Invitrogen™ pcDNA™3.1 ⁽⁺⁾ Mammalian Expression Vector

Catalog No.	Quantity
V79020	20 μg

Catalog No. V79020 Supplier Invitrogen™ Supplier No. V79020

Description

Includes

20μg of this pcDNA3.1⁽⁺⁾ vector, as well as an expression control, are supplied supercoiled and lyophilized.

Designed for high-level, constitutive expression in a variety of mammalian cell lines

• Contains Geneticin selectable marker and reverse-orientation multiple cloning site
• For use alone or in co-transfection
• Cytomegalovirus (CMV) enhancer-promoter for high-level expression
• Large multiple cloning site in forward (+) orientation
• Bovine Growth Hormone (BGH) polyadenylation signal and transcription termination sequence for enhanced mRNA stability
• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen (i.e.,COS-1 and COS-7)
• Ampicillin resistance gene and pUC origin for selection and maintenance in E. coli

Constitutive Expression, Mammalian Expression, Protein Expression, Proteins, Expression, Isolation and Analysis

Specifications

• Delivery Type: Transfection
• Promoter: CMV
• Product Type: Mammalian Expression Vector
• Selection Agent (Eukaryotic): Geneticin™ (G-418)
• Content And Storage: 20 μg of this pcDNA™3.1⁽⁺⁾ vector, as well as an expression control, are supplied supercoiled and lyophilized. Store at -20°C. Vectors are guaranteed stable for 6 months when properly stored.
• Protein Tag: Untagged
• Cloning Method: Restriction Enzyme/MCS
• Quantity: 20 μg
• Vector: pcDNA
• Product Line: pcDNA
• For Use With (Application): Constitutive Expression

Frequently Asked Questions (FAQs)

I performed stable selection but my antibiotic-resistant clones do not express my gene of interest. What could have gone wrong?

Here are possible causes and solutions:

Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.

I used a mammalian expression vector but do not get any expression of my protein. Can you help me troubleshoot?

Here are possible causes and solutions:

- Try the control expression that is included in the kit
Possible detection problem:

- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot. Protein might be degraded or truncated: Check on a Northern. Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression. Possible cloning issues: Verify clones by restriction digestion and/or sequencing.

I am using a mammalian expression vector that has the neomycin resistance gene. Can I use neomycin for stable selection in mammalian cells?

No; neomycin is toxic to mammalian cells. We recommend using Geneticin (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.

Is it okay if my construct has an ATG that is upstream of the ATG in my gene of interest? Will it interfere with translation of my gene?

Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.

What is the difference between pcDNA3.1 vectors and the pcDNA3.3-TOPO vector?

pcDNA3.1 vectors contain the core CMV promoter that is truncated before the start of transcription, whereas the pcDNA 3.3-TOPO vector has the 672 bp native CMV promoter. This native CMV promoter allows high-level gene expression with two- to five-fold higher protein yields compared to other expression vectors. pcDNA3.1 vectors are available in restriction, TOPO, and Gateway cloning versions and as untagged and epitope-tagged versions, whereas the pcDNA3.3-TOPO vector is a TOPO TA-adapted, untagged vector that can be used to express native proteins without extraneous amino acids, and is hence ideal for antibody production and structural biology.

What is the significance of the (+) and (-) designations for the pcDNA3.1, pcDNA3.1/Zeo, and pcDNA3.1/Hygro vectors?

The (+) and (-) designations refer to the orientation of the multiple cloning sites in these vectors. The availability of the cloning site in two orientations facilitates flexibility in cloning scheme design, so that if, for example, your insert must clone in as a Not I to Bam HI orientation, you may choose the (-) version of these vectors.

