If say pCru5‐IRES‐mCherry‐F2A‐Puro plasmid, the GAP promoter is used for transcription? In this case, what is the +1 nucleotide of the transcripts? I was wondering if this plasmid can be modified to study transcripts containing +1 random nucleotides in its 5'leader. eg: T7 promoter adds +1G in its transcripts, is there any such selection with this plasmid?
Hello Anjili,
As a yeast user , for me the GAP promoter is a yeast promoter. Is that this one you are using. Second, in order to answer your +1 question, I do need the sequence between the promoter and the ATG (Methionine) of your gene; Third , you can modify the 5'leader of eukaryotic origin, concerning the T7 promoter +1G can you explain with the sequences what you want to modify ?
Thanks
Thank you for your answer Edouard. A T7 promoter adds a +1G in its resulting mRNA. I was wondering if I need to create a randomer at the extreme end of the 5'TL of the mRNA from nucleotide +1, would a GAP promoter be advisable. mRNA starts with NNNNNN---- , Does GAP promoter have any +1 transcription run off?
Here is for procaryotic cells the T7 with the trancription starting at G, then you can have roughly 50 bp of 5'UTR (look at pET vectors or other vectors with T7 promoters):
From NEB website:
T7 Promoter is 5′ TAATACGACTCACTATAG 3′
T7 RNA polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G.
Instead of GAP , use the CMV promoter for eucaryotic cells.
You can use vectors with CMV promoter and T7 promoter (look at Invitrogen vectors, now ThermoFisher).
I use the T7 promoter for transcription and it begins with a TSS of +1G, I want a promoter with no set starting TSS . Would CMV promoter ideally start with +1N? if the sequence following CMV promoter for transcription is say cmv promoter- NNNNNN-GATACAG or so?
I do not catch: "I want a promoter with no set starting TSS " what do you mean ? T7 promoter does have a TSS as previously said at G position.
For CMV promoter refer to this site where you have all info on its TSS:
https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf&title=pcDNA3.1(+)%20and%20pcDNA3.1(-)
- Badges
- Science topic