基本信息
别称:pTWINI启动子:T7复制子:M13质粒分类:大肠杆菌载体;蛋白表达质粒质粒大小:7375bp原核抗性:Amp克隆菌株:DH5a培养条件:37度表达宿主:大肠杆菌培养条件:37℃,有氧,LB诱导方式:IPTG或乳糖及其类似物备注:表达质粒
质粒属性
载体宿主:大肠杆菌载体用途:蛋白表达基因种属:空载体基因类型:ORF原核抗性:Amp真核抗性:荧光蛋白:
质粒简介
pTWIN1 is an E. coli plasmid cloning vector designed for recombinant protein expression, labeling, and cyclization using the IMPACT-TWIN Kit (1). It contains the pMB1 origin of replication from pBR322 and is maintained at a similar copy number to pBR322; in addition, pTWIN1 also contains an M13 origin of replication.
The multiple cloning site (MCS) is positioned to allow translational fusion of an intein tag to the N-terminus, C-terminus, or both, of the cloned target protein. The mini-inteins encoded by pTWIN1, the Ssp DnaB intein and the Mxe GyrA intein, cleave the peptide bond at their C- and N-termini, respectively (1-4). The chitin binding domain (CBD) from B. circulans, fused to each intein, facilitates purification of the intein-target protein precursor.
Transcription of the intein tags is controlled by the inducible T7 promoter, requiring E. coli strains containing integrated copies of the T7 RNA polymerase gene [e.g. BL21(DE3)] for expression. Basal expression from the T7 promoter is minimized by the binding of the Lac repressor, encoded by the lacI gene, to the lac operator immediately downstream of the T7 promoter (5). Translation of the intein tags utilizes the translation initiation signal (Shine Dalgarno sequence) from the strongly expressed T7 gene 10 protein (φ10).