Means and methods for preparing engineered target proteins by genetic code expansion in a selective manner of the target protein

Abstract

The present invention relates to an orthogonal translation system that allows the site-specific introduction of non-canonical amino acid residues into a target protein (POI) by selectively translating the POI mRNA. Specifically, the present invention relates to assembler fusion proteins that bring an RNA-targeting polypeptide (RNA-TP) segment and an orthogonal aminoacyl-tRNA synthetase (O-RS) segment into spatial proximity with each other, either by direct linkage as an RNA-TP / O-RS fusion protein or by the action of an "assembler" fused to each of these segments as an assembler fusion protein (AFP). The present invention also relates to nucleic acid molecules containing a POI coding sequence together with a targeting nucleotide sequence capable of interacting with the RNA-TP, and AFP combinations. The present invention further relates to nucleic acid molecules, expression cassettes, and expression vectors encoding the RNA-TP / O-RS fusion protein or AFP, cells containing the same, and methods and kits for translationally preparing a POI.

Description

[Technical Field]

[0001] The present invention relates to an orthogonal translation system that enables the site-specific introduction of non-canonical amino acid (ncAA) residues onto a polypeptide of interest (POI) in a selective manner in POI mRNA. Specifically, the present invention relates to a fusion protein that brings an RNA-targeting polypeptide (RNA-TP) segment and an orthogonal aminoacyl-tRNA synthetase (O-RS) segment into spatial proximity with each other. This is achieved by combining the RNA-TP segment and the O-RS segment into the same fusion protein (RNA-TP / O-RS fusion protein) or by the action of one or more polypeptide segments that act as "assemblers" (APs) and facilitate the local concentration of assembler fusion proteins (AFPs) containing one or more APs together with the RNA-TP or O-RS segment, thereby bringing the RNA-TP segment and the O-RS segment into close proximity with each other. The present invention also relates to nucleic acid molecules and AFP combinations that contain a POI-encoding sequence together with a targeting nucleotide sequence (TN) that can interact with the RNA-TP. The present invention further relates to nucleic acid molecules, expression cassettes, and expression vectors encoding said RNA-TP / O-RS fusion proteins or AFPs, cells containing the same, as well as methods and kits for translationally preparing POIs. [Background technology]

[0002] The ability to engineer orthogonal (i.e., non-cross-reactive) translation systems site-specifically into living cells would enable the introduction of new functional groups into proteins. However, this is a challenging task because translation is a complex, multi-step process in which at least 20 different aminoacylated tRNAs, their corresponding aminoacyl-tRNA synthetases (RSs), ribosomes, and a variety of other factors work together to synthesize polypeptide chains from RNA transcripts. An ideal orthogonal system would exhibit no cross-reactivity with factors from the host machinery, minimizing its impact on the cellular housekeeping translation activity and normal physiology.

[0003] To this end, genetic code expansion (GCE) is a method that enables specific codon reprogramming. GCE allows an orthogonal (suppressive) RS (O-RS) to aminoacylate its corresponding suppressive tRNA with a non-canonical amino acid (ncAA). Typically, these ncAAs are custom-designed and possess chemical functionality that, for example, allows protein function to be photoregulated, encodes post-translational modifications, or allows the introduction of fluorescent labels for microscopy studies using click chemistry. To site-specifically introduce an ncAA into a polypeptide of interest (POI), the anticodon loop of the tRNA is selected to decode and therefore suppress one of the stop codons (see, e.g., Liu et al., Annu Rev Biochem 2010, 79:413-444; Lemke, ChemBioChem 2014, 15:1691-1694; Chin, Nature 2017, 550; 53-60). To minimize the impact on the host cell machinery, amber stop codons (corresponding tRNAs) are used to terminate endogenous proteins due to their particularly low abundance in E. coli (<10%). CUA ) are often utilized. Nevertheless, in principle, any amber codon in the genome can be suppressed, potentially leading to unwanted background suppression of non-target host proteins. When ncAA-modified proteins are recombinantly produced for in vitro applications, this background incorporation may be tolerable as long as the yield of purified full-length protein is acceptable. However, the challenge is different when the host is viewed as more than just a bioreactor vessel that can be sacrificed for the protein. To study the function of a host cell POI in situ, the physiological state of the host cell is an important factor. In that context, minimizing background incorporation of ncAAs is particularly required to ensure well-controlled experiments.

[0004] At least three successful approaches have been developed to enable orthogonal translation in E. coli, i.e., to decode specific codons only for the POI RNA rather than the entire genome. i) Orthogonal ribosomes that recognize unique Shine-Dalgarno sequences have been developed to decode quadruplet codons. These are used in place of stop codons to site-specifically encode ncAAs on the POI (see, e.g., Heumann et al., Nature 2010, 464:441:444; Orelle et al., Nature 2015, 524:119-124; Fried et al., Angew Chem 2015, 54:12791-12794). ii) Recently, genome engineering has progressed to the stage where E. coli strains can be depleted of selected native codons, providing a genetically clean (e.g., amber-free) host background for selective decoding of specific codons only on the POI. (See, e.g., Isaacs et al., Science 2011, 333:348-353; Lajoie et al., Science 2013, 342:357-360; Ostrov et al., Science 2016, 353:819-822; Wang et al., Nature 2016, 539:59-64). iii) Unique non-standard codons are designed using artificial base pairs that are encoded only in the coding sequence of the POI. This reduces the risk of non-specific readings in other parts of the genome (See, e.g., Zhang et al., Nature 2017, 551:644-647). However, due to genome complexity, transferring these orthogonal translation approaches to eukaryotes is not straightforward (See, e.g., Thompson et al., ACS Chem Biol 2018, 13:313-325). In these, in addition, amber codons are highly abundant (20% in mammalian cells).

[0005] Thus, there is a high demand for a strategy for POI-selective orthogonal translation that is versatile and works not only in well-characterized prokaryotes such as E. coli, which are relatively easy to handle and manipulate, but is also applicable to eukaryotic cells. It was therefore an object of the present invention to address this challenge. Summary of the Invention

[0006] The present inventors have discovered that spatial proximity between a POI mRNA and an O-RS, which allows for the translational introduction of an ncAA residue onto the growing polypeptide chain of the POI, creates an orthogonal translation system (OT system) that can selectively translate the POI mRNA. The present inventors have demonstrated that for various POIs, including membrane proteins, these OT systems are capable of site-specifically introducing an ncAA residue onto the POI in mammalian cells by virtue of their selectivity for the POI mRNA compared to other mRNAs in the cytoplasm that contain the same stop codon (which is used as a selector codon to encode the POI ncAA residue).

[0007] In the orthogonal translation system of the present invention, spatial proximity is achieved by including a targeting sequence (TN) on the mRNA of the POI that can selectively interact with an RNA-targeting polypeptide (RNA-TP) and linking the O-RS to such RNA-TP, which may be a fusion protein containing both the O-RS and the RNA-TP (RNA-TP / O-RS fusion protein).

[0008] In another approach, this can be achieved by the action of one or more polypeptide segments that act as "assemblers" (APs) by facilitating the local concentration of at least two assembler fusion proteins (AFPs), at least one of which contains one or more APs and RNA-TP segments, and at least one other AFP contains one or more APs and O-RS segments, thereby bringing the RNA-TP and O-RS segments (RNA-TP and O-RS are also referred to as "effectors" or "EPs") into close proximity with one another. The local concentration of AFPs allows for the formation of an assembly that can act as an artificial orthogonal translation organelle (OT assembly, also referred to herein as "OT organelle").

[0009] The inventors have demonstrated that different types of APs can be used. The first type encompasses APs that drive localized enrichment at (pre-existing) intracellular structures (e.g., microtubules or the cytoplasmic side of membranes such as the plasma or nuclear membrane, ER, mitochondria, or Golgi organelles). These are referred to as intracellular targeting polypeptide (IC-TP) segments. The second type of AP generates high local AFP concentrations by self-association (particularly by phase separation) in the cytoplasm. These are referred to herein as phase-separating polypeptide (PSP) segments. These AP types can also be combined with other polypeptide elements capable of forming multimeric structures, particularly coiled-coil heterodimers, formed by synthetic SYNZIP polypeptide pairs. Similarly, these EP types can also be combined with other polypeptide elements capable of forming multimeric structures, particularly coiled-coil heterodimers, formed by synthetic SYNZIP polypeptide pairs. Such multimerization further improves the local enrichment of AFP.

[0010] The present inventors have further discovered that AFPs that combine different AP types are particularly useful.

[0011] In yet another approach, an AFP is provided that includes both types of EP segments, i.e., RNA-TP and O-RS segments, and one or both types of AP segments, i.e., IC-TP and / or PSP segments, on a single polypeptide, i.e., fused together. Optionally, this is supplemented with a polypeptide element capable of forming multimeric structures (SYNZIP polypeptides). This offers the advantage that all elements required to create the OT system of the present invention are included on a single AFP. Therefore, in a first aspect, the present invention provides: (a) (a1) a polypeptide segment (IC-TP segment) derived from an intracellular targeting polypeptide, which targets and is therefore locally concentrated in an intracellular structural element within or directly adjacent to the cytoplasm; and (a2) a polypeptide segment (PSP segment) derived from a phase-separating polypeptide, the phase-separating polypeptide having the ability to self-associate in the cytoplasm in a manner that creates sites of high local cytoplasmic concentration; at least one first polypeptide segment acting as an assembler (AP) selected from: (b) b1) an RNA-targeting polypeptide (RNA-TP) segment, and b2) orthogonal aminoacyl-tRNA synthetase (O-RS) segment; and at least one second polypeptide segment acting as an effector (EP) selected from: wherein the polypeptide segments are functionally linked together on the AFP.

[0012] In a second aspect, the present invention relates to an assembler fusion protein (AFP) combination comprising at least two AFPs of the present invention as described herein. Preferably, the AFP combination comprises at least one AFP comprising an RNA-TP segment and at least one AFP comprising an O-RS segment. Inclusion of a first SYNZIP element on at least one AFP of the combination and a second SYNZIP element on at least another AFP of the combination represents another advantageous form of the second aspect, wherein the first and second SYNZIP elements act together by forming a heterodimeric structure.

[0013] In a third aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) at least one RNA-targeting polypeptide (RNA-TP) segment; (ii) at least one orthogonal aminoacyl-tRNA synthetase (O-RS) segment; a fusion protein (RNA-TP / O-RS fusion protein) comprising: The polypeptide segments are functionally linked on the RNA-TP / O-RS fusion protein.

[0014] In a further aspect, the present invention provides a nucleic acid molecule, or a combination of two or more nucleic acid molecules, comprising: (i) a nucleotide sequence encoding at least one RNA-TP / O-RS fusion protein of the invention described herein; or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); Includes.

[0015] In a further aspect, the present invention provides a nucleic acid molecule, or a combination of two or more nucleic acid molecules, comprising: (i) a nucleotide sequence encoding at least one AFP of the invention as described herein, or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); Includes.

[0016] In a further aspect, the present invention provides a nucleic acid molecule, or a combination of two or more nucleic acid molecules, comprising: (i) a nucleotide sequence encoding at least one AFP combination of the invention as described herein, or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); Includes.

[0017] In a further aspect, the present invention provides an expression cassette, comprising the nucleotide sequence of a nucleic acid molecule or combination of nucleic acid molecules of the invention described herein.

[0018] In certain embodiments, the present invention provides an expression cassette comprising: (i) a nucleotide sequence encoding at least one RNA-TP / O-RS fusion protein of the invention described herein; or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); Includes.

[0019] In a further particular embodiment, the present invention provides an expression cassette comprising: (i) a nucleotide sequence encoding at least one AFP of the invention as described herein, or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); Includes.

[0020] In a further particular embodiment, the present invention provides an expression cassette comprising: (i) a nucleotide sequence encoding at least one AFP combination of the invention as described herein, or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); Includes.

[0021] In a further aspect, the present invention provides an expression vector comprising at least one expression cassette of the invention described herein.

[0022] In a further aspect, the present invention provides a cell comprising at least one nucleic acid molecule or combination of nucleic acid molecules of the invention described herein. In certain embodiments, the cell comprises at least one expression cassette or at least one expression vector of the invention described herein.

[0023] In a further aspect, the present invention relates to a method for preparing a polypeptide of interest (POI) comprising one or more non-canonical amino acid (ncAA) residues on its amino acid sequence, said method comprising expressing the POI by a cell of the invention in the presence of said one or more ncAA residues, wherein the cell: (i) at least one AFP comprising an RNA-TP segment and at least one AFP comprising an O-RS segment, as described herein; (ii) a nucleotide sequence encoding the POI (CS POI ) wherein the one or more ncAA residues of the POI are encoded by a selector codon(s), (iii)CS POI a targeting nucleotide sequence (TN) functionally linked to the targeting nucleotide sequence (TN) and capable of interacting with at least one RNA-TP segment of the AFP in a cell; (iv)CS POI one or more orthogonal tRNAs carrying anticodon(s) complementary to the selector codon(s) of ncAA (O-tRNA ncAA ) molecule (the O-tRNA ncAA The molecule combines with one or more O-RS segments of an intracellular AFP to form one or more orthogonal O-RS / O-tRNAs. ncAA pair, which allows the introduction of said one or more ncAA residues onto the amino acid sequence of the POI); and the method optionally further comprises recovering the expressed POI. The at least one AFP comprising an RNA-TP segment described in (i) and the at least one AFP comprising an O-RS segment may be one AFP of the same type, i.e., an AFP comprising both an RNA-TP segment and an O-RS segment. Alternatively, the at least one AFP comprising an RNA-TP segment described in (i) and the at least one AFP comprising an O-RS segment may be different AFPs.

[0024] In a further aspect, the present invention relates to a method for preparing a polypeptide of interest (POI) comprising one or more non-canonical amino acid (ncAA) residues on its amino acid sequence, said method comprising expressing the POI by a cell of the invention in the presence of said one or more ncAA residues, wherein the cell: (i) an RNA-TP / O-RS fusion protein of the invention described herein; (ii) a nucleotide sequence encoding the POI (CS POI ) wherein the one or more ncAA residues of the POI are encoded by a selector codon(s), (iii)CS POI a targeting nucleotide sequence (TN) functionally linked to the RNA-TP / O-RS fusion protein, capable of interacting with at least one RNA-TP segment of the RNA-TP / O-RS fusion protein in a cell; (iv)CS POI one or more orthogonal tRNAs carrying anticodon(s) complementary to the selector codon(s) of ncAA (O-tRNA ncAA ) molecule (the O-tRNA ncAA The molecule, together with one or more O-RS segments of an RNA-TP / O-RS fusion protein in a cell, produces one or more orthogonal O-RS / O-tRNAs. ncAA pair, which allows the introduction of said one or more ncAA residues onto the amino acid sequence of the POI); Including, The method optionally further comprises recovering the expressed POI.

[0025] In a further aspect, the present invention relates to a method for preparing a polypeptide of interest (POI) comprising one or more non-canonical amino acid (ncAA) residues on its amino acid sequence, the method comprising: (a) expressing by a cell one or more AFPs comprising at least one RNA-TP segment and one or more AFPs comprising at least one O-RS segment, as described herein; (b) one or more orthogonal tRNAs ncAA (O-tRNA ncAA ) molecule expressed by said cell, - the orthogonal tRNA ncAA The O-RS segments of the molecule and AFP are linked to one or more orthogonal aminoacyl-tRNA synthetases / tRNAs in the cell. ncAA (O-RS / O-tRNA ncAA ) form a pair, - the O-RS / O-tRNA ncAA The pair allows the introduction of the one or more ncAA residues onto the amino acid sequence of the POI, Steps (a) and (b) may be simultaneous or sequential in any order; (c) then expressing the POI by the cell in the presence of the one or more ncAAs; - Nucleotide sequence encoding the POI (CS POI ) comprises one or more selector codons encoding the one or more ncAA residues, - the selector codon is selected from the one or more O-tRNAs ncAA Matches the anticodon of the molecule; - Said CS POI is functionally linked to the targeting nucleotide sequence (TN), and thus CS POI / TN fusion sequence, - Said CS POI a step in which the / TN fusion sequence can interact with at least one RNA-TP segment of AFP in a cell via its TN; (d) optionally recovering the expressed POI; Includes.

[0026] In a further aspect, the present invention relates to a method for preparing a polypeptide of interest (POI) comprising one or more non-canonical amino acid (ncAA) residues on its amino acid sequence, the method comprising: (a) expressing by a cell an RNA-TP / O-RS fusion protein of the invention described herein; (b) one or more orthogonal tRNAs ncAA (O-tRNA ncAA ) molecule expressed by said cell, - one or more O-RS segments of an RNA-TP / O-RS fusion protein and said orthogonal tRNA ncAA The molecule is capable of catalyzing one or more orthogonal aminoacyl-tRNA synthetases / tRNAs within the cell. ncAA (O-RS / O-tRNA ncAA ) form a pair, - the O-RS / O-tRNA ncAA The pair allows the introduction of the one or more ncAA residues onto the amino acid sequence of the POI, Steps (a) and (b) may be simultaneous or sequential in any order; (c) then expressing the POI by the cell in the presence of the one or more ncAAs; - Nucleotide sequence encoding the POI (CS POI ) comprises one or more selector codons encoding the one or more ncAA residues, - the selector codon is selected from the one or more O-tRNAs ncAA Matches the anticodon of the molecule; - Said CS POI is functionally linked to the targeting nucleotide sequence (TN), and thus CS POI / TN fusion sequence, - Said CS POI a step in which the / TN fusion sequence can interact with at least one RNA-TP segment of an RNA-TP / O-RS fusion protein in a cell via its TN; (d) optionally recovering the expressed POI; Includes.

[0027] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a nucleotide sequence encoding a polypeptide of interest (POI) (CS POI ) and (the POI is selected by the selector codon CS POI containing one or more identical or different non-canonical amino acid (ncAA) residues as encoded above), (ii) a targeting nucleotide sequence (TN), An RNA molecule containing the TN can interact with an RNA-targeting polypeptide (RNA-TP) via the TN.

[0028] In a further aspect, the present invention relates to a kit for preparing a polypeptide of interest (POI) having at least one non-canonical amino acid (ncAA) residue, the kit comprising: - at least one ncAA or a salt thereof corresponding to at least one ncAA residue of the POI; - at least one expression vector of the invention as described herein; Includes. The expression vector comprises: (i) a nucleotide sequence encoding at least one RNA-TP / O-RS fusion protein of the invention, at least one AFP of the invention, or at least one AFP combination of the invention; or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); The method comprises at least one expression cassette comprising: [Brief explanation of the drawings]

[0029] [Figure 1]This figure shows a schematic diagram of the spatial separation of components that enables orthogonal translation by decoding specific stop codons on uniquely tagged mRNAs. (A) Conventional expression of the synthetase PylRS leads to aminoacylation of its corresponding stop codon-suppressing tRNA Pyl with a custom-designed ncAA. This leads to site-specific ncAA incorporation whenever the respective stop codon is present on the mRNA of the POI. Given that many endogenous mRNAs terminate at the same stop codon, utilizing this approach in the cytoplasm potentially leads to the misincorporation of the ncAA into unwanted proteins (left box). (B) To circumvent this, the present invention allows the mRNA encoding the POI and the orthogonal aminoacyl-tRNA synthetase (e.g., PylRS) to be brought into close proximity with each other through the use of an RNA-targeting polypeptide segment (e.g., MCP) and an assembler (AP). This allows spatial concentration of all components, creating an OT assembly ("OT organelle") containing the POI-encoding mRNA, orthogonal aminoacyl-tRNA synthetase, tRNA, and ribosome (right box). Here, aminoacylated tRNAPyl is particularly available in direct proximity to the OT organelle, resulting in stop codon suppression (of the POI mRNA) occurring specifically here. This leads to selective suppression (and therefore expression) of the stop codon of the POI mRNA compared with the corresponding stop codon on mRNAs not targeted to the OT assembly. In (A), GCE occurs in a stop codon-specific manner, whereas in (B), it should occur in a stop codon- and mRNA-specific manner.

[0030] [Figure 2A] Schematic diagram of different assembler classes: B = bimolecular MCP::PylRS fusion, P1 = fusions with FUS and EWSR1, P2 = SPD5, K1 = truncated kinesin KIF13A (KIF13A1-411, ΔP390), K2 = truncated kinesin KIF16B (KIF16B1-400), and their combinations (K1::P1, K1::P2, K2::P1, K2::P2).

[0031] [Figure 2B] A schematic diagram of the dual-color reporter is shown. mRNAs encoding the fluorescent proteins GFP and mCherry, which contain stop codons at permissive sites, are expressed from a single plasmid. Each has its own CMV promoter, ensuring a consistent ratio of mRNAs in each experiment. The mCherry reporter mRNA, mRNA(mCherry)::ms2, is tagged with two MS2 RNA stem-loops ("ms2"; also referred to herein as MS2 tags). In the presence of ncAA and tRNAPyl, in the case of cytoplasmic PylRS, both GFP39 and mCherry185 stops are produced, resulting in a diagonal line in fluorescence flow cytometry (FFC) analysis (left box). However, under the same conditions, orthogonal translation in OT organelles allows selective stop codon suppression of mRNA(mCherry):ms2, resulting in an mCherry-positive and GFP-negative population (schematically depicted as vertical populations in the right box). In both schemes, untransfected HEK293T cells are represented by the lower grey circle.

[0032] [Figure 2C]The selectivity and relative efficiency of various exemplary OT systems are shown. In all experiments, the indicated constructs were coexpressed with tRNAPyl (anticodon corresponding to the indicated codon) and a dual reporter (GFP39STOP, mCherry185STOP::ms2). GCE was performed in the presence of the indicated ncAA, and cells were analyzed by FFC. Dark gray bars (normalized to cytoplasmic PylRS) represent the ~fold change in the ratio r of the mean fluorescence intensity of mCherry to GFP (derived from FFC; see Figure 2D, E) for all systems tested. Light gray bars represent the relative efficiency, defined by the mean fluorescence intensity of mCherry (derived from FFC; see Figure 2D, E) for each condition divided by the cytoplasmic PylRS control. Mean values ​​from at least three independent experiments are shown; error bars represent SEM. The box highlights the best-performing OT organelle (OTK2::P1).

[0033] [Figure 2D] Figure 1 shows the results of FFC analysis of the two-color reporter expressed by the four indicated systems and tRNAPyl in transfected HEK293T cells in the presence of the lysine derivative ncAA SCO bearing a cyclooctyne side chain. Highly selective and efficient orthogonal translation was observed for OT assembly (black arrows indicate bright, highly mCherry-positive populations). The dot plots show the totals from at least three independent experiments. The axes indicate fluorescence intensity in arbitrary units.

[0034] [Figure 2E] Shown are FFC plots of OT assemblies that selectively translate only the opal and ochre codons of the recruited mRNA(mCherry185TGA):ms2 and mRNA(mCherry185TAA):ms2, respectively.

[0035] [Figure 3]Schematic diagrams of the constructs that make up the following systems are shown: PylRS, MCP::PylRS, FUS::MCP::PylRS, and LcK::FUS::PylRS·LcK::EWS::MCP.

[0036] [Figure 4] Flow cytometry analysis of dual reporter expression by the four different systems illustrated in Figure 3. HEK293T cells were transfected with constructs encoding the dual reporter, tRNA, LcK::FUS::PylRS, and LcK::EWS::MCP, or PylRS, MCP::PylRS, FUS::MCP::PylRS, and pcDNA3.1. A total of at least three independent experiments is shown. The axis indicates fluorescence intensity in arbitrary units.

[0037] [Figure 5] A bar graph of the ratio of mean fluorescence intensity of mCherry to GFP fluorescence is shown for all lines tested. Plots represent the average of at least three biological replicates, and error bars indicate the standard error of the mean.

[0038] [Figure 6]This figure provides an overview of different approaches to generating OT organelles targeted to the surface of different intracellular structures. Different constructs were expressed, and the results of their respective fluorescence flow cytometry (FFC) analyses are shown. At the top of the figure, the dual-color reporter construct GFP39TAG·mCherry185TAG::ms2 (see also Figure 2B) applied in each of the schematically illustrated experiments A through G is illustrated, and a schematic illustration of the different target cellular compartments is shown. A control experiment performed without the effector polypeptide MCP (-MCP) is also illustrated for each of experiments A through G. A: OT organelles targeted to microtubules and obtained by expressing the system KIF16B1-400::FUS::PylRS·KIF16B1-400::EWSR1::MCP or the construct KIF16B1-400::FUS::PylRS (control). B: OT organelles targeted to the microtubule plus ends and obtained by expressing the construct EB1::FUS::MCP::PylRS or EB1::FUS::PylRS (control). C: OT organelles targeted to the plasma membrane and obtained by expressing the system LcK::FUS::PylRS·LcK::EWSR1::MCP or the construct LcK::FUS::PylRS (control). D: OT organelles targeted to the mitochondrial membrane and obtained by expressing the system TOM201-70::FUS::PylRS·TOM201-70::EWSR1::MCP or the construct TOM201-70::FUS::PylRS (control). E: OT organelles targeted to the nuclear membrane and obtained by expressing the system CG1::FUS::PylRS·CG1::EWSR1::MCP or the construct CG1::FUS::PylRS (control). F (left side): OT organelles targeted to the Golgi membrane and obtained by expressing the system EBAG91-29::FUS::PylRS·EBAG91-29::EWSR1::MCP or the construct EBAG91-29::FUS::PylRS (control). F (right side): OT organelles targeted to the Golgi membrane and obtained by expressing the system CMP Sia Tr::FUS::PylRS·CMP Sia Tr::MCP or the construct CMP Sia Tr::FUS::PylRS (control).G: OT organelles targeted to the ER membrane and obtained by expressing the system P450 2C11-27::FUS::PylRS·P450 2C11-27::EWSR1::MCP or the construct P450 2C11-27::FUS::PylRS (control).

[0039] [Figure 7] This section provides an overview of different approaches of the present invention for recruiting RNA using the interaction of different RNA loops and the respective RNA-targeting proteins. The results of respective fluorescence flow cytometry (FFC) analyses are shown and compared with the respective analyses obtained with untargeted PylRS alone. A: The ms-2-MCP system incorporates an ms2 loop into the UTR of an mRNA molecule, and recruits the mRNA to an artificial organelle via the MCP protein. B: The boxB-λN22 system incorporates a boxB loop into the UTR of an mRNA molecule, and recruits the mRNA to an artificial organelle via the λN22 protein. C: The pp7-PCP system incorporates a pp7 loop into the UTR of an mRNA molecule, and recruits the mRNA to an artificial organelle via the PCP protein.

[0040] [Figure 8]This figure illustrates a further approach of the present invention for generating OT organelles that may function on the surface of different cellular structures. Here, targeting to the plasma membrane is exemplified. This particular approach features the paired integration of the so-called synthetic heterodimeric coiled-coil peptides SYNZIP1 and SYNZIP2 fused to the system LcK::FUS::SYNZIP1::PylRS·EWSR1::SYNZIP2::MCP. Upon expression, SYNZIP1 and SYNZIP2 pair and recruit MCP to the plasma membrane-based OT organelle, which in turn enables selective orthogonal translation of subsequently recruited mRNAs containing the MS2-targeted nucleotide loop. The selective translation is illustrated by the results of the respective FFC analyses (A). In a comparative approach using the LcK::FUS::PylRS·EWSR1::SYNZIP2::MCP system lacking SYNZIP1, no translation selectivity could be observed (B). DETAILED DESCRIPTION OF THE INVENTION

[0041] Unless otherwise defined herein, scientific and technical terms used in the context of the present invention have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear. However, in the event of any possible ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0042] Unless otherwise noted, nucleotide sequences are depicted herein in a 5' to 3' orientation. Unless otherwise noted, amino acid sequences are depicted herein in an N-terminal to C-terminal orientation.

[0043] Unless otherwise specified, the polypeptide of interest (POI) translationally expressed by an OT system according to the present invention is a nucleotide sequence encoding the POI (CS POI ) contains one or more ncAA residues encoded above.

[0044] 1. Fusion Proteins

[0045] 1.1 General

[0046] The fusion proteins of the present invention can be construed in different ways.

[0047] The first type encompasses fusion proteins in which at least two types of effector polypeptides (EPs), including at least one RNA-TP and at least one O-RS, are contained in one and the same fusion protein (also referred to as RNA-TP / O-RS fusion protein).

[0048] The second type encompasses fusion proteins (also referred to as AFPs) comprising at least one assembler polypeptide (AP) and at least one type of EP selected from an RNA-TP segment and an O-RS segment. In particular, the AFP can comprise, in addition to at least one type of AP, both an RNA-TP segment and an O-RS segment, e.g., one or more RNA-TP segments and one or more O-RS segments, in any sequential order. Thus, in particular, the AFP is selected from the following fusion protein types (the segments are functionally linked in any order on the polypeptide chain; one or more segments of the same type may be in any order on the polypeptide chain):

[0049] (RNA-TP / AP) (O-RS / AP) (RNA-TP / O-RS / AP)

[0050] The AP is selected from IC-TPs and PSPs and can be composed of one or more IC-TPs and / or one or more PSPs in any sequential order. Thus, the AFP is more particularly selected from the following fusion protein types (the segments are functionally linked in any order on the polypeptide chain, and one or more segments of the same type are in any order on the polypeptide chain): (RNA-TP / IC-TP) (O-RS / IC-TP) (RNA-TP / O-RS / IC-TP)

[0051] (RNA-TP / PSP) (O-RS / PSP) (RNA-TP / O-RS / PSP)

[0052] (RNA-TP / PSP / IC-TP) (O-RS / PSP / IC-TP) (RNA-TP / O-RS / PSP / IC-TP)

[0053] The AP and / or EP may also comprise (as part of the fusion protein) hetero-oligomerizing, in particular heterodimerizing, polypeptide segments such as synthetic coiled-coil SYNZIP peptides. AFP combinations comprising such interacting SYNZIP pairs distributed among the members of said AFP combination such that each AFP comprises only one member of such interacting SYNZIP pair are particular embodiments.

[0054] The term "segment," as used herein in the context of a fusion protein, indicates that the element so designated (e.g., RNA-TP, O-RS, IC-TP, PSP, SYNZIP) is part of the fusion protein, i.e., is linked to the remainder of the fusion protein. The segments of the fusion proteins of the invention are functionally linked, i.e., linked so that they function as RNA-TP, O-RS, IC-TP, and PSP or SYNZIP, respectively. The linkage is preferably covalent, particularly a peptidic linkage.

[0055] For example, the RNA-TP segment included in the fusion protein of the invention is a segment of the fusion protein that is derived from RNA-TP and functions in the context of the fusion protein, thereby enabling the fusion protein to interact (bind) with a target RNA, and the interaction is advantageously specific. Thus, the RNA-TP segment may comprise the amino acid sequence (entire) or a functional fragment of an RNA-targeting polypeptide described herein.

[0056] Similarly, an O-RS segment included in a fusion protein of the invention is a segment of the fusion protein that is derived from an O-RS and that functions in the context of the fusion protein, thereby conferring O-RS enzymatic activity to the fusion protein, i.e., the ability to catalyze the aminoacylation of an O-tRNA with an ncAA. Thus, the O-RS segment can comprise the amino acid sequence (entire) or a functional fragment of an O-RS described herein.

[0057] The assembler fusion protein (AFP) described herein comprises at least one polypeptide segment that acts as an assembler (AP). As used herein, the term AP refers to any polypeptide segment that allows the AFP comprising said segment to be concentrated at a spatially distinct site within a living cell. Conveniently, said spatially distinct site is located within or directly adjacent to the cytoplasm of the cell, and is readily accessible to the cellular translation machinery (which includes standard aminoacylated tRNAs, translation factors, ribosomal subunits, etc.) and O-tRNA. This allows the introduction of an ncAA residue onto the POI.

