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Can anyone explain why inside plasmid mapping software the annotations are layered under each other, I am confused as to why there are essentially two layers within the map, for example in the image below.

The bottom red layer is annotated as "DsRed2" and the top yellow is "LacZ-DsRed2 fusion expressed in E. coli". Does this mean that the red layer is inserted into the original LacZ gene or some sort of mutagensis...? Any explanation would be real helpful.

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Second Plasmid design

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Third Plasmid

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Keep in mind that the plasmid map is made by someone, and probably not perfect. They're also made by programs that let you annotate everything everywhere, even overlapping stuff.

In silico, I assume that the DsRed part and the Lacz parts were present, perhaps copied from some other plasmid map including annotation. After that, someone decided it was a good idea to annotate the complete ORF (in yellow).

What I think this means in reality (just guessing from the map, which is just coarse schematic picture): there's a part of Lacz still present in the plasmid (in pink, second lane) which is fused (in frame) to the DsRed gene (red). Together this gives the fusion gene marked in yellow. Also notice the part (terminator?) in pink that's in the third lane.

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  • $\begingroup$ Thanks for this, I have added 2 more larger images of plasmids since the original was kinda small. The ORF 2 and ORF 3 (blue) in the second plasmid are under the genes (red), does that mean that simply an insert can be placed there? Also there seems to be a third green layer on top, does that mean the terminator and primer are in the same location? And just a last thing, the third plasmid has arrows in reverse, i.e. lacl and amp', what does this mean? thanks! $\endgroup$ – condo1234 Jan 5 '17 at 11:24
  • $\begingroup$ ORF2 and 3 are probably just there because of the plasmid / annotation software. There's the Ampicillin resistance gene, which is also just one of the ORFs on the plasmid. The place to insert something new would be in the multiple cloning site at the top (with all the restriction sites). Arrows running in reverse mean the gene is on the other DNA strand, "upside down". $\endgroup$ – VonBeche Jan 5 '17 at 12:02
  • $\begingroup$ If you want to more about the practicalities of a plasmid I'd suggest to look up the manual for the same (or similar) plasmid. Usually the plasmid in your lab is based on a commercial one, or you use a classic plasmid that's also commercially available. $\endgroup$ – VonBeche Jan 5 '17 at 12:04

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