pCLuc Mini-TK 2 Vector

Description

pCLuc Mini-TK 2 is a cloning vector for mammalian cells, containing a minimal promoter fragment from the HSV thymidine kinase (TK) promoter adjacent to a reporter gene, the secreted luciferase from the Ostracod Cypridina noctiluca. Cypridina luciferase (CLuc) is a 62 kDa protein with a native signal peptide at the N-terminus that allows it to be secreted from mammalian cells (1) so that CLuc activity can be detected in the culture medium of mammalian cells expressing the reporter gene. The pCLuc Mini-TK 2 Vector contains a MCS upstream of the minimal TK promoter for cloning promoter or enhancer elements. A neomycin resistance gene under the control of an SV40 promoter allows selection for stable integration of the plasmid into the mammalian cell genome using G418.

Figure 1: Cypridina Luciferase (CLuc) activity obtained from different CLuc plasmids

Figure 2: pCLuc Mini-TK 2 Vector multiple cloning site (MCS)

DNASU and Addgene are central repositories for plasmid clones and collections that may also be helpful.

Highlights

• Multiple samples can be obtained from the same transfected cells (i.e., before and after experimental treatments or at multiple time points).
• 90–95% of CLuc activity is found in the cell culture medium, with the remaining 5-10% detectable in cell lysates (Figure 1). This allows flexibility when assaying CLuc along with other co-transfected reporters.
• The activity of CLuc is high and the CLuc assay is sensitive enough to detect very small amounts of CLuc enzyme activity (Figure 2).
• CLuc does not use the same substrate as other marine luciferases (e.g. Renilla, Gaussia). Therefore, it is possible to assay both CLuc and GLuc independently in cell culture medium from cells expressing both reporters.
• The pCLuc Mini-TK 2 Vector can be transfected into cells using any standard transfection protocol.

Product Source

Advantages and Features

Features

• Polylinker upstream of Mini-TK: 20–68
• Minimal promoter from HSV-Thymidine Kinase (MiniTK): 69–137
• Kozak Consensus: 139–148
• CLuc ORF: 145–1806
• Start codon of CLuc 145–147
• Stop codon: 1804–1806
• Signal peptide: 145–198
• Synthetic poly-A site: 1815–1863
• SV40 promoter (NeoR): 2449–2784
• NeoR Orf: 2836–3630
• SV40 early polyadenylation signal: 3804–3934
• Ori: pMB1 origin of replication (complement):4376–4964
• AmpR (beta-lactamase complement): 5135–5995
• Two restriction sites are available for cloning elements downstream of the CLuc coding region. The NotI site is upstream of the polyA site and allows cloning of sequences which will become part of the CLuc mRNA. The XbaI site is downstream of the polyA site, sequences cloned into the XbaI site will not be incorporated into CLuc mRNA.
• All pLuc-2 vectors have improved poly-adenylationtranscription termination of the luciferase transcript. The polyadenylation signal is a synthetic polyadenylation sequence based on the b-globin gene (5).

Applications

Properties and Usage

Storage Temperature

Storage Conditions

10 mM Tris-HCl
1 mM EDTA
pH 7.5 @ 25°C

Sequence Files

Related Products

Companion Products

• BioLux® Cypridina Luciferase Assay Kit
• pCMV-CLuc 2 Control Plasmid
• pSV40-CLuc Control Plasmid
• pCLuc-Basic 2 Vector
• pTK-CLuc Vector
• BioLux® Gaussia Luciferase Assay Kit
• pCMV-GLuc 2 Control Plasmid
• pSV40-GLuc Control Plasmid
• pGLuc-Basic 2 Vector
• pTK-GLuc Vector
• pGLuc Mini-TK 2 Vector

References

1. Nakajima, et al. (2004). Biosci. Biotechnol. Biochem. 68, 565-570.
2. Otsuji, et al. (2004). Anal. Biochemistry. 329, 230-237.
3. Wu, et al. (2007). Biotechniques. 42, 290-292.
4. Levitt, et al. (1989). Genes Dev.. 3, 1019-1025.