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CpG-containing control plasmid for pCpGfree vectors
CpG-containing control plasmid
pCpGrich-mcs is a CpG-containing control plasmid. The CpG-free version of the murine CMV enhancer, human EF-1α promoter, ori R6K gamma, bacterial EM2K promoter and Zeocin™ resistance gene have been replaced by their wild-type counterparts. The pCpGrich-mcs plasmid contains 88 CpG dinucleotides.Back to the top
pCpGrich-mcs: CpG-containing control plasmid with a multiple cloning site (MCS)
Selectable in E. coli with Zeocin™Back to the top
ContentsBack to the top
Elements for expression in E. coli
• R6K gamma ori (wt): This origin of replication is activated by the R6K specific initiator protein π, encoded by the pir gene .
• EM7 is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli.
• Zeo (wt) selectable marker: Sh ble gene confers Zeocin™ resistance. elements for expression in mammalian cells
• Mammalian promoter combines the mouse CMV enhancer, the human elongation factor 1 alpha core promoter and 5’UTR containing a synthetic intron (SI 126).
• SV40 pAn: the Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA .
• MAR: Matrix attached regions (MARs) are sequences typically AT-rich that are able to form barriers between independently regulated domains . This plasmid contains two MARs, from the 5’ region of the human IFN-β gene and the β-globin gene. The MARs are placed between the bacterial and mammalian transcription units.
• MCS: The multiple cloning site contains several commonly used restriction sites for convenient cloning of a gene of interest.
5’ Bsr GI, Stu I, Bgl II, Acc65 I, Asp 718, Eco O109I, Bsp 120I, Nhe I 3’
Due to the presence of the R6Kγ origin of replication, pCpG plasmids can only be amplified in E. coli mutant strain expressing a pir mutant gene. They will not replicate in standard E. coli strains. Therefore, pCpG plasmids are provided with the E. coli GT115 strain, a pir mutant also deficient in Dcm methylation.
1. Wu F. et al. 1995. A DNA segment conferring stable maintenance on R6K gamma-origin core replicons. J Bacteriol. 177(22):6338-45.
2. Carswell S. & Alwine JC., 1989. Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol. Cell Biol. 10: 4248-4258.
3. Bode J. et al., 1996. Scaffold/matrix-attached regions: topological switches with multiple regulatory functions. Crit Rev Eukaryot Gene Expr. 6(2-3):115-38.