Description_x000D_ General description_x000D_ In this plasmid the beta galactosidase reporter gene is under transcriptiona l control of the strong mammalian CMV promoter and puromycin resistance is expressed from a separate expression cassette under control of the strong mammalian RSV promoter._x000D_ Promoter Expression Level: This plasmid contains the mammalian CMV promoter to drive gene expression. We have tested all of our mammalian promoters in a range of cell types and CMV is consistently the strongest in those we have studied. However there are many reports of the CMV promoter demonstrating silencing by methylation in long-term culture. For this reason we stock a range of other promoters that are compatible with this plasmid and are available on request. The RSV promoter driving reporter gene expression is one of the weakest mammalian promoters that we provide for many cell types but expression can vary significantly between cell types from different species. For example we have found that the RSV promoter shows significantly lower expression levels in human 293 cells when compared to hamster CHO cells._x000D_ Application_x000D_ 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._x000D_ 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 sIt`s 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 sIt`s that flank the gene currently in the vector._x000D_ Multiple cloning site notes: In the multiple cloning site there are two important restriction sIt`s called BsgI and BseRI sIt`s. These sIt`s 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._x000D_ BseRI and BsgI sIt`s are non-palindromic and cleave a defined number of bases away from their binding sIt`s. This allows them to cut the upstream stop codon in the gene in this plasmid regardless of the gene sequence._x000D_ Sequence_x000D_ Quick-reference Plasmid Map_x000D_ 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._x000D_ Genebank Vector Sequence File_x000D_ FASTA Vector Sequence File_x000D_ Full Plasmid Map_x000D_ Analysis Note_x000D_ To view the Certificate of Analysis for this product, please visit www.oxfordgenetics.com._x000D_ Other Notes_x000D_ Looking for more vector options to move your experiments forward faster? Consider a custom cloning vector designed and built by Oxford Genetics™. Find out more at Oxford Genetics - Sigma's partner for cloning and expression vectors for molecular biology and synthetic biology applications._x000D_ Legal Information_x000D_ Oxford Genetics is a trademark of Oxford Genetics Ltd