8/23/2023 0 Comments Ires sequences![]() The vector of claim 1, wherein the 3′ Group I intron fragment and the 5′ Group I intron fragment are from a T4 phage Td gene.ĥ. The vector of claim 1, wherein the 3′ Group I intron fragment and the 5′ Group I intron fragment are from a Cyanobacterium Anabaena sp. A vector for making circular RNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence: a) a 5′ homology arm, b) a 3′ Group I intron fragment containing a 3′ splice site dinucleotide, c) a 5′ spacer sequence, d) an internal ribosome entry site (IRES), e) a protein coding region or noncoding region, f) a 3′ spacer sequence, g) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and h) a 3′ homology arm, said vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells.ģ. Also disclosed is a method for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector.ġ. In yet another embodiment, the vector can comprise the 3′ spacer sequence, but not the 5′ spacer sequence. In another embodiment, the vector can comprise the 5′ spacer sequence, but not the 3′ spacer sequence. a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3′ spacer sequence, f) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm, said vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells.Disclosed is a vector for making circular RNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence: The preferred IRES (viral bases 273-845) and the minimum IRES (viral bases 400-836) for optimum activity are illustrated.Disclosed are methods and constructs for engineering circular RNA. It also produced nearly twice as much protein as pCITE-1, an early monocistronic iteration, harboring a suboptimal A7 sequence in a crucial structural motif The results indicate that investigators should be aware of and carefully report the sequence of their IRES in any comparative study. ![]() Compared head-to-head with dual-luciferase reporter constructs, a native EMCV IRES in a bicistronic configuration directed 8- to 10-fold more protein than a similarly configured pIRES vector. The history of this element and the experimental consequences of sequence derivations inherent to commercial IRES vectors are less well known. Inclusion of the wild-type element in monocistronic or bicistronic messenger RNAs (mRNAs) confers a high level of cap-independent translation activity to appropriately configured cistrons. The internal ribosomal entry site (IRES)from encephalomyocarditis virus (EMCV) is a popular RNA element used widely in experimental and pharmaceutical applications to express proteins in eukaryotic cells or cell-free extracts.
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