
Plasmid Info:
Plasmid Information
Product Name: pSF-CMV-Puro-NH2-STag-EKT
Product Code: OG1163
Size (bp): 6209 bp
Bacterial Antibiotic Selection: KanR
Origin and Compatibility: pUC high copy derived from pBR322
Bacterial Copy Number: 500-700 per cell
Promoter: Cytomegalovirus (CMV) immediate early promoter / Human Ubiquitin Promoter
Plasmid Purpose:
This plasmid is designed to express tagged proteins in mammalian cells either by transient transfection or by creating stable cell lines. It contains a puromycin resistance expression cassette using the human Ubiquitin promoter to drive expression and allow for the selection of cells containing the plasmid.
About the Cleavage Tag: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 EKT cleavage tag. The protein sequence of the cleavage tag is: DDDDK. Enterokinase (EKT) protease cleaves after the Lysine residue. It can cleave at other basic residues but this is dependent on protein confirmation. If a proline follows the site it will not cut. None of our products contain a proline after the site.
For more information on which cleavage tag to use see our cleavage tag guide.
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. For this reason we stock a range of other promoters that are compatible with this plasmid and are available on request.
This plasmid contains an n-terminal STag epitope tag that can be fused to a gene of interest to allow protein detection and/or purification. The sequence of the tag is: KETAAAKFERQHMDS
For more information on the methods that can be used to purify proteins please see our protein tag guide.
Sequence and Map:
Other Info:
Transcription Termination: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:
Making Protein Fusions:This plasmid has been designed to allow three types of cloning into the main MCS to join a coding sequence with the tag.
1: SnapFusion Cloning: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.
To insert your gene:
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.
2: Standard Enzymes: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.
3: Gibson cloning/InfusionHD/GeneArt Seamless/Ligase Independent Cloning (LIC) Methods:
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:
Intellectual Property StatusThis product is part of our SnapFast plasmid range, for more information on the Intellectual property status of this plasmid please click here
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①在荧光显微镜下,用波长约490 nm的紫外线激发后,即可观察到绿色荧光,直接、简捷、便于检测;
②无需任何的作用底物或共作用物,检测的灵敏度不受反应效率的影响,保证了极高的检出率;
③蛋白本身性质稳定;
④可在多种异源生物中表达且无细胞毒性;
⑤其基因片段长度较小(约717 bp),易于构建融合蛋白,且融合蛋白仍能保持荧光激发活性,为研究其他基因表达产物的分布提供了方便。
pEGFP是一种优化的突变型GFP,使其产生的荧光较普通GFP强35倍,大大增强了其报告基因的敏感度。pEGFP与其他蛋白的融合表达已有很多成功的例子,而且其N及C端均可融合,并不影响其发光。
pEGFP-N1载体上携带有EGFP蛋白表达基因,如上图质粒图谱所示。
pEGFP-N1载体具有以下几方面特点:
①从结构上看,该质粒具有很强的复制能力,可以满足随宿主细胞分裂时跟随胞质遗传给新生的子细胞,这是保证目的基因稳定表达的因素之一;
②含有高效的功能强大的启动子SV40和PCMV,可以使目的基因在增殖的细胞中稳定表达;
③具有多克隆位点,便于目的基因的插入;
④该载体具有neo基因,可以采用G418来筛选已成功转染了该载体的靶细胞。
这些特殊的结构可以实现目的基因在靶细胞内的稳定表达。向左转|向右转
完整的表达载体必须包括:
1、复制子,在细菌中扩增时所必须.
2、启动子,目的基因在细菌或细胞中转录所必须,转录了才能翻译,是谓“表达”.
3、原核筛选标记,细菌增菌所必须.
4、真核筛选标记,如果是真核表达,就是必须的.
5、多克隆位点,即酶切位点,插入目的片段的区域.
6、其他所需构件,可视实验设计情况而选用现成载体或在载体上自行添加.
另:表达的起始和终止,在目的基因上附带起始密码子和终止密码子.
