PSF-T7-T7 TERM - T7 SINGLE TERMINATOR PLASMID

PSF-T7-T7 TERM - T7 SINGLE TERMINATOR PLASMID allows transcription of RNA using the T7 bacteriophage polymerase. If you using an in vitro transcription kit the RNA produced from this vector will be uncapped and poly-adenylated making it unsuitable for mammalian cells however some kits are available that will add these features. This plasmid can also be used for inducible gene expression in bacterial cell lines that express the T7 polymerase.

This expression vector contains the T7 terminator only whereas most of our vectors contain mammalian T7 and bacterial termination signals together. This plasmid is normally only used if the presence of the mammalian and bacterial terminators is undesirable.

This molecular cloning vector can also be digested at a restriction site that is 3 prime to the end of your gene to allow run-off of the polymerase rather than exploiting the hairpin T7 terminator. If you are expressing your gene in a Vaccinia virus-free mammalian T7 system it will require an IRES upstream of your gene because of the absence of a 5 prime cap on the RNA produced please see OG85: pSF-T7-EMCV if this is required.

Cloning in a gene: PSF-T7-T7 TERM - T7 SINGLE TERMINATOR PLASMID has been designed to be compatible with a range of cloning techniques. The multiple cloning site contains a range of standard commonly used restriction sites for cloning. Using these sites genes can be inserted using standard cloning methods with DNA ligase. Other methods such as ligase independent cloning (LIC) Gibson Assembly InFusionHD or Seamless GeneArt can also be used and because all of our plasmids are based on the same backbone the same method can be used for cloning into all of our catalogue vectors.

Multiple cloning site notes: There are a few important sites within the MCS. These include the NcoI site the XbaI site and the BsgI and BseRI sites. The NcoI site contains a start codon that is immediately downstream of both a Kozak and Shine-Dalgarno ribosomal binding site. These allow for optimal positioning of genes when the start codon is placed in this location. If this is not required and you wish to use a downstream site for gene cloning you can remove the NcoI site by cleaving the plasmid with KpnI.

The XbaI site contains a stop codon. This stop codon is positioned in a specific position in relation to the BsgI and BseRI sites that are immediately downstream. When either BseRI or BsgI cleave the plasmid they produce a TA overhang from the stop codon in the XbaI site that is compatible with all of our peptide tag plasmids cut with the same sites. BseRI and BsgI sites are non-palindromic and cleave a defined number of bases away from their binding site.

Whenever we clone a gene into our multiple cloning site we always position the start and stop codon in the same positions in the MCS. If the start and ends of the genes are not compatible with NcoI and XbaI we extend the sequence to the nearest external sites but keep the start and stop codons locations consistent.

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