Systems, methods and compositions for recombinant in vitro transcription and translation utilizing thermophilic proteins

Abstract

To provide recombinant cell-free expression systems for high-yield in vitro production of biological materials.SOLUTION: A system for recombinant cell-free expression, comprises: a core recombinant protein mixture having at least the following components: a plurality of initiation factors (IFs); a plurality of elongation factors (EFs); a plurality of peptide release factors (RFs); at least one ribosome recycling factor (RRF); a plurality of aminoacyl-tRNA synthetases (RSs); and at least one methionyl-tRNA transformylase (MTF); at least one nucleic acid synthesis template; and a reaction mixture having cell-free reaction components necessary for in vitro macromolecule synthesis.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application is the benefit of U.S. Provisional Patent Application No. 62 / 833,555, filed April 12, 2019. The entire specification and drawings of the above-referenced application are incorporated herein by reference in their entirety. It is used here as a body.

[0002] The present invention provides a recombinant cell-free expression system for the in vitro production of biological substances in high yields. and methods of using the same.

[0003] Sequence Listing This application was filed electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy was generated on April 13, 2020, but Change the name to 90125.00096-Sequence-Listing_ST25.t xt and its size is 427Kbytes. [Background technology]

[0004] Cell-free expression systems (in vitro transcription / translation, cell-free protein expression, cell-free translation, or Cell-free biosynthesis (also known as cell-free biosynthesis) is a process by which researchers create functional proteins or other proteins in vitro. This refers to a molecular biology technique that allows the expression of target molecules. As well as the in vitro expression of proteins or other small molecules that are difficult to produce in vivo, Protein library hybridization for protein evolution, functional genomics, and structural studies Another advantage of this system is that it allows for high-throughput production of target proteins. Proteins can often be toxic to host cells or generally incompatible with cellular expression. and is a poor, if not completely ineffective vehicle for protein expression in vivo. This makes the use of bacterial or tissue culture cell-based systems impractical. In vitro protein expression is considerably faster than in vivo techniques based on Protein expression does not require gene transfer, cell culture, or large-scale protein purification That's why.

[0005] More specifically, cell-free expression systems allow the expression of target molecules, such as RNA species, without the use of living cells. A typical cell-free expression system produces a complex consisting of a protein and a target protein. The target gene is extracted from DNA containing one or more target genes by utilizing the biological elements / mechanisms Common components of reactions in typical cell-free expression systems include the production of cytochrome P450 receptors (CPRs), cytochrome P450 receptors (CCRs), and cytochrome P450 receptors (CCRs). Cell extracts, usually derived from cell culture lysates, provide energy sources such as ATP, amino acids, etc. , cofactors such as magnesium, and a plasmid synthesis template or linear expression The desired form of the (expression) (or synthesis) template (LET or LST) is typical. The cell extract may be prepared by lysing the cells of interest and then synthesizing the nucleic acid template with the gene of interest. by centrifugation or other sedimentation methods to remove cell walls, genomic DNA, and other debris. The remaining part of the lysate or cell extract contains the target molecule-expressing The necessary cellular machinery required for this may be contained.

[0006] Typical cell-free expression systems involve cell-free protein synthesis (CFPS). The PS system may be used in microbial cells, plant cells or other cells to produce one or more proteins of interest. Assembling catalytic components required for energy production and protein synthesis from crude animal cell lysates The crude lysate is used for the transcription of DNA to RNA, the translation of RNA to protein, and transcription. Contains elements necessary for protein folding and energy metabolism (e.g., ribosomal ribosome, aminoacyl-tRNA synthetase, translation initiation and elongation factors, ribosome termination factors, nucleotide recycling enzymes, metabolic enzymes, chaperones, foldases (fo The usual cell extracts used today are those from Escherichia coli (ECE), Reticulocytes (RRL), wheat germ (WGE), and insect cells (ICE), and It is produced by mammalian cells (MC).

[0007] Cell-free expression systems offer several advantages over traditional in vivo protein expression methods. Cell-free systems rely on the exclusive use of a single protein for most, if not all, of the metabolic resources of a cell. Furthermore, it is possible to direct the production of β-glucan in vitro without cell wall and membrane components. This is advantageous because it allows control of the synthesis environment. NA levels can be varied to reflect the codon usage of the expressed genes. The redox potential, pH or ionic strength are also much more flexible than in vivo. can be modified, but this is with little concern for cell growth or viability Furthermore, direct recovery of purified, correctly folded protein products is readily possible. is achievable.

