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View additional product information for pREP4 Mammalian Expression Vector - FAQs (V00450)
11 product FAQs found
Our episomal mammalian expression vectors (pCEP4 and pREP4) contain the Epstein Barr Virus (EBV) origin of replication (oriP) and the Epstein-Barr nuclear antigen (EBNA-1) for high-copy, transient, or stable episomal replication in human, primate, canine, and porcine cell lines. They do not bring about episomal expression in murine or rodent cell lines.
The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
All of the mentioned episomal vectors can be maintained extra-chromosomally in human, primate, and canine cell lines. They will not replicate episomally in rodent cell lines (e.g. CHO, NIH-3T3). Copy number is dependent on cell size, size of the insert, and transfection efficiency. Typically, the general range is between 1 to 30 per cell.
Note: Only pCEP4 and pREP4 are still available for purchase from Thermo Fisher Scientific as of January 2012. The other vectors have been discontinued.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The hygromycin resistance gene was obtained directly from E. coli. pCEP4, pMEP4, pREP4, pREP7, pREP10, and pEBVHis vectors have the same TK-driven hygromycin resistance gene.
Note: Only pCEP4 and pREP4 are still available for purchase from Thermo Fisher Scientific as of January 2012. The other vectors have been discontinued.
Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The hygromycin resistance gene in these vectors is driven by the TK (Thymidine Kinase) promoter and comes from E. coli.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
HAT is a compete medium supplemented with 100 micromolar hypoxanthine, 0.4 micromolar aminopterin, 16 micromolar thymidine, and 3 micromolar glycine (HAT medium) is used when using selection vectors that encode for thymidine kinase (TK). For more information on this selection method, you can refer to Current Protocols in Molecular Biology ("Red Book") or the following reference:Littlefield, JW 1964 "Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants." Science 145:709-710.
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The EBNA-1 gene in pCEP4 is expressed from a natural EBV promoter and TATA box. The promoter is not well defined, but if you BLAST the sequences upstream of the EBNA-1 ORF you will see that they line up with EBV genomic sequences.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
There is probably no termination/polyA for EBNA-1. There are only about 75 bases after the stop codon before the pUC sequence resumes with no obvious signal sequences.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.