Provided is a polypeptide tag. The amino acid sequence of the polypeptide tag is Xaa1Xaa2Xaa3PHDYNXaa4Xaa5Xaa6 (SEQ ID NO: 37), wherein in the formula, Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, and Xaa6 are each independently an amino acid or none. The polypeptide tag is used for labeling a target protein. In a second aspect, provided is a polypeptide fusion protein, comprising the following two structures: (1) any polypeptide tag according to the first aspect, and (2) a target protein connected to the polypeptide tag. Also provided are an in vitro cell-free protein synthesis system and an application thereof in in vitro protein synthesis. By constructing the polypeptide tag and a target protein as a fusion protein, the expression of the labeled target protein can be effectively increased without removing the polypeptide tag.

The invention relates to an artificial nucleic acid molecule comprising at least one open reading frame and at least one 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene. The invention further relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination. Furthermore, the invention relates to the use of a 3′-UTR element comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene for the stabilization and/or prolongation of protein expression from a nucleic acid sequence comprising such 3′-UTR element.

The invention relates to an artificial nucleic acid molecule comprising at least one open reading frame and at least one 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene. The invention further relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination. Furthermore, the invention relates to the use of a 3′-UTR element comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene for the stabilization and/or prolongation of protein expression from a nucleic acid sequence comprising such 3′-UTR element.

The present disclosure relates to methods for producing proteins, cells for producing proteins, and methods for producing cells with improved protein production. In certain embodiments, the present disclosure provides a method of producing a selected protein, the method comprising expressing the selected protein in a cell expressing a Rheb (Ras homologue enriched in brain) protein having an activity to increase protein production in the cell, and secreting the selected protein from the cell, thereby producing the protein.

The present disclosure relates to methods for producing proteins, cells for producing proteins, and methods for producing cells with improved protein production. In certain embodiments, the present disclosure provides a method of producing a selected protein, the method comprising expressing the selected protein in a cell expressing a Rheb (Ras homologue enriched in brain) protein having an activity to increase protein production in the cell, and secreting the selected protein from the cell, thereby producing the protein.

The present disclosure provides genetic constructs comprising a recombinant internal ribosome entry site (IRES), which may be used as riboswitches to modulate translation of an operably-mRNA sequence encoding a protein of interest. In other aspects, the disclosure provides recombinant cells, methods, kits and systems that utilize the same, e.g., to provide a platform for modulating the expression of essentially any protein of interest in a eukaryotic cell.

The present disclosure provides genetic constructs comprising a recombinant internal ribosome entry site (IRES), which may be used as riboswitches to modulate translation of an operably-mRNA sequence encoding a protein of interest. In other aspects, the disclosure provides recombinant cells, methods, kits and systems that utilize the same, e.g., to provide a platform for modulating the expression of essentially any protein of interest in a eukaryotic cell.

A self-circularization RNA construct that can be expressed in a DNA vector and simultaneously circularized through a self-targeting and splicing reaction to form a circRNA is disclosed. The circRNA can consist only of a gene of interest which can be a coding, non-coding, or a combination thereof. The gene of interest has the advantage of being able to rapidly express a peptide or protein. The formed circRNA has a circular structure and has a stable and high half-life because 5′ and 3′ ends are not exposed. Accordingly, functional RNA such as miRNA, anti-miRNA, siRNA, shRNA, aptamer, functional RNA for gene/RNA editing, ADAR (adenosine deaminase acting on the RNA)-recruiting RNA, mRNA vaccine, mRNA therapeutic agent, vaccine adjuvant, and CAR-T mRNA can be produced as a stable circRNA in cells.

The present disclosure relates, generally, to compositions and methods for producing, purifying, and using circular RNA.

Recombinant expression vectors are disclosed that include a control sequence for recombinant expression of proteins of interest; the control sequence combines a mCMV enhancer sequence with a rat EF-1alpha intron sequence. Some of the vectors are useful for tetracycline-inducible expression. Some of the vectors contain a 5′ PiggyBac ITR and a 3′ PiggyBac ITR to promote genomic integration into a host cell chromosome. A method of selecting a stable production cell line for manufacturing a protein of interest is also disclosed. Also disclosed are mammalian host cells comprising the inventive recombinant expression vectors and a method of producing a protein of interest, in vitro, involving the mammalian host cell.