18 Life Technologies
™
| Vector Construction and Preparation
What should I include in the insert to express my protein of interest?
Beyond the flanking restriction sites, be sure to include:
• Kozak sequence (GCCACC) just upstream of the ATG
• ATG
• Signal peptide
• cDNA sequence of gene of interest
• Stop codon
What sequencing primers can I use to verify my construct?
We strongly recommend that you analyze your recombinant expression
plasmid(s) by sequencing the promoter–gene of interest junctions before
transfecting CHO-S
®
cells (cGMP-banked) and expressing your protein(s)
of interest. For your convenience, the sequences of the primers used for
analyzing the promoter–gene of interest junctions in the recombinant
expression plasmid(s) are provided in Table 1. Note that the sequencing
primers are not supplied as part of the Freedom
®
CHO-S
®
Kit; they need to
be ordered separately and custom-synthesized.
Vector Construction and Preparation
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