■ 基本信息
啟動(dòng)子: | CMV |
復制子: | pUC |
終止子: | bGH poly(A) signal |
質(zhì)粒分類(lèi): | 哺乳系列質(zhì)粒；哺乳表達質(zhì)粒；哺乳雙框質(zhì)粒 |
質(zhì)粒大小: | 5303bp |
質(zhì)粒標簽: | C-His,C-V5 |
原核抗性: | Zeo |
真核抗性: | Zeo |
克隆菌株: | DH5a |
培養條件: | 37℃，5%CO2 |
表達宿主: | 293T等哺乳細胞 |
誘導方式: | 無(wú)需誘導，瞬時(shí)表達 |
5'測序引物: | CMV-F（CGCAAATGGGCGGTAGGCGTG） EF-1a- F (TCAAGCCTCAGACAGTGGTTC) |
3'測序引物: | Bleo-R（CTGGATGATCCTCCAGCGC） BGH（TAGAAGGCACAGTCGAGG） |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 哺乳細胞 |
質(zhì)粒用途: | 蛋白表達 |
片段類(lèi)型: | ORF
|
片段物種: | 空載體
|
原核抗性: | Zeo |
真核抗性: | Zeo
|
熒光標記: | 綠色
|
■ 質(zhì)粒簡(jiǎn)介
pBudCE4.1-hrGFP質(zhì)粒是在pBudCE4.1載體中克隆進(jìn)hrGFP綠色熒光基因。pBudCE4.1質(zhì)粒使用時(shí)，E.coli 克隆菌株時(shí)最好使用不含Tn5基因的菌株，如TOP10和DH5αT1；采用電轉法時(shí)最好用TOP10，博來(lái)霉素Zeocin 工作濃度推薦25 μg/ml ~50 μg/ml。
pBudCE4.1 載體設計用于從哺乳動(dòng)物細胞中的單個(gè)載體獨立表達兩個(gè)基因。 使用 pBudCE4.1 來(lái)產(chǎn)生穩定的哺乳動(dòng)物表達細胞系可確保細胞中每個(gè)基因的拷貝數相等。 這可以消除由于基因拷貝數的差異導致的表達變化。pBudCE4.1 可提供具有下列特征的表達盒：
? 用于高水平轉錄基因的 CMV，可選擇用于進(jìn)行快速檢測的 c-myc 表位標簽和用于進(jìn)行簡(jiǎn)單純化的 6xHis 序列
? 用于高水平表達基因的 EF-1α 啟動(dòng)子，可選擇用于快速檢測的 V5 表位標簽以及用于簡(jiǎn)單純化的 6xHis 序列
? 用于在哺乳動(dòng)物細胞和大腸桿菌 (E. coli ) 中使用選擇劑 Zeocin 進(jìn)行高效選擇的Sh ble (ZeoR) 基因
pBudCE4.1 is a 4.6 kb vector designed for simultaneous expression of two genes in mammalian cell lines. The vector contains the human cytomegalovirus (CMV) immediate-early promoter and the human elongation factor 1α-subunit (EF-1α) promoter for high-level, constitutive, independent expression of two recombinant proteins (see page 13 for more information on the EF-1α promoter). Features of the vector allow detection and purification of expressed proteins (see pages 15–16) for more information). High-level stable and transient expression studies can be carried out in most mammalian cell types. In addition to the two promoters, the vector contains the following elements:
? C-terminal peptides encoding the myc (c-myc) epitope or the V5 epitope and a polyhistidine (6xHis) metal-binding tag for detection and purification of recombinant proteins
? Zeocin? resistance gene for selection in E. coli and creation of stable, mammalian cell lines (Mulsant et al., 1988) (see pages 18–19 for more information)
? SV40 origin for episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g., COS7) pBudCE4.1/lacZ/CAT is included for use as a positive control for transfection, expression, and detection in the cell line of choice.
pBudCE4.1 is an improved version of pBudCE4. During construction of the original vector, an ATG was inadvertently created in the multiple cloning site (672–674 bp) 3′to the CMV promoter. Since it may interfere with proper translation of the cloned gene, this ATG was changed to ATT to create pBudCE4.1.
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請下載：
ZK689pBudCE4.1-hrGFP哺乳雙框質(zhì)粒.txt
