Origin of Replication Vector Set PP2378-1KT

NACRES:

NA.85

Molecular cloning often benefits from optimizing the vector used for expression.
This pack allows you to determine the optimum origin of replication for your bacterial experimental/cloning system. Four different bacterial origins are provided, each providing different plasmid copy number in bacterial cells. In general, low copy origins are used for very large plasmids, or where leakage of mammalian transgene expression may be toxic to the bacteria. The CMV promoter is available in each of these plasmids, to regulate expression in mammalian cells of any gene you insert into the MCS.This plasmid set 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.
Transcription Termination: These plasmids contains three alternative transcription terminators for mammalian bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed 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 the alternative systems.
Sequence
Analysis Note
These plasmids are sold free of reach-through rights and can be used to make commercial products. However the plasmids themselves (or derivatives) cannot be sold.

form	buffered aqueous solution
Origin of replication	BR322 (15 copies), p15A (10 copies), pUC (500 copies)
Promoter	Promoter name: CMV Promoter activity: constitutive Promoter type: mammalian
bacteria selection	ampicillin, kanamycin
shipped in	ambient
storage temp.	20°C

RIDADR	NONH for all modes of transport
Flash Point F	Not applicable
Flash Point C	Not applicable