[0008] Despite the many advantageous aspects of cell-free expression systems, they have faced several obstacles to date. These obstacles have limited the use of this system as a protein production technology. Cell-free systems derived from Escherichia coli extracts, in particular those identified in U.S. Pat. No. 7,118,883; and in a cell-free system derived from yeast extract as specified in U.S. Pat. No. 9,528,137. However, the reaction duration of active protein synthesis is short, and protein production is Slow reaction rate, small scale reaction, contains multiple disulfide bonds The limited ability to fold proteins correctly and their early development are The result is high production yields and stable ingredients. The reaction time is long and the reaction efficiency is high. There is a need for a suitable commercial cell-free expression system.

[0009] As mentioned above, cell-free systems have limitations as they lack consistency, yield, and the ability to be scaled up or down. For this reason, it is not widely used in the production of biopharmaceuticals. A cell-free system derived from E. coli that utilizes a thermostable tRNA that leads to improved production rates and high yields. Utilizing exemplary thermophilic organisms to improve the utility of the system by replacing the enzyme with proteins We have reported a cell-free system derived from the extract that not only contains the enzyme but also a novel energy regeneration system. (The novel energy regeneration system is generally described in PCT Application No. PCT / US2018 / 01 No. 2121, the specification, drawings, examples, sequence listing and claims of which are incorporated herein by reference. (which is incorporated herein by reference in its entirety).

[0010] As detailed below, the present inventors have developed a complete recombinant in vitro transcription / translation system. Although, in some embodiments, it is derived from peptides from various exemplary thermophilic bacteria. As mentioned above, currently available cell-free systems incorporate components from E. coli cell extracts. either based on adding the necessary transcription / translation machinery or based on recombinant E. coli enzymes. Various other sources of extracts have been used to improve in vitro protein production. Although many studies have reported the use of thermophilic organisms for this purpose, no complete recombinant method based on thermophilic proteins has been reported. To date, no complete recombinant expression system, including the expression system, has been reported.

[0011] As will be described in more detail below, the techniques of the present invention provide longer response duration and Truly energy efficient and robust resulting in higher production yields It overcomes the limitations of conventional cell-free expression systems while fulfilling the objectives of in vitro cell-free expression systems. Specifically, the present invention provides a method for the production of a transcription / translation-dependent gene by recombinantly expressing each protein required for transcription / translation. and therefore not encounter the large variations observed in the extractive-derived batch systems. Thermophilic bacteria allow for continuous flow with good control and precise regulation of the system without These systems include cell lines, which are used for small-scale protein production for early research applications as well as for small animal It may also be useful for medium-scale applications such as testing. The continuous flow approach in the reactor allows for large-scale production, and much This can replace current manufacturing facilities with their large footprint and personnel requirements. Summary of the Invention [Problem to be solved by the invention]

[0012] One object of the present invention is a recombinant cell-free expression system, the reaction mixture of which is Cell-free reaction components necessary for the transcription / translation machinery, providing energy and necessary for protein synthesis The recombinant cell-free medium contains all of the amino acids, nucleotides, and metabolic components In a preferred embodiment, the enzymes identified herein are expressed in various It can be sourced from various thermophilic bacteria, but this is because the source component is E. coli or This is in contrast to conventional cell-free systems, which are either yeast or other eukaryotic systems. Thermobiological strategies allow for the identification of all steps during in vitro translation, including tRNA loading. Not only does it provide greater stability during recombinant DNA synthesis (e.g., ribosomal peptide biosynthesis), but it also It is possible to improve the performance of existing systems and achieve longer uptime.

[0013] The thermophilic strategy developed by the inventors of the present invention allows for the development of a thermophilic organism that can tolerate variations in pH and salt concentration. It showed little sensitivity and was not affected by increased phosphate concentration due to ATP hydrolysis. This allows for the generation of recombinant cell-free expression systems that are unlikely to be affected by this thermophilic organism. Another advantage of the derived strategy is that the recombinant cell-free expression system of the present invention can express thermophilic aminoacyl-t It is possible to recruit different sets of tRNAs that are recognized by RNA synthetase enzymes. This results in the first complete codon coverage in a cell-free system. .

[0014] Another object of the present invention is a recombinant cell-free expression system, the reaction mixture of which contains a biological compound, Required for in vitro biosynthesis of proteins, enzymes, biosimilars or chemical modification of small molecules The recombinant cell-free expression system may include a recombinant cell-free expression system that contains all cell-free reaction components necessary for the expression of the recombinant cell-free expression system.

