Bicistronic expression vectors are used for the simultaneous translation of two genes of interest from the same mRNA transcript, involving IRES-containing vectors or 2A sequence-containing vectors.

IRES, a viral sequence of around 600-700 bp, has been widely used in bicistronic mammalian expression vectors, although its length could be a disadvantage in viral vectors with limited packaging capacity. IRES based expression vectors are characterized by the non-stoichiometric production of proteins and generally lower downstream gene expression.

2A sequence is a viral oligopeptide of approximately 20 amino acids that mediates cleavage of polypeptides during translation in eukaryotic cells. The designation “2A” refers to a specific region of the viral genome that contain the consensus motif Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro at the C-terminus. 2A sequence acts co-translationally. The mechanism of 2A sequence-mediated "self-cleavage" is that the formation of a normal peptide bond between glycine and proline residues at the C-terminal of 2A is prevented, resulting in ribosomal hopping that leads to cleavage of the nascent polypeptide. Compared with other strategies for multi-gene co-expression, 2A sequences lead to relatively high levels of downstream protein expression, and they are small in size thus bearing a lower risk of interfering with the function of co-expressed genes.

pOnebyOne™

pOnebyOne™ are efficient, accurate and flexible bicistronic mammalian expression kits that contain an expression cassette based in 2A sequence breakthrough technology. Its novel technology allows simultaneous expression of two proteins from the same mRNA. Cells transfected with bicistronic vectors ensure that if one of the proteins is present, the other one is also present.

2A sequence-based vectorFigure. 2A sequence-based vector.

Both genes must be in frame and the nascent peptide is cleaving between the glycine and proline. After the cleavage, the short peptide IGSGEGRGSLLTCGDAEENPG (21 amino acids) remains fused to the C-terminus of the protein of interest while the proline is added to the N-terminus of the reporter protein. 2A sequence used has high cleavage efficiency in some biological systems. Essential reverse primer sequence for directional cloning is underlined.

Advantages & Features

  • Complete solution: a directional cloning vector to clone and produce a protein of interest in mammalian systems.
  • Breakthrough technology: based in 2A sequence simultaneous expression of two proteins in mammalian cells (a protein of interest and reporter).
  • Highly efficient: cloning system tested with up to 4 kb inserts.
  • Time-saving cloning process: linearized vector ready-to-mix with PCR fragment.
  • Easy-to-use: facilitates the selection of positive cells expressing the recombinant gene of interest. Only check that your gene is in frame with 2A-reporter.
  • Cost avoidance: avoids the use of expensive restriction enzymes.
  • Really low experimental background: lower than 1%.
  • Convenient: available with different resistance marker cassettes.
  • Directional cloning: of PCR with the gene of interest.
  • Reporter checking: stoichiometric amount of your protein of interest and a reporter protein.
  • Risk-free: product covered by our quality 100% guarantee.

Product Range

Amerigo Scientific offers a ready-to-clone solution of your gene of interest onto a wide collection of bicistronic vectors based on 2A sequence.

  • Non-viral Mammalian Expression Kits
Product Name Resistance Promoter Reporter Protein Vector Size
pOnebyOne™ l-NEO Neomycin PCMV △NGFR 6258 bp
pOnebyOne™ ll-NEO PCMV eGFP 6141 bp
pOnebyOne™ lll-NEO PEF1α △NGFR 6568 bp
pOnebyOne™ IV-NEO PEF1α eGFP 6451 bp
pOnebyOne™ V-NEO PCMV LUC 7072 bp
pOnebyOne™ VI-NEO PEF1α LUC 7382 bp
pOnebyOne™ l-PURO Puromycin PCMV △NGFR 5926 bp
pOnebyOne™ ll-PURO PCMV eGFP 5808 bp
pOnebyOne™ lll-PURO PEF1α △NGFR 6236 bp
pOnebyOne™ IV-PURO PEF1α eGFP 6119 bp
pOnebyOne™ V-PURO PCMV LUC 6740 bp
pOnebyOne™ VI-PURO PEF1α LUC 7050 bp
pOnebyOne™ l-HYG Hygromycin PCMV △NGFR 6314 bp
pOnebyOne™ ll-HYG PCMV eGFP 6306 bp
pOnebyOne™ lll-HYG PEF1α △NGFR 6624 bp
pOnebyOne™ IV-HYG PEF1α eGFP 6615 bp
pOnebyOne™ V-HYG PCMV LUC 7128 bp
pOnebyOne™ VI-HYG PEF1α LUC 7438 bp

PCMV, citomegalovirus promoter; PEF1α, elongation factor 1 alpha promoter; △NGFR, truncated nerve growth factor receptor; eGFP, enhanced green fluorescent protein; LUC, firefly luciferase.

  • Universal Non-Viral Mammalian Expression Kits
Product Name Promoter Reporter Protein Vector Size
pOnebyOne™ FREE PCMV BGH pA 5482 bp
pOnebyOne™ FREE-2 PCMV BGH pA 5538 bp

  • Retroviral Expression Kits
Product Name Promoter Reporter Protein Vector Size
pOnebyOne™ l-Retroviral PCMV △NGFR 5811 bp
pOnebyOne™ ll-Retroviral PCMV eGFP 5696 bp
pOnebyOne™ III-Retroviral PEF1α △NGFR 6335 bp
pOnebyOne™ IV-Retroviral PEF1α eGFP 6220 bp

  • Lentiviral Expression Kits
Product Name Promoter Reporter Protein Vector Size
pOnebyOne™ l-Lentiviral PCMV △NGFR 7472 bp
pOnebyOne™ ll-Lentiviral PCMV eGFP 7353 bp
pOnebyOne™ lll-Lentiviral PEF1α △NGFR 8073 bp
pOnebyOne™ IV-Lentiviral PEF1α eGFP 7953 bp

  • Multicistronic Expression Kits
Product Name Promoter Reporter Protein Vector Size
pOnebyOne™ Multicistronic Vector PCMV eGFP 6286 bp

Applications

pOnebyOne™ Non-viral Mammalian Expression vectors

Protein Expression of intracellular, extracellular, or transmembrane in higher cells in an equimolecular ratio with a surface marker that allows quantification.

pOnebyOne™ Retroviral Expression vectors

  • Introduction of DNA in refractile transfection cell lines.
  • Co-expression of your gene of interest and a reporter gene.

pOnebyOne™ Lentiviral Expression vectors

  • Highest generation Lentiviral transfer vector to transform replicating and non-replicating cells, including stem and neuronal cells.
  • Vector of choice for short-interfering RNA (siRNA) delivery and for gene therapy.


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