This spacer as you call it is not any random sequence and it serves functions related to the mRNA translation, stability and transport. The name of this sequence is the 3'UTR (untranslated). It serves many functions, 3'UTR can be target site for miRNA, they can stabilize mRNA and protect them from degradation through protein binding, 3'UTR can also be used as protein binding sites for mRNA export.

In your case I guess you want your vector as small as possible but not having a 3'UTR is a bad idea. I woudl use the shortest UTR found in a vector that has previously been shown to work.

Hi Jonas

It is not absolutely necessary to use a spacer between your stop codon and SV40pA. The actual SV40 polyA site (AATAAA) is actually >100bp downstream of the start of the overall SV40pA sequence. The initial 100bp are important for forming 2ndary structure. As long as you keep that initial 100bp, you shouldn't have any problems. We have made vectors without any space between stop codon and the start of SV40pA.

However, as Hugo suggests, a 3'UTR can be extremely important for post-transcriptional regulation of your transgene. In vectorology, people often use WPRE, which is ~500bp. There are smaller viral 3'UTRs available, such as that of Venezuelan Equine Encephalitis Virus (VEEV). The UTR for that virus is 97bp, but functions best when using several repeats, as in the below paper. I think 2 repeats, comprising ~200p, can work ok.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933403/

In summary, you will get ok expression without a 3'UTR or spacer, but expression will be higher if you add a WPRE or VEEV.

Do you think that any of these recommendations would change if I use a Sleeping Beauty (transposon) vector? It has inverted terminal repeats (ITRs) at both sides, I was wondering if these elements (WPRE, SV40pA) could alter the function of ITRs (they are important for transposase recognition and subsequent targeted integration).

Hi Jonas,

It sounds like you know a lot about 3 UTRs and PolyA signals. If someone wants to clone their 3 UTR of interest and remove the SV40 signal extended sequence as well as the SV40 AATAAA element and further downstream SV40 cleavage site and rich U region, would the vector utilize, would the polysignal from the cloned in 3 utr of interest allow transcription termination and polyadenylation? In other words I am trying to generate a luciferase reporter in which termination and polyadenylation will be due to the cloned in 3 utr and further downstream sequence of interest, not to the SV40 elements.

Also when you say the first 100 do you mean the first 100 bps at the 5 end of the SV40 poly AATAAA or the 100 nucleotides following the stop codon?

thanks