best kozak sequence for mammalian expression

The upstream primer for generating the PCR template was designed to contain the T7 promoter for transcription by T7 RNA Polymerase ( Cat.# P2075) and the Kozak consensus sequence (CCACCAUG or CCACCAUGG) for efficient translation (6). In the in vitro translation reaction using the T nT® T7 Quick Coupled Transcription/Translation System ( Cat.# L1170, L1171, L5540), PCR products can be used as templates (6). The strongest Kozak sequence we identified is AAAAatgG, which is similar to the most frequent Kozak sequence transcriptome-wide in Drosophila, which is CAAAatgG. We then extracted genomic DNAs from cells in each bin and optimized PCR conditions for unbiased amplicon amplification 41. The consensus Kozak sequence is ACCAUGG, where AUG is the initiation codon. In 1996 Brian Seed and colleagues improved the expression and fluorescent signal of GFP in mammalian cells by 40-120 fold by heavily modifying the GFP codon sequence to reflect mammalian cell preferences. This sequence, termed a Kozak sequence, is a translation initiation enhancer. One major difference between these animals and mammals is the choice of amino acid codons. RNA templates with CCACCAUGG were translated well in a study using the Rabbit Reticulocyte Lysate System ( Cat.# L4960) in vitro translation reaction (5). To identify 5 UTRs with increased protein expression, we used FACS to sort the cell library into four bins based on GFP expression levels: top 2.5, 2.55, 510, and 0100 (unsorted). Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. Functional studies on preproinsulin and alpha-globin translation in cells indicated that a purine (usually A) in position –3 is crucial for efficient initiation of translation, and in its absence, a G at position +4 is essential (4). Subsequent mutagenesis studies and a survey of 699 vertebrate mRNAs extended the consensus sequence for translation initiation to GCCGCCACCAUGG, where the A in the underlined AUG start codon is coordinate 1 and the A at position –3 could also be a G (2) (3). Proteins that are made and function in the cytosol lack leader sequences. Leader sequence peptide is a sequence of 16 to 20 amino acids at the N-terminus of some eukaryotic proteins that determines their ultimate destination. The Kozak consensus sequence was originally defined as ACCAUGG following an analysis of the effects of single mutations surrounding the initiation codon (AUG) on translation of the preproinsulin gene (1). Commonly used leader peptide sequences for mammalian cells expression.

Best kozak sequence for mammalian expression