pCpGfree is a family of plasmids completely devoid of CpG dinucleotides. Typically, the elements required for replication and selection of the plasmid in E. coli and gene expression in mammalian cells are rich in CpG. In the pCpGfree plasmids these elements are either naturally CpG-free, were modified to remove all CpGs, or entirely synthesized.Specifications
• pCpGfree-mcs contains a multiple cloning site (MCS) featuring several commonly used restriction sites, for convenient cloning of a CpG-free gene, such as the genes provided in the pSELECT plasmid or any open reading frame or cDNA.
• pCpGfree-LacZ expresses the lacZΔCpG gene, a CpG-free allele of the LacZ gene.
• pCpGfree-mSEAP expresses the mSEAPΔCpG gene, a CpG-free allele of the mouse secreted embryonic alkaline phosphatase gene.
• pCpGfree-Lucia expresses a CpG-free allele of the novel synthetic secreted luciferase (Lucia) gene.
Selectable in E. coli with Zeocin™
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CpG-free Plasmid Backbone
pCpGfree is a family of plasmids completely devoid of CpG dinucleotides. Typically, the elements required for replication and selection of the plasmid in E. coli and gene expression in mammalian cells are rich in CpG. In the pCpGfree plasmids these elements are either naturally CpG-free, were modified to remove all CpGs, or entirely synthesized.
• Origin of replication: The E. coli R6K gamma ori has been modified to remove all CpGs. This origin is activated by the R6K specific initiator protein π, encoded by the pir gene .
• Bacterial promoter: EM2K is a CpG-free version of the bacterial EM7 promoter.
• Mammalian promoter: The CpG-free promoter combines the mouse CMV enhancer, the human elongation factor 1 alpha core promoter and 5’UTR containing a synthetic intron.
• Polyadenylation signal: The polyadenylation signal is a CpG-free form of the late SV40 polyadenylation signal.
• MAR: Matrix attached regions (MARs) are sequences typically AT-rich that are able to form barriers between independently regulated domains . pCpG plasmids contain two MARs, from the 5’ region of the human IFN-β gene or β-globin gene that were chosen because they are naturally CpG-free. The MARs are placed between the bacterial and mammalian transcription units.
• Selectable marker: The Zeocin™ resistance gene is a small gene (<400 bp) that contains numerous CpG dinucleotides. A synthetic new allele was created that contains no CpGs.
Furthermore, all Dam methylation sites (GATC) have been removed to prevent prokaryotic methylation.
Provided with the E. coli GT115 Strain
Due to the presence of the R6K gamma origin of replication, pCpGfree plasmids can only be amplified in an E. coli mutant strain expressing a pir mutant gene. They will not replicate in standard E. coli strains. Therefore, pCpGfree 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. 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.
MCS: 5’- Bsr GI, Bgl II, Acc65 I, Eco O109I, Bsp 120I, Nco I, Nhe -3’