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String of A's (15-20nt) w/out polyA signal in middle of rlentiviral vector;

Lentivirus Single-cell Sequencing

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#1 Beaver Aggie

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Posted 26 June 2018 - 07:49 AM

I know this might be a stupid question, but just want confirmation either way before going through the building and validation of a few new vectors for single cell sequencing.  

 

I am want to insert a string of A's (no polyA signal) for polyA RNA capture on a gene of interest in the middle of my lentiviral vector that also has a different promoter driving selection downstream. 

 

Basic example lentiviral vector (expression)

5'LTR--EF1a-GOI-P2A-BC(18nt)-N(280)-A(15-20)--PGK--Puro--3'LTR

 

 

I don't think this will affect the production of a full length viral transcript and packaging and might work for detecting the BC for single cell sequencing, but I am not 100% sure.

 

 

If anyone with more plasmid/lentiviral biology background can provide an answer that they believe this design would or would not work, I would greatly appreciate the input.

 

Thank you very much for your time and thoughts!


Edited by Beaver Aggie, 26 June 2018 - 07:50 AM.


#2 OldCloner

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Posted 29 June 2018 - 01:50 PM

Hi! first let me get this straight- you want a string of A's in your RNA transcript (to mimic polyadenlyation, even though no polyadenylation signal is present).  It seems to me you'd have to build a string of T's into your sequence if you want them to transcribe as A's.  But I'm not sure either. But think about it!







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