(forced expression)或过表达(overexpression),以观察基因表型,反义则是用来抑制基因表达,以观察在目的基因的表达受到抑制的情况下表型的变化
请教一下,一般做启动子报告基因检测,载体用pGL3,pGL4双荧光报告基因质粒,但pGL3,pGL4都只能做瞬时转染,我的细胞(不想用293做实验)质粒转染后都会死很多,还要加上一些处理因素(比如药物,饥饿等),细胞状态更不好了。所以想找一个慢病毒骨架的报告基因质粒。
上网查了下,SBI的pGreenFire1和Genecopia的Gluc-ON是慢病毒质粒。
1.pGreenFire1可以检测Luc和GFP,但用什么做内参呢?查了下文章,有的文章检测Luc的时候同时检测一个MTS读数做内参;另外有一篇文章用qPCR的方法检测了GFP和GAPDH,用GAPDH做内参。不确定这样是不是正确呢?
2.Gluc-ON检测的是分泌型的Gluc,载体上还有分泌型的碱性磷酸酶做内参。
不知有没有哪位以前用过这两个质粒的?文章用这两个质粒的都不多,不知投稿的时候会不会被质疑?
首先需要构建dsRNA表达载体
将这种载体导入受体细胞中后
表达产生的dsRNA在DICER酶作用下形成siRNA
引起具有相同序列的mRNA发生讲解
导致细胞或个体不能合成相应的蛋白质
所以个体会表现出功能缺失表型
构建表达载体通常选用dsRNA
需要注意的一些方面是
dsRNA序列中GC的含量要小于50%
高GC含量会降低RNAi的效果
选定的dsRNA序列应通过搜索数据库确保与其他基因无同源性
以避免对其他同源性基因表达的抑制
不同区域的dsRNA具有不同的基因沉默效果
可同时构建两个以上针对同一基因不同靶区域的dsRNA表达载体
还要充分考虑siRNA的结构特征
siRNA与mRNA的同源程度对RNAi有明显影响
启动子区或者编码区与siRNA同源的基因受siRNA抑制
但siRNA在动物细胞中对mRNA的前体没有影响
所以含非编码区序列的dsRNA不会引起RNAi
而且在构建表达载体时
经常使用U6启动子等RNA聚合酶Ⅲ能够识别的启动子序列
最后将其转入到质粒中
这里说的只是一个简单的过程
总的来说构建表达载体是个比较复杂的过程
如果要知道详细的技术
建议还是去看书吧
这里是说不清的
RNA干扰主体实验的重点在于: 按照nature的标准,一个严格的RNAi介导knockdown实验要有6个对照:
⒈转染试剂对照(监控转染及培养条件对结果的影响);
⒉nonsense siRNA对照(监控外源核酸本身对结果的影响);
⒊positive siRNA对照(监控假阴性);
⒋技术重复对照(也叫off-target 对照,也就是利用至少2个靶点的siRNA同时实验,2个siRNA互为off-target control,当两者的表型相同时,才有可能是特异性的knockdown效应);
⒌rescue 对照(knockdown之后做超表达,看是否有性状的逆转,这也是为了说明knockdown的特异性);6.全表达组扫描对照(就是转录/表达芯片扫描,以最终确定表型不是由于off-target造成)。
实际实验中,全表达组扫描对照很少有文献涉及。其它几个对照中,1,2两种对照即所谓的空白细胞对照、NC对照,基本是所有杂志都要求具备的。4,5两种对照,主要是为了解决off-target效应,选做一种即可,一般建议选5,涉及的实验即所谓的“RNA干扰回复实验”, 一般应该从mRNA水平、蛋白质水平、细胞表型水平三个层次来检测干扰效率。
mRNA水平:RT-PCR、Real-time PCR;蛋白质水平:Western-blot、ELISA、免疫组化;细胞表型水平:MTT、克隆形成实验、流式细胞检测、细胞小室实验等。RNA干扰效率在动物模型上的进一步验证(体内) 动物模型实验可以采取“体内法”和“体外法”。
体内法,即先做裸鼠成瘤模型,再将质粒或病毒导入裸鼠,检测RNA干扰效果。此法操作复杂,对质粒和病毒产品的质和量要求都较高,但是比较贴近实际,说服力较显著。体外法,即先将质粒或病毒导入肿瘤细胞,再将肿瘤细胞导入动物体内,然后检测RNA干扰效果。此法操作较简单,对质粒和病毒产品的质量要求较低,所以为大多数文献所采用。建议采用此方法来进行动物模型水平的实验。向左转|向右转
第二个问题是不是构建的启动子区均要有一个TATA盒的结构在里面,不然构建的启动子如何就能驱动LUC的表达呢?
第三个问题是,如果我们在构建时长度延至ATG之后100-200BP的启动子是否会影响LUC的表达呢?
谢谢!


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