I want to amplify the gene dosage on a large plasmid (~30 kb) in HEK293T cells by cloning a Simian Virus 40 origin of replication into it, but I've been having trouble finding if it's possible for a plasmid that large to be effectively replicated from a SV40 origin.
Searching has only given me info about the size limitations of DNA that can be packaged into a SV40 virion, but I am not using SV40 for packaging so that info isn't useful to me.
I've replicated up to 23 kb plasmids and it worked fine. I don't see a reason why a 30 kb one shouldn't work as well.
Yes, but with reduced efficiency. The SV40 origin requires the large T antigen supplied either by the plasmid or by a permissive cell line (e.g., COS cells). Plasmids larger than the ~5.2 kb SV40 genome can replicate, and in practice plasmids of 15–20 kb have been reported to replicate from the SV40 ori. However, replication efficiency decreases as plasmid size increases, and very large constructs become unstable or replicate poorly.
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