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OG39 - pSF-T3



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Sequence (txt)

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: OG39

Size (bp): 3862

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility: pUC high copy, derived from pBR322

Copy Number: 500-700 copies per cell

Promoter: T3 Promoter

Purpose: This plasmid allows transcription of un-polyadenylated and un-capped RNA using the T3 bacteriophage polymerase. Some in vitro transcription kits will enable the production poly-adenylated and capped RNA using this vector; however, this will depend on the kit used. SnapFastTM vectors do not contain an T3 terminator and so to prevent the polymerase from circling the plasmid it must be cleaved at a restriction site that is 3’ to the end of your gene to allow run-off of the polymerase. If you are expressing your gene in a vaccinia virus-free mammalian T3 system it will require an IRES upstream of your gene because of the absence of a 5’ cap on the RNA produced.

Transcription Termination: This plasmid does not contain a T3 transcription terminator. It will require linearisation at a restriction site that is downstream of your gene in order to allow transcription to stop. If producing RNA by in vitro transcription a polyA tail can be added using a commercially available polyA tailing kit. 

This plasmid does contain 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 HindIII and XbaI sites. By doing this you will be able to insert most of our other products upstream, or downstream, of your gene. 

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 plasmid has been demonstrated to express reporter genes to high levels following T3 in vitro transcription. Transcribed RNA was then capped and poly-adenylated, and transfected into the lung carcinoma cell line, A549. 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 plasmid 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.