I want to insert the CDS of my gene into the MCS of PGBKT7, but I found that the vector contains two sites NdeI(CATATG) and NcoI (CCATGG), I would like to know whether the ATG in these two sites would cause frameshift mutation of my gene that right behind them?
Actual transcription start site is from DNA -BD ATG which follows ADH promoter.Final product will be DNA-BD+C-myc+your protein(including your digestion enzyme sequence). Hence if your insert is in correct frame, no worries. ATG of used sites will be translated as inner M amino acid.
If you r using Nde1 then your gene specific primer must follow frame( 0 order-xxx xxx ATG).If you are using Nco1 then primer must have 2 additional bases from frame(1 order-NN- xxx xxx ATG). N -nucleotide in your 5' end of gene.. XXX- 5' end triplet of nucleotide( if you wish to add)
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