■ 基本信息
啟動(dòng)子: | CMV |
復(fù)制子: | pUC |
終止子: | bGH poly(A) signal |
質(zhì)粒分類: | 哺乳系列質(zhì)粒；哺乳表達(dá)質(zhì)粒；pCMV系列質(zhì)粒 |
質(zhì)粒大小: | 6210bp |
原核抗性: | Amp |
真核抗性: | Zeo |
克隆菌株: | DH5a |
培養(yǎng)條件: | 37℃，5%CO2 |
表達(dá)宿主: | 293T等哺乳細(xì)胞 |
誘導(dǎo)方式: | 無需誘導(dǎo) |
5'測(cè)序引物:
| CMV-F（CGCAAATGGGCGGTAGGCGTG）
|
3'測(cè)序引物: | BGH（TAGAAGGCACAGTCGAGG） |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 哺乳細(xì)胞 |
質(zhì)粒用途: | 蛋白表達(dá) |
片段類型: | ORF
|
片段物種: | 空載體
|
原核抗性: | Amp |
真核抗性: | Zeo
|
熒光標(biāo)記: | 綠色
|
■ 質(zhì)粒簡(jiǎn)介
pTracer-CMV2的CMV啟動(dòng)子后有MCS供插入外源基因，hEF1α啟動(dòng)子調(diào)控Cycle 3 GFP-Zeocin fusion的表達(dá)，可以做到既有熒光蛋白來做轉(zhuǎn)染指示，且熒光基因表達(dá)和外源基因表達(dá)又不會(huì)互相干擾，而且Zeocin也可以用來做穩(wěn)定性細(xì)胞篩選。另外，由于EM-7啟動(dòng)子的存在，Cycle 3 GFP-Zeocin fusion在E.coli中也可以表達(dá)，可以用來做原核熒光定位。
pTracer-CMV2 is a 6.2 kb mammalian expression vector derived from pcDNA3.1 and designed for visual detection of transformed E. coli cells and transfected mammalian cells. The vector contains the following elements:
Cycle 3-GFP, an improved GFP (Green Fluorescent Protein) gene (Crameri et al.,1996), fused to the Zeocin? resistance gene (see below) for convenient,noninvasive detection of transformed or transfected cells
? Human elongation factor 1?-subunit promoter (hEF-1?) for mammalian expression of the Cycle 3-GFP-Zeocin fusion (Goldman et al., 1996; Mizushima and Nagata, 1990)
? A synthetic bacterial promoter, EM-7, for expression of Cycle 3-GFP in E. coli
? Human cytomegalovirus immediate-early (CMV) promoter for high-level expression of your gene in a wide range of mammalian cells
? Zeocin? resistance for stable selection in mammalian cell lines
? Ampicillin and Zeocin resistance genes for selection of transformants in E. coli
The Cycle 3-GFP gene used in this vector is described in Crameri et al., 1996. In this paper, the codon usage was optimized for expression in mammalian cells and three cycles of DNA shuffling were used to generate a mutant form of GFP that has the following characteristics:
? Excitation and emission maxima that are the same as wild-type GFP (395 nm and 478 nm for primary and secondary excitation, respectively, and 507 nm for emission)
? High solubility in E. coli for visual detection of transformed cells
? >40-fold increase in fluorescent yield over wild-type GFP
? The Cycle 3-GFP gene is fused to the Zeocin? resistance marker to correlate GFP fluorescence with resistance to the antibiotic Zeocin.
Zeocin is a member of the bleomycin/phleomycin family of antibiotics isolated from Streptomyces. It shows strong toxicity against bacteria, fungi (including yeast), plants, and mammalian cell lines. It is particularly well-suited for selection of mammalian stable cell lines. Ordering information is provided below.
質(zhì)粒只保證關(guān)鍵序列正確，不保證表達(dá)效果。
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請(qǐng)下載：
ZK388pTracer-CMV2哺乳表達(dá)質(zhì)粒.txt