[0058] There are different types of polypeptide segments that function as APs in the present invention. One type of AP is a polypeptide segment derived from an intracellular targeting polypeptide (IC-TP) and that functions in the context of a fusion protein. These IC-TP segments may comprise the entire amino acid sequence of IC-TP or a functional fragment. IC-TP targets intracellular structural elements within or directly adjacent to the cytoplasm and is therefore locally concentrated therein. Examples of such structural elements include microtubules, the cytoplasmic side of membranes such as the cell membrane, nuclear membrane, mitochondrial membrane, Golgi membrane, and ER membrane.

[0059] Thus, in certain embodiments, the fusion proteins of the invention comprise at least one IC-TP segment that targets and facilitates localized concentration of the fusion protein at microtubules, particularly the plus or minus ends of microtubules. For example, dynein and kinesin (proteins of the dynein or kinesin family of proteins) and functional fragments and variants thereof can be used as IC-TPs for such functions.

[0060] In further specific embodiments, the fusion proteins of the present invention comprise at least one IC-TP segment derived from and functional as a membrane anchor. For example, the fusion proteins of the present invention comprise at least one IC-TP segment that targets the (inner) plasma membrane (particularly the cytoplasmic side of the plasma membrane) and facilitates localized concentration of the fusion protein therein. In another example, the fusion proteins of the present invention comprise at least one IC-TP segment that targets the (outer) nuclear membrane (particularly the cytoplasmic side of the nuclear membrane) and facilitates localized concentration of the fusion protein. In further specific embodiments, the fusion proteins of the present invention comprise at least one IC-TP segment that targets the outer mitochondrial membrane (particularly the cytoplasmic side of the mitochondrial membrane) and facilitates localized concentration of the fusion protein. In further specific embodiments, the fusion proteins of the present invention comprise at least one IC-TP segment that targets the outer ER membrane (particularly the cytoplasmic side of the ER membrane) and facilitates localized concentration of the fusion protein. In further specific embodiments, the fusion proteins of the invention comprise at least one IC-TP segment that targets the outer Golgi membrane (particularly the cytoplasmic side of the Golgi membrane) and facilitates localized concentration of the fusion protein. For example, transmembrane domains of membrane proteins and their functional fragments and variants can be used as IC-TPs for such functions.

[0061] Polypeptides that target, and therefore locally concentrate at, the intracellular structural elements described above are known in the art and are useful as IC-TPs in the present invention. Specific examples of suitable IC-TPs are: - optionally truncated kinesin polypeptides that constitutively move towards and are locally concentrated at the plus ends of microtubules in living cells, such as optionally truncated kinesin family member 16B (KIF16B), for example, optionally truncated Homo sapiens KIF16B (Uniprot: Q96L93), in particular a fragment covering KIF16B amino acid residues 1-400 comprising the amino acid sequence of SEQ ID NO: 20 (KIF16B 1-400or an optionally truncated form of kinesin family member 13A (KIF13A), such as an optionally truncated Homo sapiens KIF13A (Uniprot: Q9H1H9), in particular a KIF13A fragment covering amino acid residues 1-411, lacking P390, comprising the amino acid sequence of SEQ ID NO: 22 (KIF13A 1-411,Δ390 the microtubule tip-binding protein polypeptide EB1, which binds to growing microtubule plus ends (Nehlig A, Molina A, Rodrigues-Ferreira S, Honore S, Nahmias C. Regulation of end-binding protein EB1 in the control of microtubule dynamics. Cell Mol Life Sci. 2017;74(13):2381-2393. doi:10.1007 / s00018-017-2476-2) (Uniprot:Q15691), thus targeting organelles to microtubule plus ends, and comprising the amino acid sequence of SEQ ID NO: 302; - a polypeptide targeting the outer mitochondrial membrane derived from a transmembrane protein, such as, for example, an optionally truncated version of mitochondrial outer membrane translocase 20 (TOMM20), e.g., an optionally truncated version of Homo sapiens TOMM20 (Uniprot: Q15388), in particular a fragment covering amino acid residues 1-70 of TOMM20 comprising the amino acid sequence of SEQ ID NO: 24 (TOMM20 1-70 ); - cell membrane-targeted polypeptides derived from transmembrane proteins such as lymphocyte-specific protein tyrosine kinase (LcK; e.g., Mus musculus LcK, Uniprot: P06240), CD4 (e.g., Mus musculus CD4, Uniprot: P06332), FRB (similar to Homo sapiens mTOR; Uniprot: P42345), CD28 (e.g., Mus musculus CD28, Uniprot: P31041), and combinations thereof, in particular polypeptides comprising the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30; - CG1, a nucleoporin polypeptide that binds to the cytoplasmic side of the nuclear pore complex (Fernandez-Martinez J, Kim SJ, Shi Y, et al. Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform. Cell. 2016; 167(5): 1215-1228. e25. doi: 10.1016 / j.cell.2016.10.028) (also called Nup42) (Uniprot: O15504). Targets to the cytoplasmic side of the nuclear membrane. Contains the amino acid sequence of SEQ ID NO: 304; - a polypeptide EBAG9 of a Golgi membrane protein with one transmembrane helix (Engelsberg A, Hermosilla R, Karsten U, Schulein R, Dorken B, Rehm A. The Golgi protein RCAS1 controls cell surface expression of tumor-associated O-linked glycan antigens. J Biol Chem. 2003; 278(25): 22998-23007. doi: 10.1074 / jbc.M301361200 (Uniprot: O00559) which is targeted to the cytoplasmic side of the Golgi membrane and comprises the amino acid sequence of SEQ ID NO: 292 (full length) or the first 29 N-terminal amino acid residues of SEQ ID NO: 294; or a polypeptide CMP Sia Tr of a Golgi protein with 10 transmembrane helices (Eckhardt M, Gotza B, Gerardy-Schahn R. Membrane topology of the mammalian CMP-sialic acid transporter. J Biol Chem. 1999;274(13):8779-8787. doi:10.1074 / jbc.274.13.8779) (Uniprot:P78382). Targets to the cytoplasmic side of the Golgi membrane. Contains the amino acid sequence of SEQ ID NO: 296; - a polypeptide fragment of the endoplasmic reticulum resident protein P450 2C1 (Fazal FM, Han S, Parker KR, et al. Atlas of Subcellular RNA Localization Revealed by APEX-Seq. Cell. 2019;178(2):473-490.e26.doi:10.1016 / j.cell.2019.05.027) (Uniprot:P78382), which targets the cytoplasmic side of the ER membrane. In particular, a fragment comprising the first 27 (SEQ ID NO:298) or first 29 (SEQ ID NO:300) amino acid residues at the N-terminus; - the transmembrane protein stomatin-like protein 3 (SLP-3) (membrane containing the amino acid sequence of SEQ ID NO: 310; aa 1-59 (Homo sapiens, Uniprot: Q8TAV4). It is localized in the plasma membrane and vesicle membranes (Lapatsina L, Jira JA, Smith ES, et al. Regulation of ASIC channels by a stomatin / STOML3 complex located in a mobile vesicle pool in sensory neurons. Open Biol. 2012; 2(6): 120096. doi:10.1098 / rsob.120096); and functional fragments and variants of these polypeptides, which can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid of the polypeptide from which they are derived.

[0062] Another type of AP is a polypeptide segment derived from a phase-separating polypeptide (PSP) and functions in the context of a fusion protein. PSPs are polypeptides that have the ability to self-assemble in the cytoplasm of cells, creating sites of high local concentration in the cytoplasm. Specifically, PSPs can drive phase separation (especially liquid-liquid phase separation), leading to the formation of membraneless compartments in the cytoplasm. The compartments can take the form of droplets, aggregates, condensates, or dense phases. In particular, PSPs encompass intrinsically disordered proteins (IDPs), an important class of proteins that drive phase separation (see, e.g., Alberti et al., Bioessays 2016, 38:959-968 and references cited therein, e.g., Patel et al., Cell 2015, 162:1066-1077; Han et al., Cell 2012, 149:768-779; Kato et al., Cell 2012, 149:753-767). There are three distinct classes of ICPs. Each protein, or its functional fragment or variant, can be used as a PSP in the present invention. One prominent class of IDPs contains so-called prion-like domains. These are uncharged and contain polar amino acid residues (Q, N, S, G) interspersed with aromatic residues (F, Y). See, e.g., Malinovska et al., Biochim Biophys Acta 2013, 1834:918-931; Alberti et al., 2009, Cell 137:146-158; Malinovska et al., Prion 2015, 9:339-346. Another class of IDPs is also characterized by low sequence complexity, but frequently contains acidic and basic amino acid side chains. For example, RGG repeat-containing IDPs such as Ddx4. See Nott et al., Cell 2015, 57:936-947. Specific examples of suitable IC-TPs include: - Spindle defect protein 5 (SPD5) (e.g. Caenorhabditis elegans SPD5; Uniprot: P91349), in particular a polypeptide comprising the amino acid sequence of SEQ ID NO: 32; - fusion sarcoma (FUS) (e.g. Homo sapiens FUS; Uniprot: P35637), in particular a polypeptide comprising the amino acid sequence of SEQ ID NO: 34; - Ewing's sarcoma breakpoint region 1 (EWSR1) (e.g., Homo sapiens EWSR1; Uniprot: Q01844), in particular a polypeptide comprising the amino acid sequence of SEQ ID NO: 36; - ATP-dependent RNA helicase laf-1 (RGG domain, 1-168, LAF-1 membrane. Contains the amino acid sequence of SEQ ID NO: 308) (Caenorhabditis elegans, Uniprot: D0PV95) (Schuster BS, Reed EH, Parthasarathy R, et al. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. Nat Commun. 2018;9(1):2985. Published 2018 Jul 30. doi:10.1038 / s41467-018-05403-1); and functional fragments and variants of these polypeptides, but are not limited to these. The functional fragments and variants may comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid of the polypeptide from which they are derived.

[0063] The number of APs included in the fusion proteins of the present invention is not particularly limited. That is, the fusion proteins may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more APs, which may be the same or different. Fusion proteins of the present invention comprising at least one AP selected from an IC-TP segment and at least one AP selected from a PSP segment are particularly preferred. Similarly, the number of RNA-TP segments is not particularly limited and can be independently selected from 1, 2, 3, 4, 5, or more, such as 6, 7, 8, 9, or 10, different or identical RNA-TP segments. Similarly, the number of O-RS segments is not particularly limited and can be independently selected from 1, 2, 3, 4, 5, or more, such as 6, 7, 8, 9, or 10, different or identical O-RS segments. This applies to both AFP and RNA-TP / O-RS fusion proteins. Naturally, the number of segments in the fusion proteins of the present invention affects their size. While not particularly limited, the size is typically less than 3,500 amino acid residues, e.g., less than 3,000 amino acid residues.

[0064] The order of the segments in the fusion proteins of the present invention is also not particularly limited. Therefore, the RNA-TP, O-RS, and / or AP segments can be functionally linked in any order. An example of an RNA-TP / O-RS fusion protein structure (containing both types of EP segments) is: [RNA-TP] x -[O-RS] y [O-RS] y -[RNA-TP] x wherein x and y are, independently of each other, integers selected from 1, 2, 3, 4, and 5; "-" indicates a peptidic linkage. [RNA-TP] for x ≥ 2 x can include the same or different RNA-TP segments. [O-RS] where y≧2 ycan include the same or different O-RS segments.

[0065] An example of an RNA-TP / O-RS fusion protein structure is: [IC-TP] m -[EP] o [EP] o -[IC-TP] m [PSP] n -[EP] o [EP] o -[PSP] n [IC-TP] m -[EP] o -[PSP] n [PSP] n -[EP] o -[IC-TP] m [IC-TP] m -[PSP] n -[EP] o [EP] o -[PSP] n -[IC-TP] m [PSP] n -[IC-TP] m -[EP] o [EP] o -[IC-TP] m -[PSP] n wherein m, n, and o are independently integers selected from 1, 2, 3, 4, or 5, or selected from 1, 2, 3, 4, 5, 6, and "-" indicates a peptidic linkage.

[0066] In a preferred embodiment, "m" is the integer 1.

[0067] In another preferred embodiment, "n" is an integer selected from 1 and 2.

[0068] In yet another preferred embodiment, "o" is an integer selected from 1, 2, 3, 4, 5, or 6 when EP is selected from RNA-TP.

[0069] In yet another preferred embodiment, "o" is an integer selected from 1 or 2 when EP is selected from O-RS.

[0070] In yet another preferred embodiment of the RNA-TP / O-RS fusion protein structure, at least one ICT-TP preferably takes the C- or N-terminal position in the polypeptide chain.

[0071] In yet another preferred embodiment of the RNA-TP / O-RS fusion protein structure, at least one EP preferably takes the C- or N-terminal position on the polypeptide chain.

[0072] In yet another preferred embodiment of the RNA-TP / O-RS fusion protein structure, at least one ICT-TP occupies either the C- or N-terminal position on the polypeptide chain, and at least one EP occupies either the N- or C-terminal position on the polypeptide chain, respectively, and any PSP, if present in such a structure, is located within the polypeptide chain.

[0073] [IC-TP] for m ≥ 2 m can include the same or different IC-TP segments. Preferably, IC-TPs with the same functional group (targeting the same type of cellular structure (e.g., the same membrane type or organelle type)) are applied. [PSP] where n≧2 n can include the same or different PSP segments. o≧2 [EP] o can encompass the same or different EPs. [EP] o Where different EPs are included, for example, at least one EP may be an RNA-TP segment and at least one may be an O-RS segment.

[0074] The fusion proteins of the present invention provide an orthogonal translation (OT) system, in which one or more O-RS (segments) required for the introduction of one or more ncAA residues into a POI are spatially adjacent to at least one RNA-targeting polypeptide (RNA-TP) segment. The POI mRNA contains at least one targeting nucleotide sequence (TN) that can interact with at least one RNA-TP segment of the fusion protein of the OT system. The interaction is conveniently specific. The RNA-TP segment of the fusion protein of the present invention is preferably an mRNA-targeting polypeptide segment. The RNA-TP segment of the fusion protein and the TN of the POI mRNA are conveniently selected so as to specifically interact (bind) with each other. Suitable pairs of RNA-TP segments and TNs for this purpose can be selected from coat proteins of RNA viruses and nucleic acid motifs bound by the coat proteins. Such viral coat proteins and RNA motifs bound by the proteins are known in the art.

[0075] Specific examples of suitable RNA-TPs are: - MCP (coat protein of the Enterobacteriaceae phage MS2), in particular a polypeptide comprising the amino acid sequence of SEQ ID NO: 14; - λ N22 (the 22 amino acid RNA binding domain of lambda phage antiterminator protein N). In particular, a polypeptide comprising the amino acid sequence of SEQ ID NO: 16; - PCP (coat protein of bacteriophage PP7. Wu B, Chao JA, Singer RH. Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys J. 2012; 102(12): 2936-2944. doi:10.1016 / j.bpj.2012.05.017), in particular a polypeptide comprising the amino acid sequence of SEQ ID NO: 306; and functional fragments and variants of these polypeptides, which may comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid of the polypeptide from which they are derived.

[0076] Specific examples of suitable TNs are: - Enterobacteriaceae phage MS2 RNA stem loop, in particular a polynucleotide having an RNA sequence that corresponds to (is encoded by) the nucleotide (DNA) sequence of SEQ ID NO: 17; - BoxB (λ phage RNA stem loop. N22 In particular, a polynucleotide having an RNA sequence corresponding to (encoded by) the nucleotide (DNA) sequence of SEQ ID NO: 18; - bacteriophage pp7 RNA stem loop (Wu B, Chao JA, Singer RH. Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys J. 2012; 102(12): 2936-2944. doi:10.1016 / j.bpj.2012.05.017), in particular a polynucleotide having an RNA sequence corresponding to (encoded by) the nucleotide (DNA) sequence of SEQ ID NO: 289 or SEQ ID NO: 290; and functional fragments and variants thereof, but are not limited to these. The functional fragments and variants may comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity with the polynucleotide sequence from which they are derived.

[0077] Such TN can be used as a single copy segment or as a multiple copy segment composed of more than one repeat unit of TN, for example 2, 3, 4, 5, 6, or more.

[0078] MCP specifically interacts with MS2 RNA stem loops. Thus, where the RNA-TP segment(s) of the fusion protein(s) comprise (consists of) a functional segment derived from an MCP, the mRNA of the POI conveniently comprises one or more MS2 RNA stem loops, e.g., 2, 3, 4, 5, or 6 MS2 RNA stem loops. N22 specifically interacts with BoxB. Therefore, the RNA-TP segment(s) of the fusion protein(s) interacts with λ N22 Where the RNA-TP segment(s) of the fusion protein(s) comprise (or consist of) a functional segment derived from PCP, the POI mRNA conveniently comprises one or more BoxB motifs, for example, 1, 2, 3, 4, 5, or 6 or more BoxB motifs. PCP specifically interacts with pp7 RNA stem-loops. Thus, where the RNA-TP segment(s) of the fusion protein(s) comprise (or consist of) a functional segment derived from PCP, the POI mRNA conveniently comprises one or more pp7 RNA stem-loops, for example, 2, 3, 4, 5, or 6 or more pp7 RNA stem-loops.

[0079] Several RSs have been used for genetic code expansion, including Methanococcus jannaschii tyrosyl-tRNA synthetase, E. coli tyrosyl-tRNA synthetase, E. coli leucyl-tRNA synthetase, pyrrolysyl-tRNA synthetase from certain Methanosarcina (e.g., M. mazei, M. barkeri, M. acetivorans, M. thermophila), Methanococcoides (M. burtonii), or Desulfitobacterium (D. hafniense). Corresponding orthogonal RS / tRNA pairs have been used to genetically encode various functionalities on polypeptides (Chin, Annu Rev Biochem 2014, 83:379-408; Chin et al., J Am Chem Soc 2001, 124:9026; Chin et al., Science 2003, 301:964; Nguyen et al., J Am Chem Soc 2009, 131:8720; Yanagisawa et al., Chem Biol 2008, 15:1187). Depending on the cell used to translate the POI, these RSs can be used as O-RSs in the present invention.

[0080] The pyrrolysyl-tRNA synthetase (PylRS) that can be used in the methods and fusion proteins of the present invention may be wild-type or engineered PylRS. Examples of wild-type PylRS include, but are not limited to, PylRS from archaea and eubacteria, such as Methanosarcina maize, Methanosarcina barkeri, Methanococcoides burtonii, Methanosarcina acetivorans, Methanosarcina thermophila, and Desulfitobacterium hafniense. Engineered PylRSs are described, for example, by Neumann et al. (Nat Chem Biol 2008, 4:232), Yanagisawa et al. (Chem Biol 2008, 15:1187), and EP 2192185 A1. The efficiency of genetic code expansion using PylRS can be increased by modifying the amino acid sequence of PylRS so that it is not targeted to the nucleus. To this end, the nuclear localization signal (NLS) can be removed from the PylRS or can be overridden by introducing a suitable nuclear export signal (NES). The PylRS used in the fusion proteins and methods of the invention can be a PylRS lacking an NLS and / or containing an NES, e.g., as described in WO2018 / 069481.

[0081] Thus, examples of O-RS segment(s) that can be used in the fusion proteins of the invention are: Methanococcus jannaschii tyrosyl-tRNA synthetase Escherichia coli tyrosyl-tRNA synthetase Escherichia coli leucyl-tRNA synthetase Methanosarcina mazeii pyrrolysyl-tRNA synthetase Methanosarcina barkeri pyrrolysyl-tRNA synthetase Methanosarcina acetivorans pyrrolysyl-tRNA synthetase Methanosarcina thermophila pyrrolysyl-tRNA synthetase Methanococcoides burtonii pyrrolysyl-tRNA synthetase Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase and functional (i.e., enzymatically active) fragments and variants of these polypeptides, which may comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the aminoacyl-tRNA synthetase from which they are derived.

[0082] Specific examples of O-RS segments useful in the present invention derived from M. mazei pyrrolysyl-tRNA synthetase are: - PylRS AF (Methanosarcina mazei pyrrolysyl-tRNA synthetase double mutant: Y306A, Y384F; Uniprot: Q8PWY1), for example, an O-RS segment comprising the amino acid sequence of SEQ ID NO: 8; - PylRS AA (Methanosarcina mazei pyrrolysyl-tRNA synthetase double mutant: N346A, C348A; Uniprot: Q8PWY1), for example, an O-RS segment comprising the amino acid sequence of SEQ ID NO: 10; - PylRS AAAF (Methanosarcina mazei pyrrolysyl-tRNA synthetase quadruple mutant: Y306A, N346A, C348A, Y384F; Uniprot: Q8PWY1), for example, an O-RS segment comprising the amino acid sequence of SEQ ID NO: 12; an O-RS segment derived from Methanosarcina mazei pyrrolysyl tRNA mutant (L305M, Y306L, L309S, N346S, C348M) IFRS1, for example, an O-RS segment comprising the amino acid sequence of SEQ ID NO: 224; - an O-RS segment derived from Methanosarcina mazei pyrrolysyl tRNA mutant (Y306M, L309G, C348T) CbzRS, for example, an O-RS segment comprising the amino acid sequence of SEQ ID NO: 226; - an O-RS segment derived from Methanosarcina mazei pyrrolysyl tRNA mutant (A302S) CpkRS, for example, an O-RS segment comprising the amino acid sequence of SEQ ID NO: 228; an O-RS segment derived from the Methanosarcina mazei pyrrolysyl tRNA mutant (A302T, Y384F, N346V, C348W, V401L) OMeRS, for example, an O-RS segment comprising the amino acid sequence of SEQ ID NO: 236; and functional (i.e., enzymatically active) fragments and variants of these polypeptide segments, which may comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the aminoacyl-tRNA synthetase from which they are derived.

[0083] According to certain embodiments, the wild-type and mutant M. mazei PylRS described herein are used for aminoacylation of tRNA with ncAA as described in WO2012 / 104422 or WO2015 / 107064. Exemplary ncAAs for this purpose include, but are not limited to, 2-amino-6-(cyclooct-2-yn-1-yloxycarbonylamino)hexanoic acid (SCO), 2-amino-6-(cyclooct-2-yn-1-yloxyethoxycarbonylamino)hexanoic acid, 2-amino-6[(4E-cyclooct-4-en-1-yl)oxycarbonylamino]hexanoic acid (TCO), 2-amino-6[(2E-cyclooct-2-en-1-yl)oxycarbonylamino]hexanoic acid (TCO*), 2-amino-6-(prop-2-ynoxycarbonylamino)hexanoic acid (PrK), and 2-amino-6-(9-biocyclo[6.1.0]non-4-ynylmethoxycarbonylamino)hexanoic acid (BCN).

[0084] In another embodiment of the present invention, the above-mentioned AP (IC-TP and PSP) segments and / or the above-mentioned EP (RNA-TP and O-RS) segments can be combined, independently of one another, with further natural or, more particularly, synthetic protein segments that induce and control macromolecular interactions. In particular, such additional protein segments are functionally fused to the polypeptide chain of the AFP of the present invention. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, but preferably one, such protein segments can be functionally fused to the AFP alone. Fusion to the AFP polypeptide chain ensures that the activity of the other polypeptide segments, AP and EP, is not substantially affected, in particular inhibited (i.e., AP and EP remain functional), while the ability of the additional polypeptide segment to induce and control macromolecular interactions is maintained. The literature describes so-called SYNZIP peptides that form multimeric structures. SYNZIPs, which have the ability to form specific heterodimeric coiled-coil protein structures, are of particular interest in the context of the present invention. Such SYNZIPs are pairs of synthetic peptides that can interact with each other and are used to direct and control macromolecular interactions. Non-limiting examples are the SYNZIP 1:2; SYNZIP 3:4; and SYNZIP 5:6 pairs. According to the present invention, the heterospecific coiled-coil pair SYNZIP2:SYNZIP1 (SYNZIP1: SEQ ID NO: 312, SYNZIP2: SEQ ID NO: 314, SYNZIP3: SEQ ID NO: 316, SYNZIP4: SEQ ID NO: 318) described by Reinke, A.W., Grant, R.A., Keating, A.E. (2010) J Am Chem Soc 132 6025-6031, as well as functional fragments and variants of these SYNZIP polypeptides, which may comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acids of the polypeptide from which they are derived, are particularly preferred.Because their use as a pair is required to induce macromolecular interactions, these SYNZIPs are preferably used as pairs in the AFP combinations described herein, and the interaction of such SYNZIP pairs integrated on different AFP fusion proteins can further support the formation of OT organelles according to the present invention.

[0085] In yet another embodiment of the present invention, the fusion proteins of the present invention can be further modified by the introduction (fusion) of at least one so-called "epitope tag," i.e., a short oligopeptide sequence that serves as an antibody binding site, useful for detecting / quantifying the expressed fusion product of the present invention. Non-limiting examples of such tags are: VSV-G: vesicular stomatitis virus glycoprotein epitope tag (SEQ ID NO: 680) HA: human influenza hemagglutinin epitope tag (SEQ ID NO: 682) Myc: human c-Myc proto-oncogene epitope tag (SEQ ID NO: 684)

[0086] 1.2 Specific Examples of AFP Constructs of the Invention

[0087] Each individual exemplified construct can be interpreted in either the N->C or C->N orientation. The illustrated scheme is given in the N->C orientation.

[0088] Segment block where m, n, y, or x is an integer > 1 [IC-TP] m , [PSP] n , [O-RS] y , and [RNA-TP] x In this case, the repeating segments within such a block may be identical or different, preferably identical.

[0089] The segments that apply to it are [IC-TP], [PSP], [O-RS], and [RNA-TP]. x, and [SYNZIP] can be prepared from each of the example segments listed in Section 1.1 above.

[0090] 1.2.1. Monofunctional AFP targeting intracellular structures

[0091] 1.2.1.1 Monofunctional AFPs (i.e., containing one type of EP) targeted to intracellular structures

[0092] Preferred examples of these are:

[0093] [IC-TP] m -[O-RS] y wherein m=1 or 2, preferably 1; y=1 or 2, preferably 1;

[0094] [IC-TP] m -[RNA-TP] x where m=1 or 2, preferably 1; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4;

[0095] [IC-TP] m -[PSP] n -[O-RS] y wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; y=1 or 2, preferably 1;

[0096] [IC-TP] m -[PSP] n -[RNA-TP] x wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4;

[0097] [IC-TP] m -[O-RS1] y -[PSP] n -[O-RS2] ywherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; independently of each other, y=1 or 2, preferably 1; O-RS1 and O-RS2 are identical or different, preferably identical;

[0098] [IC-TP] m -[PSP1] n -[O-RS] y -[PSP2] n wherein m=1 or 2, preferably 1; n are independently 1, 2 or 3, preferably 1 or 2; y are independently 1 or 2, preferably 1; PSP1 and PSP2 are the same or different;

[0099] [IC-TP] m -[RNA-TP1] x -[PSP] n -[RNA-TP2] x wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; independently of each other, x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; RNA-TP1 and RNA-TP2 are identical or different, preferably identical;

[0100] [IC-TP] m -[PSP1] n -[O-RS1] y -[PSP2] n -[O-RS2] y wherein m=1 or 2, preferably 1; n are independently 1, 2 or 3, preferably 1 or 2; y=1 or 2, independently 1; O-RS1 and O-RS2 are identical or different, preferably identical; PSP1 and PSP2 are identical or different;

[0101] [IC-TP] m -[PSP1] n -[RNA-TP1] x -[PSP2] n -[RNA-TP2] xwherein m=1 or 2, preferably 1; independently of each other n=1, 2, or 3, preferably 1 or 2; independently of each other x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; RNA-TP1 and RNA-TP2 are the same or different; and PSP1 and PSP2 are the same or different.

[0102] 1.2.1.2 Intracellular structure-targeted bifunctional AFPs (including two forms of EP)

[0103] Individually preferred examples are:

[0104] [IC-TP] m -[O-RS] y -[RNA-TP] x wherein m=1 or 2, preferably 1; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0105] [IC-TP] m -[RNA-TP] x -[O-RS] y wherein m=1 or 2, preferably 1; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0106] [IC-TP] m -[PSP] n -[O-RS] y -[RNA-TP] x wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0107] [IC-TP] m -[PSP] n -[RNA-TP] x -[O-RS] y wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0108] [IC-TP] m -[O-RS] y -[PSP] n -[RNA-TP] x wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0109] [IC-TP] m -[RNA-TP] x -[PSP] n -[O-RS] y wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0110] [IC-TP] m -[PSP1] n -[O-RS] y -[PSP2] n -[RNA-TP] x wherein m=1 or 2, preferably 1; n is independently from each other and n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1; PSP1 and PSP2 are the same or different;

[0111] [IC-TP] m -[PSP1] n -[RNA-TP] x -[O-RS1] y -[PSP2] n -[O-RS2] y wherein m=1 or 2, preferably 1; independently of each other, n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; independently of each other, y=1 or 2, preferably 1; PSP1 and PSP2 are the same or different; O-RS1 and O-RS2 are the same or different, preferably the same;

[0112] [IC-TP] m -[PSP1] n -[O-RS1] y -[PSP2] n -[O-RS2] y -[RNA-TP] x wherein m=1 or 2, preferably 1; independently of each other, n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4, and independently of each other, y=1 or 2, preferably 1; PSP1 and PSP2 are the same or different; and O-RS1 and O-RS2 are the same or different, preferably the same.

[0113] 1.2.2. No monofunctional AFP targeted to intracellular structures

[0114] These are the same AFPs listed above in section 1.2.1, with the only exception that segment [IC-TP] is missing but segment [PSP] is retained.

[0115] SYNZIP Variants

[0116] These are the same AFPs listed above in sections 1.2.1 and 1.2.2, with the only exception that at least one of the segments [IC-TP], [PSP], [O-RS2], or [RNA-TP] is supplemented at the N- or C-terminus with a SYNZIP element. The AFP can contain 1, 2, 3, 4, or 5, preferably 1 or 2, identical or different, preferably identical SYNZIPs. Non-limiting examples of such molecules are:

[0117] 1.2.3.1 Monofunctional SYNZIP AFP

[0118] Preferred examples of these are:

[0119] [PSP] n -[SYNZIP]-[O-RS] ywhere y=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2;

[0120] [PSP] n -[SYNZIP]-[RNA-TP] x where x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; n=1, 2, or 3, preferably 1 or 2;

[0121] [IC-TP] m -[SYNZIP]-[O-RS] y wherein m=1 or 2, preferably 1; y=1 or 2, preferably 1;

[0122] [IC-TP] m -[SYNZIP]-[RNA-TP] x where m=1 or 2, preferably 1; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4;

[0123] [IC-TP] m -[PSP] n -[SYNZIP]-[O-RS] y wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; y=1 or 2, preferably 1;

[0124] [IC-TP] m -[PSP] n -[SYNZIP]-[RNA-TP] x where m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; and x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4.