What is the difference between pcDNA3 and pcDNA3.1 vectors?

pcDNA3 is no longer available from Thermo Fisher Scientific but has been directly replaced by pcDNA3.1, which was derived from pcDNA3. The center of the multiple cloning site (MCS) within the original pcDNA3 vector contained homology to a hairpin mRNA structure and involved the Eag I, Not I, and both BstXI sequences. This hairpin would only have affected expression of genes cloned downstream of the Not I site, if at all. To address this issue, some sequences were removed, including the Eag I site, and the BstXI sequences were slightly modified to reduce homology. A 32-base fragment from pcDNA3 (between bases 995 and 1026), which contains the Sp6 primer site, was also removed and 11 bases were inserted in its place, adding another PmeI restriction site into the MCS of pcDNA3.1.

Here are the links to the vector sequences for pcDNA3, pcDNA3.1(+), and pcDNA3.1(−):
http://tools.thermofisher.com/content/sfs/vectors/pcdna3_seq.txt
http://tools.thermofisher.com/content/sfs/vectors/pcdna3_1p_seq.txt
http://tools.thermofisher.com/content/sfs/vectors/pcdna3.1-_seq.txt

Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression?

We offer pJTI R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.

I am working with a mouse cell line and would like to express my gene at high levels using one of your vectors with the CMV promoter. Do you foresee any problems with this approach?

The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.

Do I need to include a consensus Kozak sequence when I clone my gene of interest into one of your mammalian expression vectors?

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.

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

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.

Will the T7 promoter in mammalian expression vectors (for example, pcDNA3.1) function in prokaryotes for expression of proteins?

No. While transcripts will usually be made, there is no ribosome binding site (RBS) or Shine-Dalgarno sequence to initiate translation. Furthermore, these vectors do not contain a prokaryotic transcription terminator.

Which enzyme is recommended for the linearization of the pcDNA3.1 vector in order to generate stable transfectants?

Although not required, if you would like to linearize pcDNA3.1 vector prior to stable transfection, Pvu I or Sca I are single-cutter enzymes that cut within the ampicillin resistance gene and should work well.

What is the SV40 poly A recognition site for the Neomycin resistance gene in pcDNA3.1 vector?

pcDNA3.1 vector, as with most vectors which contain what is described as the SV40 polyA region, actually contains 3 polyA signals, two on one strand and one on the other. Wild type SV40 virus has transcription going in both directions at different parts of its cycles (early and late), and transcripts from both directions get polyadenylated in the small region that contains these three signals.

In pcDNA3.1 vector, the SV40 poly A recognition site for the Neomycin resistance gene is AAATAAA and is located at 3148 and 3177 in the SV40 Poly A.

Why do I see expression of my protein in bacteria with pCDNA3.1?

It has been reported that pCDNA3.1 may have a cryptic E. coli promoter that will often express your gene in E. coli when your gene is in the correct orientation for eukaryotic expression. The level of toxicity to E. coli will depend on your particular gene product and on the efficiency of translation initiation. This problem can be solved by placing a prokaryotic transcription terminator at the Hind III site, upstream of your gene. Eukaryotic expression will not be altered by this additional sequence.

What enzymes could be used to cut the Neomycin resistance cassette out of the pcDNA3.1 vector?

Here's a cloning scheme for cutting out the Neomycin resistance cassette from pcDNA3.1 (-) vector, including the SV40 promoter and SV40 polyA signal:

At bp 1726 use either: Asp 700I, Mro XI, or XmnI (blunt). These enzymes also cut at 5107.
At bp 3237 use either: Bss NAI, Bst 1107I, or Bst 217I (blunt). Isolate the 1.5 kb fragment.

Can you tell me the difference between a Shine-Dalgarno sequence and a Kozak sequence?

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.

I sequenced one of your vectors after PCR amplification and observed a difference from what is provided online (or in the manual). Should I be concerned?

Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.

Are your vectors routinely sequenced?

No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.

How was the reference sequence for your vectors created?

Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.

What is the consensus Kozak sequence and what is the function of the Kozak sequence?

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.

For Research Use Only. Not for use in diagnostic procedures.