pOnebyOne™ FREE Bicistronic Mammalian Expression Kit
pOnebyOne™ FREE is an extremely efficient, accurate, and flexible vector to clone your PCR genes and express your proteins of interest.
pOnebyOne™ FREE allows co-expression of two genes of interest driven by human cytomegalovirus early promoter. It is based in 2A sequence from Thosea asigna Virus (TaV) which is flanked by two multiple cloning sites. T2A sequence leads to the highest level of protein expression at the second gene position compared to IRES sequence.
The mechanism of 2A self-cleavage is a ribosome skipping process where the ribosome skips the formation of a glycil-prolyl peptide bond at the C-terminus of 2A. Stable mammalian cells could be selected by neomycin resistance..
Both genes of interest must be in frame with T2A and between them. Successful skipping results in translation of two proteins: 2A upstream protein is attached to the 2A sequence (GSGEGRGSLLTCGDVEENPG) at C-terminus while 2A downstream protein is attached to one Pro at the N-terminus.
Advantages & Features
• Complete solution: a cloning vector to clone and produce two proteins of interest
• Breakthrough technology: based in 2A sequence simultaneous expression of two proteins in mammalian cells (for example, a protein of interest and reporter)
• Highly efficient: tested with up to 4 kb inserts
• Accurate: proven performance for most common cell lines
• Flexible: allows transfection in difficult cell lines
• Convenient: available with different resistance marker cassettes
• Reporter checking: stoichiometric amount of your protein of interest and a reporter protein
• Protein expression of intracellular, extracellular or transmembrane higher cells in an equimolecular to a surface marker that allows quantification
• Cytomegalovirus Promoter: 232-887
• Multiple Cloning site 1 (MCS 1): 895-967
• T2A from Thosea asigna virus: 968-1030
• Multiple Cloning site 2 (MCS 2): 1031-1065
• BGH Polyadenylation Sequence: 1082-1306
• f1 Origin: 1362-1780
• Neomycin Resistance Cassette: 1785-3288
• Neomycin Resistance Gene (ORF): 2190-2981
• pUC Origin: 3671-4341
• Ampicillin Resistance Cassette (complementary strand): 4486-5480
• Ampicillin Resistance Gene (ORF): 4489-5346
*Developed and manufactured by Canvax Biotechnology, S.L under contractual agreement for AG Scientific, Inc.