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InvivoGen has engineered a fusion between the firefly luciferase (Luc) and the Sh ble genes. The firefly Luc gene is a highly sensitive reporter gene and thus is ideal for detecting low-level gene expression. The Sh ble gene confers Zeocin™ resistance. Both genes have been modified and contain no CpGs.
The Luc::Sh fusion gene exhibits a higher luciferase activity and enables a better and faster selection of Zeocin™ resistant clones. The synthetic Luc::Sh fusion gene is provided in the expression plasmid pSelect-zeo which is selectable with Zeocin™ in mammalian and E.coli cells.
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• 20 μg of pSELECT-zeo-LucSh plasmid provided as lyophilized DNA
• 4 pouches of E. coli Fast-Media® Zeo (2 TB and 2 Agar)
First expression cassette
• hEF1-htLV prom is a composite promoter comprising the Elongation Factor-1alpha (EF-1α) core promoter [1 ]and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat . The EF-1α promoter exhibits a strong activity and yields long lasting expression of a transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to enhance stability of RNA.
• LucSh: Synthetic LucSh fusion gene (LucSh-ΔCpG): InvivoGen has engineered a fusion between the firefly luciferase gene and the Sh ble gene conferring Zeocin™ resistance. Both genes have been modified and contain no CpG, whereas their wildtype counterparts contain 95 and 50 CpG motifs respectively. This fusion exhibits a higher luciferase activity and enables a better and faster selection of Zeocin™ resistant clones.
• SV40 pAn: the Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA .
• ori: a minimal E. coli origin of replication to limit vector size, but with the same activity as the longer Ori.
Second expression cassette
• CMV enh/prom: The human cytomegalovirus immediate-early gene 1 promoter/enhancer was originally isolated from the Towne strain and was found to be stronger than any other viral promoters.
• EM7 is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli.
• Zeo: Resistance to Zeocin™ is conferred by the Sh ble gene from Streptoalloteichus hindustanus The Sh ble gene is driven by the CMV enhancer/promoter in tandem with the bacterial EM7 promoter allowing selection in both mammalian cells and E. coli.
• ßGlo pAn: The human beta-globin 3’UTR and polyadenylation sequence allows efficient arrest of the transgene transcription .
1. Kim, D.W. et al. (1990). Gene 2: 217-223.
2. Takebe, Y. et al. (1988). Mol. Cell Biol. 1: 466-472.
3. Carswell, S. & Alwine, J.C. (1989). Mol. Cell Biol. 10: 4248-4258.
4. Yu J & Russell JE. (2001). Mol Cell Biol, 21(17):5879-88.