pCMV-CREB

pCMV-CREB

载体基本信息

出品公司:	Ybscience
载体名称:	pCMV-CREB 、 CREB Dominant-Negative Vector
质粒类型:	信号通路分析载体；显性抑制载体
克隆方法:	--
启动子:	CMV
载体大小:	4.9kb
5' 测序引物及序列:	--
3' 测序引物及序列:	--
载体标签:	--
载体抗性:	卡那霉素
筛选标记:	Neomycin
克隆菌株:	DH5a 或 HB101
宿主细胞（系）:	--
备注:	pCMV-CREB载体是信号通路分析载体； CREB是信号转导蛋白，CREB的突变体是显性负性突变体，抑制信号转导过程。
稳定性:	瞬表达
组成型/诱导型:	组成型
病毒/非病毒:	非病毒

 

载体质粒图谱和多克隆位点信息

载体简介

 

载体描述
CREB (CRE-binding protein) is a member of the leucine zipper family of transcription factors and forms both a homodimer with itself and heterodimers with other leucine zipper proteins (1, 2). CREB also has a kinase-inducible domain, which contains consensus phosphorylation sites for several kinases, such as protein kinase A (1, 2). The CREB Dominant-Negative Vector Set consists of three vectors:
pCMV-CREB Vector onstitutively expresses the human wild-type (wt) CREB protein. pCMV-CREB133 Vector expresses a mutant variant of the human CREB protein that contains a serine to alanine mutation corresponding to amino acid 133 in the mutant mouse CREB protein. This mutation blocks phosphorylation of CREB, thus preventing transcription.

pCMV-KCREB Vector expresses a mutant variant of the human CREB protein that contains mutations in its DNA-binding domain. KCREB acts as a dominant repressor by forming an inactive dimer with CREB, blocking its ability to bind cAMP-regulated enhancer element (CRE).
These proteins are expressed at high levels from the constitutive CMV promoter. The SV40 polyadenylation sequence directs proper processing of the 3' end of the mRNAs. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor) consisting of the SV40 early promoter, the Tn5 neomycin/kanamycin resistance gene, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gen eallows kanamycin selection in E. coli and neomycin
selection in eukaryotic cells. The vector backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

使用
The CREB Dominant-Negative Vector Set can be used to monitor signal transduction pathways related to CREB. In conjunction with reporter systems (such as our cis-acting reporter vectors), you can monitor CREB-induced transcription by assaying for the reporter. For example, CREB activation can be measured in cells cotransfected with pCMV-CREB and pCRE-d2EGFP (Cat.No. 631802) by treating the transfected cells with forskolin and observing EGFP expression by FACS analysis or fluorescence microscopy. Cells expressing dominant-negative CREB133 or KCREB will not respond to this stimulus, so induced transcription by forskolin is inhibited. For more information about Pathway Profiling Vectors, visit our web site at www.clontech.com.
These vectors can be transfected into mammalian cells using any standard method. Stable transformants can be selected using G418(3).

Propagation in E. coli
 Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM101 or XL1-Blue.
 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
 E. coli replication origin: pUC
 Copy number: ~500
 Plasmid incompatibility group: pMB1/ColE1