Form: buffered aqueous solution
Origin of replication
pUC (500 copies)
- Promoter name: CMV
- Promoter activity: constitutive
- Promoter type: mammal
Reporter gene location
- 2nd promoter
Reporter gene: GFP
Sent in: environment
Storage temperature: −20 ° C
Molecular cloning often benefits from the optimization of the vector used for expression. This package allows you to determine the best configuration for the green fluorescence protein reporter gene (a non-Aequorea variant called Dasher GFP) and to optimize expression for your specific needs. Each component plasmid contains the green fluorescent reporter regulated in a different way – in the MCS under the CMV promoter, under an IRES or regulated by its own chosen promoter, or regulated by independent promoters immediately downstream of the MCS (sharing a polyA with the gene inserted into the MCS) or in a different part of the plasmid.
This package should allow you to compare different strategies for green fluorescence expression and choose the one that best suits your needs. This set of plasmids has been designed to be compatible with a variety of cloning techniques. The multiple cloning site contains a variety of standard restriction sites commonly used for cloning. Using these sites, genes can be inserted using standard DNA ligase cloning methods. Other methods can also be used, such as Gibson Assembly InFusionHD Ligase Independent Cloning (LIC) or Seamless GeneArt and since all of our plasmids are based on the same backbone, the same method can be used for cloning in all of our vectors catalogue.
Notes on various cloning sites: There are some 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 a Kozak and Shine-Dalgarno ribosomal binding site. These allow optimal positioning of genes when the start codon is placed in this location. If it is not necessary and you want 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 placed at a specific position in relation to the BsgI and BseRI sites that are immediately downstream. When BseRI or BsgI cleave the plasmid, they produce a stop codon TA overhang at the XbaI site that is compatible with all of our peptide tag plasmids cut with the same sites. The BseRI and BsgI sites are non-palindromic and cleave a defined number of bases from their binding site. Whenever we clone a gene at our multiple cloning sites, we always place the start and stop codon at the same positions in the MCS. If the start and end of the genes are not compatible with NcoI and XbaI, we extend the sequence to the closest outer sites, but keep the start and stop codon locations consistent.
Transcription termination: These plasmids contain three alternative transcription terminators for the expression of bacteria and bacteriophages (T7) in mammals. This means that you only need to change the promoter to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in alternative systems.
To view the Certificate of Analysis for this product, visit www.oxfordgenetics.com.
Looking for more vector options to move your experiments forward faster? Consider a custom cloning vector designed and built by Oxford Genetics ™. Learn more at Oxford Genetics, Sigma’s partner for cloning and expression vectors for molecular biology and synthetic biology applications.
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