Hi all,

 

I use the plasmid "pBabe" for the first time. Does anyone know where the start codon for protein expression is located? Is it in the the beginning of "truncated Gag" or it is the "ATG" in the incerted cDNA sequence? Thank you very much.

We need more info. What kind of vector is pBabe? Was it made in a lab or bought from a company? Do you know the sequence? Is this an eukaryotic- or prokaryotic-based study? Etc etc. Most eukaryotic genes begin translation with the ATG (usually defined by a Kozak consensus sequence), but is oftentimes posttranslationally modified. There is so much info out there. More info from you would help. Also, consult a good genetics textbook as it would definetly help you.

 

Newty

It really depends on how you want to use the vector. Most plasmids do come with a start codon in them upstream of the promoter elements on the plasmid. You can either clone your gene in upstream of the start codon, in which case your gene is translated using the ATG in pBABE and your protein is expressed with a leader sequence at the n-terminus, or you can clone your gene in downstream of the start codon, in which case the ATG of the gene acts as your site. If you choose the former option, you can be sure that your protein will be expressed (as long as you clone it into the correct reading frame). If you choose the latter option, you will have a better quality protein (since you will not have the N-terminal leader sequence which could potentially mess with the structure and function of your protein) but you cannot be sure that you protein will be well expressed.

 

Try looking up the manual for pBABE from whatever company produces that plasmid. It should have more relevant information about that vector including the sequence around the start codon in the plasmid.

Many thanks. I have got it. The start condon is the first ATG in the inserted DNA sequence.