PSF-MINCMV-BLAST - BLASTICIDIN SELECTION MINIMAL CMV VECTOR

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Cloning in a gene: This plasmid contains a gene within the main multiple cloning site (NotI-ClaI). Any plasmid that we sell where the gene is this configuration will be located in the exact same position in relation to the start and stop codon of the gene. The only exceptions to this rule are fusions proteins where the fusion gene may be positioned at the front or end of the MCS to allow gene fusion.

By positioning all of our genes in the same location it allows them to be transferred between plasmids using the same cloning method and restriction sites regardless of the plasmid being used from our product range. Inserting a new gene into this plasmid should be easily possible using a range of standard restriction enzyme sites that flank the gene currently in the vector.

Multiple cloning site notes: In the multiple cloning site there are two important restriction sites called BsgI and BseRI sites. These sites both cut the DNA at the same position and cleave the stop codon of the gene in the multiple cloning site in this plasmid thereby producing a TA overhang. This overhang is compatible with any of our peptide or reporter fusion tag plasmids also cut with either of these enzymes. This allows seamless C-terminal fusions to be made with the gene in this multiple cloning site using a single cloning step from our C-terminal peptide and reporter tag product range. Normally the easiest method is to clone the C-terminal tag from our other plasmid products into this plasmid using BsgI or BseRI and the downstream ClaI restriction site.

BseRI and BsgI sites are non-palindromic and cleave a defined number of bases away from their binding sites. This allows them to cut the upstream stop codon in the gene in this plasmid regardless of the gene sequence.

Here the blasticidin resistance gene is under transcriptional regulation of the minimal CMV promoter giving only low level expression in most mammalian cells. Expression of the resistance gene can be regulated by inserting additional regulatory components upstream of the promoter using a specially-positioned MCS for example sites that respond to specific transcription factors or to externally-applied drugs such as doxycycline. In this way sophisticated constructs can be prepared that provide blasticidin resistance only under certain pre-defined conditions.

Promoter Expression Level: This plasmid contains the minimal CMV immediate early promoter with a multiple cloning site immediately upstream that allows transcription factor binding sites to be inserted. This allows tissue specific or physiologically responsive promoters to be created.

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