[0125] 1.2.3.2 Bifunctional SYNZIP AFP

[0126] Preferred examples of these are:

[0127] [IC-TP] m -[O-RS] y -[SYNZIP]-[RNA-TP]x wherein m=1 or 2, preferably 1; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0128] [IC-TP] m -[RNA-TP] x -[SYNZIP]-[O-RS] y wherein m=1 or 2, preferably 1; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0129] [IC-TP] m -[PSP] n -[SYNZIP]-[O-RS] y -[RNA-TP] x wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0130] [IC-TP] m -[PSP] n -[SYNZIP]-[RNA-TP] x -[O-RS] y wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1;

[0131] [IC-TP] m -[PSP] n -[SYNZIP a ]-[O-RS] y -[SYNZIP b ]-[RNA-TP] x where m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1; SYNZIP a and SYNZIP b are the same or different, preferably the same;

[0132] [IC-TP] m -[PSP] n -[SYNZIP a ]-[RNA-TP] x -[SYNZIP b ]-[O-RS] y where m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1; SYNZIP a and SYNZIP b are the same or different, preferably the same;

[0133] [IC-TP] m -[PSP1] n -[SYNZIP]-[RNA-TP] x -[O-RS1] y -[PSP2] n -[O-RS2] y wherein m=1 or 2, preferably 1; n=1, 2, or 3, preferably 1 or 2; x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4; y=1 or 2, preferably 1; PSP1 and PSP2 are the same or different; and O-RS1 and O-RS2 are the same or different, preferably the same.

[0134] 1.2.4. Monofunctional Fusion Proteins

[0135] Preferred examples of these are:

[0136] [SYNZIP]-[O-RS], where y=1 or 2, preferably 1 y ;

[0137] [SYNZIP]- [RNA-TP], where x=1, 2, 3, 4, 5, or 6, preferably 2, 3, or 4. x ; is.

[0138] Since IC-TP and PSP are absent here, they can preferably be used in combination with AFP molecules containing at least one C-TP and / or PSP segment.

[0139] 1.3 Examples of individual fusion proteins

[0140] Highly specific examples of fusion proteins of the present invention and specific combinations thereof are listed below in Tables 1, 2, and 3, the contents of which also form part of the general disclosure of this specification, and the contents thereof will not be repeated explicitly and verbatim in the general part herein. The disclosure of Tables 1 and 2 in their respective columns entitled "Fusion protein(s) comprising an O-RS and an RNA-TP segment" is considered to be disclosed independently of the contents of the other columns of Tables 1 and 2 that refer to specific reports and host cell lines.

[0141] 2. Functional Fragments and Mutants

[0142] Described herein are fragments and variants of specific RNA-TPs, O-RSs, IC-TPs, PSPs, TNs, and SYNZIPs that are functional (i.e., possess the RNA-binding activity of the parent RNA-TP, the intracellular structure targeting activity of the parent IC-TP, the self-assembly activity of the parent PSP, the RNA-TP-binding activity of the parent TN, the enzymatic activity of the parent O-RS, or the heterodimeric coiled-coil formation ability of the parent SYNZIP, respectively). Such fragments and variants can be characterized by the minimum degrees of sequence identity described herein. The amino acid or nucleotide sequence identity refers to identity over the entire length of the amino acid or nucleotide sequence so characterized, respectively. Percentage identity values ​​can be determined based on BLAST alignments, the blastp algorithm (protein-protein BLAST), or using the Clustal method, as known in the art (Higgins et al., Comput Appl. Biosci. 1989, 5(2):151-1).

[0143] Fragments and variants of specific RNA-TP, O-RS, IC-TP, SYNZIP, or PSP useful in the present invention retain the relevant function of the parent polypeptide (binding, self-assembly, or enzymatic activity, respectively) and can be obtained, for example, by conservative amino acid substitutions, i.e., substitutions with different amino acid residues having similar biochemical properties (e.g., charge, hydrophobicity, and size) known in the art. Typical examples are substitution of Leu with Ile or vice versa, Asp with Glu or vice versa, Asn with Gln or vice versa, and others.

[0144] 3. Orthogonal Translation, tRNA, and POI Coding Sequences

[0145] The term "translation system" generally refers to the set of components necessary to incorporate a naturally occurring amino acid onto a growing polypeptide chain (protein). Components of a translation system can include, for example, ribosomes, tRNA, aminoacyl-tRNA synthetase, mRNA, and the like. An aminoacyl-tRNA synthetase (RS) is an enzyme that can aminoacylate a tRNA with an amino acid or amino acid analog. The RS used in the process of the present invention is responsible for aminoacylation of a tRNA with the corresponding ncAA, i.e., tRNA ncAAcan be aminoacylated. As used herein, the term "orthogonal" refers to a translation system element (e.g., an orthogonal tRNA (O-tRNA) and / or an orthogonal aminoacyl-tRNA synthetase (O-RS)) that is used with reduced efficiency by a translation system (e.g., a cell) of interest. "Orthogonal" means that the O-tRNA or O-RS is unable to function or functions with reduced efficiency, e.g., less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or, e.g., less than 1% efficiency, with an endogenous RS or endogenous tRNA, respectively, of the translation system of interest. For example, the O-tRNA is aminoacylated in the translation system of interest by any endogenous RA of the translation system with reduced or even zero efficiency when compared to aminoacylation of the endogenous tRNA by the endogenous RS. In another example, an O-RS aminoacylates any endogenous tRNA in a translation system of interest with reduced or even zero efficiency compared to aminoacylation of the endogenous tRNA by the endogenous RS. Specifically, the term "orthogonal translation system" or "OT system" as used herein refers to an O-RS / O-tRNA system that allows for the introduction of an ncAA residue onto a growing polypeptide chain. ncAA Used to refer to translation systems that use pairs.

[0146] O-RS / O-tRNA used in the present invention ncAA The pair preferably has the following properties: O-tRNA ncAA is preferentially aminoacylated by the O-RS with an ncAA. ncAA The orthogonal pair functions in a translation system of interest (e.g., a cell) such that the O-tRNA is used to incorporate the ncAA residue into the growing polypeptide chain of the POI. Incorporation occurs in a site-specific manner. Specifically, the O-tRNA ncAA recognizes a selector codon (e.g., an amber, ochre, or opal stop codon) on the mRNA encoding the POI. The term "preferentially aminoacylates" means that the O-RS aminoacylates the O-tRNA with the unnatural amino acid with, e.g., about 50% efficiency, about 70% efficiency, about 75% efficiency, about 85% efficiency, about 90% efficiency, about 95% efficiency, or about 99% or greater efficiency, as compared to the endogenous tRNA or amino acids in the translation system (e.g., cell) of interest. The unnatural amino acid is incorporated into a growing polypeptide chain with high fidelity, e.g., greater than about 75% efficiency for a given selector codon, greater than about 80% efficiency for a given selector codon, greater than about 90% efficiency for a given selector codon, greater than about 95% efficiency for a given selector codon, or greater than about 99% or greater efficiency for a given selector codon.

[0147] tRNAs that can be used to be aminoacylated by a fusion protein of the invention comprising at least one O-RS segment derived from M. mazei pyrrolysyl-tRNA synthetase include, but are not limited to, M. mazei pyrrolysyl-tRNAs and functional variants thereof, in which the anticodon is an anticodon for a selector codon, such as, for example, the CUA anticodon for the amber stop codon TAG, the UCA anticodon for the opal stop codon TGA, and the UUA anticodon for the ochre stop codon TAA. An example of such a pyrrolysyl-tRNA is SEQ ID NO: 4 (tRNA Pyl,CUA ), SEQ ID NO: 5 (tRNA Pyl,UCA ), or SEQ ID NO: 6 (tRNA Pyl,UUA ) ). A further non-limiting example of a suitable tRNA is the following, derived from the pyrrolysyl tRNA of M. mazei:

[0148] tRNA pyl,CGA Pyrrolidinyl tRNA (serine codon), SEQ ID NO: 229 tRNA pyl,CGG Pyrrolysyl tRNA (proline codon), SEQ ID NO: 230 tRNA pyl,UAA Pyrrolysyl tRNA (leucine codon), SEQ ID NO: 231 tRNApyl,UAG Pyrrolysyl tRNA (leucine codon), SEQ ID NO: 232 tRNA pyl,CCG Pyrrolysyl tRNA (arginine codon), SEQ ID NO: 233 tRNA pyl,AUA Pyrrolysyl tRNA (isoleucine codon), SEQ ID NO: 234 As used herein, the term "selector codon" refers to a codon that is selected from an O-tRNA during the translation process. ncAA The term refers to a codon recognized (i.e., bound) by a POI. The term is also used for the corresponding codon on a polypeptide-coding sequence of a polynucleotide that is not a messenger RNA (mRNA), e.g., a DNA plasmid. The novel OT system described herein allows for orthogonal translation of a POI in a manner that is selective for the mRNA of said POI relative to other mRNAs present in the cytoplasm of a cell. Nevertheless, it is preferred that the selector codon is a codon of low abundance in the cell selected for expression, e.g., a codon of low abundance in naturally occurring eukaryotic cells. The novel OT system recognizes the POI mRNA, O-RS, and tRNA ncAA The selector codon is preferably a sense codon. In a preferred embodiment, the selector codon is a codon that is not recognized by endogenous tRNAs of the cell used to prepare the POI.

[0149] O-tRNA ncAA The anticodon of binds to the selector codon on the mRNA (the mRNA of the POI), thus site-specifically incorporating the ncAA onto the growing chain of the polypeptide (POI) encoded by said mRNA. Examples of selector codons that are useful in the new OT system described herein are: - nonsense codons, such as stop codons, e.g., amber (UAG), ochre (UAA), and opal (UGA) codons; - codons consisting of more than three bases (e.g., four-base codons); - codons derived from natural or unnatural base pairs; and - including but not limited to sense codons. Where a selector codon is used that is a sense codon (i.e., a naturally occurring three-base codon), it is preferred that the endogenous translation system of the cell used for POI expression according to the methods of the invention does not use (or only uses to a limited extent) said naturally occurring three-base codon, for example, a cell that lacks or has reduced abundance of a tRNA that recognizes the naturally occurring three-base codon, or a cell in which the naturally occurring three-base codon is a rare codon. Particularly preferred is the use of one or more stop codons, such as one or more of amber, ochre, and opal, as the selector codon of the invention.

[0150] Any number of selector codons, such as one or more, two or more, or more than three selector codons, can be introduced into a polynucleotide encoding a desired polypeptide (target polypeptide POI). The POI can carry two or more ncAA residues. The ncAA residues can be the same and encoded by the same type of selector codon, or they can be different and encoded by different selector codons. An anticodon has the reverse complementary sequence of the corresponding codon.

[0151] Suppressor tRNAs are tRNAs (e.g., O-tRNAs) that modify the reading of messenger RNA (mRNA) by a given translation system (e.g., a cell). ncAA ) Suppressor tRNAs can read, for example, stop codons, four-base codons, or rare codons.

[0152] The O-tRNA is preferentially aminoacylated by an O-RS (rather than an endogenous synthetase) and can decode a selector codon as described herein. The O-RS recognizes an O-tRNA, e.g., with an extended anticodon loop, and preferentially aminoacylates the O-tRNA with an ncAA. The O-tRNA and O-RS used in the methods and / or fusion proteins of the invention can be naturally occurring or can be derived by mutation of naturally occurring tRNAs and / or RSs from various organisms. In various embodiments, the tRNA and RS are derived from at least one organism. In other embodiments, the tRNA is derived from a naturally occurring or mutated naturally occurring tRNA from a first organism, and the RS is derived from a naturally occurring or mutated naturally occurring RS from a second organism. Suitable (orthogonal) tRNA / RS pairs can be selected from a library of mutant tRNAs and RSs, for example, based on the results of library screening. Alternatively, suitable tRNA / RS pairs can be heterologous tRNA / synthetase pairs that are transferred into the translation system from a source species. Preferably, the cells used as the translation system are different from the source species. Methods for evolving tRNA / RS pairs are described, for example, in WO 02 / 085923 and WO 02 / 06075. Conventional site-directed mutagenesis can be used to introduce a selector codon into the coding sequence of the POI.

[0153] 4. Nucleic acid molecules

[0154] The present invention also relates to nucleic acid molecules (single- or double-stranded DNA and RNA sequences, e.g., cDNA, mRNA) comprising a nucleotide sequence encoding at least one of the fusion proteins of the invention and / or a nucleotide sequence complementary thereto, or a combination of such nucleic acid molecules.

[0155] Furthermore, the present invention provides a method for the preparation of a polypeptide comprising: (i) a nucleotide sequence encoding at least one POI (CS POI ) and (the POI is selected by the selector codon CS POI(ii) a nucleic acid molecule (single- or double-stranded DNA and RNA sequences, e.g., cDNA, mRNA) or a combination of such nucleic acid molecules comprising a targeting nucleotide sequence (TN) as described herein (wherein the RNA version of said TN can interact with an RNA-targeting polypeptide (RNA-TP) via said TN).

[0156] The nucleic acid molecules of the present invention may additionally comprise untranslated sequences at the 3' and / or 5' ends of the coding gene region. TNs are preferably located at the 3' end of the nucleic acid molecule encoding POI(s). For example, nucleic acid molecules of the present invention encoding POI(s) can be prepared by introducing at least one TN into the 3' untranslated region (particularly the 3' end thereof) using conventional cloning techniques known in the art.

[0157] The nucleic acid molecules of the present invention can additionally include untranslated sequences at the 3' and / or 5' ends of the coding gene region.

[0158] The present invention further relates to, in particular, recombinant expression constructs or expression cassettes, which contain the nucleic acid sequences of the nucleic acid molecules or combinations of nucleic acid molecules of the invention described herein under the genetic control of regulatory nucleic acid sequences. Thus, an expression cassette of the invention comprises a nucleic acid sequence encoding at least one POI (plus TN) or at least one fusion protein of the invention, and / or a nucleic acid sequence complementary thereto. The present invention also relates to, in particular, recombinant vectors comprising at least one of these expression constructs (expression vectors).

[0159] An expression cassette typically comprises a promoter sequence 5' (upstream) of and operably linked to the nucleic acid sequence encoding the POI(s) or fusion protein(s) to be expressed, a terminator sequence 3' (downstream) of the coding sequence, and optionally additional regulatory elements. Examples of such additional regulatory elements include, but are not limited to, targeting sequences, enhancers, polyadenylation signals, selection markers, amplification signals, origins of replication, and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0160] In addition to these regulatory sequences, the natural control of these sequences may still be present in front of the actual structural gene, and optionally, the gene may be genetically modified so that the natural control is switched off and gene expression is increased. However, the nucleic acid construct may also be a simpler construct, i.e., no additional regulatory signals are inserted in front of the coding sequence, and the natural promoter, along with its control, is not removed. Instead, the natural regulatory sequences are mutated so that the control no longer occurs and gene expression is increased.

[0161] "Operative" linkage of elements of a nucleic acid molecule, such as a promoter, a polypeptide-coding sequence, a terminator, or a regulatory element, means that these elements are positioned so that the coding sequence can be transcribed and any regulatory elements can exert their control of said transcription. This can be achieved by direct linkage of the elements on one and the same nucleic acid molecule. However, such direct linkage is not necessarily required. Gene control sequences, such as enhancer sequences, can exert their function on a target sequence from more remote locations or even from other DNA molecules. A preferred arrangement is one in which the nucleic acid sequence to be transcribed is located downstream of the promoter sequence (i.e., at its 3' end), such that the two sequences are covalently joined together. The distance between the promoter sequence and the nucleic acid sequence to be expressed can be less than 200 base pairs, less than 100 base pairs, or less than 50 base pairs.

[0162] For cellular expression, the expression cassette is advantageously inserted into an expression vector. The expression vector is selected according to the cell to be used for expression. This allows optimal expression of the coding nucleotide sequence by the cell. Vectors are well known to those skilled in the art and are described, for example, in "Cloning Vectors" (Pouwels PH et al., Eds., Elsevier, Amsterdam-New York-Oxford, 1985). Examples of expression vectors include, but are not limited to, plasmids, viral vectors (phages), such as SV40, CMV, baculovirus, and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. See, for example, the book "Cloning Vectors" (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0444904018). These vectors can replicate autonomously in (host) cells or can replicate chromosomally. An expression vector comprising at least one expression cassette of the present invention represents a further aspect of the present invention.

[0163] For expression of a POI by a cell according to the invention, it is possible, for example, to introduce a nucleic acid molecule encoding the POI (e.g., an expression vector of the invention) into the cell. Alternatively, an existing gene of the cell can be modified to include a selector codon at the amino acid position where the POI is intended to carry an ncAA residue. Methods for introducing (recombinant) nucleic acid molecules encoding a polypeptide into a cell or for modifying an existing gene of a cell are known in the art.

[0164] The term "expression" in the context of the present invention describes the production of a polypeptide encoded by a corresponding nucleic acid sequence in a cell. The term "expression" is also used for the production of a tRNA molecule encoded by a nucleic acid sequence in a cell.

[0165] The nucleic acid molecules of the present invention, including the expression cassettes and expression vectors of the present invention, can be prepared using conventional cloning techniques known in the art. Conventional recombination and cloning techniques are used, for example, as described in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0166] The nucleic acid molecules or combinations of nucleic acid molecules of the invention, including the expression cassettes and expression vectors of the invention, can be isolated, for example, by methods known in the art.

[0167] An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid, and further can be essentially free of other cellular material or culture medium when it is produced by recombinant techniques, or free of chemical precursors or other chemicals when it is chemically synthesized.

[0168] Nucleic acid molecules according to the present invention can be isolated using standard techniques of molecular biology and the sequence information provided according to the present invention. For example, cDNA can be isolated from a suitable cDNA bank using standard hybridization techniques with one of the specifically disclosed complete sequences or a segment thereof as a hybridization probe (e.g., as described in Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Furthermore, nucleic acid molecules containing one of the disclosed sequences or a segment thereof can be isolated by polymerase chain reaction using oligonucleotide primers constructed based on this sequence. The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides according to the present invention can be produced by standard synthetic methods, for example, using an automated DNA synthesizer.

[0169] 5. ncAA and post-translational POI modifications

[0170] The abbreviation "ncAA" generally refers to a non-standard or unnatural amino acid or amino acid residue that is not one of the 22 naturally occurring proteinogenic amino acids. Numerous ncAAs are well known in the art (see, e.g., Liu et al., Annu Rev Biochem 2010, 79:413-444; Lemke, ChemBioChem 2014, 15:1691-1694). The term "ncAA" also refers to amino acid derivatives, such as α-hydroxy acids (rather than α-amino acids). Such derivatives have also been shown to be translationally incorporable. See, e.g., Ohta et al., 2008, ChemBioChem 9:2773-2778. Thus, the meaning of terms such as "aminoacylate" or "aminoacylation," as used herein, is not limited to RS-catalyzed ligation of tRNA with α-amino acids, but also includes RS-catalyzed ligation of tRNA with ncAA derivatives, such as α-hydroxy acids.

[0171] Particularly preferred ncAAs for use in the present invention are those that can be further post-translationally modified, for example, using click chemistry reactions, including strain-promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC; see, e.g., Devaraj et al., Angew Chem Int Ed Engl 2009, 48:7013), and cycloadditions between strained cycloalkynyl groups (or strained cycloalkynyl analog groups having one or more ring atoms not connected by a triple bond replaced by an amino group) with azides, nitrile oxides, nitrones, and diazocarbonyl reagents (see, e.g., Sanders et al., J Am Chem Soc 2010, 133:949; Agard et al., J Am Chem Soc 2004, 126:15046), such as strain-promoted alkyne-azide cycloaddition (SPAAC). Such a click reaction allows for ultrafast and mutually orthogonal covalent site-specific coupling of an ncAA labeling group of a target polypeptide with a suitable group of a coupling partner molecule. Pairs of linking and labeling groups that can react via the above-mentioned click reaction are known in the art. Examples of ncAAs suitable for use in the present invention that contain a linking group include, but are not limited to, ncAAs ("unnatural amino acids," "UAAs") described, for example, in WO 2012 / 104422 and WO 2015 / 107064. Optionally substituted strained alkynyl groups include, but are not limited to, optionally substituted trans-cyclooctenyl groups, such as those described in WO 2012 / 104422 and WO 2015 / 107064. Optionally substituted strained alkenyl groups include, but are not limited to, optionally substituted cyclooctynyl groups, such as those described in WO 2012 / 104422 and WO 2015 / 107064. Optionally substituted tetrazinyl groups include, but are not limited to, those described in WO2012 / 104422 and WO2015 / 107064.

[0172] The ncAAs used in the context of the present invention can be used in the form of their salts. The salts of ncAAs described herein refer to acid or base addition salts, particularly addition salts with physiologically tolerated acids or bases. Physiologically tolerated acid addition salts can be formed by treating the base form of ncAAs with appropriate organic or inorganic acids. ncAAs containing acidic protons can be converted to their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Salts of the carboxyl group of ncAAs can be produced in a manner known in the art and include inorganic salts, such as sodium, calcium, ammonium, iron, and zinc salts, as well as salts with organic bases, such as amines, e.g., triethanolamine, arginine, lysine, piperidine, and the like. ncAAs can also be used in the form of acid addition salts, such as salts with mineral acids, e.g., hydrochloric acid or sulfuric acid, and salts with organic acids, e.g., acetic acid and oxalic acid. The ncAAs and their salts useful in the present invention also include their hydrates and solvent addition forms, such as hydrates, alcoholates, and the like.

[0173] A physiologically tolerated acid or base is one that is tolerated by the translation system used to prepare the POI having the ncAA residue, and is substantially non-toxic to, for example, eukaryotic cells.

[0174] The ncAAs and their salts useful in the context of the present invention can be prepared analogously to methods well known in the art and described, for example, in the various publications cited herein.

[0175] The nature of the coupling partner molecule depends on the intended use. For example, the target polypeptide can be coupled to a molecule suitable for imaging methods or functionalized by coupling to a biologically active molecule. For example, in addition to the linking group, the coupling partner molecule can be a dye (e.g., a fluorescent, luminescent, or phosphorescent dye, such as dansyl, coumarin, fluorescein, acridine, rhodamine, silicon rhodamine, BODIPY, or cyanine dye), a molecule that can emit fluorescence upon contact with a reagent, a chromophore (e.g., phytochrome, phycobilin, bilirubin, etc.), a radioactive label (e.g., a radioactive form of hydrogen, fluorine, carbon, phosphorus, sulfur, or iodine, such as tritium, 18 F, 11 C. 14 C. 32 P, 33 P, 33 S, 35 S, 11 In, 125 I, 123 I, 131 I, 212 B. 90 Y, or 186The label may include, but is not limited to, a group selected from the group consisting of Rh), MRI-sensitive spin labels, affinity tags (e.g., biotin, His tags, Flag tags, strep tags, sugars, lipids, sterols, PEG linkers, benzylguanine, benzylcytosine, or cofactors), polyethylene glycol groups (e.g., branched PEG, linear PEG, PEGs of different molecular weights, etc.), photocrosslinkers (e.g., p-azidoiodoacetanilide), NMR probes, X-ray probes, pH probes, IR probes, resins, solid supports, and biologically active compounds (e.g., synthetic drugs). Suitable biologically active compounds include, but are not limited to, cytotoxic compounds (e.g., cancer chemotherapeutic compounds), antiviral compounds, biological response modifiers (e.g., hormones, chemokines, cytokines, interleukins, etc.), agents that affect microtubules, hormone-modulating agents, and steroid compounds. Specific examples of useful coupling partner molecules include, but are not limited to, members of receptor / ligand pairs; members of antibody / antigen pairs; members of lectin / carbohydrate pairs; members of enzyme / substrate pairs; biotin / avidin; biotin / streptavidin; and digoxin / antidigoxin.

[0176] The ability of certain ncAA residues (labeling groups) to be covalently coupled in situ to conjugation partner molecules (binding groups) can be used to detect target polypeptides bearing such ncAA residue(s) in eukaryotic cells or tissues expressing the target polypeptide, particularly via the click reaction described herein, as well as to study the distribution and fate of the target polypeptide. Specifically, the methods of the present invention for preparing a POI by expression in (e.g., eukaryotic) cells can be combined with super-resolution microscopy (SRM) to detect the POI within cells or tissues of such cells. Several SRM methods are known in the art and can be adapted to utilize click chemistry to detect target polypeptides expressed by eukaryotic cells of the present invention. Specific examples of such SRM methods include DNA-PAINT (DNA point accumulation for imaging in nanoscale topography; e.g., as described by Jungmann et al., Nat Methods 11:313-318, 2014), dSTORM (direct stochastic optical reconstruction microscopy), and STED (stimulated emission depletion) microscopy.

[0177] 6. Translational preparation of POI by cells

[0178] The OT system provided by the present invention allows for the translational preparation of a POI by a cell.

[0179] The cells used to prepare a POI according to the present invention may be prokaryotic cells. Alternatively, the cells used to prepare a POI according to the present invention may be eukaryotic cells. The cells used to prepare a POI according to the present invention may be individual cells, such as, for example, a unicellular microorganism, or a cell line derived from the cells of a multicellular organism. Alternatively, the cells used to prepare a POI according to the present invention may be present in (and be part of) a tissue, an organ, a body part, or a whole multicellular organism. Thus, the method of the present invention for preparing a POI can be carried out with individual cells or cell cultures, or with tissues or tissue cultures, organs, body parts, or (whole) multicellular organisms.

[0180] Eukaryotic cells are often difficult to handle and manipulate compared to prokaryotes such as E. coli, and are therefore inaccessible or less accessible to known approaches for POI-selective orthogonal translation, such as those described in the "Background" section above. Thus, the OT systems and methods of the present invention are particularly advantageous when used for POI expression by eukaryotic cells (including, for example, unicellular and multicellular eukaryotic organisms, and eukaryotic cell lines).

[0181] In principle, any prokaryotic or eukaryotic cell can be used to prepare a POI according to the method of the present invention. Microorganisms such as bacteria, fungi, or yeasts, as well as eukaryotic cells such as mammalian cells, insect cells, yeast cells, and plant cells can be used. Eukaryotic cells, especially mammalian cells, are particularly preferred.

[0182] The cells used to prepare the POI according to the present invention contain a nucleotide sequence encoding the POI (CS POI ), and the ncAA residue(s) of the POI are encoded by a selector codon(s). POI is functionally linked to one or more targeting sequences (TN). POIand TN(s). The cell further comprises one or more fusion proteins of the invention, wherein the fusion protein(s) comprises at least one O-RS segment and at least one RNA-TP segment. The O-RS and RNA-TP can be on separate fusion proteins of the invention (e.g., AFP). Alternatively, the O-RS and RNA-TP can be on one and the same fusion protein of the invention (e.g., RNA-TP / O-RS fusion protein or AFP). Through (at least one of) the TN(s), the mRNA can interact (bind) with at least one of the RNA-TP segments of the fusion protein of the invention within the cell. ... POI one or more orthogonal tRNAs carrying anticodon(s) for the selector codon(s) of ncAA Molecule (O-tRNA ncAA The O-tRNA ncAA The molecule and one or more of the O-RS segments of the fusion protein are coupled to one or more orthogonal O-RS / O-tRNAs in a cell. ncAA This allows the introduction of ncAA residue(s) into the amino acid sequence of the POI (which is translationally prepared).

[0183] CSs with RNA-TP segment(s) POI and interaction of mRNA containing TN(s), O-tRNA with ncAA through O-RS segment(s). ncAA The translational preparation of the POI, including aminoacylation of the POI and the introduction of the ncAA residue(s), is thought to occur in the cytoplasm of the cell, more specifically in the OT assembly (OT organelle), in the presence of the ncAA.

[0184] CS POI and mRNA containing TN(s) (mRNA POI) can be produced from a recombinant construct (e.g., an expression vector) introduced into a cell. Alternatively, one or more endogenous genes of a cell can be modified to include one or more selector codons and one or more TNs. Techniques for introducing recombinant constructs into cells and methods for modifying endogenous genes of a cell are well known in the art.

[0185] The tRNA of the present invention ncAA The molecules and fusion proteins can be produced from recombinant constructs (eg, expression vectors) that are introduced into cells.

[0186] The expression vectors according to the invention can be used to produce recombinant cells which can be used to prepare the POI using the methods of the invention. Advantageously, the recombinant vectors according to the invention described above are introduced into suitable cells and expressed.

[0187] The cells used to prepare the POI as described herein may contain the fusion protein(s), tRNA ncAA The POI may be prepared by introducing into a cell a nucleotide sequence encoding the molecule(s) and the POI, which may be located on separate nucleic acid molecules (vectors) or on the same nucleic acid molecule (e.g., vector), in any combination, and introduced into the cell in combination or sequentially.

[0188] Preferably, conventional cloning and transfection techniques known to those skilled in the art, such as co-precipitation, protoplast fusion, electroporation, viral-mediated gene delivery, lipofection, microinjection, or others, are used to introduce the described nucleic acid molecules into the respective cells. Suitable techniques are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0189] In the methods of the present invention, the cells used for POI expression are grown or cultured by methods known to those skilled in the art. Depending on the cell type, liquid media can be used for the culture. The culture can be batch, semi-batch, or continuous. Nutrients can be present at the start of the culture or can be supplied later in a semi-continuous or continuous manner.

[0190] The expressed POI can be purified by known techniques, such as molecular sieve chromatography (gel filtration), e.g., Q-Sepharose chromatography, ion exchange chromatography, and hydrophobic chromatography, as well as other common protein purification techniques, such as ultrafiltration, crystallization, salting out, dialysis, and native gel electrophoresis. Suitable methods are described, for example, in Cooper, T.G., Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, New York, or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0191] To isolate the POI, it may be advantageous to link the POI to a tag that can effect easier purification. This can be achieved by linking the corresponding tag coding sequence to the CS. POI This can be achieved by introducing the tag into the protein. Suitable tags for protein purification are well known in the art and include, for example, histidine tags (e.g., His6 tags) and epitopes that can be recognized as antigens by antibodies (e.g., as described by Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press). These tags can serve to attach the protein to a solid support, such as a polymer matrix, which can be used, for example, as packing for a chromatography column, or on a microtiter plate or some other support.

[0192] A tag linked to a POI can also serve to detect the POI. Tags for protein detection are well known in the art and include, for example, fluorescent dyes, enzymatic markers that form a detectable reaction product after reaction with a substrate, and others.

[0193] To prepare a POI according to the methods of the invention, expression can be achieved by culturing cells in the presence of one or more ncAAs corresponding to the ncAA residue(s) of the POI (which can conveniently be included in the culture medium) for a suitable period of time to allow translation of the POI (and optionally the fusion protein and / or tRNA of the invention). ncAA Depending on the nucleic acid(s) encoding the gene(s), it may be necessary to induce expression by adding a compound that induces transcription, such as arabinose, isopropyl β-D-thiogalactoside (IPTG), or tetracycline, to enable transcription.

[0194] After translation, the POI can optionally be recovered from the translation system. To this end, the POI can be recovered and purified to either partial or substantial homogeneity according to procedures used and known by those skilled in the art. Unless the target polypeptide is secreted into the culture medium, recovery usually requires cell disruption. Methods for cell disruption are well known in the art and include physical disruption by, for example, (ultrasonic) sonication, liquid shear disruption (e.g., with a French press), mechanical methods (e.g., using a blender or grinder), or freeze-thaw cycles, as well as chemical lysis using agents that disrupt lipid-lipid, protein-protein, and / or protein-lipid interactions (e.g., detergents), and combinations of physical disruption techniques with chemical lysis. Standard procedures for purifying polypeptides from cell lysates or culture medium are also well known in the art and include, for example, ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, gel electrophoresis, etc. If desired, a protein refolding step can be used to produce a correctly folded mature protein. High-performance liquid chromatography (HPLC), affinity chromatography, or other suitable methods can be employed for final purification steps where high purity is desired. Antibodies raised against the polypeptides of the invention can be used as purification reagents, i.e., for affinity-based purification of the polypeptides. Various purification / protein refolding methods are well known in the art, including, for example, those provided in Scopes, Protein Purification, Springer, Berlin (1993); and Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press (1990); and references cited therein.

