■ 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；哺乳雙框質(zhì)粒 |
質(zhì)粒大小: | 5303bp |
質(zhì)粒標(biāo)簽: | C-His,C-V5 |
原核抗性: | Zeo |
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(wú)需誘導(dǎo)，瞬時(shí)表達(dá) |
5'測(cè)序引物: | CMV-F（CGCAAATGGGCGGTAGGCGTG） EF-1a- F (TCAAGCCTCAGACAGTGGTTC) |
3'測(cè)序引物: | Bleo-R（CTGGATGATCCTCCAGCGC） 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: | Zeo |
真核抗性: | Zeo
|
熒光標(biāo)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(zhuǎn)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(shè)計(jì)用于從哺乳動(dòng)物細(xì)胞中的單個(gè)載體獨(dú)立表達(dá)兩個(gè)基因。 使用 pBudCE4.1 來(lái)產(chǎn)生穩(wěn)定的哺乳動(dòng)物表達(dá)細(xì)胞系可確保細(xì)胞中每個(gè)基因的拷貝數(shù)相等。 這可以消除由于基因拷貝數(shù)的差異導(dǎo)致的表達(dá)變化。pBudCE4.1 可提供具有下列特征的表達(dá)盒：
? 用于高水平轉(zhuǎn)錄基因的 CMV，可選擇用于進(jìn)行快速檢測(cè)的 c-myc 表位標(biāo)簽和用于進(jìn)行簡(jiǎn)單純化的 6xHis 序列
? 用于高水平表達(dá)基因的 EF-1α 啟動(dòng)子，可選擇用于快速檢測(cè)的 V5 表位標(biāo)簽以及用于簡(jiǎn)單純化的 6xHis 序列
? 用于在哺乳動(dòng)物細(xì)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(qǐng)下載：
ZK689pBudCE4.1-hrGFP哺乳雙框質(zhì)粒.txt
