Dear Researchers,
I'm trying to construct a vector (using pcDNA3.1) with two different reporter genes with two different promoter sequences of target genes. I have two different constructs with two reporter genes. I have planned to clone the regions in a single vector in two different directions. Can I use the same poly A seq or the Antibiotic resistance polyA sequence in case of opposite directions? Or a single poly A sequence is sufficient if the promoter is cloned in the same direction as the other. Please see the attached image file. The vector I use is pcDNA 3.1 which is modified with GFP and cloned with a promoter seq. The same vector is used to replace GFP by RFP. Can Anyone help me out in sorting with this? Will be glad to have a clear solution to these queries. Thank you in advance :)
The same polyA can be used as many times as you want in either direction until you do not arrange them side by side, which will cause some problem for sequencing confirmation. Two promoter-genes share the same polyA does not always work. I prefer the use promoter-gene-terminator cassette for each gene.
Bicistonic constructs are sometimes tricky. The two promoter may interfere each other if you want them to have differential regulation. If they are not differentially regulated, you may consider also using an IRES construct. However, the two genes may have different expression efficiency in translation. The nice thing with IRES is the single transcript for both cDNAs. Your construct design should work too but there is little advantage over two separated constructs with different selection markers.
In principle yes, although you have to take in account the possibility that the bacteria recombine the plasmid on these homologous sequences.
As stated by Bill Chi Shun Ho above, the expression itself is difficult to control. If you want equal expression, use the (P)2A sequence system which makes a single mRNA being translated equall. The IRES system works too, but protein content will be different and not controllable.
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