[0195] As mentioned, those skilled in the art will recognize that after synthesis, expression, and / or purification, a polypeptide may possess a conformation that differs from the desired conformation of the polypeptide. For example, polypeptides produced by prokaryotic systems are often optimized by exposure to chaotropic agents to achieve proper folding. For example, during purification from cell lysates, expressed polypeptides are optionally denatured and then renatured. This can be achieved, for example, by solubilizing the protein in a chaotropic agent such as guanidine HCl. Generally, it may be desirable to denature and reduce the expressed polypeptide and then refold the polypeptide into a preferred conformation. For example, guanidine, urea, DTT, DTE, and / or chaperonins can be added to the translation product of interest. Methods for reducing, denaturing, and renaturing proteins are well known to those skilled in the art. Polypeptides can be refolded, for example, in a redox buffer containing oxidized glutathione and l-arginine. Polypeptides produced by the methods of the invention are also described. Such polypeptides can be prepared by the methods of the invention that utilize the OT system described herein.

[0196] 7. Kit

[0197] The present invention also provides a kit for preparing a POI having at least one non-canonical amino acid (ncAA) residue. The kit of the present invention may include at least one expression vector for at least one fusion protein of the present invention. The fusion protein(s) encoded by the expression vector(s) of the kit may include at least one O-RS segment and at least one RNA-TP segment. The kit may further include at least one ncAA or a salt thereof corresponding to at least one ncAA residue of the POI. Conveniently, the O-RS segment is capable of aminoacylating a tRNA with at least one ncAA. The kit may further include an orthogonal tRNA. ncAA (O-tRNA ncAA The kit may include at least one expression vector for a targeting nucleotide sequence (TN), such that an RNA molecule comprising the TN can interact with an RNA-targeting polypeptide (RNA-TP) via the TN. Conveniently, the TN is a sequence that, when present on an RNA molecule, can interact with at least one RNA-TP segment of a fusion protein(s) encoded by the expression vector(s) included in the kit. The kit may further include at least one reporter construct encoding an easily detectable (e.g., fluorescent) reporter polypeptide having at least one non-canonical amino acid (ncAA) residue, such that mRNA transcribed from the construct comprises the TN described herein.

[0198] The kits of the present invention can be used in the methods of the present invention for preparing the ncAA residue-containing POIs described herein.

[0199] Specific Embodiments

[0200] The present invention further provides the following non-limiting embodiments E1 to E50.

[0201] E1: an assembler fusion protein (AFP) comprising: (a) (a1) a polypeptide segment (IC-TP segment) derived from an intracellular targeting polypeptide (the intracellular targeting polypeptide targets and is therefore locally concentrated in an intracellular structural element within or directly adjacent to the cytoplasm); and (a2) a polypeptide segment (PSP segment) derived from a phase-separating polypeptide, the phase-separating polypeptide having the ability to self-assemble in the cytoplasm of a cell in a manner that creates sites of high local cytoplasmic concentration; and at least one first polypeptide segment acting as an assembler (AP) selected from: (b) b1) an RNA-targeting polypeptide (RNA-TP) segment, and b2) orthogonal aminoacyl-tRNA synthetase (O-RS) segment; and at least one second polypeptide segment acting as an effector (EP) selected from: Including, The polypeptide segments are functionally linked to each other on the AFP.

[0202] E2: AFP of E1, comprising at least two APs, preferably at least one IC-TP segment and at least one PSP segment.

[0203] E3: An AFP of E1 or E2 having one of the following structures (N- to C-terminus): [IC-TP] m -[EP] o [EP] o -[IC-TP] m [PSP] n -[EP] o [EP] o -[PSP] n [IC-TP] m-[EP] o -[PSP] n [PSP] n -[EP] o -[IC-TP] m [IC-TP] m -[PSP] n -[EP] o [EP] o -[PSP] n -[IC-TP] m [PSP] n -[IC-TP] m -[EP] o [EP] o -[IC-TP] m -[PSP] n wherein m, n, and o are, independently of one another, integers selected from 1, 2, 3, 4, or 5, and "-" indicates a peptidic linkage.

[0204] E4: An AFP of any one of E1 to E3, wherein at least one EP is selected from an RNA-TP segment.

[0205] E5: An AFP of any one of E1 to E3, wherein at least one EP is selected from an O-RS segment.

[0206] E6: An AFP selected from any one of E1 to E3, which comprises at least one EP selected from the RNA-TP segment and at least one EP selected from the O-RS segment.

[0207] E7: An AFP of any one of E1 to E6, comprising at least one IC-TP segment selected from dynein and kinesin, and fragments and mutants of dynein and kinesin, which retain the ability to target and be concentrated at the plus or minus ends of microtubules.

[0208] E8: An AFP of any one of E1 to E6, comprising at least one IC-TP segment selected from a transmembrane domain of a membrane protein and functional fragments and mutants of the transmembrane domain, which retains the ability to target and be concentrated on the cytoplasmic side of a membrane, particularly a membrane selected from the cell membrane, the nuclear membrane, and the mitochondrial membrane.

[0209] E9: Any one of AFPs E1 to E8: - KIF16B comprising the amino acid sequence of SEQ ID NO: 20 1-400 or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 20; - KIF13A comprising the amino acid sequence of SEQ ID NO: 22 1-411 , Δ390, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 22; TOMM20 comprising the amino acid sequence of SEQ ID NO: 24 1-70 or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 24; - LcK comprising the amino acid sequence of SEQ ID NO: 26, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 26; - FRB-CD28 comprising the amino acid sequence of SEQ ID NO: 28, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 28; - FUS-CD28 comprising the amino acid sequence of SEQ ID NO: 30, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 30; - EB1 comprising the amino acid sequence of SEQ ID NO: 302, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 303; - CG1 comprising the amino acid sequence of SEQ ID NO: 304, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 304; - EBAG9 comprising the amino acid sequence of SEQ ID NO: 292 (full length), or having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 292; or a functional fragment or variant thereof comprising the first N-terminal 29 amino acid residues of SEQ ID NO: 294; or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 294; - CMP Sia Tr comprising the amino acid sequence of SEQ ID NO: 296, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 296; and - P450 2C1 targeted to the cytoplasmic side of the ER membrane, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity therewith, in particular a fragment comprising the first 27 (SEQ ID NO: 298) or the first 29 (SEQ ID NO: 300) amino acid residues at the N-terminus; or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% amino acid sequence identity therewith to SEQ ID NO: 298 or 300; The IC-TP segment includes at least one selected from:

[0210] E10: An AFP of any one of E1 to E9, comprising at least one PSP segment selected from an intrinsically disordered protein (IDP), particularly a prion-like domain, and functional fragments and variants of an IDP or prion-like domain, which retain the ability to self-associate in the cytoplasm of a cell in a manner that creates sites of high local cytoplasmic concentration.

[0211] E11: Any one of AFPs E1 to E10: - SPD5 comprising the amino acid sequence of SEQ ID NO: 32, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 32; - FUS comprising the amino acid sequence of SEQ ID NO: 34, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 34; and - EWSR1 comprising the amino acid sequence of SEQ ID NO: 36, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 36; The PSP segment comprises at least one PSP segment selected from:

[0212] E12: An AFP of any one of E1 to E11, which contains at least one RNA-TP segment and is selected from the RNA-binding segment of a viral coat protein, and functional fragments and mutants of the RNA-binding segment of a viral coat protein that retain the ability to specifically interact with a viral RNA motif.

[0213] E13: AFP of any one of E1 to E12: - an MCP comprising the amino acid sequence of SEQ ID NO: 14, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14; - λ comprising the amino acid sequence of SEQ ID NO: 16N22 or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 16; and - a PCP comprising the amino acid sequence of SEQ ID NO: 306, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 306; The RNA-TP segment comprises at least one selected from:

[0214] E14: Any one of AFPs E1 to E13: - Methanococcus jannaschii tyrosyl-tRNA synthetase; - Escherichia coli tyrosyl-tRNA synthetase; - Escherichia coli leucyl-tRNA synthetase; - Methanosarcina mazeii pyrrolysyl-tRNA synthetase; - Methanosarcina barkeri pyrrolysyl-tRNA synthetase; - Methanosarcina acetivorans pyrrolysyl-tRNA synthetase; - Methanosarcina thermophila pyrrolysyl-tRNA synthetase; - Methanococcoides burtonii pyrrolysyl-tRNA synthetase; - Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase; and their functional fragments and variants that retain aminoacyl-tRNA synthetase enzymatic activity; The O-RS segment comprises at least one O-RS segment selected from:

[0215] E15: AFP of any one of E1 to E14: - PylRS comprising the amino acid sequence of SEQ ID NO: 8 AF or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:8; - PylRS comprising the amino acid sequence of SEQ ID NO: 10 AA or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 10; - PylRS comprising the amino acid sequence of SEQ ID NO: 12 AAAF or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 12; - IFRS1 comprising the amino acid sequence of SEQ ID NO: 224, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 224; - a CbzRS comprising the amino acid sequence of SEQ ID NO: 226, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 226; - a CpkRS comprising the amino acid sequence of SEQ ID NO: 228, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 228; and - an OMeRS comprising the amino acid sequence of SEQ ID NO: 236, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 236; The O-RS segment comprises at least one O-RS segment selected from:

[0216] E16: An assembler fusion protein (AFP) combination, comprising at least two AFPs from any one of E1 to E15.

[0217] E17: An AFP combination of E16, comprising at least one first AFP comprising at least one RNA-TP segment and at least one second AFP comprising at least one O-RS segment.

[0218] E18: A fusion protein (RNA-TP / O-RS fusion protein): (i) at least one RNA-targeting polypeptide (RNA-TP) segment; (ii) at least one orthogonal aminoacyl-tRNA synthetase (O-RS) segment; Including, The polypeptide segments are functionally linked to each other on the RNA-TP / O-RS fusion protein.

[0219] E19: An RNA-TP / O-RS fusion protein of E18 having one of the following structures (N- to C-terminus): [RNA-TP] x-[O-RS] y [O-RS] y- [RNA-TP] x wherein x and y are, independently of one another, integers selected from 1, 2, 3, 4, and 5, and "-" denotes a peptidic linkage.

[0220] E20: An RNA-TP / O-RS fusion protein of E18 or E19, comprising at least one RNA-TP segment, selected from an RNA-binding segment of a viral coat protein and functional fragments and variants of an RNA-binding segment of a viral coat protein that retain the ability to specifically interact with a viral RNA motif.

[0221] E21: An RNA-TP / O-RS fusion protein of any one of E18 to E20, comprising: - an MCP comprising the amino acid sequence of SEQ ID NO: 14, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14; - λ comprising the amino acid sequence of SEQ ID NO: 16 N22 or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 16; and - a PCP comprising the amino acid sequence of SEQ ID NO: 306, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 306; The RNA-TP segment comprises at least one selected from:

[0222] E22: An RNA-TP / O-RS fusion protein of any one of E18 to E21: - Methanococcus jannaschii tyrosyl-tRNA synthetase; - Escherichia coli tyrosyl-tRNA synthetase; - Escherichia coli leucyl-tRNA synthetase; - Methanosarcina mazeii pyrrolysyl-tRNA synthetase; - Methanosarcina barkeri pyrrolysyl-tRNA synthetase; - Methanosarcina acetivorans pyrrolysyl-tRNA synthetase; - Methanosarcina thermophila pyrrolysyl-tRNA synthetase; - Methanococcoides burtonii pyrrolysyl-tRNA synthetase; - Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase; and their functional fragments and variants that retain aminoacyl-tRNA synthetase enzymatic activity; The O-RS segment comprises at least one O-RS segment selected from:

[0223] E23: An RNA-TP / O-RS fusion protein of any one of E18 to E22, comprising: - PylRS comprising the amino acid sequence of SEQ ID NO: 8 AF or a functional fragment or variant thereof having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 8; - PylRS comprising the amino acid sequence of SEQ ID NO: 10 AA or a functional fragment or variant thereof having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 10; - PylRS comprising the amino acid sequence of SEQ ID NO: 12 AAAFor a functional fragment or variant thereof having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 12; - IFRS1 comprising the amino acid sequence of SEQ ID NO: 224, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 224; - a CbzRS comprising the amino acid sequence of SEQ ID NO: 226, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 226; - a CpkRS comprising the amino acid sequence of SEQ ID NO: 228, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 228; and - an OMeRS comprising the amino acid sequence of SEQ ID NO: 236, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 236; The O-RS segment comprises at least one O-RS segment selected from:

[0224] E24: A nucleic acid molecule or a combination of two or more nucleic acid molecules: (i) a nucleotide sequence encoding at least one AFP from any one of E1 to E15, or at least one AFP combination from E16 or E17; or (ii) a nucleic acid sequence complementary to the nucleotide sequence of (i); (iii) both (i) and (ii); Includes.

[0225] E25: A nucleic acid molecule or a combination of two or more nucleic acid molecules: (i) a nucleotide sequence encoding at least one RNA-TP / O-RS fusion protein of any one of E18 to E23; or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); Includes.

[0226] E26: An expression cassette comprising the nucleotide sequence of a nucleic acid molecule or combination of nucleic acid molecules of E24 or E25.

[0227] E27: An expression vector comprising at least one expression cassette of E26.

[0228] E28: A cell comprising at least one nucleic acid molecule or combination of nucleic acid molecules of E24 or E25, at least one expression cassette of E26, or at least one expression vector of E27.

[0229] E29: Eukaryotic cells, E28 cells.

[0230] E30: E28 cells, which are mammalian cells.

[0231] E31: A cell of any one of E28 to E30, comprising at least one expression cassette comprising the nucleotide sequence of at least one nucleic acid molecule or combination of nucleic acid molecules of E24, or the nucleic acid molecule or combination of nucleic acid molecules, or at least one expression vector comprising the expression cassette.

[0232] E32: A cell of E31, comprising a nucleotide sequence encoding at least one AFP of any one of E1 to E3 and E7 to E15, which comprises at least one EP selected from the RNA-TP segment and at least one EP selected from the O-RS segment, or which is complementary to a nucleotide sequence encoding the same.

[0233] E33: A cell of E31, comprising a nucleotide sequence encoding at least one AFP of any one of E1 to E3 and E7 to E15 that includes at least one EP selected from the RNA-TP segment, and at least one AFP of any one of E1 to E3 and E7 to E15 that includes at least one EP selected from the O-RS segment, or that is complementary to a nucleotide sequence encoding the same.

[0234] E34: A cell of any one of E28 to E30, comprising at least one expression cassette comprising the nucleotide sequence of at least one nucleic acid molecule or combination of nucleic acid molecules of E25, or the nucleic acid molecule or combination of nucleic acid molecules, or at least one expression vector comprising the expression cassette.

[0235] E35: The cell of any one of E28 to E34, wherein the cell expresses at least one AFP, at least one AFP combination, or at least one RNA-TP / O-RS fusion protein, respectively, encoded by the nucleotide sequence of said nucleic acid molecule or combination of nucleic acid molecules.

[0236] E36: A method for preparing a polypeptide of interest (POI) comprising one or more non-canonical amino acid (ncAA) residues on its amino acid sequence, the method comprising expressing the POI in the presence of said one or more ncAAs by a cell of any one of E31 to E33, wherein the cell: (i) the nucleotide sequence encoding the POI (CS POI ) wherein the one or more ncAA residues of the POI are encoded by a selector codon(s), (ii)CS POI a targeting nucleotide sequence (TN) functionally linked to the targeting nucleotide sequence (TN) and capable of interacting with at least one RNA-TP segment of the AFP in a cell; (iii)CS POI one or more orthogonal tRNAs carrying anticodon(s) complementary to the selector codon(s) of ncAA (O-tRNA ncAA ) molecule (the O-tRNA ncAA The molecule comprises one or more orthogonal O-RS / O-tRNAs that can be combined with one or more O-RS segments of at least one AFP in a cell to allow the introduction of the one or more ncAA residues into the amino acid sequence of the POI. ncAA form pairs); Including, The method optionally further comprises recovering the expressed POI.

[0237] E37: A method for preparing a polypeptide of interest (POI) comprising one or more non-canonical amino acid (ncAA) residues on its amino acid sequence, the method comprising expressing the POI in the presence of said one or more ncAAs by a cell of E35, wherein the cell: (i) the nucleotide sequence encoding the POI (CS POI ) wherein the one or more ncAA residues of the POI are encoded by a selector codon(s), (ii)CS POI a targeting nucleotide sequence (TN) functionally linked to the RNA-TP / O-RS fusion protein, capable of interacting with at least one RNA-TP segment of the RNA-TP / O-RS fusion protein in a cell; (iii)CS POI one or more orthogonal tRNAs carrying anticodon(s) complementary to the selector codon(s) of ncAA (O-tRNA ncAA ) molecule (the O-tRNA ncAAThe molecule comprises one or more orthogonal O-RS / O-tRNAs that, together with one or more O-RS segments of the RNA-TP / O-RS fusion protein in a cell, enable the introduction of said one or more ncAA residues onto the amino acid sequence of the POI. ncAA form pairs); Including, The method optionally further comprises recovering the expressed POI.

[0238] E38: A method for preparing a polypeptide of interest (POI) comprising one or more non-canonical amino acid (ncAA) residues on its amino acid sequence, said method comprising: (a) expressing by a cell one or more AFPs of any one of E1 to E3 and E7 to E15 that include at least one RNA-TP segment, and one or more AFPs of any one of E1 to E3 and E7 to E15 that include at least one O-RS segment; (b) one or more orthogonal tRNAs ncAA (O-tRNA ncAA ) molecule expressed by said cell, - the orthogonal tRNA ncAA The O-RS segments of the molecule and AFP are linked to one or more orthogonal aminoacyl-tRNA synthetases / tRNAs in the cell. ncAA (O-RS / O-tRNA ncAA ) form a pair, - the O-RS / O-tRNA ncAA The pair allows the introduction of the one or more ncAA residues onto the amino acid sequence of the POI, Steps (a) and (b) may be simultaneous or sequential in any order; (c) then expressing the POI by the cell in the presence of the one or more ncAAs; - Nucleotide sequence encoding the POI (CS POI ) comprises one or more selector codons encoding the one or more ncAA residues, - the selector codon is selected from the one or more O-tRNAs ncAA Matches the anticodon of the molecule; - Said CS POI is functionally linked to the targeting nucleotide sequence (TN), and thus CS POI / TN fusion sequence, - Said CS POI the / TN fusion sequence is capable of interacting with at least one RNA-TP segment of AFP in a cell via its TN; (d) optionally recovering the expressed POI; Includes.

[0239] E39: A method for preparing a polypeptide of interest (POI) comprising one or more non-canonical amino acid (ncAA) residues on its amino acid sequence, said method comprising: (a) expressing an RNA-TP / O-RS fusion protein at any one of E18 to E23 by a cell; (b) one or more orthogonal tRNAs ncAA (O-tRNA ncAA ) molecule expressed by said cell, - one or more O-RS segments of an RNA-TP / O-RS fusion protein and said orthogonal tRNA ncAA The molecule is capable of catalyzing one or more orthogonal aminoacyl-tRNA synthetases / tRNAs within the cell. ncAA (O-RS / O-tRNA ncAA ) form a pair, - the O-RS / O-tRNA ncAA The pair allows the introduction of the one or more ncAA residues onto the amino acid sequence of the POI, Steps (a) and (b) may be simultaneous or sequential in any order; (c) then expressing the POI by the cell in the presence of the one or more ncAAs; - Nucleotide sequence encoding the POI (CS POI ) comprises one or more selector codons encoding the one or more ncAA residues, - the selector codon is selected from the one or more O-tRNAs ncAA Matches the anticodon of the molecule; - Said CS POI is functionally linked to the targeting nucleotide sequence (TN), and thus CS POI / TN fusion sequence, - Said CS POI the / TN fusion sequence is capable of interacting with at least one RNA-TP segment of the RNA-TP / O-RS fusion protein in a cell via its TN; (d) optionally recovering the expressed POI; Includes.

[0240] E40: The method of any one of E36 to E39, wherein TN is selected from viral RNA motifs that are bound by viral coat proteins, and functional fragments and variants thereof that retain the ability to be bound by viral coat proteins.

[0241] E41: The method of any one of E36 to E40, wherein TN is: - an MS2 RNA stem loop comprising an RNA sequence encoded by the nucleotide sequence of SEQ ID NO: 17, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 17; - BoxB comprising an RNA sequence encoded by the nucleotide sequence of SEQ ID NO: 18, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 18, and - a pp7 RNA stem loop that exists in at least two different versions and that comprises an RNA sequence that is encoded by a nucleotide sequence of a polynucleotide that has an RNA sequence that corresponds to (is encoded by) the nucleotide (DNA) sequence of SEQ ID NO: 289 or SEQ ID NO: 290, or a functional fragment or variant thereof that has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 289 or 290, is selected from.

[0242] E42: The method of any one of E36 to E41, wherein the selector codon(s) encoding the ncAA residue(s) of the POI are selected from amber, ochre, and opal stop codons.

[0243] E43: A nucleic acid molecule comprising: (i) a nucleotide sequence encoding a polypeptide of interest (POI) (CS POI ) and (the POI is selected by the selector codon CS POI containing one or more non-canonical amino acid (ncAA) residues as encoded above), (ii) a targeting nucleotide sequence (TN) (an RNA molecule comprising the TN can interact with an RNA-targeting polypeptide (RNA-TP) via the TN); Includes.

[0244] A nucleic acid molecule of E44:E43, wherein TN is selected from viral RNA motifs that are bound by viral coat proteins, and functional fragments and variants thereof that retain the ability to be bound by viral coat proteins.

[0245] E45: A nucleic acid molecule of E43 or E44, wherein TN is: - an MS2 RNA stem loop comprising an RNA sequence encoded by the nucleotide sequence of SEQ ID NO: 17, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 17; - BoxB comprising an RNA sequence encoded by the nucleotide sequence of SEQ ID NO: 18, or a functional fragment or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 18; and - a pp7 RNA stem loop that exists in at least two different versions and that comprises an RNA sequence that is encoded by a nucleotide sequence of a polynucleotide that has an RNA sequence that corresponds to (is encoded by) the nucleotide (DNA) sequence of SEQ ID NO: 289 or SEQ ID NO: 290, or a functional fragment or variant thereof that has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 289 or 290, is selected from.

[0246] E46: The nucleic acid molecule of any one of E43 to E45, wherein the selector codon(s) encoding the ncAA residue(s) of the POI are selected from amber, ochre, and opal stop codons.

[0247] E47: A kit for preparing a polypeptide of interest (POI) having at least one non-canonical amino acid (ncAA) residue, the kit comprising: - at least one ncAA or a salt thereof corresponding to at least one ncAA residue of the POI; - at least one expression vector for E27; Includes.

[0248] E48: The kit of E47, wherein the expression vector encodes a fusion protein comprising at least one O-RS segment and at least one RNA-TP segment.

[0249] E49: A kit of E47 or E48, further comprising an orthogonal tRNA ncAA (O-tRNA ncAA ) molecule.

[0250] E50: A kit of any one of E47 to E49, further comprising at least one expression vector comprising a multiple cloning site and a targeting nucleotide sequence (TN), wherein the RNA molecule comprising the TN can interact with an RNA-targeting polypeptide (RNA-TP) via the TN.

[0251] Any one of the above embodiments also encompasses the following modifications: the above-mentioned AP (i.e., IC-TP and PSP) segments and / or EP (RNA-TP or O-RS) segments can be further combined with synthetic protein segments that induce and control macromolecular interactions. One or more, preferably one, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, protein segments can be functionally fused into the AFP of the present invention. SYNZIPs, which have the ability to form heterodimeric coiled-coil protein structures, are particularly interesting in the context of the present invention. Such SYNZIPs are pairs of synthetic peptides that can interact with each other and are used to induce and control macromolecular interactions. Non-limiting examples are the SYNZIP 1:2; SYNZIP 3:4; and SYNZIP 5:6 pairs. According to the present invention, the heterospecific coiled-coil pair SYNZIP2:SYNZIP1 (SYNZIP1: SEQ ID NO: 312, SYNZIP2: SEQ ID NO: 314, SYNZIP3: SEQ ID NO: 316, SYNZIP4: SEQ ID NO: 318) described by Reinke, AW, Grant, RA, Keating, AE (2010) J Am Chem Soc 132 6025-6031, as well as functional fragments and variants of these SYNZIP polypeptides, are particularly preferred. The functional fragments and variants may comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid of the polypeptide from which they are derived.

[0252] The present invention is further illustrated by the following non-limiting examples. [Example]

[0253] method

[0254] (A) Cell culture, transfection, and ncAA feeding

[0255] HEK293T cells (ATCC CRL-3216) and COS-7 cells (ATCC CRL-1651) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, 41965-039) supplemented with 1% penicillin-streptomycin (Sigma, 10,000 U / ml penicillin, 10 mg / ml streptomycin, 0.9% NaCl), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Life Technologies), and 10% FBS (Sigma). Cells were cultured at 37°C in a 5% CO atmosphere and passaged every 2–3 days for up to 15–20 passages.

[0256] In both cases, cells were seeded 15–20 h before transfection to reach 70–80% confluency at the time of transfection. Flow cytometry was performed in 24-well plates with plastic bottoms (Nunclon Delta Surface ThermoScientific). Immunofluorescence labeling and FISH were performed on 24-well plates with glass bottoms (Greiner Bio-One) or on 4-well Lab-Tek #1.0 borosilicate coverglasses (ThermoFisher).

[0257] HEK293T cells were transfected with polyethyleneimine (PEI, Sigma-Aldrich) using 3 μg of PEI per μg of DNA. COS-7 cells were transfected with JetPrime reagent (PeqLab) at a 1:2 ratio according to the manufacturer's recommendations.

[0258] For amber suppression assays, cells were cultured at POI TAG Vector, tRNA Pyl The cells were transfected with the synthetase, and MCP or mock constructs at a ratio of 1:1:1:1. Four to six hours after transfection, the medium was replaced with fresh ncAA-containing medium.

[0259] All stock and working solutions of ncAAs used were prepared as described by Nikic et al. (Nat Protoc 10(5):780-791, 2015). SCO (cyclooctyne lysine, SiChem SC-8000) was used at a final concentration of 250 μM. 3-Iodophenylalanine (Chem-Impex International Inc.) was used at a final concentration of 1 mM. SCO was used as a PylRS AF (Y306A, Y384F) is efficiently recognized by PylRS (see Plass et al., Angew Chem 2011, 50:3878-3881). AA (C346A, N348A) (see Wang et al., ACS Chem Biol 2013, 8:405-415).

[0260] (B) Flow cytometry

[0261] HEK293T cells were harvested one day after transfection, resuspended in 1x PBS, and passed through a 100µm nylon mesh. Co-transfection for flow cytometry was performed with 1.2µg total DNA at a ratio of 1:1:1:1: - a reporter plasmid encoding the POI (a stop codon encoding the amino acid position to be occupied by the ncAA), - a tRNA with an anticodon that matches (i.e., is the reverse complement of) the stop codon on the POI coding sequence Pyl (From now on, we will simply refer to tRNA Pyl (referred to as "plasmid"); - a plasmid encoding PylRS or a functional variant thereof, respectively, and - either a plasmid encoding an MCP fusion polypeptide or a mock plasmid, was carried out by. The cell culture medium was replaced with fresh medium containing the ncAA to be incorporated into the POI 4–6 h after transfection and left until the time of harvest.

[0262] Data acquisition and analysis were performed using an LSR Fortessa SORP Cell Analyzer (Becton, Dickinson and Company) and FlowJo software (FlowJo). First, cells were gated by cell type using forward scatter area (FSC-A) and side scatter area (SSC-A) parameters. Single cells were then identified based on SSC-A and side scatter width (SSC-W). Each FFC plot shown is the sum of three independent biological replicates, from which the mean and SEM were calculated. At least 130,000 single cells were analyzed per condition. GFP fluorescence was acquired in the 488-530 / 30 channel, and mCherry fluorescence was acquired in the 561-610 / 20 channel.

[0263] (C) PylRS immunostaining and imaging, fluorescence in situ hybridization (FISH).

[0264] For immunolabeling experiments, cells were rinsed with 1x PBS, fixed with 2% paraformaldehyde in 1x PBS for 10 min at RT, rinsed again with 1x PBS, and then permeabilized with 0.5% Triton X in 1x PBS for 15 min at RT. After rinsing the permeabilized cell samples twice with 1x PBS, the samples were incubated in blocking solution (3% BSA in 1x PBS for 90 min at room temperature) for 90 min, and then with 1 μg / ml primary antibody (polyclonal rat anti-PylRS, prepared as described by Nikic et al. (Angew Chem Int Ed Engl 2016, 55(52):16172-16176), and / or polyclonal rabbit anti-MCP (Merck, ABE76), and / or monoclonal rabbit anti-RPL26L1 antibody (EPR8478, Abcam, ab137046)) in blocking solution overnight at 4°C. The next day, cell samples were rinsed with 1x PBS and incubated with 2 μg / ml secondary antibody (chicken anti-rat IgG (H+L) cross-adsorbed Alexa Fluor 594-conjugated antibody (Thermo Fisher Scientific, A-21471) and / or goat anti-rabbit IgG (H+L) cross-adsorbed Alexa Fluor 647-conjugated F(ab')2 (Thermo Fisher Scientific, A-21246)) in blocking solution for 60 min at RT. DNA was stained with Hoechst 33342 (1 μg / ml in 1x PBS) for 10 min at RT. When only DNA was stained, cells were fixed and permeabilized as described above and then stained with Hoechst 33342 (1 μg / ml in 1x PBS) for 10 min at RT. Finally, cells were rinsed twice with 1x PBS.

[0265] FISH experiments were performed one day after transfection, similar to the FISH experiments described by Nikic et al. (Angew Chem Int Ed Engl 2016, 55(52):16172-16176). The hybridization protocol from Pierce et al. (Methods Cell Biol 122:415-436, 2014) was adapted for 24-well plates.

[0266] tRNA Pyl For imaging of IgG-only, the hybridization probe 5'-CTAACCCGGCTGAACGGATTTAGAGTCCATTCGATC-3' (5'-end Cy5-labeled; SEQ ID NO: 1) was used at 0.25 μM. After four washes with SSC and one with TN buffer (0.1 M TrisHCl, 150 mM NaCl), cells were incubated with 3% BSA in TN buffer for 1 h at RT prior to standard immunofluorescence labeling as described above.

[0267] tRNA Pyl For imaging of both the MS2 RNA stem-loop sequence and the tRNA PylA hybridization probe for the MS2 RNA stem-loop sequence (5'-CTGCAGACATGGGTGATCCTCATGTTTTCTA-3'; SEQ ID NO: 3) labeled at the 5' end with Alexa Fluor 647 was used at 0.75 μM. After four washes with SSC, cells were incubated in blocking buffer (0.1 M TrisHCl, 150 mM NaCl, 1x blocking reagent (Sigma 11096176001) for 1 h at RT. Next, cells were incubated with fluorescein-conjugated sheep anti-digoxigenin Fab (Sigma 11207741910) at a 1:200 dilution in blocking buffer overnight at 4°C. The next day, cells were washed three times for 5 min with Tween buffer (0.1 M TrisHCl, 150 mM NaCl, 0.5% Tween 20). DNA was stained with Hoechst 33342 (1 μg / ml in 1x PBS) for 10 min at RT.

[0268] Confocal images were acquired with a Leica SP8 STED 3X microscope equipped with a 63x / 1.40 oil immersion objective using the following laser lines for excitation: 405 nm for Hoechst 33342, 488 nm for fluorescein and GFP, 548 nm for mOrange, 594 nm for Alexa Fluor 594, and 647 nm for Alexa Fluor 647 and Cy5. Emission light was collected by a HyD detector at 420–500 nm and 605–680 nm, respectively.

