with your protein of interest. The pNLF Vector series uses using traditional cloning with a multiple cloning site (MCS) to generate N- or C-terminal fusions to the full-length Nluc protein with the pNLF1-N or pNLF1-C [CMV/Hygro] Vectors or attach secreted Nluc to the N-terminus of the protein of interest with pNLF1-secN [CMV/Hygro] Vector. Also make NanoLuc protein fusions with the pF Vector series: Generate N- or C-terminal Nluc fusion proteins using the Flexi Vector Cloning System—a directional cloning method based on two rare-cutting restriction enzymes, SgfI and PmeI, that provides a rapid, efficient and high-fidelity way to transfer protein-coding regions between a variety of Flexi Vectors without the need to resequence.

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pmirGLO Dual-Luciferase miRNA Target Expression Vector Open/Close Add
Reporter activity correlates with miRNA activity
Optimized luc2 reporter gene provides highest expression while the Renilla luciferase gene provides normalization
The moderate-strength PGK promoter provides more biologically relevant analysis not possible with strong promoters

The pmirGLO Vector is designed to quantitatively evaluate microRNA (miRNA) activity by the insertion of miRNA target sites downstream or 3' of the firefly luciferase gene (luc2). Firefly luciferase is the primary reporter gene; reduced firefly luciferase expression indicates the binding of endogenous or introduced miRNAs to the cloned miRNA target sequence. This vector is based on Promega dual-luciferase technology, with luc2 used as the primary reporter to monitor mRNA regulation and Renilla luciferase (hRluc-neo) acting as a control reporter for normalization and selection.

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pGEM-luc DNA Open/Close Add
Remove the firefly luciferase gene using the unique restriction enzyme sites
Use the reporter gene to analyze transcriptional activity, mRNA processing, protein structure/function, or label cells and viruses

The pGEM-luc Vector is a cassette vector designed to be a source of the luc gene encoding firefly luciferase, which is found in the pGL2 Vectors. The plasmid is not intended for the expression of luciferase in eukaryotic or prokaryotic cells. The pGEM-luc Vector was constructed by positioning the luciferase gene (luc) in the center of the multiple cloning region of the pGEM-11Zf(-) Vector, providing a number of unique restriction sites at both ends of the gene. Sites that are surrounded by parentheses are not unique, as additional sites for each also exist in the luciferase gene. Note also that using HindIII or NsiI to clone the luciferase gene will include upstream ATG codons, which may reduce the efficiency of expression in eukaryotes. The luciferase cassette does not contain the prokaryotic Shine-Delgarno sequence for bacterial expression.

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NanoLuc Stability Sensors for Cell Signaling Open/Close Add
Ready-to-use constructs are predesigned, optimized and tested for low endotoxin levels
C-terminal fusion of HIF1A NanoLuc® luciferase reporter in pNLF1-HIF1A [CMV/neo] Vector
NRF2 vector system has a pKEAP1-expressing vector for regulating intracellular NRF2 levels and pNLF1-NRF2 [CMV/neo] Vector expressing a C-terminal fusion with NanoLuc® luciferase

The rate of protein turnover is tightly regulated for many signaling proteins involved in oncogenesis and response to cellular stress. Protein stabilization and subsequent accumulation occurs in response to changing cellular conditions resulting in activation of downstream transcriptional events. The NanoLuc Stability Sensors are ready-to-use vector systems that utilize the advantages of the NanoLuc luciferase reporter to enable stability studies of two key signaling proteins, HIF1A and NRF2, providing a method to directly measure this primary signaling event. HIF1A Vector System: The HIF1A Vector System enables simple quantification of intracellular HIF1A protein levels to study the dynamics of this signaling protein in mediating cellular response to hypoxia. It contains a vector encoding NanoLuc fused to the C-terminus of the HIF1A protein under control of the CMV promoter plus Transfection Carrier DNA to allow for titratable intracellular fusion protein expression. NRF2 Vector System: The NRF2 Vector System enables simple quantification of intracellular NRF2 protein levels to study the dynamics of this signaling protein in mediating cellular response to oxidative stress. It contains a vector encoding NanoLuc fused to the C-terminus of the NRF2 protein under the control of the CMV promoter, a pKEAP1-expressing vector for proper regulation of intracellular NRF2 levels and Transfection Carrier DNA for titratable intracellular fusion protein expression.

