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IRES Expression Plasmids

OG185 - pSF-T7-EMCV


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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.

Product Code: OG185

Size (bp): 4378

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility: pUC high copy, derived from pBR322   

Copy Number: 500-700 copies per cell

Promoter: T7 bacteriophage Promoter

Purpose: This vector allows transcription of un-polyadenylated and un-capped RNA using the T7 bacteriophage polymerase. This plasmid also contains the internal ribosome entry site (IRES) from Encephalomyocarditis virus (ECMV) between the promoter and the multiple cloning site (MCS). It is designed to be used to express genes using eukaryotic T7 expression systems, such as those used for the recovery of a variety of viruses. This vector is not designed for the expression of two proteins from the same mRNA. For this purpose, we have designed pSF-CMV-MCS-IRES-PciI, where the PciI site contains a start codon in the correct position for the IRES to express efficiently. In this vector there is an NcoI site (which contains an ATG start codon) in the correct position to enable efficient expression of a gene in the MCS.    

Transcription Termination: This plasmid contains three alternative transcription terminators for mammalian, bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.

Inserting a Gene: Each restriction site in this vector has a purpose and allows the insertion of specific pre-designed DNA sequences. To insert a gene, we recommend using any sites between (and including) the NcoI and XbaI sites. By doing this you will be able to insert most of our other products upstream, or downstream, of your gene. Click here to read more about the purpose of each restriction site.

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

Quality Validation: This vector has been demonstrated to express reporter genes to high levels following transfection of the plasmid into T7 expressing mammalian cells. The start codon of these reporters was positioned within the NcoI site and the stop codon was positioned within the XbaI site.

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:

  • 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 SnapFast system and to retain a high level of antibiotic resistance. It has been validated in E.coli.

Restriction site notes:

  • Bgl2, KpnI, SwaI and PmeI each cut the vector at two sites positioned to flank the promoter, start codon, origin, and KanR, respectively.
  • 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.
  • ECMV IRES – The IRES has been modified to remove a BseRI site, however, it still retains a PciI, HindIII and a KpnI site. Structural predictions suggest that the HindIII and KpnI sites cannot be ablated, however, the normal functions of these two sites in the SnapFastTM vector are redundant in this plasmid because of the positioning of the IRES. We are attempting to ablate the PciI site. If this does not reduce activity, this plasmid will eventually be replaced by the PciI ablated version.