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IRES Expression Plasmids



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Product Code: OG298

Quantity Provided: 5µg

Size (bp): 5468

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 chloramphenicol acetyl transferase (CAT) reporter gene under the control of the internal ribosome entry site (IRES) from Encephalomyocarditis virus (EMCV). 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 CAT reporter gene.   

Quality Validation: This vector has been demonstrated to express reporter genes (other than CAT) to high levels under the CMV promoter, and express CAT to high levels under the downstream IRES. The level of expression of the second gene (CAT) is significantly lower (approximately 20-30-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 CAT reporter gene placed under the control of the IRES was inserted into the PciI site and the NheI site of pSF-CMV-EMCV-PciI. Reporter gene expression 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.

Intellectual Property Status: According to our IP-friendly policy, this plasmid is sold free of reach-through rights on any derivatives you may create.

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.  
  •     EMCV IRES – The IRES has been modified to remove a BseRI site and a PciI site, however, it still retains HindIII and a KpnI sites. Structural predictions suggest that the HindIII and KpnI sites cannot be ablated without significantly modifying the hairpin structure of the IRES.  
  •     CAT Reporter Gene – The CAT gene has been modified to remove an NcoI site and an EcoRI site to ensure compatibility with all of our products. These changes did not alter the amino acid sequence of the CAT protein.

Restriction site notes:

  •     Bgl2, SwaI, and PmeI each cut the vector at two sites positioned to flank the promoter, origin, and KanR, respectively. KpnI cuts at three positions in this plasmid.
  •     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.