pBudCE4.1-hrGFP质粒是在pBudCE4.1载体中克隆进hrGFP绿色荧光基因。pBudCE4.1质粒使用时，E.coli 克隆菌株时最好使用不含Tn5基因的菌株，如TOP10和DH5αT1；采用电转法时最好用TOP10，博来霉素Zeocin 工作浓度推荐25 μg/ml ~50 μg/ml。
pBudCE4.1 载体设计用于从哺乳动物细胞中的单个载体独立表达两个基因。 使用 pBudCE4.1 来产生稳定的哺乳动物表达细胞系可确保细胞中每个基因的拷贝数相等。 这可以消除由于基因拷贝数的差异导致的表达变化。pBudCE4.1 可提供具有下列特征的表达盒：
• 用于高水平转录基因的 CMV，可选择用于进行快速检测的 c-myc 表位标签和用于进行简单纯化的 6xHis 序列
• 用于高水平表达基因的 EF-1α 启动子，可选择用于快速检测的 V5 表位标签以及用于简单纯化的 6xHis 序列
• 用于在哺乳动物细胞和大肠杆菌 (E. coli ) 中使用选择剂 Zeocin 进行高效选择的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.
信息
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