pCpGfree-vitro Neomycin

PLASMID FEATURES
pCpGfree-vitro is a new family of expression vectors completely devoid of CpG dinucleotides that are selectable in mammalian cells. Similarly to the other pCpGfree plasmids (i.e. pCpGfree-lacZ, pCpGfree-mcs, and pCpGfree-siRNA), all the elements required for replication and selection of the plasmids in bacteria, and gene expression in mammalian cells have been modified to remove all CpG dinucleotides.

• Composite CpG-free promoter combining the mouse CMV enhancer, the human elongation factor 1α core promoter and 5’UTR containing a synthetic intron (I 126). This composite promoter yields high and ubiquitous expression of the gene cloned into the mcs.
• Multiple cloning site (MCS) or CpG-free allele of the lacZ gene. This reporter gene can be easily subcloned and replaced by a gene of interest.
• CpG-free polyadenylation signals (pAn): The polyadenylation signals utilized are CpG-free versions of the SV40 late and human β-globin polyadenylation signals. These polyA enable efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.
• CpG-free matrix attached regions (MARs) are AT-rich sequences that are able to form barriers between independent expression cassettes.
• CpG-free Neo resistance gene (Neo-∆CpG): The CpG-free Neo gene is active both in E. coli and mammalian cells and confers resistance to Kanamycin in E. coli and G418 in mammalian cells.
• CpG-free SV40 promoter works in tandem with a bacterial promoter located within a synthetic intron (I-EC2K). This composite promoter drives the expression of the resistance gene in both mammalian cells and E. coli.
• CpG-free E. coli R6K gamma origin of replication: This origin is activated by the R6K specific initiator protein π, encoded by the pir gene. Expression of the pir gene is necessary for the replication and amplification of pCpGvitro plasmids. E. coli GT115 strain expresses a pir mutant gene that allows higher plasmid copy number.