Hi there,

The plasmid you are using is a mamalian expression vector so it contains everything for the proper expression of the cDNA you clone in it.

Dear Dominque Liger,

Thank you for the information. I will design the construct accordingly then.

Best regards,

Jeremy

Dear Jeremy

are you using pcdna 3.4 topo cloning?

to guarantee protein expression you need to add at least the kozac sequence (that correspond to the bacterial rbs) in you forward primer as reported in the manual ( the cacc sequence) moreover pcdna3.4 in this way you will produce untagged protein intraccellular, if you would like to direct your protein in the surnatant for example you have also to add a leader

sequence in your forward primer and in case you wuold like also to add an his tag you need to add also it ib the primers. in this case i suggest to you to add the his at the c term in the reverse primer before this sequence do not disturb the protein secretion and your forward primer remain acceptable in terms of lenght.. because a forward primer containing the atg + leader peptide + his tag + annealing region is probably too long.

ps: pay attention thay is quite common in topo cloning to have gene insertion in the opposite direction. Screen more colonies with primers that allow to you to discriminate it.

good luck

Manuele

Dear Manuele Martinelli,

Thank you for your suggestion. It is really informative and practical to me.

I am planning to add in CD33 (MPLLLLLPLLWAGALA) as leader peptide at the N-terminus the targeted protein.

Thank you again for the great suggestion. Appreciated.

Regards,

Jeremy