PSF-CMV-PGK-FLUC - DUAL PROMOTER LUCIFER

Description_x000D_ General description_x000D_ This plasmid contains two promoters that terminate transcription at the same poly-adenylation signal allowing the expression of two genes from one expression cassette where the second gene is the Photinus pyralis luciferase (Firefly FLuc) reporter gene. The second promoter (PGK) is approximately 10-fold weaker than the upstream CMV promoter in most commonly used cell lines._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._x000D_ Application_x000D_ 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 sIt`s for cloning. Using these sIt`s 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._x000D_ Multiple cloning site notes: There are a few important sIt`s within the MCS. These include the NcoI site the XbaI site and the BsgI and BseRI sIt`s. 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._x000D_ The XbaI site contains a stop codon. This stop codon is positioned in a specific position in relation to the BsgI and BseRI sIt`s 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 sIt`s. BseRI and BsgI sIt`s are non-palindromic and cleave a defined number of bases away from their binding site._x000D_ 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 sIt`s but keep the start and stop codons locations consistent._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