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IRES Expression Plasmids

OG293 - pSF-CMV-FMDV-Neo/G418

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This plasmid is compatible with all of the other DNA sequences available on this site, which are all sold in the same backbone. This flexibility allows you to create any vector you require, with a variety of functions, simply by compiling the relevant sections from our product range.

Vector: pSF-CMV-FMDV-Neo/G418 

Product Code: OG293

Quantity Provided: 5µg

Size (bp): 5511                 

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility:        pUC high copy, derived from pBR322   

Copy Number:             500-700 copies per cell

Promoter: Cytomegalovirus (CMV) immediate early promoter. 

Purpose: This vector allows the expression of two genes from one vector, where the second gene produced from the mRNA is the neomycin (G418) (G418) antibiotic resistance gene (Neo) under the control of the internal ribosome entry site (IRES) from Encephalomyocarditis virus (ECMV). Transcription is driven by the CMV promoter. There is a multiple cloning site immediately downstream of the CMV promoter which is then followed by the IRES element driving the Neo gene.     

G418 Selection notes: The neo gene induces resistance to kanamycin, neomycin and G418 in bacterial cells, and G418 in mammalian cells. Neomycin cannot be used to select eukaryotic cells, because it is ineffective at inhibiting eukaryotic ribosomes. For this reason, the plasmid must be selected in mammalian cells using G418 (otherwise known as Geneticin). In addition the neo gene is under IRES control and is not designed to express in bacteria, hence selection in bacteria should use kanamycin, with resistance conferred by the gene encoded between the PmeI sites (labelled KanR).  

Quality Validation: This vector has been demonstrated to express reporter genes to high levels under the CMV promoter, and express Neo to high levels under the control of the downstream IRES. The level of expression of the second gene (Neo) is significantly lower (approximately 30-40-fold) than the level of expression achieved from the CMV promoter using cap-dependent mechanism. However, the level of expression from the IRES under the CMV promoter in this vector is still higher than many other constitutive promoters, such as SV40, using cap-dependent translation mechanisms in a range of cell lines. The reporter genes under the CMV promoter were inserted into the NcoI site and XbaI site, with the start codon and stop codon residing within each of these sites, respectively. The Neo resistance gene placed under the control of the IRES was inserted into the PciI site and the NheI site of pSF-CMV-FMDV-PciI. Reporter gene expression and antibiotic resistance was validated in 293 cells.   

Quality Sequencing Analysis: This vector has been fully sequenced using primers F1-F10. The sequences and details of these primers are available through our website.

Genetic Modifications to standard parts: 
•    CMV promoter – An NcoI site has been ablated in the CMV promoter. This change does not reduce expression. 
•    KanR cassette – This KanR resistance region is not a wild type sequence, it has been modified to remove all restriction sites that conflict with the SnapFastTM system and to retain a high level of antibiotic resistance. It has been validated in E.coli.  
•    Neomycin (G418) resistance Gene – The Neo/G418 gene has been modified to remove NcoI, EagI, PstI and SphI sites to ensure compatibility with all of our products. These are all conservative changes and do not change the amino acid sequence.   

Restriction site notes: 
•    Bgl2, SwaI, and PmeI each cut the vector at two sites positioned to flank the promoter, origin, and KanR, respectively. 
•    The FMDV IRES contains a range of sites that conflict with the SnapFast system, including KpnI (2 additional sites) XbaI (1 site), Eag1 (1 site), Hind3 (1 site) and BseRI (1 site). It is unlikely that all of these sites can be removed because of the complex structural nature of the IRES RNA.
•    BsgI and BseRI recognise non-palindromic DNA sequences and cleave upstream of their recognition sites, within the stop codon that is found inside the XbaI restriction site. However, BseRI also cuts within the IRES in this plasmid.