[0269] Ribosome immunofluorescence images were taken on an Olympus Fluoroview FV3000 microscope equipped with a 60x / 1.40 oil immersion objective using the following laser lines for excitation: 488 nm for GFP, 594 nm for Alexa Fluor 594, and 640 nm for Alexa Fluor 647.

[0270] Images were processed using FIJI software.

[0271] (D) Constructs, cloning, and mutagenesis

[0272] Two different fluorescent protein reporters (dual-color reporters) were cloned onto the pBI-CMV1 vector (Clontech 631630), with one protein in one multiple cloning site and the other reporter in the other multiple cloning site. The CDS of one of the reporters encoded an mRNA carrying two MS2 RNA stem-loops ("MS2 tags") fused to the 3' untranslated region, whereas the mRNA encoded by the other reporter was not MS2-tagged.

[0273] GFP reporter for amber suppression studies 39TAG and mCherry 185TAG was used as an N-terminal fusion with an NLS. Similar constructs were prepared for ochre and opal suppression studies (GFP and GFP, respectively). 39TAA and mCherry 185TAA , GFP 39TGA and mCherry 185TGA by).

[0274] NLS::GFP 39TAG ::MS2 tag reporter:NLS::GFP 39TAG was cloned along with two copies of the MS2 RNA stem-loop onto the pBI-CMV1 vector as a reporter for successful amber suppression in imaging experiments.

[0275] For the study of multiple amber codon suppression, a second multiple cloning site containing a GFP-free reporter (e.g., mCherry) is used. 39,149TAG and GFP 39,149,182TAG The pBI-CMV construct was prepared.

[0276] Further non-limiting examples of GFPs that are applicable in the context of the present invention are: GFP 66TAG GFP with amber site (SEQ ID NO: 238) GFP 66TCG GFP with serine site (SEQ ID NO: 240) GFP 66CCG GFP with proline site (SEQ ID NO: 242) GFP 66CTA GFP with leucine site (SEQ ID NO: 244) GFP 66TTA GFP with leucine site (SEQ ID NO: 246) GFP 66ATA GFP with isoleucine site (SEQ ID NO: 248) GFP 66CGG GFP with arginine site (SEQ ID NO: 250) GFP 39TCG GFP with serine site (SEQ ID NO: 252) GFP 39CCG GFP with proline site (SEQ ID NO: 254) GFP 39CTA GFP with leucine site (SEQ ID NO: 256) GFP 39CGG GFP with arginine site (SEQ ID NO: 258) GFP 39TCG LCK-GFP with serine site (SEQ ID NO: 278) GFP 39CCG LCK-GFP with proline site (SEQ ID NO: 280) GFP 39CTA LCK-GFP with leucine site (SEQ ID NO: 282) Extended GFP 39TCG GFP 66CCG GFP with a serine site at position 39 genetically fused to (SEQ ID NO: 284) Extended GFP 39CCG GFP 66TCG GFP with a proline site at position 39 genetically fused to (SEQ ID NO: 286) Extended GFP 39CTA GFP 66TCG GFP with a leucine site at position 39 genetically fused to (SEQ ID NO: 288).

[0277] Further non-limiting examples of mCherry that are applicable in the context of the present invention are: mCherry 72TAG mCherry with amber site (SEQ ID NO: 260) mCherry 72TCG mCherry with serine site (SEQ ID NO: 262) mCherry 72CCG mCherry with a proline site (SEQ ID NO: 264) mCherry 72CTA mCherry with leucine site (SEQ ID NO: 266) mCherry 72TTA mCherry with leucine site (SEQ ID NO: 268) mCherry 72ATA mCherry with an isoleucine site (SEQ ID NO: 270) mCherry 185TCG mCherry with serine site (SEQ ID NO: 272) mCherry 185CCG mCherry with a proline site (SEQ ID NO: 274) mCherry 185CTA mCherry (SEQ ID NO: 276) with a leucine site.

[0278] Further non-limiting examples of mCherry constructs containing different TN loops that are applicable in the context of the present invention are: mCherry 190TAG -2xPP7 mCherry with amber site and 2xpp7 loop (SEQ ID NO: 216) mCherry 190TAG -4xPP7 mCherry with amber site and 4xpp7 loop (SEQ ID NO: 218) mCherry 190TAG -6xPP7 mCherry with amber site and 6xpp7 loop (SEQ ID NO: 220) H2B-mCherry 190TAG-2xMS2 Human histone H2B1-J type (Uniprot: P06899) ​​(SEQ ID NO: 222) fused to mCherry with an amber site and 2xms2 loops.

[0279] They can be fused to the polypeptide chain of any of the AFP molecules described herein, particularly at positions on the fusion molecule that do not interfere with the function of any one of the other polypeptide segments (AP and EP) of the AFP molecule. Examples of such epitope-tagged AFP molecules are shown below.

[0280] Constructs for OT assembly were prepared as follows: tRNA Pyl was cloned under the control of the human U6 promoter, and all other constructs were cloned under the CMV promoter in the pcDNA3.1 (Invitrogen V86020) vector. The MCP protein was cloned from Addgene plasmid #31230, and FUS was cloned from Addgene plasmid #26374. All FUS fusions used amino acids 1-478 (S108N), replacing the C-terminal NLS region with a Flag tag. All RS fusions used the previously reported efficient NES::PylRS. AF (Y306A, Y384F) sequence was used (see, e.g., Nikic et al., Angew Chem Int Ed Engl 2016, 55(52):16172-16176). AA (N346A, C348A) was cloned by site-directed mutagenesis starting from wild-type PylRS. The SPD5 gene was ordered from Genewiz, and MCP and PylRS were cloned by restriction cloning. AF It was fused to KIF13A. 1-411 and KIF16B 1-400 was cloned from human cDNA and inserted into pcDNA3.1 by restriction cloning. 1-411 The P390 of MCP and PylRS was deleted by site-directed mutagenesis. AF , EWSR1::MCP, FUS::PylRS AF, FUS::PylRS AA , SPD5::MCP, and SPD5::PylRS AF and KIF13A 1-411 , ΔP390 and KIF16B -400 Fusions were assembled by Gibson assembly (see Gibson et al., Nat Methods 2009, 6:343-345).

[0281] Construct for differential imaging experiments: Nup153-EGFP 149TAG and Vim 116TAG To selectively express -mOrange, one gene was first inserted onto pBI-CMV1 with an MS2 tag (compare Nikic et al., Angew Chem Int Ed Engl 2016, 55(52):16172-16176). Then, the other gene was inserted without an MS2 tag. Nup153::EGFP 149TAG and Vim 116TAG Vim on a pBI vector with the ::mOrange::MS2 tag 116TAG By replacing -mOrange, INSR 676TAG ::mOrange is merged with the MS2 tag, INSR 676TAG: ::mOrange on one cassette and Nup153::EGFP 149TAG on the other cassette.

[0282] Multicistronic amber suppression vector for COS-7 cell experiments: Because COS-7 cells have a lower transfection efficiency, we generated a multicistronic vector harboring components of the OT assembly. To construct the multicistronic amber suppression vector, we transfected a tRNA 1 vector under the control of the human U6 promoter. Pyl The first copy of the AFP CDS KIF16B::FUS::PylRS was inserted into the pBI-CMV1 vector by Gibson assembly. AFFinally, the AFP CDS KIF16B::EWSR1::MCP was inserted by Gibson assembly. Alternatively, the NES::PylRS under the CMV promoter was inserted. AF and tRNA under the human U6 promoter Pyl A previously published pcDNA3.1-based construct expressing U6-tRNA (see Nikic et al., Angew Chem Int Ed Engl 2016, 55(52):16172-16176) was used. Alternatively, U6-tRNA Pyl , KIF16B::FUS::PylRS AF , and KIF16B::EWSR1::MCP, or NES::PylRS AF The construct was inserted into the pDonor vector (GeneCopoeia). The sequence information for each of the AFPs used in the following experiments can be obtained from the sequence listing below.

[0283] Example 1 - RNA-TP / O-RS fusion and AFP containing a single AP

[0284] We engineered OT assemblies ("OT organelles", Figure 1) with the following components:

[0285] i) In the mRNA targeting system, two MS2 RNA stem-loops (MS2 tags) were fused to a selected mRNA encoding a POI to generate an mRNA::ms2 fusion. The MS2 tag specifically binds to the MS2 bacteriophage coat protein (MCP) (see Bertrand et al., Mol Cell 1998, 2:437-445), which would therefore form a stable and specific mRNA::ms2-MCP complex in cells. The MS2 tag was always fused to the 3' untranslated region (3'UTR) of the mRNA. This ensured that translation would not result in a trace of the final POI.

[0286] ii) tRNA / RS suppression pair. The orthogonal tRNA / RS pair (tRNA Pyl / PylRS) was chosen because it allows the encoding of over 200 ncAAs with diverse functionalities into proteins using GCE in multiple cell types and species, including E. coli, mammalian cells, and even live mice (see, e.g., Liu et al., Annu Rev Biochem 2010, 79:413-444; Lemke, ChemBioChem 2014, 15:1691-1694; Chin, Nature 2017, 550; 53-60).

[0287] iii) The assembler (AP) was the main component required for forming OT assemblies. The purpose of the assembler was to create membrane-free structures in the form of dense phases, aggregates, droplets, or condensates. In this, the mRNA::ms2-MCP complex assembles tRNA Pyl / PylRS pair in close proximity.

[0288] The simplest strategy tested was a bimolecular fusion of MCP::PylRS (designated B; Figure 2). In addition, strategies predicted to generate larger assemblies were tested. All of these assembly systems consisted of an assembler fusion to PylRS coexpressed with an assembler fusion to MCP. Assembler::PylRS·assembler::MCP was predicted to form large aggregates (coexpression is represented herein by a "·"). One tested assembly strategy was based on protein phase separation, and one was based on kinesin assembly, abbreviated herein as P and K, respectively (Figure 2A). Furthermore, for each P and K approach, two different molecular designs (P1, P2, and K1, K2, respectively) were tested. These are summarized as follows:

[0289] P1. Previous studies have established the ability of the fusion sarcoma (FUS) and Ewing sarcoma breakpoint region 1 (EWSR1) proteins to form mixed droplet-like structures through phase separation. Both contain prion-like denaturation domains that facilitate phase separation into liquid, gel, and solid states (see, e.g., Altmeyer et al., Nat Commun 2015, 6:8088; Patel et al., Cell 2015, 162:1066-1077). In the phase-separated state, these proteins are locally highly enriched (by several orders of magnitude) relative to the remaining soluble fraction in the cytoplasm. FUS was fused to PylRS, and EWSR1 was fused to MCP. This was predicted to lead to the formation of droplets highly enriched in MCP and PylRS. P1 is represented as FUS::PylRS·EWSR1::MCP.

[0290] P2. The Caenorhabditis elegans protein spindle-defective protein 5 (SPD5) has been shown to phase separate into particularly large (several micron-sized) droplets (see Woodruff et al., Cell 2017, 169:1066-1077, e1010). In the phase-separated state, SPD5 becomes highly enriched locally (by several orders of magnitude) compared to the remaining soluble fraction in the cytoplasm. Proteins fused to SPD5 were predicted to condense into droplets. Similar to the FUS-EWSR1 droplets, PylRS fused to SPD5 and MCP fused to SPD5 were predicted to be highly enriched. P2 is represented as SPD5::PylRS·SPD5::MCP.

[0291] K1. Certain kinesin truncations move constitutively toward the plus ends of microtubules in living cells (Soppina et al., Proc Natl Acad Sci USA 2014, 111:5562-5567). One such truncated kinesin is KIF13A. 1-411,ΔP390 It was predicted that PylRS and MCP, respectively fused to and co-expressed with this kinesin truncation, would be locally concentrated due to spatial targeting to the microtubule plus ends.1-411,ΔP390 ::PylRS·KIF13A 1-411,ΔP390 It is expressed as ::MCP.

[0292] K2. Similar to K1, a truncated kinesin, KIF16B 1-400 K2 was also tested. 1-400 ::PylRS·KIF16B 1-400 It is expressed as ::MCP.

[0293] To evaluate these assemblers for facilitating functional orthogonal translation of MS2-tagged mRNAs, a dual reporter construct was designed, in which GFP and mCherry variants are co-expressed from two different expression cassettes from one plasmid, ensuring that the mRNA ratio between them is constant in all experiments. A stop codon was inserted at a permissive site to GFP at position 39 (GFP 39終止 ), introduced into mCherry at position 185 (mCherry 185終止 (Figure 2B). Only if the stop codon is successfully suppressed will the corresponding green or red fluorescent protein be produced. Transfected cells (unless otherwise noted) Pyl The GFP and mCherry mRNAs (and ncAA were always present) were analyzed by fluorescence flow cytometry (FFC). Settings were adjusted so that when GFP and mCherry were expressed from this plasmid using the conventional cytoplasmic PylRS system, which cannot distinguish between mRNAs, an approximately diagonal line was produced in the FFC plot. Selective and functional OT organelles should selectively express mCherry only when the MS2 tag is fused to the 3'UTR of mCherry mRNA, leading to the appearance of a vertical line in the cytometry plot (Figure 2B). Unless otherwise reported, all experiments were performed using tRNA PylThe experiments were performed in the presence of the widely used and well-characterized lysine derivative ncAA SCO, whose side chain carries a cyclooctyne moiety that can be used in various click chemistry reactions to install diverse chemical groups onto proteins. As previously reported, this ncAA is effectively encoded by the Y306A, Y384F double mutant of PylRS (for simplicity, this mutant will be referred to as PylRS herein unless otherwise specified) (see Nikic et al., Angew Chem 2014, 53:2245-2249; Plass, Angew Chem 2012, 51:4166-4170; Plass et al., Angew Chem 2011, 50:3878-3881). Omission of the ncAA served as a standard negative control and did not result in expression of GFP or mCherry.

[0294] The performance of each OT system was evaluated according to its selectivity and relative efficiency. Selectivity is defined as the ratio r of the mean mCherry FFC signal divided by the mean GFP signal. The final value is expressed as ~-fold selectivity relative to that of the cytoplasmic PylRS. Relative efficiency is defined as the mean mCherry signal of each system divided by the mean mCherry signal of the cytoplasmic PylRS system, which serves as the reference (defined here as 100%). All results for selectivity (dark gray positive bars) and efficiency (light gray negative bars) are summarized in the bar graph in Figure 2C. Selected FFC data are also shown in Figure 2D.

[0295] The simplest strategy B (MCP fused to PylRS) showed a selectivity gain of approximately 1.5-fold (Fig. 2C). OT system P1 (based on the phase separation of FUS / EWSR1) had a somewhat lower selectivity gain (Fig. 2C, D). P2 system (based on SPD5) showed an approximately 2-fold selectivity gain (Fig. 2C). A 2-fold increase in selectivity was observed with K1 (Fig. 2C). K2 system behaved similarly (Fig. 2C, D). Overall, the selectivity gain was relatively small, but robustly detectable and distinguishable from a simple drop in efficiency. The observed selectivity effect (data not shown) was due to the titration of amber suppression efficiency (specifically, 0.48 ng, 2.4 ng, 12 ng, 60 ng, or 300 ng of tRNA, respectively). Pyl The results were robust across a range of ncAA aminoacylation activities (i.e., tRNAPyl / PylRS in the presence of an ncAA) and constructs used, indicating that bringing ncAA aminoacylation activity (i.e., tRNAPyl / PylRS in the presence of an ncAA) into direct proximity of a target mRNA represents a route to more selective codon suppression.

[0296] Example 2 - AFP containing a combination of two APs

[0297] AFPs containing the AP combinations described in Example 1 were tested in a similar manner. They were: K1::P1= KIF13A 1-411,ΔP390 ::FUS::PylRS·KIF13A 1-411,ΔP390 ::EWSR1::MCP, K2::P1=KIF16B 1-400 ::FUS::PylRS·KIF16B 1-400 ::EWSR1::MCP, K1::P2=KIF13A 1-411,ΔP390 ::SPD5::PylRS·KIF13A 1-411,ΔP390 ::SPD5::MCP, K2::P2=KIF16B 1-400 ::SPD5::PylRS·KIF16B 1-400 ::SPD5::MCP.

[0298] For all combinations, at least a 5-fold selectivity gain was observed, supporting orthogonal translation. The best performing of these systems was KIF16B 1-400 Based on the FUS / EWSR1 fusion with K2::P1, we observed an 8-fold selectivity (box in Figure 2C), which was also directly evident from the FFC data, in which a bright mCherry-positive cell population was clearly retained and GFP expression was minimal (arrow in Figure 2D).

[0299] Example 3 - AFPs containing combinations of APs including membrane-targeted APs

[0300] APs derived from phase separation polypeptides (PSPs), FUS, and EWSR1 (also referred to herein as EWS), optionally fused to a SYNZIP segment, and different APs LcK, EB1, CG1, EBAG9, which act as membrane targeting signals. 全長 , EBAG9 1-29 , CMP Sia Tr, P450 2C1 1-27 , and P450 2C1 1-29 AFPs containing the following combinations were tested in a similar manner to Example 2.

[0301] LcK is a plasma membrane targeting signal (Resh, Bba-Mol Cell Res 1999, 1451:1-16), which post-translationally adds an amphipathic helix to the POI. In these experiments, the AFPs LcK::FUS::PylRS and LcK::EWSR1::MCP were coexpressed in HE293T cells (see Figures 3 and 6C). Testing this system with the same dual reporter resulted in a dramatic shift in signal and strong selectivity for the expression of MS2-tagged mCherry exclusively compared to the control PylRS. See Figures 4 and 5, which show a 26-fold selectivity gain compared to the control. IF and FISH for MCP, PylRS, and tRNA show a clear membrane signal with the appearance of occasional droplet-like structures and complete colocalization of all components. Without wishing to be bound by theory, we hypothesize that targeting the OT system to membranes results in the confinement of components to a 2D surface (i.e., a film), offering even greater spatial segregation than cytoplasmic droplets. Following this cumulative effect of two combined assembler strategies (LcK for membrane targeting and FUS / EWSR1 for droplet generation), we demonstrated that the presence of the FUS / EWSR1 "assembler" is not a requirement for LcK fusion (and thus, a membrane-anchored system) to obtain selective amber suppression (data not shown). Nevertheless, combining LcK targeting with FUS / EWSR1 resulted in higher selectivity of the system. Furthermore, swapping the MS2 tag on the fluorescent reporter was found to yield swapped selectivity in FFC data, highlighting the selective (orthogonal) translation of MS2-tagged mRNA.

[0302] For further LcK-based experiments, the AFP constructs LcK::FUS::SYNZIP1::PylRS and EWSR1::SYNZIP2::MCP were coexpressed in HE293T cells (see Figure 8A). Testing this system with the same dual reporter resulted in a dramatic shift in the signal exclusively for MS2-tagged mCherry and strong selectivity of expression. Upon expression, SYNZIP1 and 2 pair and recruit MCP to plasma membrane-based OT organelles. In a comparative approach where AFP constructs LcK::FUS::PylRS and EWSR1::SYNZIP2::MCP were coexpressed, lacking SYNZIP1, no translational selectivity could be observed (see Figure 8B).

[0303] EB1 is a microtubule plus-end targeting signal (Nehlig A, Molina A, Rodrigues-Ferreira S, Honore S, Nahmias C. Regulation of end-binding protein EB1 in the control of microtubule dynamics. Cell Mol Life Sci. 2017;74(13):2381-2393. doi:10.1007 / s00018-017-2476-2). In these experiments, the AFP constructs EB1::PylRS with EB1::MCP, EB1::FUS::PylRS with EB1::EWSR1::MCP, or EB1::FUS::MCP were expressed in HE293T cells. Testing of this system with the same dual reporter resulted in a signal shift and strong selectivity for the expression of exclusively MS2-tagged mCherry compared to the control PylRS. See Figure 6B.

[0304] CG1 is a nuclear envelope targeting signal (Kim SJ, Fernandez-Martinez J, Nudelman I, et al. Integrative structure and functional anatomy of a nuclear pore complex. Nature. 2018;555(7697):475-482. doi:10.1038 / nature26003). In these experiments, the AFP constructs CG1::FUS::PylRS and CG1::EWSR1::MCP were coexpressed in HE293T cells. Testing of this system with the same dual reporter resulted in a signal shift and strong selectivity for the expression of exclusively MS2-tagged mCherry compared to the control PylRS. See Figure 6E.

[0305] EBAG9 全長 and EBAG9 1-29is a Golgi membrane targeting signal (Engelsberg A, Hermosilla R, Karsten U, Schulein R, Dorken B, Rehm A. The Golgi protein RCAS1 controls cell surface expression of tumor-associated O-linked glycan antigens. J Biol Chem. 2003;278(25):22998-23007. doi:10.1074 / jbc.M301361200). In these experiments, the AFP construct EBAG9 1-29 ::FUS::PylRS and EBAG9 1-29 ::EWSR1::MCP was co-expressed in HE293T cells. Testing of this system with the same dual reporter resulted in a signal shift and strong selectivity for the expression of exclusively MS2-tagged mCherry compared to the control PylRS (see Figure 6F (left)).

[0306] CMP Sia Tr is a Golgi membrane targeting signal (Eckhardt M, Gotza B, Gerardy-Schahn R. Membrane topology of the mammalian CMP-sialic acid transporter. J Biol Chem. 1999;274(13):8779-8787. doi:10.1074 / jbc.274.13.8779). In these experiments, the AFP constructs CMP Sia Tr::FUS::PylRS and CMP Sia Tr::MCP were coexpressed in HE293T cells. Testing of this system with the same dual reporter resulted in a signal shift and strong selectivity for the expression of exclusively MS2-tagged mCherry compared to the control PylRS. See Figure 6F (right).

[0307] P450 2C1 1-27is an ER membrane targeting signal (Fazal FM, Han S, Parker KR, et al. Atlas of Subcellular RNA Localization Revealed by APEX-Seq. Cell. 2019;178(2):473-490.e26.doi:10.1016 / j.cell.2019.05.027). In these experiments, the AFP construct P450 2C1 1-27 ::FUS::PylRS and P450 2C1 1-27 ::EWSR1::MCP or P450 2C1 1-29 ::FUS::MCP::PylRS was co-expressed in HE293T cells. Testing of this system with the same dual reporter resulted in a signal shift and strong selectivity for the expression of exclusively MS2-tagged mCherry compared to the control PylRS (see Figure 6G).

[0308] Example 4 - Verification that the selectivity gain is specific to the interaction between mRNA MS2 tag and MCP

[0309] To verify that the observed selectivity gain was specific to the interaction of the MCP segment with the MS2 tag of mRNA, we characterized all OT systems by expressing RS assembler fusions of each OT system without MCP. As expected, selective orthogonal translation of MS2-tagged mRNA was not observed in these cases (see Figure 6A to G). In addition, we performed reporter reversal by moving the MS2 tag from mCherry to a GFP cassette on the dual-color reporter. This reversed the selectivity of the system to dominant GFP expression, as expected (data not shown). This established that the OT system selectively acts on MS2-tagged RNA.

[0310] Example 5 - Introducing multiple ncAAs on the same POI

[0311] GCE can also be used to introduce multiple ncAAs onto the same POI (see, e.g., Liu et al., Annu Rev Biochem 2010, 79:413-444; Lemke, ChemBioChem 2014, 15:1691-1694; Chin, Nature 2017, 550; 53-60). However, very few publications have reported on the suppression of more than one, i.e., two or three, codons on the same protein in eukaryotes. This is because the yields are typically poor compared to single-codon suppression (see Xiao et al., Angew Chem 2013, 52:14080-14083; Schmied et al., J Am Chem Soc 2014, 136:15577-15583; Zhang et al., Biochem Biophys Res Co 2017, 489:490-496). Notably, even double and triple amber proteins were still suppressed by the OT organelle (data not shown).

[0312] Example 6-OT with 3-iodophenylalanine

[0313] To ensure that other ncAAs could also be translated by OT assembly, we tested another structurally distinct ncAA (3-iodophenylalanine), which is a phenylalanine derivative instead of a lysine derivative (e.g., SCO) and is encoded by a different PylRS mutant (N346A, C348A) (see Wang et al., ACS Chem Biol 2013, 8:405-415). Consistent results were also observed with this system (Figure 2C).

[0314] Example 7 - OT with different selector codons

[0315] Because opal and ochre codons are highly abundant in eukaryotic genomes (52% opal and 28% ochre in the human genome), amber codons are by far the most used for GCE in eukaryotes. In addition, a genomic approach to orthogonal translation by removing these codons throughout the eukaryotic genome is even more challenging than amber codons and is currently beyond the state of the art. However, in the OT system of the present invention, tRNA Pyl Simple mutations in the anticodon loop of the POI and in the respective codons on the MS2-tagged POI-encoding mRNA allow for orthogonal translation of these codons. FFC analysis revealed that our OT system offers freedom of choice for the stop (selector) codon (Figure 2C, E). In fact, opal suppression proved to be the best-performing system, demonstrating an 11-fold increase in selectivity. Ochre suppression still demonstrated a 5-fold increase in selectivity at 20% efficiency.

[0316] Example 8 - Orthogonal translation of proteins for different cellular compartments

[0317] OT goes beyond "simple" reporters K2::P1 To visualize the power of the system (the best performing amber-suppressed OT system in terms of selectivity and efficiency), we aimed to demonstrate the differential expression of human nucleoporin 153 (Nup153) versus cytoskeletal vimentin. Nup153 is located in the nuclear pore complex and is over 1500 amino acids long. Thus, its mRNA is approximately six times larger than that of the fluorescent protein reporter used above. For this experiment, we used the previously described C-terminal GFP fusion (Nup153::EGFP) carrying an amber mutation. 149TAG ) was used, which produced characteristic nuclear envelope staining in confocal images only when amber suppression was successful (see Nikic et al., Angew Chem 2016, 55:16172-16276). 149TAG The gene was tagged at the mRNA level with an MS2 tag (nup153::egfp 149TAG::ms2), vimentin (cytoskeletal protein) containing an amber codon at position 116 fused to mOrange (Vim 116TAG OT was co-expressed from the same plasmid as OT (mOrange). Expression in HEK293T cells resulted in the production of both proteins in the presence of cytoplasmic PylRS, which displayed characteristic nuclear envelope and cytoskeletal staining, respectively. K2::P1 With this assembly, only Nup153::GFP was visible (selective nuclear rim staining in confocal images of co-transfected HEK293T cells). Consistent results were also observed in COS-7 cells. Swapping the MS tag for vimentin reversed the effect, resulting in a loss of Vim. 116TAG Only ::mOrange was visible (observed in both COS-7 and HEK293T cell experiments). K2::P1 showed that the ATPases act on dramatically different mRNAs.

[0318] Example 9 - Orthogonal translation of a transmembrane protein

[0319] Transmembrane protein is OT K2::P1 It was also shown that membrane protein expression can be selectively expressed using mOrange. Membrane protein expression represents another layer of translational complexity because ribosomes require binding to the endoplasmic reticulum during translation, where proteins are co-translationally inserted into the membrane. In this experiment, a fusion of insulin receptor 1 (INSR) with an amber codon at position 676 with mOrange was expressed. 676TAG The construct used was Nup153::mOrange, which localizes to the plasma membrane and produces characteristic plasma membrane staining in HEK293T cells (see Nikic et al., Angew Chem 2014, 53:2245-2249). This construct was tagged with an MS2 tag in the 3'UTR, resulting in Nup153::EGFP. 149TAG The constructs were then cloned onto a single double-cassette plasmid in the presence of the cytoplasmic PylRS system or in the presence of OT K2::P1The assembly was expressed by HEK293T cells in the presence of either OT. K2::P1 Selective expression of MS2-tagged proteins in the presence of assembly and INSR 676TAG The expected plasma membrane localization of ::mOrange was observed (data not shown), further supporting the potential of the present OT system to participate in complex membrane-associated translation processes.

[0320] Example 10 - Spatial distribution of elements of the OT system within cells

[0321] The spatial distribution of AFPs, especially PylRS, within cells was assessed using immunofluorescence (IF). In addition, fluorescence in situ hybridization (FISH) was performed to detect the expression of tRNAs. Pyl In contrast to the dual-color reporter used in the FFC experiments above, in all IF / FISH experiments, a single-color NLS-GFP fused to an MS2 tag was used. 39TAG Reporter (nls-gfp 39TAG We used a fluorescent dye (GFP) to identify cells active for amber suppression (which produces green nuclei when amber suppression is successful, helping to optimize distinguishable color channels). IF and FISH staining showed that, in contrast to cytoplasmic PylRS, the P1 system forms small intracellular assembler::PylRS droplets (data not shown). This indicated the occurrence of phase separation. tRNA Pyl K1::P1, K2::P1, K1::P2, K2::P2) resulted in the formation of large, micron-sized, organelle-like structures in the cytoplasm. These structures were localized to a few or even one location per cell in most cases. For the combined assemblers, mRNA::ms2, tRNAPyl , assembler::PylRS, and assembler::MCP all co-localized in organelle-like structures. The combination of the two assembler strategies, namely, phase separation coupled with spatial targeting by kinesin truncation, resulted in the best containment and the highest selectivity enhancement as determined by FISH and IF. This was consistent with the results for tRNA Pyl This is consistent with the hypothesis that higher spatial segregation and therefore higher local concentrations of PylRS and mRNA correlate with higher selectivity.

[0322] Ribosomes were stained to identify the OT K2::P1 IF staining of the ribosomal protein RPL26L1 revealed that OT cells colocalize with the ribosomal assembly. K2::P1 We found strong colocalization with organelles (data not shown), demonstrating ribosome recruitment due to binding to translating mRNA::ms2. High ribosome mobility also explains why the membrane protein INSR (construct:INSR 676TAG This may explain why it was possible to successfully express ::mOrange::ms2).

[0323] Without wishing to be bound by theory, experimental results suggest that tRNA Pyl This strongly suggests that selective orthogonal translation occurs in close proximity, and potentially even within, the OT assembly, with a set of ribosomes near or fully immersed within an enriched pool of tRNA. Pyl It is OT due to its affinity to the assembler::PylRS. K2::P1 The ribosomes are recruited to the assemblies, readily co-distribute into droplets, and can be aminoacylated with their corresponding ncAAs. Meanwhile, the assembler::MCP recruits MS2-tagged mRNAs, which in turn attract ribosomes and co-distribute them into the dense phase formed by the dual assembler system (K2::P1 = KIF16B::FUS::PylRS and KIF16B::EWSR1::MCP), maintaining access to other translation factors for translation to function. tRNAPyl Other ribosomes in the cytoplasm that are not exposed to the ribosome carry out their standard function to terminate translation whenever they encounter a stop codon.

[0324] Example 11 - Further OT systems

[0325] In addition to the OT system described in the previous examples, various other OT systems have been tested and found to allow selective orthogonal translation of the reporter (i.e., POI). A summary of these experiments is shown in Table 1 below. Unless otherwise noted, the cytoplasmic NES-PylRS system, as previously described by Nikic et al. (Angew Chem Int Ed Engl 2016, 55(52):16172-16176), but with the corresponding AF, AA, or AAAF mutations, was used as a non-specific reference (negative control). All experiments were performed using codon-specific tRNAs. Pyl and in the presence of the ncAA corresponding to the PylRS mutant.

