Plasmid Info:

Plasmid Purpose:

A cyan fluorescent protein (CFP) reporter gene plasmid that allows for the expression of a transgene alongside the CFP gene. The CFP variant in this plasmid was developed by DNA2.0 and is called Frosty CFP. Expression of the reporter gene is mediated by the TPI promoter whilst the expression of a gene of interest would be controlled by the TEF1 promoter. These are both endogenous yeast promoters that require no induction for activity. They demonstrate approximately equal expression levels. The plasmid also contains the URA3 selection cassette which encodes an essential component for uracil synthesis. This means the that plasmid can be maintained in media that is deficient in uracil using yeast strains that are defective for the URA3 cassette.

This plasmid contains the yeast translation elongation factor 1 promoter. It is the strongest promoter that we provide for expression in Saccharomyces cerevisiae. It also contains the strong yeast constitutive triosephosphate isomerase (TPI1) gene promoter to drive the expression of the reporter gene. The TPI promoter demonstrates similar levels of expression to the translation elongation factor 1 promoter (TEF-1).

Sequence and Map:

Other Info:

This plasmid contains three alternative transcription terminators for yeast 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.

Cloning:

This 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.

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.

IP Status:

This product is part of our SnapFast plasmid range, for more information on the Intellectual property status of this plasmid please click here