Plasmids are closed circles of double-stranded DNA,
ranging in size from 1 to 200 kilobases, found in many bacteria and in a few eukaryotic cells. They frequently carry genes
conferring antibiotic resistance; infective drug resistance, originally discovered in Shigella, is due to plasmids. Plasmids are
widely used as carriers of cloned genes, for example the E.
coli plasmid pBR322, and numbers of such plasmids may be
amplified by treating the donor cells with chloramphenicol,
which stops host cell DNA synthesis but not that of the plas_x0002_mid. Some bacteriophages may exist either as integrated or
free copies in the cell, and either case can be termed plasmids.
Yeast cells have been found to harbour some plasmids, and the
Ti plasmid of Agrobacterium can survive in either bacterial or
plant cells.
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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.
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Expression in bacterial cells. The RecA promoter is a very strong constitutive promoter. It is approximately 650 x stronger than the constitutive AraBAD promoter that we also sell. The RecA promoter is normally regulated by the repressor LexA. In this promoter this binding site has been ablated to enable constitutive expression. This plasmid is used when the T7 terminator and the SV40 poly-adenylation sites that are found within most of our vectors are not required.
Promoter Expression Level: This plasmid contains a constitutive bacterial promoter that does not require induction. It is the strongest bacterial promoter we sell and this can cause solubility and expression problems with some proteins. We also offer a range of other bacterial promoters that are compatible with this plasmid and are available on request.
