PSF-TEFI-CEN6/ARS4-URA3 - LOW COPY ORIGIN YEAST PLASMID

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Cloning in a gene: PSF-TEFI-CEN6/ARS4-URA3 - LOW COPY ORIGIN YEAST 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.

This is a low copy Saccharomyces cerevisiae yeast plasmid that allows for the plasmid to be maintained in cells at an approximate frequency of 1-2 copies per cell. These plasmids are not typically used for protein production because expression is signficantly lower than using the standard 2 micron origin which has a much higher copy number. This plasmid can still be used when large DNA inserts are required and low levels of expression of genes are needed. The plasmid contains the URA3 selection cassette which allows the plasmid to be maintained in cells that are defective for this gene. Cells carrying the plasmid can then be grown in media that is deficient in uracil.

Promoter Expression Level: This expression vector contains the yeast translation elongation factor 1 promoter. It is the strongest promoter that we provide for expression in Saccharomyces cerevisiae.

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