pEF4/myc-His A, B, and C BioVector NTCC质粒载体菌种细胞基因保藏中心

pEF4/myc-His A, B, and C BioVector NTCC质粒载体菌种细胞基因保藏中心
pEF4-myc-His A,B,C哺乳动物细胞表达载体，带有hEF-1alpha启动子可在多种细胞中高效表达，C端myc和6xHis标签，Zeocin细胞筛选标记，Amp抗性，可在携带SV40或SV40 large T抗原的细胞株中过表达。
Description of the System
pEF4/myc-His A, B, and C are 5.9 kb vectors designed for overproduction of
recombinant proteins in mammalian cell lines. Features of the vectors allow
purification and detection of expressed proteins
. High-level stable and transient expression can be carried out in most mammalian cells. The vectors contain the following elements:
•1. Human elongation factor 1-subunit (hEF-1) promoter for high-level
expression across a broad range of species and cell types (Goldman et al.,
1996) (Mizushima and Nagata, 1990) (see page 11 for more information).
• 2.Three reading frames to facilitate in-frame cloning with a C-terminal peptide
encoding the myc epitope and a polyhistidine (6×His) metal-binding tag.
•3. Zeocin™ resistance gene for selection of stable cell lines* (Mulsant et al., 1988)
(see page 15 for more information).
• 4.Episomal replication in cell lines that are latently infected with SV40 or that
express the SV40 large T antigen (e.g. COS7).
The control plasmid, pEF4/myc-His/lacZ is included for use as a positive control
for transfection, expression, and detection in the cell line of choice.

Experimental Outline
Use the following outline to clone and express your gene of interest in pEF4/mycHis.

• Consult the multiple cloning sites described on pages 3–5 to determine which
vector (A, B, or C) should be used to clone your gene in frame with the Cterminal
myc epitope and the polyhistidine tag.
• Ligate your insert into the appropriate vector and transform into E. coli. Select
transformants on 50 to 100 ug/mL ampicillin or 25 to 50 ug/mL Zeocin™ in
Low Salt LB. For more information.
• Analyze your transformants for the presence of insert by restriction digestion.
• Select a transformant with the correct restriction pattern and use sequencing
to confirm that your gene is in frame with the C-terminal peptide.
• Transfect your construct into the cell line of choice using your own method of
transfection. Generate a stable cell line, if desired.
• Test for expression of your recombinant gene by western blot analysis or
functional assay. For antibodies to the myc epitope or the C-terminal
polyhistidine tag

【Supplier来源】BioVector NTCC Inc.
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【Website网址】 http://www.biovector.net