[0326] [Table 1-1]

[0327] [Table 1-2]

[0328] [Table 1-3]

[0329] [Table 1-4]

[0330] Example 12 - Further OT systems

[0331] In addition to the OT system described in the previous examples, various similar OT systems differing in mRNA targeting components were tested and found to allow selective orthogonal translation of the reporter (i.e., POI). A summary of these experiments is shown in Table 2 below. The results are shown in Figures 7A, B, and C. The cytoplasmic NES-PylRS system previously described by Nikic et al. (Angew Chem Int Ed Engl 2016, 55(52):16172-16176) was used as a nonspecific reference (negative control). All experiments were performed using codon-specific tRNAs. Pyl and in the presence of the ncAA corresponding to the PylRS mutant.

[0332] Table 2: OT strains tested [Table 2] The results are shown in Figures 7A, B, and C.

[0333] Example 13 - Further OT fusion constructs tested

[0334] In addition to the OT system described in the previous examples, various other OT fusion constructs were prepared and tested and found to allow selective orthogonal translation of the reporter (i.e., POI). A summary of the constructs tested is shown in Table 3 below. Unless otherwise noted, a cytoplasmic NES-PylRS system, as previously described by Nikic et al. (Angew Chem Int Ed Engl 2016, 55(52):16172-16176), but with the corresponding AF, AA, or AAAF mutation, or one of the PylRS mutants CpkRS, CbzRS, IFRS1, and OMeRS, was used as a nonspecific reference (negative control).

[0335] All experiments were performed using codon-specific tRNAs Pyland in the presence of non-standard amino acids corresponding to PylRS mutants (e.g., cyclopropene-L-lysine and CpkRS, N(epsilon)-benzyloxycarbonyl-L-lysine and CbzRS, 3-iodo-L-phenylalanine and IFRS-1, and 4-methoxy-L-phenylalanine and OMeRS).

[0336] All constructs contained the ms2 loop, λ for each reporter MCP. N22 The BoxB loop was tested for PCP, and the pp7 loop was tested for PCP.

[0337] In all fusion constructs, the synthetases should be freely interchangeable.

[0338] It is important to note that in the SYNZIP construct, SYNZIP1 pairs with SYNZIP2, and SYNZIP3 pairs with SYNZIP4. In principle, all other described SYNZIPs should function similarly (https: / / pubs.acs.org / doi / pdf / 10.1021 / ja907617a).

[0339] [Table 3-1]

[0340] [Table 3-2]

[0341] [Table 3-3]

[0342] [Table 3-4]

[0343] Abbreviation

[0344] "-" or "::" symbol for peptidic linkage “·” symbol representing a polypeptide combination AP Polypeptide segments acting as assemblers AFP assembler fusion protein BSA Bovine serum albumin BoxB λ N22 Specific binding site of lambda phage RNA stem loop CbzRS Methanosarcina mazei PylRS(Y306M,L309G,C348T) CDS coding sequence CG1 (Nup42) nucleoporin protein for targeting to the nuclear envelope CMPSiaTr: A CMP sialic acid transporter for targeting to Golgi membranes CpkRS Methanosarcina mazei PylRS(A302S EB1 protein for targeting to microtubule plus ends EBAG9: A receptor-binding cancer antigen expressed on SiSo cells EBAG9 FL EBAG9 full-length protein for targeting to Golgi membranes EBAG9 1-29 EBAG9 amino acid residues 1-29 (N-terminus) for targeting to Golgi membranes EGFP 149TAG Enhanced green fluorescent protein encoded by an amber codon (TAG) at amino acid position 149 EP Polypeptide segment that acts as an effector ER endoplasmic reticulum EWSR1 Ewing sarcoma breakpoint region 1 (also referred to herein as EWS) FBS fetal bovine serum FFC Fluorescence Flow Cytometry FISH Fluorescence in situ hybridization FRB-CD28: A synthetic membrane targeting domain derived from the transmembrane proteins CD4, FRB (similar to mTOR), and CD28. FSC-A forward scatter area FUS sarcoma fusion FUS-CD28: A synthetic membrane-targeted fusion polypeptide derived from CD4, FUS, and CD28. GCE Genetic Code Extension GFP Green Fluorescent Protein GFP 39TAA Green fluorescent protein, where amino acid position 39 is encoded by the ochre codon (TAA) GFP 39TAG Green fluorescent protein encoded by an amber codon (TAG) at amino acid position 39 GFP 39TGA Green fluorescent protein encoded by the opal codon (TGA) at amino acid position 39 GFP 39,149TAG Green fluorescent protein encoded by amber codons (TAG) at amino acid positions 39 and 149, respectively GFP 39,149,182TAG Green fluorescent protein encoded by amber codons (TAG) at amino acid positions 39, 149, and 182, respectively. IC-TP Intracellular Targeting Polypeptide IDP Intrinsically Disordered Proteins IFRS1 Methanosarcina mazei PylRS(L305M,Y306L,L309S,N346S,C348M) INSR insulin receptor INSR 676TAG The insulin receptor is encoded by an amber codon (TAG) at amino acid position 676. iRFP Near-infrared fluorescent protein KIF13A Kinesin family member 13A—Unless otherwise specified herein, “KIF13A” specifically refers to a fragment covering amino acid residues 1-411 of KIF13A lacking P390 (KIF13A 1-411,ΔP390 ) KIF16B Kinesin family member 16B—Unless otherwise specified herein, “KIF16B” specifically refers to a fragment covering amino acid residues 1-400 of KIF16B (KIF16B 1-400 ) λ N22 The 22-amino acid RNA-binding domain of lambda phage antiterminator protein N Post-translational modification site for plasma membrane targeting of LcK lymphocyte-specific protein tyrosine kinase mCherry 185TAG mCherry, where amino acid position 185 is encoded by an amber codon (TAG) MCP MS2 bacteriophage coat protein MLC membraneless compartment MS2 Enterobacteriaceae phage MS2 Two MS2 RNA stem-loops fused to the 3' untranslated region (or its coding sequence) of the MS2-tag mRNA ms2 MS2 tag ncAA non-standard amino acids NLS nuclear localization sequence Nup153 nucleoporin 153 O-RS orthogonal aminoacyl-tRNA synthetase OMeRS Methanosarcina mazei PyrRS(A302T,Y384F,N346V,C348W,V401L) OT assembly: spatially concentrated components of the GCE machinery in a membraneless assembly that can act as an artificial orthogonal translation (OT) organelle. P450 2C1 1-27 P450 2C1 residues 1-27 (N-terminus) for ER membrane targeting PBS Phosphate-buffered saline Bacteriophage coat protein for targeting against PCP pp7 loop tag PEI Polyethyleneimine POI Polypeptide of Interest (= Target Polypeptide) POI TAGPOI (or its coding sequence) containing amino acid residues encoded by amber (TAG) pp7 loop tag from RNA bacteriophage pp7 PSP Phase-separating Polypeptide PylRS pyrrolysyl-tRNA synthetase PylRS AA Mutant M. mazei pyrrolysyl-tRNA synthetase containing the amino acid substitutions N346A and C348A. PylRS AF Mutant M. mazei pyrrolysyl-tRNA synthetase containing the amino acid substitutions Y306A and Y384F. PylRS AAAF Mutant M. mazei pyrrolysyl-tRNA synthetase containing amino acid substitutions Y306A, N346A, C348A, and Y384F RNA-TP RNA-targeting polypeptide RS aminoacyl-tRNA synthetase RT room temperature SCO Cyclooctyne Lysine SEM Standard error of the mean SSC Saline-Sodium Citrate (Buffer) SSC-A side scatter area SSC-W side scatter width SPD5 spindle-defective protein 5 SYNZIP1 Synthetic Coiled-Coil Peptide 1 SYNZIP2 Synthetic Coiled-Coil Peptide 2 SYNZIP3 Synthetic Coiled-Coil Peptide 3 SYNZIP4 Synthetic Coiled-Coil Peptide 4 TOMM20 Mitochondrial outer membrane translocase 20 TOMM20 1-70 A fragment covering amino acid residues 1-70 of TOMM20 tRNA PylA tRNA that is coupled to a pyrrolidinyl or another non-standard amino acid residue by a wild-type or modified PylRS and preferably has an anticodon that is the reverse complement of the selector codon for site-specific incorporation of the (non-standard) amino acid residue onto the POI. Pyl Depending on which of these was used as the selector codon on the POI coding sequence, the stop codon amber (tRNA Pyl,CUA ), ochre (tRNA Pyl,UUA ), or Opal (tRNA Pyl,UCA ) had an anticodon for 3'UTR 3' untranslated region Vim 116TAG Vimentin is encoded by an amber codon (TAG) at amino acid position 116

[0345] array

[0346] The next section presents the sequences of the polypeptides and polynucleotides described herein.

[0347] Nucleic acid sequences are written in the 5' to 3' direction, protein sequences are written from the N to C terminus.

[0348] Array-Set 1 1. Hybridization probes tRNA labeled with Cy5 at the 5' end Pyl Hybridization probes CTAACCCGGCTGAACGGATTTAGAGTCCATTCGATC (SEQ ID NO: 1) tRNA labeled with digoxigenin at the 5' end Pyl Hybridization probes CTAACCCGGCTGAACGGATTTAGAGTCCATTCGATC (SEQ ID NO: 2) Hybridization probe for MS2 RNA stem-loop sequence labeled with Alexa Fluor 647 at the 5' end CTGCAGACATGGGTGATCCTCATGTTTTCTA (SEQ ID NO: 3)

[0349] 2. tRNA tRNA Pyl,CUA DNA sequence of (Amber codon Methanosarcina mazei pyrrolysyl tRNA; anticodon is underlined) GGAAACCTGATCATGTAGATCGAATGGACT CTA AATCCGTTCAGCCGGGTTAGATTCCCGGGGTTTCCG (SEQ ID NO: 4) tRNA Pyl,UCA DNA sequence of (opal codon Methanosarcina mazei pyrrolysyl tRNA; anticodon is underlined) GGAAACCTGATCATGTAGATCGAATGGACT TCA AATCCGTTCAGCCGGGTTAGATTCCCGGGGTTTCCG (SEQ ID NO: 5) tRNA Pyl,UUA DNA sequence of (Methanosarcina mazei pyrrolysyl tRNA at ochre codon; anticodon is underlined) GGAAACCTGATCATGTAGATCGAATGGACT TTA AATCCGTTCAGCCGGGTTAGATTCCCGGGGTTTCCG (SEQ ID NO: 6)

[0350] 3. O-RS PylRS AF (Methanosarcina mazei pyrrolysyl-tRNA synthetase double mutant: Y306A, Y384F; Uniprot: Q8PWY1)

[0351] DNA:

[0352] protein: MACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO:8) PylRS AA (Methanosarcina mazei Pyrrolysyl-tRNA synthetase double mutant: N346A, C348A; Uniprot: Q8PWY1)

[0353] DNA:

[0354] protein: MACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLAFAQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 10) PylRS AAAF (Methanosarcina mazei Pyrrolidyl tRNA synthetase quadruple mutant: Y306A, N346A, C348A, Y384F; Uniprot: Q8PWY1)

[0355] DNA:

[0356] protein: ACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTR TKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESEL LSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLAFAQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 12)

[0357] 4. RNA-TP MCP (coat protein of enterobacterial phage MS2)

[0358] DNA: GCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCA TCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTAC (SEQ ID NO: 13)

[0359] protein: ASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 14)

[0360] λ N22 (22 amino acid RNA-binding domain of lambda phage antiterminator protein N) DNA: ATGGACGCACAAACACGACGACGTGAGCGTCGCGCTGAGAAACAAGCTCAATGGAAAGCTGCAAAC (SEQ ID NO: 15) protein: MDAQTRRRERRAEKQAQWKAAN (SEQ ID NO: 16)

[0361] 5. TN DNA sequence of the enterobacterial phage MS2 RNA stem-loop. ACATGAGGATCACCCATGT (SEQ ID NO: 17) The DNA sequence of Box B (lambda phage RNA stem loop, λN22 specific binding site of GCCCTGAAAAAGGGC (SEQ ID NO: 18)

[0362] 6. IC-TP KIF16B 1-400 (Homo sapiens kinesin family member 16B fragment covering amino acid residues 1-400; Uniprot:Q96L93)

[0363] DNA:

[0364] protein: MASVKVAVRVRPMNRREKDLEAKFIIQMEKSKTTITNLKIPEGGTGDSGRERTKTFTYDFSFYSADTKSPDYVSQEMVFKTLGTDVVKSAFEGYNACVFAYGQTGSGKSYTMMGNSGDSGLIPRICEGLFSRINETTRWDEASFRTEVSYLEIYNERVRDLLRRKSSKTFNLRVREHPKEGPYVEDLSKHLVQNYGDVEELMDAGNINRTTAATGMNDVSSRSHAIFTIKFTQAKFDSEMPCETVSKIHLVDLAGSERADATGATGVRLKEGGNINKSLVTLGNVISALADLSQDAANTLAKKKQVFVPYRDSVLTWLLKDSLGGNSKTIMIATISPADVNYGETLSTLRYANRAKNIINKPTINEDANVKLIRELRAEIARLKTLLAQGNQIALLDSPT (SEQ ID NO: 20)

[0365] KIF13A 1-411,ΔP390 (Homo sapiens kinesin family member 13A fragment covering amino acid residues 1-411, where P390 is deleted; Uniprot:Q9H1H9)

[0366] DNA:

[0367] protein: MSDTKVKVAVRVRPMNRRELELNTKCVVEMEGNQTVLHPPPSNTKQGERKPPKVFAFDYCFWSMDESNTTKYAGQEVVFKCLGEGILEKAFQGYNACIFAYGQTGSGKSFSMMGHAEQLGLIPRLCCALFKRISLEQNESQTFKVEVSYMEIYNEKVRDLLDPKGSRQSLKVREHKVLGPYVDGLSQLAVTSFEDIESLMSEGNKSRTVAATNMNEESSRSHAVFNIIITQTLYDLQSGNSGEKVSKVSLVDLAGSERVSKTGAAGERLKEGSNINKSLTTLGLVISSLADQAAGKGKSKFVPYRDSVLTWLLKDNLGGNSQTSMIATISPAADNYEETLSTLRYADRAKRIVNHAVVNEDPNAKVIRELREEVEKLREQLSQAEAMKAELKEKLEESEKLIKELTVTWE (SEQ ID NO: 22)

[0368] TOMM20 1-70 (Homo sapiens mitochondrial outer membrane translocase 20 fragment covering amino acid residues 1-70; Uniprot:Q15388) DNA: ATGGTGGGTCGGAACAGCGCCATCGCCGCCGGTGTATGCGGGGCCCTTTTCATTGGGTACTGCATCTACTTCGACCGCAAAAGACGAAGTGACCCCAACTTCAAGAACAGGCTTCGAGAACGAAGAAAGAAACAGAAGCTTGCCAAGGAGAGAGCTGGGCTTTCCAAGTTACCTGACCTTAAAGATGCTGAAGCTGTTCAGAAATTCTTC (SEQ ID NO: 23) protein: MVGRNSAIAAGVCGALFIGYCIYFDRKRRSDPNFKNRLRERRKKQKLAKERAGLSKLPDLKDAEAVQKFF (SEQ ID NO: 24)

[0369] LcK (post-translational modification site for plasma membrane targeting of Mus musculus lymphocyte-specific protein tyrosine kinase; Uniprot:P06240) DNA: GGCTGCGTGTGCAGCAGCAACCCCGAGGGTACCGAGCTC (SEQ ID NO: 25) protein: (The same part is underlined in P06240) GCVCSSNPE GTEL (SEQ ID NO: 26)

[0370] FRB-CD28 (a membrane-targeted synthetic fusion polypeptide derived from Mus musculus CD4 (Uniprot: P06332), FRB (similar to Homo sapiens mTOR; Uniprot: P42345), and Mus musculus CD28 (Uniprot: P31041)

[0371] DNA: ATGTGCCGAGCCATCTCTCTTAGGCGCTTGCTGCTGCTGCTGCTGCAGCTGTCACAACTCCTAGCTGTCACTCAAGGGATGCTCGAGATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAACATCCTTTAA TCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCTCCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCTCAAAGACTAGAACCGGTAAGCTTTTTGGGCACTGGTCGTGGTTGCTGGAGTCCTGTTTTGTTATGGCTTGCTAGTGACAGTGGCTCTTTGTGTT (SEQ ID NO: 27)

[0372] protein: MCRAISLRRLLLLLLQLSQLLAVTQGMLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTRTGKLFWALVVVAGVLFCYGLLVTVALCV (SEQ ID NO: 28)

[0373] FUS-CD28 (a membrane-targeted synthetic fusion polypeptide derived from Mus musculus CD4 (Uniprot: P06332), Homo sapiens sarcoma fusion (Uniprot: P35637), and Mus musculus CD28 (Uniprot: P31041)

[0374] DNA:

[0375] protein: MCRAISLRRLLLLLLQLSQLLAVTQGMLMASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGGRGRGGSGGGGGGGGGGYNRSSGGYEPRGRGGGRGGRGGMGGSDRGGFNKFGGPRDQGSRHDSEQDNSDNNTIFVQGLGENVTIESVADYFKQIGIIKTNKKTGQPMINLYTDRETGKLKGEATVSFDDPPSAKAAIDWFDGKEFSGNPIKVSFATRRADFNRGGGNGRGGRGRGGPMGRGGYGGGGSGGGGRGGFPSGGGGGGGQQRAGDWKCPNPTCENMNFSWRNECNQCKAPKPDGPGGGPGGSHMGGNYGDDRRGGRGGGTGKLFWALVVVAGVLFCYGLLVTVALCV (SEQ ID NO: 30)

[0376] 7. PSP SPD5 (Caenorhabditis elegans spindle defect protein 5; Uniprot: P91349)

[0377] DNA:

[0378] protein:

[0379] FUS (Homo sapiens sarcoma fusion; Uniprot:P35637)

[0380] DNA:

[0381] protein: MASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSS YGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGGRGRGGSGGGGGGGGGGYNRSSGGYEPRG RGGGRGGRGGMGGSDRGGFNKFGGPRDQGSRHDSEQDNSDNNTIFVQGLGENVTIESVADYFKQIGIIKTNKKTGQPMINLYTDRETGKLKGEATVSFDDPPSAKAAIDWFDGKEFSGNPIKVSFATRRADFNRGGGNGRGGRGRGGPMGRGGYGGGGSGGGGRGGFPSGGGGGGGQQRAGDWKCPNPTCENMNFSWRNECNQCKAPKPDGPGGGPGGSHMGGNYGDDRRGGRGG (SEQ ID NO: 34)

[0382] EWSR1 (Homo sapiens Ewing's sarcoma breakpoint region 1; Uniprot:Q01844)

[0383] DNA:

[0384] protein: MASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDVSYTQAQTTATYGQTAYATSYGQPPTGYTTPTAPQAYSQPVQGYGTGAYDTTTATVTTTQASYAAQSAYGTQPAYPAYGQQPAATAPTRPQDGNKPTETSQPQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSTQPTSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQAPSQYSQQSSSYGQQSSFRQDHPSSMGVYGQESGGFSGPGENRSMSGPDNRGRGRGGFDRGGMSRGGRGGGRGGMGSAGERGGFNKPGGPMDEGPDLDLGPPVDPDEDSDNSAIYVQGLNDSVTLDDLADFFKQCGVVKMNKRTGQPMIHIYLDKETGKPKGDATVSYEDPPTAKAAVEWFDGKDFQGSKLKVSLARKKPPMNSMRGGLPPREGRGMPPPLRGGPGGPGGPGGPMGRMGGRGGDRGGFPPRGPRGSRGNPSGGGNVQHRAGDWQCPNPGCGNQNFAWRTECNQCKAPKPEGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGPGGMFRGGRGGDRGGFRGGRGMDRGGFGGGRRGGPGGPPGPLMEQ(SEQ ID NO: 36)

[0385] 8. AFP EWSR1-MCP

[0386] DNA:

[0387] protein: MASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDVSYTQAQTTATYGQTAYATSYGQPPTGYTTPTAPQAYSQPVQGYGTGAYDTTTATVTTTQASYAAQSAYGTQPAYPAYGQQPAATAPTRPQDGNKPTETSQPQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSTQPTSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQAPSQYSQQSSSYGQQSSFRQDHPSSMGVYGQESGGFSGPGENRSMSGPDNRGRGRGGFDRGGMSRGGRGGGRGGMGSAGERGGFNKPGGPMDEGPDLDLGPPVDPDEDSDNSAIYVQGLNDSVTLDDLADFFKQCGVVKMNKRTGQPMIHIYLDKETGKPKGDATVSYEDPPTAKAAVEWFDGKDFQGSKLKVSLARKKPPMNSMRGGLPPREGRGMPPPLRGGPGGPGGPGGPMGRMGGRGGDRGGFPPRGPRGSRGNPSGGGNVQHRAGDWQCPNPGCGNQNFAWRTECNQCKAPKPEGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGPGGMFRGGRGGDRGGFRGGRGMDRGGFGGGRRGGPGGPPGPLMEQDYKDDDDKGTEQKLISEEDLGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY(SEQ ID NO: 38)

[0388] FUS-MCP

[0389] DNA:

[0390] protein: MASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGGRGRGGSGGGGGGGGGGYNRSSGGYEPRGRGGGRGGRGGMGGSDRGGFNKFGGPRDQGSRHDSEQDNSDNNTIFVQGLGENVTIESVADYFKQIGIIKTNKKTGQPMINLYTDRETGKLKGEATVSFDDPPSAKAAIDWFDGKEFSGNPIKVSFATRRADFNRGGGNGRGGRGRGGPMGRGGYGGGGSGGGGRGGFPSGGGGGGGQQRAGDWKCPNPTCENMNFSWRNECNQCKAPKPDGPGGGPGGSHMGGNYGDDRRGGRGGDYKDDDDKGTEQKLISEEDLGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY(SEQ ID NO:40)

[0391] FUS-PylRS AF

[0392] DNA:

[0393] protein:

[0394] MCP-PylRS AF

[0395] DNA:

[0396] protein: MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGTGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 44)

[0397] SPD5 - MCP

[0398] DNA:

[0399] protein:

[0400] SPD5-PylRS AF

[0401] DNA:

[0402] protein:

[0403] KIF16B-FUS-PylRS AF

[0404] DNA:

[0405] protein:

[0406] KIF16B-VSV-G-FUS-PylRS AF

[0407] DNA:

[0408] protein:

[0409] KIF16B-FUS-PylRS AA

[0410] DNA:

[0411] protein:

[0412] KIF16B-FUS-PylRS AAAF

[0413] DNA:

[0414] protein:

[0415] KIF16B-EWSR1-MCP

[0416] DNA:

[0417] protein:

[0418] KIF16B-FUS-4xλ N22 -PylRS AF

[0419] DNA:

[0420] protein:

[0421] KIF16B-FUS-MCP-PylRS AF

[0422] DNA:

[0423] protein:

[0424] KIF16B-PylRS AF

[0425] DNA:

[0426] protein: (SEQ ID NO: 64)

[0427] KIF16B-MCP

[0428] DNA:

[0429] protein: MASVKVAVRVRPMNRREKDLEAKFIIQMEKSKTTITNLKIPEGGTGDSGRERTKTFTYDFSFYSADTKSPDYVSQEMVFKTLGTDVVKSAFEGYNACVFAYGQTGSGKSYTMMGNSGDSGLIPRICEGLFSRINETTRWDEASFRTEVSYLEIYNERVRDLLRRKSSKTFNLRVREHPKEGPYVEDLSKHLVQNYGDVEELMDAGNINRTTAATGMNDVSSRSHAIFTIKFTQAKFDSEMPCETVSKIHLVDLAGSERADATGATGVRLKEGGNINKSLVTLGNVISALADLSQDAANTLAKKKQVFVPYRDSVLTWLLKDSLGGNSKTIMIATISPADVNYGETLSTLRYANRAKNIINKPTINEDANVKLIRELRAEIARLKTLLAQGNQIALLDSPTYTDIEMNRLGKGAPGSAGSAAGSGMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 66)

[0430] KIF16B - MCP - PylRS AF

[0431] DNA:

[0432] protein:

[0433] KIF16B-SPD5-PylRS AF

[0434] DNA:

[0435] protein:

[0436] KIF16B-SPD5-MCP

[0437] DNA: AATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAA (sequence number 71)

[0438] protein:

[0439] KIF16B-SPD5-MCP-PylRS AF

[0440] DNA:

[0441] protein:

[0442] KIF16B-SPD5-4xλ N22 -PylRS AF

[0443] DNA:

[0444] protein:

[0445] KIF13A-FUS-PylRS AF

[0446] DNA:

[0447] protein:

[0448] KIF13A-FUS-PylRS AA

[0449] DNA:

[0450] protein:

[0451] KIF13A-FUS-PylRS AAAF

[0452] DNA:

[0453] protein:

[0454] KIF13A-EWSR1-MCP

[0455] DNA:

[0456] protein:

[0457] KIF13A-SPD5-PylRS AF

[0458] DNA:

[0459] protein:

[0460] KIF13A-SPD5-MCP

[0461] DNA: ATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAA (sequence number 87)

[0462] protein:

[0463] KIF13A-FUS-MCP-PylRS AF

[0464] DNA:

[0465] protein:

[0466] KIF13A-PylRS AF

[0467] DNA:

[0468] protein: (query number 92)

[0469] KIF13A-MCP

[0470] DNA:

[0471] protein: MSDTKVKVAVRVRPMNRRELELNTKCVVEMEGNQTVLHPPPSNTKQGERKPPKVFAFDYCFWSMDESNTTKYAGQEVVFKCLGEGILEKAFQGYNACIFAYGQTGSGKSFSMMGHAEQLGLIPRLCCALFKRISLEQNESQTFKVEVSYMEIYNEKVRDLLDPKGSRQSLKVREHKVLGPYVDGLSQLAVTSFEDIESLMSEGNKSRTVAATNMNEESSRSHAVFNIIITQTLYDLQSGNSGEKVSKVSLVDLAGSERVSKTGAAGERLKEGSNINKSLTTLGLVISSLADQAAGKGKSKFVPYRDSVLTWLLKDNLGGNSQTSMIATISPAADNYEETLSTLRYADRAKRIVNHAVVNEDPNAKVIRELREEVEKLREQLSQAEAMKAELKEKLEESEKLIKELTVTWEYTDIEMNRLGKGAPGSAGSAAGSGMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 94)

[0472] TOMM20-EWSR1-MCP

[0473] DNA:

[0474] protein: MVGRNSAIAAGVCGALFIGYCIYFDRKRRSDPNFKNRLRERRKKQKLAKERAGLSKLPDLKDAEAVQKFFMASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDVSYTQAQTTATYGQTAYATSYGQPPTGYTTPTAPQAYSQPVQGYGTGAYDTTTATVTTTQASYAAQSAYGTQPAYPAYGQQPAATAPTRPQDGNKPTETSQPQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSTQPTSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQAPSQYSQQSSSYGQQSSFRQDHPSSMGVYGQESGGFSGPGENRSMSGPDNRGRGRGGFDRGGMSRGGRGGGRGGMGSAGERGGFNKPGGPMDEGPDLDLGPPVDPDEDSDNSAIYVQGLNDSVTLDDLADFFKQCGVVKMNKRTGQPMIHIYLDKETGKPKGDATVSYEDPPTAKAAVEWFDGKDFQGSKLKVSLARKKPPMNSMRGGLPPREGRGMPPPLRGGPGGPGGPGGPMGRMGGRGGDRGGFPPRGPRGSRGNPSGGGNVQHRAGDWQCPNPGCGNQNFAWRTECNQCKAPKPEGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGPGGMFRGGRGGDRGGFRGGRGMDRGGFGGGRRGGPGGPPGPLMEQDYKDDDDKGTEQKLISEEDLGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY(SEQ ID NO: 96)

[0475] TOMM20-EWSR1-HA-MCP

[0476] DNA:

[0477] protein: MVGRNSAIAAGVCGALFIGYCIYFDRKRRSDPNFKNRLRERRKKQKLAKERAGLSKLPDLKDAEAVQKFFMASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDVSYTQAQTTATYGQTAYATSYGQPPTGYTTPTAPQAYSQPVQGYGTGAYDTTTATVTTTQASYAAQSAYGTQPAYPAYGQQPAATAPTRPQDGNKPTETSQPQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSTQPTSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQAPSQYSQQSSSYGQQSSFRQDHPSSMGVYGQESGGFSGPGENRSMSGPDNRGRGRGGFDRGGMSRGGRGGGRGGMGSAGERGGFNKPGGPMDEGPDLDLGPPVDPDEDSDNSAIYVQGLNDSVTLDDLADFFKQCGVVKMNKRTGQPMIHIYLDKETGKPKGDATVSYEDPPTAKAAVEWFDGKDFQGSKLKVSLARKKPPMNSMRGGLPPREGRGMPPPLRGGPGGPGGPGGPMGRMGGRGGDRGGFPPRGPRGSRGNPSGGGNVQHRAGDWQCPNPGCGNQNFAWRTECNQCKAPKPEGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGPGGMFRGGRGGDRGGFRGGRGMDRGGFGGGRRGGPGGPPGPLMEQAIAYPYDVPDYAGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY(SEQ ID NO: 98)

[0478] TOMM20-FUS-PylRS AF

[0479] DNA:

[0480] protein:

[0481] TOMM20-FUS-V5-PylRS AF

[0482] DNA:

[0483] protein:

[0484] TOMM20-FUS-PylRS AA

[0485] DNA:

[0486] protein:

[0487] TOMM20-FUS-V5-PylRS AA

[0488] DNA:

[0489] protein:

[0490] TOMM20-FUS-PylRS AAAF

[0491] DNA:

[0492] protein:

[0493] TOMM20-FUS-V5-PylRS AAAF

[0494] DNA:

[0495] protein:

[0496] TOMM20-EWSR1-L N22

[0497] DNA:

[0498] protein: (SEQ ID NO: 112)

[0499] TOMM20-EWSR1-Myc-λ N22

[0500] DNA:

[0501] protein: (SEQ ID NO: 114)

[0502] TOMM20-3xMCP-PylRS AF

[0503] DNA:

[0504] protein: (query number 116)

[0505] TOMM20-FUS-3xMCP-PylRS AF

[0506] DNA:

[0507] protein:

[0508] TOMM20-FUS-3xMCP-PylRS AAAF

[0509] DNA:

[0510] protein:

[0511] TOMM20-FUS-4xλ N22 -PylRS AF

[0512] DNA:

[0513] protein:

[0514] TOMM20-FUS-4xλ N22 -PylRS AAAF

[0515] DNA:

[0516] protein:

[0517] LcK-EWSR1-MCP

[0518] DNA:

[0519] protein: (SEQ ID NO: 126)

[0520] LcK-EWSR1-4xλ N22

[0521] DNA:

[0522] protein: MGCVCSSNPEGTELASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDVSYTQAQTTATYGQTAYATSYGQPPTGYTTPTAPQAYSQPVQGYGTGAYDTTTATVTTTQASYAAQSAYGTQPAYPAYGQQPAATAPTRPQDGNKPTETSQPQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSTQPTSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQAPSQYSQQSSSYGQQSSFRQDHPSSMGVYGQESGGFSGPGENRSMSGPDNRGRGRGGFDRGGMSRGGRGGGRGGMGSAGERGGFNKPGGPMDEGPDLDLGPPVDPDEDSDNSAIYVQGLNDSVTLDDLADFFKQCGVVKMNKRTGQPMIHIYLDKETGKPKGDATVSYEDPPTAKAAVEWFDGKDFQGSKLKVSLARKKPPMNSMRGGLPPREGRGMPPPLRGGPGGPGGPGGPMGRMGGRGGDRGGFPPRGPRGSRGNPSGGGNVQHRAGDWQCPNPGCGNQNFAWRTECNQCKAPKPEGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGPGGMFRGGRGGDRGGFRGGRGMDRGGFGGGRRGGPGGPPGPLMEQDYKDDDDKGTEQKLISEEDLLATMDAQTRRRERRAEKQAQWKAANPPLDGAGAGAGAGAGAGGLATMDAQTRRRERRAEKQAQWKAANPPLDGAGAGAGAGAGAGGLATMDAQTRRRERRAEKQAQWKAANPPLDGAGAGAGAGAGAGGLATMDAQTRRRERRAEKQAQWKAANPPLESRGPVKPADQPRLCLLVASHLLFAPPPCLP(SEQ ID NO: 128)

[0523] LcK-FUS-PylRS AF

[0524] DNA:

[0525] protein: (SEQ ID NO: 130)

[0526] LcK-FUS-MCP-PylRS AF

[0527] DNA:

[0528] protein:

[0529] LCK-FUS-3xMCP-PylRS AF

[0530] DNA:

[0531] protein:

[0532] LcK-FUS-PylRS AAAF

[0533] DNA:

[0534] protein: (SEQ ID NO: 136)

[0535] LcK-PylRS AF

[0536] DNA:

[0537] protein: MGCVCSSNPEGTELACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSTARARRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPF RELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL(SEQ ID NO:138)

[0538] LcK-PylRS AAAF

[0539] DNA:

[0540] protein: MGCVCSSNPEGTELACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSTARARRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPF RELESELLSRRKKDLQQIYAEERENYLGKLEREITRFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLAFAQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL(SEQ ID NO:140)

[0541] LcK-MCP

[0542] DNA: ATGGGCTGCGTGTGCAGCAGCAACCCCGAGGGTACCGAGCTCGAGCAGAAGCTGATCTCAGAGGAGGACCTGGGCGCCCCCGGCTCCGCCGGCTCCGCCGCCGGCTCCGGCGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAA(SEQ ID NO: 141)

[0543] protein: MGCVCSSNPEGTELEQKLISEEDLGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY(SEQ ID NO: 142)

[0544] FRB-CD28-FUS-PylRS AF

[0545] DNA:

[0546] protein:

[0547] FRB-CD28-FUS-PylRS AA

[0548] DNA:

[0549] protein:

[0550] FRB-CD28-EWSR1-MCP

[0551] DNA:

[0552] protein: (SEQ ID NO: 148)

[0553] FRB-CD28-EWSR1-4xλN22

[0554] DNA:

[0555] protein: (SEQ ID NO: 150)

[0556] FUS-CD28-FUS-PylRS AF

[0557] DNA:

[0558] protein:

[0559] FUS-CD28-FUS-PylRS AA

[0560] DNA:

[0561] protein:

[0562] FUS-CD28-FUS-MCP-PylRS AF

[0563] DNA:

[0564] protein:

[0565] FUS-CD28-FUS-MCP-PylRS AA

[0566] DNA:

[0567] protein:

[0568] FUS-CD28-EWSR1-MCP

[0569] DNA:

[0570] protein:

[0571] FUS-CD28-EWSR1-4xλ N22

[0572] DNA:

[0573] protein:

[0574] FRB-CD28-FUS-MCP-PylRS AA

[0575] DNA:

[0576] protein:

[0577] FRB-CD28-FUS-MCP-PylRS AF

[0578] DNA:

[0579] protein:

[0580] FUS-MCP-PylRS AF

[0581] DNA:

[0582] protein:

[0583] SPD5-MCP-PylRS AF

[0584] DNA: CATCTGGAGGAGTTTACCATGCTGAACTTTTGCCAAATGGGTTCAGTTGTACTCGTGAGAACCTGGAAAGCATCATCACCGATTTTCTGAACCACCTGGGCATTGACTTCAAAATTGTGGCGACAGCTGTATGGTGTTTGGCGACACCCTGGATGTCATGCACGGCGACCTGGAACTGTCTAGTGCCGTTGGGCCCAATCCCGCTGGATCGTGATGGGTATCGACAAACCTTGGATCGGTGCGGGTTTTGGTCTGGACGTCTGCTGAAAGTAAAACACGACTTCAAGAACATCAAACGTGCTGCAGTTCCGAGTCCTATTACAATGGTATTTCACTAACCGTTAA (sequence number 169)

[0585] protein:

[0586] 9. POI (reporter) and control GFP 39TAG (GFP with amino acid position 39 encoded by amber)

[0587] DNA: (Amber codons are underlined) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGATTACAAAGATGATGATGATAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACC TAGGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAA(SEQ ID NO: 171)

[0588] protein: ( X indicates a non-classical amino acid) MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT XGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 172)

[0589] GFP 39TAG -2xMS2 (GFP with two MS2 stem loops) 39TAG )

[0590] DNA: (The MS2 stem-loop and amber codon are underlined.) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCAGCGATTACAAAGATGATGATGATAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACC TAGGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGGATCCTAAGGTACCTAATTGCCTAGAAA ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 173)

[0591] protein: ( X indicates a non-classical amino acid) MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT XGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 174)

[0592] mOrange

[0593] DNA: ATGGTGAGCAAGGGCGAGGAGAATAATATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCACCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCTTTCAGACCGCTAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCTCTTCACCTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTCAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTACGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCACTGCAGGACGGCGAGTTCATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAGGATGAGGCTGAAGCTGAAGGACGGCGGCCACTACACCTCCGAGGTCAAGACCACCTACAAGGCCAAGAAGTCCGTGCAGCTGCCCGGCGCCTACATCGTCGGCATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA(SEQ ID NO: 175)

[0594] protein: MVSKGEENNMAIIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGFQTAKLKVTKGGPLPFAWDILSPLFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNYEDGGVVTVTQDSSLQDGEFIYKVKMRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIRMRLKLKDGGHYTSEVKTTYKAKKSVQLPGAYIVGIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK (SEQ ID NO: 176)

[0595] iRFP (near-infrared fluorescent protein)

[0596] DNA: GAAGGATCCGTCGCCAGGCAGCCTGACCTCTTGACCTGCGACGATGAGCCGATCCATATCCCCGGTGCCATCCAACCGCATGGACTGCTGCTCGCCCTCGCCGCCGACATGACGATCGTTGCCGGCAGCGACAACCTTCCCGAACTCACCGGACTGGCGATCGGCGCCCTGATCGGCCGCTCTGCGGCCGATGTCTTCGACTCGGAGACGCACAACCGTCTGACGATCGCCTTGGCCGAGCCCGGGGCGGCCGTCGGAGCACCGATCACTGTCGGCTTCACGATGCGAAAGGACGCAGGCTTCATCGGCTCCTGGCATCGCCATGATCAGCTCATCTTCCTCGAGCTCGAGCCTCCCCAGCGGGACGTCGCCGAGCCGCAGGCGTTCTTCCGCCGCACCAACAGCGCCATCCGCCGCCTGCAGGCCGCCGAAACCTTGGAAAGCGCCTGCGCCGCCGCGGCGCAAGAGGTGCGGAAGATTACCGGCTTCGATCGGGTGATGATCTATCGCTTCGCCTCCGACTTCAGCGGCGAAGTGATCGCAGAGGATCGGTGCGCCGAGGTCGAGTCAAAACTAGGCCTGCACTATCCTGCCTCAACCGTGCCGGCGCAGGCCCGTCGGCTCTATACCATCAACCCGGTACGGATCATTCCCGATATCAATTATCGGCCGGTGCCGGTCACCCCAGACCTCAATCCGGTCACCGGGCGGCCGATTGATCTTAGCTTCGCCATCCTGCGCAGCGTCTCGCCCGTCCATCTGGAATTCATGCGCAACATAGGCATGCACGGCACGATGTCGATCTCGATTTTGCGCGGCGAGCGACTGTGGGGATTGATCGTTTGCCATCACCGAACGCCGTACTACGTCGATCTCGATGGCCGCCAAGCCTGCGAGCTAGTCGCCCAGGTTCTGGCCTGGCAGATCGGCGTGATGGAAGAG(SEQ ID NO: 177)

[0597] protein: EGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE (SEQ ID NO: 178)

[0598] mCherry 185TAG (mCherry with amino acid position 185 encoded by amber)

[0599] DNA: (Amber codons are underlined) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAG TAG CCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCATCATCATCATCATCATTAA(SEQ ID NO: 179)

[0600] protein: ( X is a non-classical amino acid) MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIP DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK X PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 180)

[0601] mCherry 185TAG -2xMS2 (mCherry with two MS2 RNA stem loops) 185TAG )

[0602] DNA: (The MS2 stem-loop and amber codon are underlined.) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAG TAG CCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCATCATCATCATCATCATTAAAGATCCTAAGGTACCTAATTGCCTAGAAA ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 181)

[0603] protein: ( X indicates a non-classical amino acid) MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIP DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK X PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 182)

[0604] mCherry 185TAG -4xBoxB (mCherry with four BoxB loops) 185TAG )

[0605] DNA: (BoxB stem-loop and amber codon are underlined) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAG TAG CCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCATCATCATCATCATCATTAAAGATCCTAAGGTACC GCCCTGAAAAAGGGC TCGA GCCCTGAAAAAGGGC AATT GCCCTGAAAAAGGGC GTCGAC GCCCTGAAAAAGGGC (SEQ ID NO: 183)

[0606] protein: ( X indicates a non-classical amino acid) MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIP DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK X PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 184)

[0607] GFP 39TAA -2xMS2 (GFP with amino acid position 39 encoded by ochre and two MS2 RNA stem loops)

[0608] DNA: (The MS2 stem-loop and ochre codon are underlined.) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCAGCGATTACAAAGATGATGATGATAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACC TAAGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTGAGGATCCTAAGGTACCTAATTGCCTAGAAA ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 185)

[0609] protein: ( X indicates a non-classical amino acid) MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT XGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 186)

[0610] mCherry 185TAA -2xMS2 (mCherry with amino acid position 185 encoded by ochre and two MS2 RNA stem loops)

[0611] DNA: (The MS2 stem-loop and ochre codon are underlined.) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAG TAA CCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCATCATCATCATCATCATTGAAGATCCTAAGGTACCTAATTGCCTAGAAA ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 187)

[0612] protein: ( X indicates a non-classical amino acid) MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIP DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK X PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 188)

[0613] GFP 39TGA -2xMS2 (GFP with amino acid position 39 encoded by opal and two MS2 RNA stem loops)

[0614] DNA: (The MS2 stem-loop and opal codon are underlined.) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCAGCGATTACAAAGATGATGATGATAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACC TGAGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGGATCCTAAGGTACCTAATTGCCTAGAAA ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 189)

[0615] protein: ( X indicates a non-classical amino acid) MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT XGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 190)

[0616] mCherry 185TGA -2xMS2 (mCherry with amino acid position 185 encoded by opal and two MS2 RNA stem-loops)

[0617] DNA: (The MS2 stem-loop and opal codon are underlined.) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAG TGA CCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCATCATCATCATCATCATTAAAGATCCTAAGGTACCTAATTGCCTAGAAA ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 191)

[0618] protein: ( X indicates a non-classical amino acid) MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIP DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK X PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 192)

[0619] Nup153 (Homo sapiens nucleoporin 153; Uniprot:P49790)

[0620] DNA:

[0621] protein:

[0622] Vim 116TAG (Homo sapiens vimentin with amino acid position 116 encoded by amber; Uniprot:P08670)

[0623] DNA: (amber codons are underlined) ATGTCCACCAGGTCCGTGTCCTCGTCCTCCTACCGCAGGATGTTCGGCGGCCCGGGCACCGCGAGCCGGCCGAGCTCCAGCCGGAGCTACGTGACTACGTCCACCCGCACCTACAGCCTGGGCAGCGCGCTGCGCCCCAGCACCAGCCGCAGCCTCTACGCCTCGTCCCCGG GCGGCGTGTATGCCACGCGCTCCTCTGCCGTGCGCCTGCGGAGCAGCGTGCCCGGGGTGCGGCTCCTGCAGGACTCGGTGGACTTCTCGCTGGCCGACGCCATCAACACCGAGTTCAAGAACACCCGCACCAACGAGAAGGTGGAGCTGCAGGAGCTGAATGACCGCTTCGCC TAG

[0624] protein: ( X indicates a non-classical amino acid) MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFA X YIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLE (SEQ ID NO: 196)

[0625] INSR 676TAG (Homo sapiens insulin receptor; Uniprot:P06213)

[0626] DNA: (Amber codons are underlined) TAG

[0627] protein: ( X indicates a non-classical amino acid) MGTGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVE DNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPK VCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIR TSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCL X(SEQ ID NO: 198)

[0628] Nup153-EGFP 149TAG

[0629] DNA: (Amber codons are underlined) TAG GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTT CGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO: 199)

[0630] protein: ( X indicates a non-classical amino acid) X VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 200)

[0631] Nup153-EGFP 149TAG -MS2

[0632] DNA: (The MS2 stem-loop and amber codon are underlined.) TAG GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTT CGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGACTCTAGATCATAATCAGC ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 201)

[0633] protein: ( X indicates a non-classical amino acid) X VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 202)

[0634] Vim 116TAG -mOrange

[0635] DNA: (Amber codons are underlined) ATGTCCACCAGGTCCGTGTCCTCGTCCTCCTACCGCAGGATGTTCGGCGGCCCGGGCACCGCGAGCCGGCCGAGCTCCAGCCGGAGCTACGTGACTACGTCCACCCGCACCTACAGCCTGGGCAGCGCGCTGCGCCCCAGCACCAGCCGCAGCCTCTACGCCTCGTCCCCGG GCGGCGTGTATGCCACGCGCTCCTCTGCCGTGCGCCTGCGGAGCAGCGTGCCCGGGGTGCGGCTCCTGCAGGACTCGGTGGACTTCTCGCTGGCCGACGCCATCAACACCGAGTTCAAGAACACCCGCACCAACGAGAAGGTGGAGCTGCAGGAGCTGAATGACCGCTTCGCC TAG

[0636] protein: ( X indicates a non-classical amino acid) MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFA X (SEQ ID NO: 204)

[0637] Vim 116TAG -mOrange-MS2

[0638] DNA: (The MS2 stem-loop and amber codon are underlined.) ATGTCCACCAGGTCCGTGTCCTCGTCCTCCTACCGCAGGATGTTCGGCGGCCCGGGCACCGCGAGCCGGCCGAGCTCCAGCCGGAGCTACGTGACTACGTCCACCCGCACCTACAGCCTGGGCAGCGCGCTGCGCCCCAGCACCAGCCGCAGCCTCTACGCCTCGTCCCCGGGCGGCGTGTATGCCACGCGCTCCTCTGCCGTGCGCCTGCGGAGCAGCGTGCCCGGGGTGCGGCTCCTGCAGGACTCGGTGGACTTCTCGCTGGCCGACGCCATCAACACCGAGTTCAAGAACACCCGCACCAACGAGAAGGTGGAGCTGCAGGAGCTGAATGACCGCTTCGCC TAG ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 205)

[0639] protein: ( X indicates a non-classical amino acid) MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFA X (SEQ ID NO: 206)

[0640] INSR 676TAG -EGFP

[0641] DNA:(Amber codons are underlined) TAG

[0642] protein: ( X indicates a non-classical amino acid) MGTGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVE DNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPK VCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIR TSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCL X(SEQ ID NO: 208)

[0643] INSR 676TAG -EGFP-MS2

[0644] DNA: (The MS2 stem-loop and amber codon are underlined.) TAG ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 209)

[0645] protein: ( X indicates a non-classical amino acid) MGTGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVE DNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPK VCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIR TSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCL X(SEQ ID NO: 210)

[0646] INSR 676TAG -mOrange-MS2

[0647] DNA: (The MS2 stem-loop and amber codon are underlined.) TAG ACATGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 211)

[0648] protein: ( X indicates a non-classical amino acid) MGTGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVE DNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPK VCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIR TSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCL X(SEQ ID NO: 212)

[0649] INSR 676TAG-iRFP-MS2

[0650] DNA: (The MS2 stem-loop and amber codon are underlined.) TAG ACA TGAGGATCACCCATGT CTGCAGGTCGACTCTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 213)

[0651] protein: ( X indicates a non-classical amino acid) MGTGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVE DNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPK VCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIR TSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCL X

[0652] Array-Set 2 1. Further components: mCherry 190TAG -2xPP7 mCherry with an amber site and two PP7 loops (TAG codon, PP7 Loop )

[0653] DNA: ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGTGAGCAAGGGCGAGGAGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCTACGAGGGCACCCAGACCGC CAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCCGGTGATCGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCT CCCTGCAGGACGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCACTTCCCCTCCGACGGCCCCGTAATGCAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCCAAGACC ACCTACAAGGCCAAGTAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCATCATCATCATCATTAAAGATCCTAAGGTACAATTGCCTAGAAA GGAGCAGACGATATGGCGTCGCTCC CTGCAGGTCGACTCTAGAAA CCAGCAGAGCATATGGGCTCGCTGG( sequence 215)

[0654] protein: MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKXPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH* (SEQ ID NO: 216)

[0655] mCherry 190TAG -4xPP7 mCherry with amber site and four PP7 loops (TAG codon, PP7 Loop )

[0656] DNA: ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGTGAGCAAGGGCGAGGAGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCTACGAGGGCACCCAGACCGC CAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCCGGTGATCGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCT CCCTGCAGGACGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCACTTCCCCTCCGACGGCCCCGTAATGCAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCCAAGACC ACCTACAAGGCCAAGTAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCATCATCATCATCATTAAAGATCCTAAGGTACAATTGCCTAGAAA GGAGCAGACGATATGGCGTCGCTCC CTGCAGGTCGACTCTAGAAA CCAGCAGAGCATATGGGCTCGCTGG CTGGGTACAATTGCCTAGAAA GGAGCAGACGATATGGCGTCGCTCC CTGCAGGTCGACTCTAGAAA CCAGCAGAGCATATGGGCTCGCTGG( sequence number 217)

[0657] protein: MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKXPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH* (SEQ ID NO: 218) mCherry 190TAG -6xPP7 contains an amber region and six PP7 loops

[0658] mCherry (TAG codon, PP7 loop)

[0659] DNA: ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGTAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCATCATCATCATCATCATTAAAGATCCTAAGGTACAATTGCCTAGAAA GGAGCAGACGATATGGCGTCGCTCC CTGCAGGTCGACTCTAGAAA CCAGCAGAGCATATGGGCTCGCTGG CTGGGTACAATTGCCTAGAAA GGAGCAGACGATATGGCGTCGCTC CCTGCAGGTCGACTCTAGAAA CCAGCAGAGCATATGGGCTCGCTG GCTGGGTACAATTGCCTAGAAA GGAGCAGACGATATGGCGTCGCTCC CTGCAGGTCGACTCTAGAAA CCAGCAGAGCATATGGGCTCGCTGG(SEQ ID NO: 219)

[0660] protein: MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKXPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH* (SEQ ID NO: 220)

[0661] H2B-mCherry 190TAG -2xMS2 Human histone H2B 1-J type (Uniprot:P06899) ​​fused to mCherry with an amber site and a 2xms2 loop (TAG codon, ms2 loop )

[0662] DNA: ACATGAGGATCACCCATGT CTGCACCTCGACACTAGAAA ACATGAGGATCACCCATGT (SEQ ID NO: 221)

[0663] protein: MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAITKYTSAKDPPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKXPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH* (SEQ ID NO: 222)

[0664] IFRS1 Methanosarcina mazei PylRS(L305M,Y306L,L309S,N346S,C348M)

[0665] DNA:

[0666] protein: DKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQ QIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNMLNYSRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLSFMQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO:224)

[0667] CbzRS Methanosarcina mazei PylRS(Y306M,L309G,C348T)

[0668] DNA:

[0669] protein: DKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQ QIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLMNYGRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFTQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLESSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO:226)

[0670] CpkRS Methanosarcina mazei PylRS(A302S)

[0671] DNA:

[0672] protein: DKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAP KPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQ QIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLSPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL* (SEQ ID NO: 228)

[0673] tRNA pyl,CGA Pyrrolidinyl-tRNA (serine codon, anticodon in bold) (Methanosarcina mazei)

[0674] [ka]

[0675] tRNA pyl,CGG Pyrrolysyl-tRNA (proline codon, anticodon in bold) (Methanosarcina mazei)

[0676] [ka]

[0677] tRNA pyl,UAAPyrrolysyl-tRNA (leucine codon, anticodon in bold) (Methanosarcina mazei)

[0678] [ka]

[0679] tRNA pyl,UAG Pyrrolysyl-tRNA (leucine codon, anticodon in bold) (Methanosarcina mazei)

[0680] [ka]

[0681] tRNA pyl,CCG Pyrrolysyl-tRNA (arginine codon, anticodon in bold) (Methanosarcina mazei)

[0682] [ka]

[0683] tRNA pyl,AUA Pyrrolysyl tRNA (isoleucine codon, anticodon is purple) (Methanosarcina mazei)

[0684] [ka]

[0685] OMeRS pyrrolysyl-tRNA synthetase mutants: A302T, Y384F, N346V, C348W, V401L (Methanosarcina mazei)

[0686] DNA:

[0687] protein: MACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTR TKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESEL LSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLTPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLVFWQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSALVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 236)

[0688] GFP 66TAG GFP with amber sites

[0689] DNA: (MS2 stem loop, Amber Codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGG ACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC TAGGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGCGGCCGCATCGATTATAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT(SEQ ID NO: 237)

[0690] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTXGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 238)

[0691] GFP 66TCG GFP with serine site

[0692] DNA: (MS2 stem loop, serine codon) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGG ACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC TCGGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGCGGCCGCATCGATTATAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT(SEQ ID NO: 239)

[0693] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTXGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 240)

[0694] GFP 66CCG GFP with proline site

[0695] DNA: (MS2 stem loop, Proline codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGG ACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC CCGGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGCGGCCGCATCGATTATAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 241)

[0696] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTXGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 242)

[0697] GFP 66CTA GFP with leucine moiety

[0698] DNA: (MS2 stem loop, leucine codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGG ACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC CTAGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGCGGCCGCATCGATTATAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT(SEQ ID NO: 243)

[0699] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTXGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 244)

[0700] GFP 66TTA GFP with leucine moiety

[0701] DNA: (MS2 stem loop, leucine codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGG ACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC TTAGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGCGGCCGCATCGATTATAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 245)

[0702] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTXGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 246)

[0703] GFP 66ATA GFP with isoleucine moiety

[0704] DNA: (MS2 stem loop, Isoleucine codon ) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGATTACAAAGATGATGATGATAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACC ATAGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGCGGCCGCATCGATTATAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 247)

[0705] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTXGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 248)

[0706] GFP 66CGG GFP with arginine moieties

[0707] DNA: (MS2 stem loop, Arginine codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGG ACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC CGGGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAAAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 249)

[0708] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTXGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 250)

[0709] GFP 39TCG GFP with serine site

[0710] DNA: (MS2 stem loop, serine codon) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC TCGGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCATAAGGATCCTAAGGATCCTAATTGCCTAGAAAACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 251)

[0711] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHHK (SEQ ID NO: 252)

[0712] GFP 39CCG GFP with proline site

[0713] DNA: (MS2 stem loop, Proline codon ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC CCGGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAAAGATCCTAAGGATCCTAATTGCCTAGAAAACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 253)

[0714] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHHK (SEQ ID NO: 254)

[0715] GFP 39CTA GFP with leucine moiety

[0716] DNA: (MS2 stem loop, leucine codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC CTAGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAAAGATCCTAAGGATCCTAATTGCCTAGAAAACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 255)

[0717] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHHK (SEQ ID NO: 256)

[0718] GFP 39CGG GFP with arginine moieties

[0719] DNA: (MS2 stem loop, Arginine codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC CGGGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAAAGATCCTAAGGATCCTAATTGCCTAGAAAACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCCATGTC (SEQ ID NO: 257)

[0720] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHHK (SEQ ID NO: 258)

[0721] mCherry 72TAG mCherry with amber sites

[0722] DNA: (MS2 stem loop, Amber Codon ) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGATTACAAAGATGATGATGATAAAGTGGCGTACGCGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAG GTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATG TAG(SEQ ID NO: 259)

[0723] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVAYAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMXGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 260)

[0724] mCherry 72TCG mCherry with serine site

[0725] DNA: (MS2 stem loop, Serine codon ) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGATTACAAAGATGATGATGATAAAGTGGCGTACGCGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAG GTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATG TCG(SEQ ID NO: 261)

[0726] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVAYAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMXGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 262)

[0727] mCherry 72CCG mCherry with a proline site

[0728] DNA: (MS2 stem loop, Proline codon ) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGATTACAAAGATGATGATGATAAAGTGGCGTACGCGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAG GTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATG CCG(SEQ ID NO: 263)

[0729] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVAYAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMXGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 264)

[0730] mCherry 72CTA mCherry with leucine site

[0731] DNA: (MS2 stem loop, leucine codon ) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGATTACAAAGATGATGATGATAAAGTGGCGTACGCGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAG GTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATG CTA(SEQ ID NO: 265)

[0732] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVAYAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMXGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 266)

[0733] mCherry 72TTA mCherry with leucine site

[0734] DNA: (MS2 stem loop, leucine codon ) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGATTACAAAGATGATGATGATAAAGTGGCGTACGCGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAG GTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATG TTA(SEQ ID NO: 267)

[0735] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVAYAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMXGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 268)

[0736] mCherry 72ATA mCherry with an isoleucine site

[0737] DNA: (MS2 stem loop, Isoleucine codon ) ATGGGCCGCCTGGAAAGCACCCCGCCGAAAAAAAAACGCAAAGTGGAAGATAGCGCGAGCGATTACAAAGATGATGATGATAAAGTGGCGTACGCGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAG GTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATG ATA(SEQ ID NO: 269)

[0738] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASDYKDDDDKVAYAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMXGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 270)

[0739] mCherry 185TCG mCherry having a serine site

[0740] DNA: ( serine codon) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCAGCGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGAGCCCCCAGTTCATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACAGCAGCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCAGCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCAGCAGCGAGCGCATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGCGCCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTGAAGACCACCTACAAGGCCAAG TCG CCCGTGCAGCTGCCCGGCGCCTACAACGTGAACATCAAGCTGGACATCACCAGCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCGCCGAGGGCCGCCACAGCACCGGCGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAA (SEQ ID NO: 271)

[0741] protein:X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKXPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 272)

[0742] mCherry 185CCG mCherry with a proline site

[0743] DNA: ( Proline codon) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCAGCGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGAGCCCCCAGTTCATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACAGCAGCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCAGCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCAGCAGCGAGCGCATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGCGCCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTGAAGACCACCTACAAGGCCAAG CCG CCCGTGCAGCTGCCCGGCGCCTACAACGTGAACATCAAGCTGGACATCACCAGCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCGCCGAGGGCCGCCACAGCACCGGCGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAA(SEQ ID NO: 273)

[0744] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKXPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 274) mCherry 185CTA mCherry with leucine site

[0745] DNA: ( leucine codon) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCAGCGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGAGCCCCCAGTTCATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACAGCAGCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCAGCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCAGCAGCGAGCGCATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGCGCCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTGAAGACCACCTACAAGGCCAAG CTA CCCGTGCAGCTGCCCGGCGCCTACAACGTGAACATCAAGCTGGACATCACCAGCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCGCCGAGGGCCGCCACAGCACCGGCGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAA(SEQ ID NO: 275)

[0746] protein: X indicates a non-classical amino acid MGRLESTPPKKKRKVEDSASVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKXPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHHHHHH (SEQ ID NO: 276)

[0747] GFP 39TCG LCK-GFP with serine site

[0748] DNA :(MS2 stem loop, Serine codons are red , LCK is blue) ATGGGCTGTGTGCAGCAGCAACCCCGAGGGTACCGAGCTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC TCGGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAAAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 277)

[0749] protein: X indicates a non-classical amino acid MGCVCSSNPEGTELVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 278)

[0750] GFP 39CCG LCK-GFP with a proline site

[0751] DNA: (MS2 stem loop, Proline codon , LCK ) ATGGGCTGCGTGTGCAGCAGCAACCCCGAGGGTACCGAGCTC GTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC CCGGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAAAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 279)

[0752] protein: X indicates a non-classical amino acid MGCVCSSNPEGTELVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 280)

[0753] GFP 39CTA LCK-GFP with leucine moiety

[0754] DNA:( MS2 stem loop, leucine codon , LCK ) ATGGGCTGCGTGTGCAGCAGCAACCCCGAGGGTACCGAGCTC GTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC CTAGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAAAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT(SEQ ID NO: 281)

[0755] protein: X indicates a non-classical amino acid MGCVCSSNPEGTELVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH (SEQ ID NO: 282)

[0756] Extended GFP 39TCG GFP 66CCG GFP with a serine site at position 39 genetically fused to

[0757] DNA:( MS2 stem loop, Serine codon , Proline codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC TCGGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGGGAGCACCAGGAAGTGCTGGTTCTGCTGCTGGTAGTGGAGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC CCGGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCACCACCACCACCACCACTAAAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT (SEQ ID NO: 283)

[0758] protein: X indicates a non-classical amino acid (SEQ ID NO: 284)

[0759] Extended GFP 39CCG GFP 66TCG GFP with a proline site at position 39 genetically fused to

[0760] DNA: (MS2 stem loop, Proline codon, serine codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC CCGGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGGGAGCACCAGGAAGTGCTGGTTCTGCTGCTGGTAGTGGAGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC TCGGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGCGGCCGCATCGATTATAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT(SEQ ID NO: 285)

[0761] protein: X indicates a non-classical amino acid (SEQ ID NO: 286)

[0762] Extended GFP 39CTA GFP 66TCG GFP with a leucine moiety at position 39 genetically fused to

[0763] DNA:( MS2 stem loop, leucine codon , Serine codon ) ATGGGCCGCCTGGAGAGCACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACAGCGCCACGACTACAAGGACGACGACGACAAGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACC CTAGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGGGAGCACCAGGAAGTGCTGGTTCTGCTGCTGGTAGTGGAGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTATGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACC TCGGGCGTGCAGTGTTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCATCACCATCACCATCACTAAGCGGCCGCATCGATTATAAGCTTTGTACACCTAGAAAACATGAGGATCACCCATGTCTGCACCTCGACACTAGAAAACATGAGGATCACCCATGT(SEQ ID NO: 287)

[0764] protein: X indicates a non-classical amino acid (SEQ ID NO: 288)

[0765] pp7 Bacteriophage PP7 RNA stem-loop: (version 1) DNA: GGAGCAGACGATATGGCGTCGCTCC (SEQ ID NO: 289)

[0766] pp7 Bacteriophage PP7 RNA stem-loop: (version 2) DNA sequence DNA: CCAGCAGAGCATATGGGCTCGCTGG (SEQ ID NO: 290)

[0767] EBAG9: A receptor-binding cancer antigen expressed on SiSo cells (Homo sapiens, Uniprot: O00559)

