Analysis Note

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

Application

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.

General description

This plasmid contains a Puromycin (Puro) resistance expression cassette under the transcriptional control of the Ubiquitin (Ub) promoter that is designed as an add-on to any of our other plasmids. The Ub Puro cassette is flanked by AscI sites but is not designed to be split up. If you would like the Puromycin gene alone or the Ub promoter alone please see the mammalian antibiotic selection gene section or the promoter section on our website. This plasmid can be used to force recombination into the genome of mammalian cells following transfection and selection although this method can be inefficient. It can also be used conjunction with the SV40 origin of replication in cells expressing the SV40 large T antigen (such as 293T cells) to make stable cell lines following transfection and selection. 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.

Sequence

To view sequence information for this product, please visit the product page