Plasmids 101: Restriction Cloning
Type
Blog Post
... the start of the gene to end up just downstream of the promoter found in the backbone. Ideally, the backbone... enzyme cut sites (restriction sites) downstream of the promoter as part of a multiple cloning site (MCS...place your gene in the proper orientation downstream of the promoter, you can add an EcoRI site just 5’ of... leaving the 5’ end of your gene just downstream of the promoter and placing the gene in the proper orientation...easily orient your gene insert with respect to the promoter.
For example, let’s say your plasmid backbone ... on the left side of the image below. It has a promoter (blue arrow) followed by the restriction sites...of the backbone plasmid (see below). You should treat your digested backbone plasmid with a phosphatase...