the author might have better describe the problem more clearly. if the author wants the single plasmid carrying both the miRNA target site and GFP reporter gene, it will be easy to purchase (https://www.cellbiolabs.com/sites/default/files/E04AE5B2-3048-812A-2E3082B609FBBB72.pdf ). If the author wants the single plasmid carrying the miRNA transcription cassette and GFP reporter gene, it can be constructed by gene assembly.

Hi Dr. @Ma Dejun,

Thank you for the suggestion. I am planning to design/purchase a single plasmid that carries both the miRNA and GFP reporter gene. And after successful transfection, I hope the miRNA carried by the plasmid can be stably integrated into the host cell genome for stable transfection. The reporter gene is to check the transfection efficiency/success post-transfection.

Therefore in this case, is gene assembly the option for me?

Zx Chong Hi, I have got your thoughts. In this case, you could use the gibson assembly for your purpose. It is quite simple to get what you want. No commercial plasmid carrying both the miRNA transcription cassette and GFP reporter gene was available now.

Thank you Dr. Ma for the enlightenment. If I am unable to get a single plasmid that contains both miRNA transcription cassette and reporter gene, I will try to use different miRNA plasmid and reporter gene plasmid to co-transfect the cells.

Thank you again =)

Good luck with the assembly option. But co-transfection of 2 plasmids is not really an issue, just a question of optimization.

In my opinion, it is not necessary to generate such an artificial and maybe not well working and possibly too complex contruct. I would propose to use standardized, optimized and often veryfied protocolls/techniques via co-transfecting two different vectors - which offers the chance to get relyable results or to identify potential sources for not optimal results.

Thank you Sandra Lagauzère and Andreas Eisenreich for the suggestions. I will try to look for the possibilities of co-transfection. =)