|MadSci Network: Molecular Biology|
When subcloning a gene of interest into a plasmid for gene expression, the directionality of cloning is of extreme importance. In order for your gene to transcribed into mRNA correctly, your gene must be situated with its 5' end following a promoter (use of the correct promoter is very important and depends on which organism you are trying to express your protein). Remember, DNA is is transcribed into mRNA in the 5' to 3' direction, so your start (ATG) codon must follow the promoter. In some instances, there are promoters in plasmids flanking the multiple cloning sites and RNA transcript can be made in either direction, depending on which RNA polymerase is used (this is mostly used for in vitro transcription/translation reactions).
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