[0015] Another object of the present invention is to provide a method for the preparation of vaccines, biological products, In vitro transcription, translation and biochemistry of proteins, enzymes and biosimilars Continuous flow bioreactors for synthesis and enzymatic biosynthesis or chemical modification of small molecules The present invention may include methods, systems, and apparatus for a computer system.

[0016] Another object of the present invention is to provide a method for the preparation of recombinant cell-free expression reaction mixtures comprising: Or, a plurality of isolated nucleotide coding sequences may be included. Alternatively, the one or more nucleotide coding sequences may be from a thermophilic or other bacterium. In a preferred embodiment, the nucleotide coding sequence comprises an initiator nucleotide coding sequence. code sequence, elongation factor nucleotide coding sequence, release factor nucleotide coding sequence, ribosomal Chromatin recycling factor nucleotide coding sequence, aminoacyl-tRNA synthetase Nucleotide coding sequence, and methionyl-tRNA transformylase nucleotide Additional nucleotide coding sequences may include, but are not limited to, The term "RNA polymerase nucleotide coding sequence" is used herein to refer to the RNA polymerase nucleotide coding sequence as well as to the inorganic Incorporated References for Polyphosphate Energy Regeneration Systems PCT Application No. PCT / US2 The nucleotide coding sequences identified in Application No. 018 / 012121 ("the '121 application") The columns may be mentioned.

[0017] Another object of the present invention is to provide a method for the preparation of a method for the preparation of a bacterial cell comprising the steps of: An expression vector having one or more isolated nucleotide coding sequences operably linked thereto. In some embodiments, the nucleotide coding sequence can be used to generate a gene encoding a nucleotide sequence that is capable of encoding a gene for a selected bacterium. The vector may be optimized for expression in a genomic DNA sequence.

[0018] Another object of the present invention is to provide a method for the production of proteins that can be further isolated and included in a recombinant cell-free expression reaction mixture. The expression of the nucleotide coding sequences identified herein produces proteins. In a preferred embodiment, the expressed proteins include an initiation factor protein, an elongation factor protein, and a nucleotide sequence. Length factor protein, release factor protein, ribosome recycling factor protein, Aminoacyl-tRNA synthetase protein and methionyl-tRNA transformase Additional nucleotide coding sequences may include, but are not limited to, the amylase protein. The inorganic polyphosphate enzymes incorporated herein by reference are also known as ribonucleotides. Proteins and compounds identified in the '121 application related to energy regeneration systems obtain.

[0019] Another object of the present invention may include a continuous flow recombinant cell-free expression device. In an embodiment, the continuous flow recombinant cell-free expression device is a continuous flow operation. In vitro transcription and in vitro characterization of biological products, proteins, enzymes, and biosimilars for in vitro translation and in vitro biosynthesis and enzymatic biosynthesis or chemical modification of small molecules This may include the use of hollow fibers and hollow fiber-based bioreactors as an exchange medium for microbial bioreactors. [Means for solving the problem]

[0020] Further objects of the present invention may include one or more of the following preferred embodiments: 1. A system for recombinant cell-free expression comprising: -Contains at least the following ingredients: -Multiple initiation factors (IFs); -Multiple elongation factors (EFs); -Multiple peptide release factors (RFs); - at least one ribosome recycling factor (RRF); - multiple aminoacyl-tRNA synthetases (RS); and -has at least one methionyl-tRNA transformylase (MTF); R recombinant core protein mixture, at least one nucleic acid synthesis template, a reaction mixture having the cell-free reaction components necessary for in vitro macromolecule synthesis, Including, - the components are located in a bioreactor configured for cell-free expression of macromolecules; A type of system.

[0021] 2. The components of the recombinant core protein mixture are recombinant core protein mixtures derived from bacteria. 2. The system of embodiment 1, comprising:

[0022] 3. The recombinant core protein mixture derived from bacteria is a recombinant core protein mixture. and a recombinant core protein mixture, at least one component of which is derived from a thermophilic bacterium. Hmm, system of embodiment 2.

[0023] 4. The thermophilic bacterium is a thermophilic bacterium of the family Bacillaceae, or a geochemical Any one of embodiments 2 and 3, comprising a thermophilic bacterium, Geobacillus. Two systems.