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NanoLuc Reporter Vector with NF-kappaB Response Element Open/Close Add
Responds to changes in NF-κB expression in cells
Includes an optimized NanoLuc® luciferase gene that codes for a reporter protein with a shorter half-life (NlucP)
Hygromycin resistance selectable marker means choosing transient transfection or generating a stable cell line

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. For use as a genetic reporter, multiple forms of NanoLuc luciferase have been configured to meet differing experimental objectives. NanoLuc-PEST (NlucP) present in pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] Vector closely couples protein expression to changes in transcriptional activity and increased signal-to background ratios. Use the pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] Vector to assess the response to cellular changes in the nuclear factor kappaB. 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 NlucP gene is codon optimized and has removed many potential regulatory elements or other undesirable features (such as common restriction enzyme sites).

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Monster Green Fluorescent Protein phMGFP Vector Open/Close Add
Visualize low-level expression in situ using fluorescence microscopy, imagers or FACS®
Create fusion proteins for imaging and localization studies using standard FITC detection
Transfect at high efficiencies for precloning confirmation studies

The phMGFP Vector contains the open reading frame for the Monster Green Fluorescent Protein cloned into a mammalian expression vector. The Monster Green Fluorescent Protein is encoded by an improved synthetic version of the green fluorescent protein gene originally cloned from Montastrea cavernosa (Great Star Coral). The synthetic gene (hMGFP) expresses a 26kDa protein that shows improved fluorescence intensity compared to the native gene. Furthermore, the hMGFP gene has been codon optimized and cleared of most consensus sequence transcription factor binding sites to ensure reliability and high levels of expression. The Monster Green Fluorescent Protein encoded by the hMGFP gene is an ideal fluorescent reporter, providing high-level fluorescence and reducing cytotoxicity. Monster Green Fluorescent Protein generally fluoresces at least 20% brighter than other commercially available green fluorescent proteins (GFPs) and also reduces cytotoxicity, offering flexibility when working with transient and stable expression assays.

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Coincidence Reporter Vectors Open/Close Add
Firefly and NanoLuc® luciferase have dissimilar compound interference profiles for better identifying more false-positives versus individually
Using two different transcriptional reporters reduces false hit rates and increases the identification of true biological hits
luc2 and NlucP provide a bright reporter combination compatible with low-copy-number and plate scale-up, and provide greater signal-to-background ratios

Luciferase-based reporter-gene assays remain a useful and powerful method of high-throughput compound screening. However, false hits that result from direct interaction of compounds with the luciferase reporter can result in unnecessary follow-up efforts. The pNLCoI Vectors comprise a second-generation coincidence reporter vector system that allow expression of both firefly luciferase (luc2) and NanoLuc Luciferase fused to a PEST destabilization domain (NlucP) from the same mRNA transcript. The stoichiometric expression of both luciferases is achieved by use of the P2A sequence from porcine teschovirus-1, which promotes a ribosomal skip and expression of the two unfused enzymes with distinct compound interaction profiles. When used in high-throughput compound screening, false hits caused by direct interaction with one or the other luciferases can be distinguished from true hits that show a similar response for both, reducing workload associated with follow-up screens. The pNLCoI Vectors are designed for use with the Nano-Glo Dual-Luciferase Reporter (NanoDLR) Assay System, which allows sequential detection of firefly and NanoLuc Luciferase in activity in the same sample. Both reagents provide stable glow-type luminescence signals with half-lives of approximately two hours allowing batch processing of samples and amenable to assays or screens in 96-, 384- or 1,536-well plate formats. Potent inhibition of firefly luciferase coupled with the high-intensity luminescence of NanoLuc luciferase maximizes sensitivity for detection of both reporters.

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