Mitochondria localization Addgene pPD96.32

I would like to use the fire vector pPD96.32, which is said to have a gfp and mitochondrial leader sequence. However, on the addgene map of this plasmid (Addgene pPD96_32 - Sequence Analyzer), the mitochondria leader sequence is not annotated. Has anyone used this plasmid and knows what the sequence is or does anyone know how I can figure this out?
Isabel (I am a novice at cloning, so any advice is welcome)

This isn’t tremendously helpful, but back before Addgene, back before there were annotated maps available, I would look at the documentation for the Fire vector kits and then with that as a guide I would directly compare the sequences of related plasmids to see what elements had been inserted to make a different version, and to see what restriction enzymes were used for the insertion. In this case you could compare the sequences of PD95.75 and PD96.32 (which are the same except for the mitochondrial localization tag), find the sequence unique to PD96.32, look to see what restriction sites border it (look for a 6 nucleotide sequence that’s the same after reverse-complementation), and BLAST the unique sequence to see where it came from and what was added to it to insert it in the plasmid.

Hi Isabel,

A few days ago I had the same problem as you, but I think I managed to figure out the sequence.
If I got it right, the annotation that is shown on the Addgene webpage is for both MTS (Mitochondria Targeting Sequence) and GFP (you can see it in a sequence view mode).
In such a case first 29 aa, or 87 bp, is the MTS (sequence: atggcactcctgcaatcacgtctcctgctatccgcacctcgtcgtgccgctgcaactgcaagagcttcatccgtaccggtagaaaaa). MTS, after import into the mitochondria, should be cleaved off after 16th aa by MPP.
The rest, based on the amino acid sequence, is GFP. Although I don’t know why it shows TAA stop codon after 163 aa…
Just keep in mind that I have not cloned anything with it, I am about to do it, and hopefully, in January I can let you know whether I was right.


mturek - did you account for the synthetic introns when you translated the GFP sequence in your sequence analyzer software? If not, that’s likely why. The introns are always the same in all the Fire Vectors, as far as I can tell. They are listed below.

intron 1: gtaagtttaaacatatatatactaactaaccctgattatttaaattttcag
intron 2:gtaagtttaaacagttcggtactaactaaccatacatatttaaattttcag
intron 3: gtaagtttaaacatgattttactaactaactaatctgatttaaattttcag


Thank you for the feedback! Michal, I would like to know if it is right. I did some more looking around yesterday and found one of the original papers from the Fire Lab and this is what was said in regards to mitochondrial localization: “Mitochondrial matrix localization signal: An N-terminal mitochondrial matrix localization signal from chicken mitochondrial aspartate aminotransferase [18] was synthesized and incorporated into the gfp expression cassette. This signal was sufficient to localize gfp to mitochondrial structures in body wall muscle, in hypodermis, and in embryos. At high expression levels, residual staining was observed in the cytoplasm. Although quantitative comparisons have not been performed, it appears that the mitochondrial gfp constructs may be somewhat brighter than their nuclear or cytoplasmic equivalents.” Hope this might help you too!

Thank you Steve for your suggestion regarding introns, you were right, I did not account for them. Now it all makes sense.
Isabel, I will let you know about the result of my experiment and thank you for that additional information regarding MTS, it is really helpful.