pcDNA3.1载体图谱及多克隆位点详细信息-Biovector中国质粒基因库
2014-02-17 17:06阅读：
中国质粒载体菌株细胞株基因保藏中心-Biovector Science
Lab,Inc.可提供数万种质粒载体、菌株、基因和细胞株,并可提供实验技术外包服务,如基因克隆、质粒构建、蛋白原核、真核表达及纯化、病毒包装、基因沉默RNAi等服务。作为美国质粒载体菌株细胞株基因保藏中心中国分库及唯一办事处，Biovector
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电邮Biovector@163.com
电话1890I268599
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Inc. 质粒 载体 索取
报价 价格 优惠 售价
出售 图谱 序列 酶切位点
转化 构建 求购 购买
订购sequence map plasmid vector gene strain order
ship 抗性 resistance restriction
site
使用pCDNA3.1进行载体构建需要注意的问题有：
1.pcDNA3.1（+）和pcDNA3.1（-）不是融合蛋白表达载体。你在进行载体构建设计的时候，插入的片段序列必须自带ATG起始密码子和TAG（或TGA，TAA）终止密码子。
2.pcDNA3.1载体在进行载体构建设计的时候必须包含Kozak序列。下面我们给出了一个针对pcDNA3.1的Kozak序列为(G/A)NN
ATG,需要注意的是其他的文献当中的Kozak序列也是可以的，但是在-3位的G或A和+4位的G对于蛋白的正确表达具有很重要的作用和意义。
pcDNA3.1(+) and pcDNA3.1(-) are
5.4 kb
vectors derived from pcDNA3 and
designed for high-level stable and transient expression in
mammalian hosts. High-level stable and non-replicative transient
expression can be carried out in most mammalian cells.
pcDNA3.1载体的序列包含以下几个部分：
*
Human cytomegalovirus
immediate-early (
CMV)
promoter
for high-level expression in a wide range of
mammalian cells
*
Multiple cloning
sites in the forward (+) and reverse
(-) orientations to facilitate cloning
*
Neomycin resistance
gene for selection of stable cell
lines
*
Episomal replication in cells
lines that are latently infected with
SV40 or
that express the SV40 large T antigen (e.g. COS-1, COS-7)
The control plasmid, pcDNA3.1/CAT, is included for use as a
positive control for transfection and expression in the cell line
of choice.
适用的菌株：
Many E. coli strains are suitable for the propagation of this
vector including
TOP10F′,
DH5α-T1 , and
TOP10. We recommend that you
propagate vectors containing inserts in E. coli strains that are
recombination deficient (recA) and endonuclease A-deficient
(endA).
使用pcDNA3.1载体的分子克隆
试验流程：
1.
Consult the multiple cloning
sites
to design a strategy to clone your gene into
pcDNA3.1.
2.
Ligate your insert into the
appropriate vector and transform into E. coli. Select transformants
on LB plates containing 50 to 100 μg/ml ampicillin.
3.
Analyze your transformants
for the presence of insert by restriction digestion.
4.
Select a transformant with
the correct restriction pattern and use sequencing to confirm that
your gene is cloned in the proper orientation.
5.
Transfect your construct into
the mammalian cell line of interest using your own method of
choice. Generate a stable cell line, if desired.
6.
Test for expression of your
recombinant gene by western blot analysis or functional
assay.
pCDNA3.1 质粒图谱和多克隆位点详细信息如下：
