PSF-TEFI-TPI1-DAGFP-URA3 - GFP YEAST REPORTER PLASMID

A green fluorescent reporter vector expression in Saccharomyces cerevisiae yeast cells. The plasmid is designed to enable the co-expression of a gene of interest alongside the GFP reporter. The plasmid is selected for in yeast using the URA3 cassette which allows stable maintanence provided that the cells are grown in uracil deficient media and the cell line is defective for the URA3 gene. The GFP reporter was developed by DNA2. 0 and is called Dasher GFP. The promoter driving GFP expression (TPI1) is of similar strength to the TEF-1 promoter that is upstream of the main multiple cloning site.

Promoter Expression Level: 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).

Cloning in a gene: 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.

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.

Please select the file type you require. For reference most cloning programs will import a .gb (Genbank) file and will show all of the plasmids features automatically when downloaded and imported.

Genebank Vector Sequence File

FASTA Vector Sequence File

Full Plasmid Map

To view the Certificate of Analysis for this product, please visit www.oxfordgenetics.com.

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