Plasmid Info:

Plasmid Purpose:

This plasmid is designed to express tagged proteins in E.coli. The plasmid contains a constitutive promoter (OXB20) derived from the region upstream of the E.coli RecA gene. It does not require induction or any additional components for activity. It is the strongest of the bacterial promoters that we provide and this high level of expression can cause expression problems with some proteins with poor solubility. For this reason we sell a range of bacterial promoters with different expression levels (OXB1(low)>OXB20(high)) that can be provided with the peptide tags in this plasmid on request.

This plasmid also encodes a protease cleavage site that is designed to be positioned between your gene of interest and the tag to allow the removal of the tag following protein purification or isolation. This plasmid contains a 3C cleavage tag. The protein sequence of the cleavage tag is: LEVLFQ?GP. Human Rhinovirus (HRV) 3C Protease is a highly specific protease that cleaves between the Glu and Gly residues of its recognition site. It is often produced with the trademname 'PreScission protease'.

For more information on which cleavage tag to use see our cleavage tag guide.

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.

FLAG is a registered trademark of the Sigma Aldrich Corporation.

This plasmid contains an n-terminal Flag epitope tag that can be fused to a gene of interest to allow protein detection and/or purification. The sequence of the tag is: DYKDDDDK

For more information on the methods that can be used to purify proteins please see our protein tag guide.

Sequence and Map:

Other Info:

This plasmid 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.

Cloning:

This plasmid has been designed to allow three types of cloning into the main MCS to join a coding sequence with the tag.

If you would like to fuse your coding sequence to the tag with minimal additional bases you can use our SnapFusion technology. This process involves amplifying your gene by PCR to add specific restriction sites onto the ends. When these sites are cut they produce an overhang that is compatible with this plasmid cut with BseRI or BsgI.

1: Amplify your gene with primers designed using this spreadsheet
2: Cut the plasmid with either BseRI or BsgI.*
3: Cut your gene with the enzyme you added using the spreadsheet (any of AcuI BpmI BpuEI BseRI BsgI EciI).
4: Clone the gene into the plasmid using DNA ligase.

Using this method with an N-terminal tag plasmid will result in the tag coding sequence immediately followed by your genes ATG start codon at the join. This results in a seamless fusion of the two sequences with no extra bases being added. Using this method on C-terminal tag plasmids will convert your genes stop codon into a TAC (Tyr Y) codon followed by the plasmid tag coding sequence. This results in no extra bases between your gene and the tag. See the diagram below for more information.

*Please note that insect expression plasmids cannot be cut with BsgI only BseRI because of unavoidable conflicting sites in the backbone. Also Yeast plasmids cannot be cut with BseRI because of unavoidable restriction sites in the backbone.

Using this technique will create a gene fragment that can be ligated into any or our >1500 peptide and reporter tag plasmids. If you use one of the other techniques below (Gibson InFusion Seamless or LIC) you will need new primers for every vector you clone into because the arms of homology will change according to the tag plasmid you are cloning into.

If you find that your gene sequence has sites in it that make using this cloning strategy difficult you can still use one of the alternative methods below (e.g. standard cloning or Gibson cloning).

Open the Primer Design Tool to help you design primers for cloning your gene in our SnapFusion technique.

If you are not concerned about leaving a few extra bases between the tag coding sequence and your gene you can clone your gene into the vector using standard cloning restriction enzymes. This strategy will require you to choose which enzymes you want to use to clone your gene.

Open the Primer Design Tool which provides primers with different enzyme choices positioning your gene as close to the tag as possible in each case. Please note that standard enzymes will always leave additional nucleotides between your gene and the tag but using the spreadsheet will ensure the tag and gene are in frame.

These cloning techniques use reagents sold by other companies and allow you to fuse sequences together using enzymes that chew back the DNA to leave overlapping ends/overhangs. The subsequent method of joining the DNA depends on the kit used. To use one of these techniques you can either design your own primers or you can use the spreadsheet below to help with the design.

Open the Primer Design Tool to help you design primers for cloning your gene using Gibson assembly InfusionHD GeneArt Seamless cloning or Ligase Independent Cloning (LIC) techniques.

IP Status:

This product is part of our SnapFast plasmid range, for more information on the Intellectual property status of this plasmid please click here

Oxford Genetics公司位于英国牛津，由来自生物各领域中具有多年基因工程经验的专家组建而成，专注于为全球生物不同领域的科研用户和生物技术工业用户提供多种DNA表达载体。 其所有产品基于一个中心质粒骨架(SnapFast )SnapFast 载体经过精心设计，不同的构建中配以不同的DNA元件，在特异酶切位点更换需要的元件，可进行上千种改造，满足各种生物学研究的需要。该公司还为科学家提 供万能克隆平台和DNA插入匹配库，有针对哺乳动物，细菌和噬菌体等多个系统的各种元件，包括启动子、报告基因、抗性标记、标签蛋白、核糖体位点、复制起 点、多克隆位点、融合蛋白、信号肽、终止子及其他DNA元件(SV40原点、P2A肽段、CMV增强子、小启动子等)。所有的元件都可以在不同载体间互换 使用，并且和很多市场上应用的克隆载体和穿梭载体都兼容，利于基因片段的转移。该系统非常灵活、高效、经济、知识产权友好，可解决目前基因工程研究中普遍 存在的克隆问题。