[0768] DNA: ATGGCCATCACCCAGTTTCGGTTATTTAAATTTTGTACCTGCCTAGCAACAGTATTCTCATTCCTAAAGAGATTAATATGCAGATCTGGCAGAGGACGGAAATTAAGTGGAGACCAAATAACTTTGCCAACTACAGTTGATTATTCATCAGTTCCTAAGCAGACAGATGTTGAAGAGTGGACTTCCTGGGATGAAGATGCACCCACCAGTGTAAAGATCGAAGGAGGGAATGGGAATGTGGCAACACAACAAAATTCTTTGGAACAACTGGAACCTGACTATTTTAAGGACATGACACCAACTATTAGGAAAACTCAGAAAATTGTTATTAAGAAGAGAGAACCATTGAATTTTGGCATCCCAGATGGGAGCACAGGTTTCTCTAGTAGATTAGCAGCTACACAAGATCTGCCTTTTATTCATCAGTCTTCTGAATTAGGTGACTTAGATACCTGGCAGGAAAATACCAATGCATGGGAAGAAGAAGAAGATGCAGCCTGGCAAGCAGAAGAAGTTCTGAGACAGCAGAAACTAGCAGACAGAGAAAAGAGAGCAGCCGAACAACAAAGGAAGAAAATGGAAAAGGAAGCACAACGGCTAATGAAGAAGGAACAAAACAAAATTGGTGTGAAACTTTCA(SEQ ID NO: 291)

[0769] protein: MAITQFRLFKFCTCLATVFSFLKRLICRSGRGRKLSGDQITLPTTVDYSSVPKQTDVEEWTSWDEDAPTSVKIEGGNGNVATQQNSLEQLEPDYFKDMTPTIRKTQKIVIKKREPLNFGIPDGSTGFSSRLAATQDLPFIHQSSELGDLDTWQENTNAWEEEEDAAWQAEEVLRQQKLADREKRAAEQQRKKMEKEAQRLMKKEQNKIGVKLS(SEQ ID NO: 292)

[0770] EBAG9 1-29: Receptor-binding cancer antigen expressed on SiSo cells (Homo sapiens, Uniprot: O00559) DNA: ATGGCCATCACCCAGTTTCGGTTATTTAAATTTTGTACCTGCCTAGCAACAGTATTCTCATTCCTAAAGAGATTAATATGCAGATCT (SEQ ID NO: 293) protein: MAITQFRLFKFCTCLATVFSFLKRLICRS (SEQ ID NO: 294)

[0771] CMP-SaTr / SLC35A1: CMP-sialic acid transporter (Homo sapiens, Uniprot: P78382)

[0772] DNA:

[0773] protein: MAAPRDNVTLLFKLYCLAVMTLMAAVYTIALRYTRTSDKELYFSTTAVCITEVIKLLLSVGILAKETGSLGRFKASLRENVLGSPKELLKLSVPSLVYAVQNNMAFLALSNLDAAVYQVTYQLKIPCTALCTVLMLNRTLSKLQWVSVFMLCAGVTLVQWKPAQATKVVVEQNPLLGFGAIAIAVLCSGFAGVYFEKVLKSSDTSLWVRNIQMYLSGIIVTLAGVYLSDGAEIKEKGFFYGYTYYVWFVIFLASVGGLYTSVVVKYTDNIMKGFSAAAAIVLSTIASVMLFGLQITLTFALGTLLVCVSIYLYGLPRQDTTSIQQGETASKERVIGV (SEQ ID NO: 296)

[0774] P450 2C1 1-27 : Cytochrome P450 2C1 (Oryctolagus cuniculus, Uniprot: P00180) DNA: ATGGACCCCGTGGTCGTGCTGGGCCTGTGCCTGTCATGCCTGCTGCTGCTGAGCCTGTGGAAGCAGAGCTACGGCGGAGGC (SEQ ID NO: 297) protein: MDPVVVLGLCLSCLLLLSLWKQSYGGG (SEQ ID NO: 298)

[0775] P450 2C1 1-29 : Cytochrome P450 2C1 (Oryctolagus cuniculus, Uniprot: P00180) DNA: ATGGACCCCGTGGTCGTGCTGGGCCTGTGCCTGTCATGCCTGCTGCTGCTGAGCCTGTGGAAGCAGAGCTACGGCGGAGGCAAGCTG (SEQ ID NO: 299) protein: MDPVVVLGLCLSCLLLLSLWKQSYGGGKL (SEQ ID NO: 300)

[0776] EB1: Microtubule-associated protein RP / EB family member 1 (Homo sapiens, Uniprot: Q8WQ86)

[0777] DNA: ATGGCAGTGAACGTATACTCAACGTCAGTGACCAGTGATAACCTAAGTCGACATGACATGCTGGCCTGGATCAATGAGTCTCTGCAGTTGAATCTGACAAAGATCGAACAGTTGTGCTCAGGGGCTGCGTATTGTCAGTTTATGGACATGCTGTTCCCTGGCTCCATTGCCTTGAAGAAAGTGAAATTCCAAGCTAAGCTAGAACACGAGTACATCCAGAACTTCAAAATACTACAAGCAGGTTTTAAGAGAATGGGTGTTGACAAAATAATTCCTGTGGACAAATTAGTAAAAGGAAAGTTTCAGGACAATTTTGAATTCGTTCAGTGGTTCAAGAAGTTTTTCGATGCAAACTATGATGGAAAAGACTATGACCCTGTGGCTGCCAGACAAGGTCAAGAAACTGCAGTGGCTCCTTCCCTTGTTGCTCCAGCTCTGAATAAACCGAAGAAACCTCTCACTTCTAGCAGTGCAGCTCCCCAGAGGCCCATCTCAACACAGAGAACCGCTGCGGCTCCTAAGGCTGGCCCTGGTGTGGTGCGAAAGAACCCTGGTGTGGGCAACGGAGATGACGAGGCAGCTGAGTTGATGCAGCAGGTCAACGTATTGAAACTTACTGTTGAAGACTTGGAGAAAGAGAGGGATTTCTACTTCGGAAAGCTACGGAACATTGAATTGATTTGCCAGGAGAACGAGGGGGAAAACGACCCTGTATTGCAGAGGATTGTAGACATTCTGTATGCCACAGATGAAGGCTTTGTGATACCTGATGAAGGGGGCCCACAGGAGGAGCAAGAAGAGTAT(SEQ ID NO: 301)

[0778] protein: MAVNVYSTSVTSDNLSRHDMLAWINESLQLNLTKIEQLCSGAAYCQFMDMLFPGSIALKKVKFQAKLEHEYIQNFKILQAGFKRMGVDKIIPVDKLVKGKFQDNFEFVQWFKKFFDANYDGKDYDPVAARQGQETAVAPSLVAPALNKPKKPLTSSSAAPQRPISTQRTAAAPKAGPGVVRKNPGVGNGDDEAAELMQQVNVLKLTVEDLEKERDFYFGKLRNIELICQENEGENDPVLQRIVDILYATDEGFVIPDEGGPQEEQEEY (SEQ ID NO: 302)

[0779] CG1: nucleoporin NUP42 (Homo sapiens, Uniprot: O15504)

[0780] DNA:

[0781] protein: MAICQFFLQGRCRFGDRCWNEHPGARGAGGGRQQPQQQPSGNNRRGWNTTSQRYSNVIQPSSFSKSTPWGGSRDQEKPYFSSFDSGASTNRKEGFGLSENPFASLSPDEQKDEKKLLELEGIVKDMEVWESSGQWMFSVYSPVKKKPNISGFTDISPEELRLEYHNFLTSNNLQSYLNSVQRLINQWRNRVNELKSNLISTKVALLSDVKDGVNQAAP AFGFGSSQAATFMSPGFPVNNSSSDNAQNFSFKTNSGFAAASSGSPAGFGSSPAFGAAASTSSGISTSAPAFGFGKPEVTSAASFSFKSPAASSFGSPGFSGLPASLATGPVRAPVAPAFGGGSSVAGFGSPGSHSHTAFSKPSSDTFGNSSISTSLSASSSIIATDNVLFTPRDKLTVEELEQFQSKKFTLGKIPLKPPPLELLNV(sequence number 304)

[0782] PCP:シトクロムP450 2C1(Oryctolagus cuniculus,Uniprot:P00180)

[0783] DNA: TCCAAAACCATCGTTCTTTCGGTCGGCGAGGCTACTCGCACTCTGACTGAGATCCAGTCCACCGCAGACCGTCAGATCTTCGAAGAGAAGGTCGGGCCTCTGGTGGGTCGGCTGCGCCTCACGGCTTCGCTCCGTCAAAACGGAGCCAAGACCGCGTATCGCGTCAACCTAAAACTGGATCAGGCGGACGTCGTTGATTCCGGACTTCCGAAAGTGCGCTACACTCAGGTATGGTCGCACGACGTGACAATCGTTGCGAATAGCACCGAGGCCTCGCGCAAATCGTTGTACGATTTGACCAAGTCCCTCGTCGCGACCTCGCAGGTCGAAGATCTTGTCGTCAACCTTGTGCCGCTGGGCCGT(SEQ ID NO: 305)

[0784] protein: SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR(SEQ ID NO: 306)

[0785] LAF-1: ATP-dependent RNA helicase laf-1 (RGG domain, 1-168) (Caenorhabditis elegans, Uniprot: D0PV95)

[0786] DNA: ATGGAAAGCAACCAGAGCAACAACGGCGGCTCTGGCAACGCCGCTCTGAACAGAGGCGGCAGATACGTGCCCCCCCACCTGAGAGGAGGCGACGGCGGCGCCGCCGCCGCTGCATCTGCCGGCGGAGATGACAGAAGAGGCGGAGCCGGAGGCGGCGGCTATAGACGGGGAGGCGGAAACAGCGGCGGCGGAGGCGGAGGCGGCTACGACAGAGGCTACAACGACAACCGGGACGACCGGGACAACAGAGGCGGCAGCGGCGGATACGGCAGAGATCGAAACTACGAGGACAGAGGCTACAATGGCGGAGGCGGAGGCGGCGGCAACCGGGGCTACAACAACAACAGAGGAGGCGGCGGCGGCGGCTACAACCGCCAGGACAGAGGCGATGGCGGATCTAGCAATTTCAGCAGAGGCGGCTACAACAACCGGGACGAGGGCAGCGACAACAGAGGCAGCGGAAGAAGCTACAACAATGACCGGAGAGATAATGGCGGAGATGGC(SEQ ID NO: 307)

[0787] protein: MESNQSNNGGSGNAALNRGGRYVPPHLRGGDGGAAAAASAGGDDRRGGAGGGGYRRGGGNSGGGGGGGYDRGYNDNRDDRDNRGGSGGYGRDRNYEDRGYNGGGGGGGNRGYNNNRGGGGGGYNRQDRGDGGSSNFSRGGYNNRDEGSDNRGSGRSYNNDRRDNGGDG(SEQ ID NO: 308)

[0788] SLP3: Stomatin-like protein 3, aa 1-59 (Homo sapiens, Uniprot: Q8TAV4) DNA: ATGGATTCTAGGGTGTCTTCACCTGAGAAGCAAGATAAAGAGAATTTCGTGGGTGTCAACAATAAACGGCTTGGTGTATGTGGCTGGATCCTGTTTTCCCTCTCTTTCCTGTTGGTGATCATTACCTTCCCCATCTCCATATGGATGTGCTTGAAGATCATTAAGGAGTATGAACGT (SEQ ID NO: 309) protein: MDSRVSSPEKQDKENFVGVNNKRLGVCGWILFSLSFLLVIITFPISIWMCLKIIKEYER (SEQ ID NO: 310)

[0789] SYNZIP1: Synthetic coiled-coil peptide 1 (synthetic) DNA: AATCTGGTGGCCCAGCTGGAAAACGAGGTGGCCAGCCTGGAAAACGAGAACGAAACCCTGAAGAAAAAAGAACCTGCACAAGAAGGACCTGATCGCCTACCTGGAAAAGGAAATCGCCAACCTGAGAAAGAAGATCGAGGAA (SEQ ID NO: 311) protein: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEE (SEQ ID NO: 312)

[0790] SYNZIP2: Synthetic coiled-coil peptide 1 (synthetic) DNA: GCTAGAAACGCCTACCTGAGAAAGAAAATCGCCAGACTGAAGAAGGACAACCTGCAGCTGGAAAGAGACGAGCAGAACCTGGAAAAGATCATCGCCAACCTCAGAGATGAGATCGCCAGACTGGAAAACGAGGTGGCCAGCCACGAGCAG (SEQ ID NO: 313) protein: ARNAYLRKKIARLKKDNLQLERDEQNLEKIIANLRDEIARLENEVASHEQ (SEQ ID NO: 314)

[0791] SYNZIP3: Synthetic coiled-coil peptide 1 (synthetic) DNA: AATGAGGTGACCACCCTGGAAAACGACGCCGCCTTCATCGAGAACGAGAACGCCTACCTGGAAAAAGAGATCGCCAGACTGAGAAAGGAAAAGGCCGCTCTGCGGAACAGACTGGCCCACAAGAAG (SEQ ID NO: 315) protein: NEVTTLENDAAFIENENAYLEKEIARLRKEKAALRNRLAHKK (SEQ ID NO: 316)

[0792] SYNZIP4: Synthetic coiled-coil peptide 1 (synthetic) DNA: CAAAAGGTGGCTGAACTGAAAAATAGAGTGGCCGTGAAGCTGAACCGGAACGAGCAGCTGAAGAACAAGGTGGAAGAGCTGAAGAACAGAAACGCTACCTGAAGAATGAGCTGGCCACCCTGGAAAACGAGGTGGCCAGACTGGAAAACGACGTGGCCGAG (SEQ ID NO: 317) protein: QKVAELKNRVAVKLNRNEQLKNKVEELKNRNAYLKNELATLENEVARLENDVAE (SEQ ID NO: 318)

[0793] 2. Further fusion proteins: EBAG9 1-29 ::FUS::PylRS(AF)

[0794] DNA:

[0795] protein:

[0796] EBAG9::PylRS(AF)

[0797] DNA:

[0798] protein: MAITQFRLFKFCTCLATVFSFLKRLICRSGRGRKLSGDQITLPTTVDYSSVPKQTDVEEWTSWDEDAPTSVKIEGGNGNVATQQNSLEQLEPDYFKDMTPTIRKTQKIVIKKREPLNFGIPDGSTGFSSRLAATQDLPFIHQSSELGDLDTWQENTNAWEEEEDAAWQAEEVLRQQKLADREKRAAEQQRKKMEKEAQRLMKKEQNKIGVKLSGADYKDDDDKGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO: 322)

[0799] EBAG9::FUS::PylRS(AF)

[0800] DNA:

[0801] protein:

[0802] EBAG9::MCP

[0803] DNA:

[0804] protein: MAITQFRLFKFCTCLATVFSFLKRLICRSGRGRKLSGDQITLPTTVDYSSVPKQTDVEEWTSWDEDAPTSVKIEGGNGNVATQQNSLEQLEPDYFKDMTPTIRKTQKIVIKKREPLNFGIPDGSTGFSSRLAATQDLPFIHQSSELGDLDTWQENTNAWEEEEDAAWQAEEVLRQQKLADREKRAAEQQRKKMEKEAQRLMKKEQNKIGVKLSAIAYPYDVPDYAGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY* (SEQ ID NO: 326)

[0805] EBAG9::EWSR1::MCP

[0806] DNA:

[0807] protein: *(SEQ ID NO: 328)

[0808] EBAG9 1-29 ::PylRS(AF)

[0809] DNA:

[0810] protein: MAITQFRLFKFCTCLATVFSFLKRLICRSGADYKDDDDKGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO: 330)

[0811] EBAG9 1-29 ::FUS::PylRS(AF)

[0812] DNA:

[0813] protein:

[0814] EBAG9 1-29 ::MCP

[0815] DNA: ATGGCCATCACCCAGTTTCGGTTATTTAAATTTTGTACCTGCCTAGCAACAGTATTCTCATTCCTAAAGAGATTAATATGCAGATCTGCGATCGCATATCCCTATGATGTGCCGGATTATGCTGGAGCACCAGGAAGTGCTGGTTCTGCTGCTGGTAGTGGAGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAA(SEQ ID NO: 333)

[0816] protein: MAITQFRLFKFCTCLATVFSFLKRLICRSAIAYPYDVPDYAGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY*(SEQ ID NO: 334)

[0817] EBAG9 1-29 ::EWSR1::MCP

[0818] DNA:

[0819] protein: *(SEQ ID NO: 336)

[0820] EBAG9::EWSR1::4xλ N22

[0821] DNA:

[0822] protein:

[0823] EBAG9 1-29 ::EWSR1::4xλ N22

[0824] DNA:

[0825] protein: *(SEQ ID NO: 340)

[0826] EBAG9::PylRS(AA)

[0827] DNA:

[0828] protein: MAITQFRLFKFCTCLATVFSFLKRLICRSGRGRKLSGDQITLPTTVDYSSVPKQTDVEEWTSWDEDAPTSVKIEGGNGNVATQQNSLEQLEPDYFKDMTPTIRKTQKIVIKKREPLNFGIPDGSTGFSSRLAATQDLPFIHQSSELGDLDTWQENTNAWEEEEDAAWQAEEVLRQQKLADREKRAAEQQRKKMEKEAQRLMKKEQNKIGVKLSGADYKDDDDKGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLAFAQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO: 342)

[0829] EBAG9::PylRS(AAAF)

[0830] DNA:

[0831] protein: MAITQFRLFKFCTCLATVFSFLKRLICRSGRGRKLSGDQITLPTTVDYSSVPKQTDVEEWTSWDEDAPTSVKIEGGNGNVATQQNSLEQLEPDYFKDMTPTIRKTQKIVIKKREPLNFGIPDGSTGFSSRLAATQDLPFIHQSSELGDLDTWQENTNAWEEEEDAAWQAEEVLRQQKLADREKRAAEQQRKKMEKEAQRLMKKEQNKIGVKLSGADYKDDDDKGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLAFAQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO: 344)

[0832] EBAG9::FUS::PylRS(AA)

[0833] DNA:

[0834] protein:

[0835] EBAG9::FUS::PylRS(AAAF)

[0836] DNA:

[0837] protein:

[0838] EBAG9 1-29 ::FUS::PylRS(AA)

[0839] DNA:

[0840] protein:

[0841] EBAG9 1-29 ::FUS::PylRS(AAAF)

[0842] DNA:

[0843] protein:

[0844] EBAG9 1-29 ::FUS::MCP::PylRS(AF)

[0845] DNA:

[0846] protein:

[0847] CG1::ArrowRS(AF)

[0848] DNA:

[0849] protein: *(SEQ ID NO: 356)

[0850] CG1::PylRS(AA)

[0851] DNA:

[0852] protein: *(SEQ ID NO: 358)

[0853] CG1::PylRS(AAAF)

[0854] DNA:

[0855] protein: *(SEQ ID NO: 360)

[0856] CG1::FUS::PylRS(AA)

[0857] DNA:

[0858] protein:

[0859] CG1::FUS::PylRS(AAAF)

[0860] DNA:

[0861] protein:

[0862] CG1::MCP

[0863] DNA:

[0864] protein: *(SEQ ID NO: 366)

[0865] CG1::EWSR1::MCP

[0866] DNA:

[0867] protein:

[0868] CG1::FUS::ArrowRS(AF)

[0869] DNA:

[0870] protein:

[0871] CG1::FUS::MCP::PylRS(AF)

[0872] DNA:

[0873] protein:

[0874] CMP-SaTr::PylRS(AF)

[0875] DNA:

[0876] protein: MAAPRDNVTLLFKLYCLAVMTLMAAVYTIALRYTRTSDKELYFSTTAVCITEVIKLLLSVGILAKETGSLGRFKASLRENVLGSPKELLKLSVPSLVYAVQNNMAFLALSNLDAAVYQVTYQLKIPCTALCTVLMLNRTLSKLQWVSVFMLCAGVTLVQWKPAQATKVVVEQNPLLGFGAIAIAVLCSGFAGVYFEKVLKSSDTSLWVRNIQMYLSGIIVTLAGVYLSDGAEIKEKGFFYGYTYYVWFVIFLASVGGLYTSVVVKYTDNIMKGFSAAAAIVLSTIASVMLFGLQITLTFALGTLLVCVSIYLYGLPRQDTTSIQQGETASKERVIGVGADYKDDDDKGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO: 374)

[0877] CMP-SaTr::PylRS(AA)

[0878] DNA:

[0879] protein: MAAPRDNVTLLFKLYCLAVMTLMAAVYTIALRYTRTSDKELYFSTTAVCITEVIKLLLSVGILAKETGSLGRFKASLRENVLGSPKELLKLSVPSLVYAVQNNMAFLALSNLDAAVYQVTYQLKIPCTALCTVLMLNRTLSKLQWVSVFMLCAGVTLVQWKPAQATKVVVEQNPLLGFGAIAIAVLCSGFAGVYFEKVLKSSDTSLWVRNIQMYLSGIIVTLAGVYLSDGAEIKEKGFFYGYTYYVWFVIFLASVGGLYTSVVVKYTDNIMKGFSAAAAIVLSTIASVMLFGLQITLTFALGTLLVCVSIYLYGLPRQDTTSIQQGETASKERVIGVGADYKDDDDKGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLAFAQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO: 376)

[0880] CMP-SaTr::PylRS(AAAF)

[0881] DNA:

[0882] protein: MAAPRDNVTLLFKLYCLAVMTLMAAVYTIALRYTRTSDKELYFSTTAVCITEVIKLLLSVGILAKETGSLGRFKASLRENVLGSPKELLKLSVPSLVYAVQNNMAFLALSNLDAAVYQVTYQLKIPCTALCTVLMLNRTLSKLQWVSVFMLCAGVTLVQWKPAQATKVVVEQNPLLGFGAIAIAVLCSGFAGVYFEKVLKSSDTSLWVRNIQMYLSGIIVTLAGVYLSDGAEIKEKGFFYGYTYYVWFVIFLASVGGLYTSVVVKYTDNIMKGFSAAAAIVLSTIASVMLFGLQITLTFALGTLLVCVSIYLYGLPRQDTTSIQQGETASKERVIGVGADYKDDDDKGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLAFAQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO: 378)

[0883] CMP-SaTr::FUS::PylRS(AA)

[0884] DNA:

[0885] protein:

[0886] CMP-SaTr::FUS::PylRS(AAAF)

[0887] DNA:

[0888] protein:

[0889] CMP-SaTr::MCP

[0890] DNA:

[0891] protein: MAAPRDNVTLLFKLYCLAVMTLMAAVYTIALRYTRTSDKELYFSTTAVCITEVIKLLLSVGILAKETGSLGRFKASLRENVLGSPKELLKLSVPSLVYAVQNNMAFLALSNLDAAVYQVTYQLKIPCTALCTVLMLNRTLSKLQWVSVFMLCAGVTLVQWKPAQATKVVVEQNPLLGFGAIAIAVLCSGFAGVYFEKVLKSSDTSLWVRNIQMYLSGIIVTLAGVYLSDGAEIKEKGFFYGYTYYVWFVIFLASVGGLYTSVVVKYTDNIMKGFSAAAAIVLSTIASVMLFGLQITLTFALGTLLVCVSIYLYGLPRQDTTSIQQGETASKERVIGVAIAYPYDVPDYAGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY*(SEQ ID NO: 384)

[0892] CMP-SaTr::EWSR1::MCP

[0893] DNA:

[0894] protein:

[0895] CMP-SaTr::PylRS(AF)EWSR1::4xλ N22

[0896] DNA:

[0897] protein:

[0898] CMP-SaTr::FUS::PylRS(AF)

[0899] DNA:

[0900] protein:

[0901] P450 2C1 1-27 ::ArrowRS(AF)

[0902] DNA:

[0903] protein: MDPVVVLGLCLSCLLLLSLWKQSYGGGAIAGKPIPNPLLGLDSTGAPGSAGSAAGSGACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*(SEQ ID NO: 392)

[0904] P450 2C1 1-27 ::MCP

[0905] DNA: ATGGACCCCGTGGTCGTGCTGGGCCTGTGCCTGTCATGCCTGCTGCTGCTGAGCCTGTGGAAGCAGAGCTACGGCGGAGGCGCGATCGCATATCCCTATGATGTGCCGGATTATGCTGGAGCACCAGGAAGTGCTGGTTCTGCTGCTGGTAGTGGAGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAA(SEQ ID NO: 393)

[0906] protein: MDPVVVLGLCLSCLLLLSLWKQSYGGGAIAYPYDVPDYAGAPGSAGSAAGSGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY*(SEQ ID NO: 394)

[0907] P450 2C1 1-27 ::FUS::PylRS(AF)

[0908] DNA:

[0909] protein:

[0910] P450 2C1 1-27 ::EWSR1::MCP

[0911] DNA:

[0912] protein: 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*(SEQ ID NO: 398)

[0913] P450 2C1 1-27 ::FUS::MCP::PylRS(AF)

[0914] DNA:

[0915] protein:

[0916] P450 2C1 1-29 ::FUS::MCP::PylRS(AF)

[0917] DNA:

[0918] protein:

[0919] EB1::ArrowRS(AF)

[0920] DNA:

[0921] protein: MAVNVYSTSVTSDNLSRHDMLAWINESLQLNLTKIEQLCSGAAYCQFMDMLFPGSIALKKVKFQAKLEHEYIQNFKILQAGFKRMGVDKIIPVDKLVKGKFQDNFEFVQWFKKFF...

Claims

1. An assembler fusion protein (AFP), comprising: (a) (a1) a polypeptide segment (IC-TP segment) derived from an intracellular targeting polypeptide, which targets and is therefore locally concentrated to an intracellular structural element within or directly adjacent to the cytoplasm; and (a2) a polypeptide segment (PSP segment) derived from a phase-separated polypeptide, the phase-separated polypeptide having the ability to self-associate in the cytoplasm of a cell in a manner that creates sites of high local concentration in the cytoplasm; At least one first polypeptide segment acting as an assembler (AP) selected from (b) b1) an RNA-targeting polypeptide (RNA-TP) segment, and b2) an orthogonal aminoacyl-tRNA synthetase (O-RS) segment; At least one second polypeptide segment (EP) acting as an effector selected from Including, The polypeptide segments are operably linked on the AFP. Assembler Fusion Proteins (AFPs).

2. 2. An assembler fusion protein (AFP) combination comprising at least two different AFPs according to claim 1, At least a first AFP comprises an EP selected from an RNA-TP segment; at least a second AFP comprises an EP selected from an O-RS segment; A combination of assembler fusion proteins, wherein said polypeptide segments are operably linked in each of said AFP polypeptide chains.

3. An assembler fusion protein (AFP) according to claim 1, comprising at least two different EPs as defined in claim 1 within the same polypeptide chain, at least one EP is an RNA-TP segment and at least one other EP is an O-RS segment; An assembler fusion protein, wherein the polypeptide segments are operably linked within the AFP.

4. A fusion protein (RNA-TP / O-RS fusion protein), (i) at least one RNA targeting polypeptide (RNA-TP) segment; (ii) at least one orthogonal aminoacyl-tRNA synthetase (O-RS) segment; Including, the polypeptide segments are operably linked to each other on the RNA-TP / O-RS fusion protein; Fusion proteins (RNA-TP / O-RS fusion proteins).

5. (i) a nucleotide sequence encoding at least one AFP according to claim 1 or 3 or a combination of at least one AFP according to claim 2; or (ii) a nucleic acid sequence complementary to the nucleotide sequence of (i); (iii) both (i) and (ii); A nucleic acid molecule or a combination of two or more nucleic acid molecules comprising:

6. (i) a nucleotide sequence encoding at least one RNA-TP / O-RS fusion protein according to claim 4; or (ii) a nucleic acid sequence complementary to (i); or (iii) both (i) and (ii); A nucleic acid molecule or a combination of two or more nucleic acid molecules comprising:

7. 7. An expression cassette comprising the nucleotide sequence of a nucleic acid molecule or a combination of nucleic acid molecules according to claim 5 or claim 6.

8. An expression vector comprising at least one expression cassette according to claim 7.

9. A cell comprising at least one nucleic acid molecule or a combination of nucleic acid molecules according to claim 5 or claim 6, at least one expression cassette according to claim 7, or at least one expression vector according to claim 8.

10. 10. The cell of claim 9, comprising a nucleotide sequence that encodes or is complementary to a nucleotide sequence encoding at least one AFP of claim 1 or 3, the AFP comprising (i) at least one EP selected from an RNA-TP segment and (ii) at least one EP selected from an O-RS segment.

11. The cell of claim 9, comprising a nucleotide sequence that encodes or is complementary to a nucleotide sequence that encodes a combination of at least two AFPs described in claim 1 or 2, wherein one of the at least two AFPs comprises at least one RNA-TP segment and another of the at least two AFPs comprises at least one O-RS segment.

12. 10. The cell of claim 9, comprising a nucleotide sequence that encodes or is complementary to a nucleotide sequence that encodes at least one RNA-TP / O-RS fusion protein of claim 4.

13. 1. A method for preparing a polypeptide of interest (POI) that contains one or more non-standard amino acid (ncAA) residues on its amino acid sequence, comprising: The method comprises expressing a POI by a cell according to any one of claims 9 to 11 in the presence of said one or more ncAAs, The cells: (i) a nucleotide sequence encoding a POI (CSPOI), wherein the one or more ncAA residues of the POI are encoded by a selector codon(s); (ii) a targeting nucleotide sequence (TN) that is operably linked to the CSPOI and capable of interacting with at least one RNA-TP segment of the AFP in the cell; and (iii) one or more orthogonal tRNAs having an anticodon(s) complementary to the selector codon(s) of the CSPOI; ｎｃＡＡ (O-tRNA ｎｃＡＡ )molecule; Including, The O-tRNA ｎｃＡＡ The molecule comprises one or more orthogonal O-RS / O-tRNAs in combination with one or more O-RS segments of at least one of the AFPs in the cell. ｎｃＡＡ pairs are formed, which allow the introduction of said one or more ncAA residues onto the amino acid sequence of the POI.

14. The method further comprises recovering the expressed POI. The method of claim 13.

15. 1. A method for preparing a polypeptide of interest (POI) that contains one or more non-standard amino acid (ncAA) residues on its amino acid sequence, comprising: The method comprises expressing a POI by the cell of claim 12 in the presence of said one or more ncAAs, The cells: (iv) a nucleotide sequence encoding a POI (CSPOI), wherein the one or more ncAA residues of the POI are encoded by a selector codon(s); (v) a targeting nucleotide sequence (TN) that is operably linked to the CSPOI and capable of interacting with at least one RNA-TP segment of the RNA-TP / O-RS fusion protein in a cell; and (vi) one or more orthogonal tRNAs having an anticodon(s) complementary to the selector codon(s) of the CSPOI. ｎｃＡＡ (O-tRNA ｎｃＡＡ )molecule; Including, The O-tRNA ｎｃＡＡ The molecule is coupled with one or more O-RS segments of an RNA-TP / O-RS fusion protein in a cell to produce one or more orthogonal O-RS / O-tRNAs. ｎｃＡＡ pairs are formed, which allow the introduction of said one or more ncAA residues onto the amino acid sequence of the POI.

16. The method further comprises recovering the expressed POI. The method of claim 15.

17. (i) a nucleotide sequence (CSPOI) encoding a polypeptide of interest (POI), said POI comprising one or more non-canonical amino acid (ncAA) residues encoded on the CSPOI by a selector codon; (ii) a targeting nucleotide sequence (TN); Including, The RNA molecule comprising the TN can interact with an RNA-targeting polypeptide (RNA-TP) via the TN; Nucleic acid molecule.

18. The kit includes: - at least one ncAA or a salt thereof corresponding to at least one ncAA residue of the POI; - at least one expression vector according to claim 8, Including, A kit for preparing a polypeptide of interest (POI) having at least one non-standard amino acid (ncAA) residue.

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