Plasmid Design and Construction
The baseline mRNA production construct was designed with 5’ and 3’ UTR
sequences from Xenopus beta-globin, directly flanking a human codon
optimised eGFP sequence. All DNA sequences containing stabilisation
features of interest flanking the UTRs and coding sequence were
synthesized and cloned into pET-24b (Novagen) by Twist Bioscience.
Sequences were inserted using the BamHI and XhoI restriction sites,
placing the GOI downstream of the T7 promoter and lac operon, and
removing the ribosome binding site from the original plasmid. High copy
number plasmids were constructed by amplifying the mRNA encoding region
from the original pET29b vector by PCR, and inserting it into pRNA128A,
a vector with the ColE1 origin of replication. Synthetic triple
terminator constructs were assembled using DNA fragments purchased from
Genewiz. The fragments consisted of the T3 and E. coli endogenous
rrnB T1 terminators, and were ligated into vectors using the XhoI and
BlpI restriction sites, directly upstream of the T7 terminator.
Sequences of all DNA parts are available in Supplementary table 1.