[0024] 5. The Geobacillus thermophilic bacterium is Geobacill us subterraneus, and Geobacillus stearother 5. The strain of embodiment 4, selected from the group consisting of:

[0025] 6. The recombinant core protein mixture derived from bacteria is a recombinant core protein mixture. At least one of the components is a non-thermophilic bacterium or a combination of a non-thermophilic bacterium and a thermophilic bacterium. 2. The system of embodiment 1, comprising a recombinant core protein mixture derived from a combination of

[0026] 7. The non-thermophilic bacteria include Escherichia coli. Form 6 system.

[0027] 8. The multiple initiation factors (IFs) include multiple initiation factors from thermophilic bacteria, in an embodiment System of type 1.

[0028] 9. The multiple initiation factors derived from thermophilic bacteria are IF1, IF2, IF3, or any of them. 9. The system of any one of embodiments 1 and 8, comprising a fragment or variant of any of

[0029] 10. The multiple initiation factors comprise the amino acid sequence of SEQ ID NOs: 2, 4, 6, 70, 72, and 74. 1, wherein the sequence is selected from a group of sequences, or sequences having at least 90% sequence identity. , 8 and 9.

[0030] 11. The multiple elongation factors (EFs) include multiple elongation factors derived from thermophilic bacteria. Form 1 system.

[0031] 12. The multiple elongation factors from thermophilic bacteria include EF-G, EF-Tu, EF-Ts, EF-4, EF-P, or a fragment or variant of either thereof. and one of 11 systems.

[0032] 13. The multiple elongation factors are selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16, 76, 78, 80, 82, and 84, or a group of amino acid sequences having at least 90% sequence identity 13. The system of any one of embodiments 1, 11 and 12, wherein the system is selected from the sequence:

[0033] 14. The multiple peptide release factors (RFs) are derived from thermophilic bacteria or Bacillus llus) The system of embodiment 1, comprising a plurality of peptide release factors derived from bacteria.

[0034] 15. The multiple peptide release factors from thermophilic bacteria are RF1, RF2, and RF3. 15. Any one of embodiments 1 and 14, comprising: Two systems.

[0035] 16. The multiple peptide terminators are peptides consisting of SEQ ID NOs: 18, 20, 22, 86, and 88. or sequences having at least 90% sequence identity. A system of any one of forms 1, 14 and 15.

[0036] 17. The ribosome recycling factor (RRF) is a ribosome recycling factor derived from thermophilic bacteria. 2. The system of embodiment 1, comprising a recycling factor.

[0037] 18. The ribosome recycling factor is derived from Geobacillus 18. The line of any one of embodiments 1 and 17, wherein the line is derived from

[0038] 19. The ribosome recycling factor has the amino acid sequence SEQ ID NO: 14 and 90 or at least ribosome recycling factors that follow sequences with at least 90% sequence identity. The system of any one of embodiments 1, 17 and 18.

[0039] 20. The multiple aminoacyl-tRNA-synthetases (RS) are derived from thermophilic bacteria or bacteria. An embodiment comprising multiple aminoacyl-tRNA synthetases from E. coli. System of type 1.

[0040] 21. Several aminoacyl-tRNA synthetases are known: AlaRS, ArgRS, and As nRS;AspRS;CysRS;GlnRS;GluRS;GlyRS;HisRS; IleRS;LeuRS;LysRS;MetRS;PheRS(a);PheRS(b ); ProRS; SerRS; ThrRS; TrpRS; TyrRS; and ValRS, 21. Any one of embodiments 1 and 20, comprising: System of.

[0041] 22. The aminoacyl-tRNA-synthetases are selected from the group consisting of SEQ ID NOs: 26, 28, 32 , 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, 98, 100, 102, 104, 106, 108 , 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 and 130, or a group of amino acid sequences having at least 90% sequence identity. 22. The system of any one of embodiments 1, 20 and 21, selected from the series:

[0042] 23. The methionyl-tRNA transformylase (MTF) is derived from a thermophilic bacterium. 2. The system of embodiment 1, comprising a methionyl-tRNA transformylase.

[0043] 24. The methionyl-tRNA transformylase is derived from Geobacillus 24. The line of embodiments 1 and 23, wherein the line is derived from S. illus.

[0044] 25. Methionyl-tRNA transformylase has the amino acid sequence SEQ ID NO: 68 and 1 32, or a methionyl-tRNA tRNA tRNA sequence with at least 90% sequence identity. 25. The system of any one of embodiments 1, 23 and 24, comprising a phosphodiesterase.

[0045] 26. The system of embodiment 1, wherein the nucleic acid synthesis template comprises a DNA template.

[0046] 27. The DNA template is: - at least one target sequence operably linked to a promoter, optionally including a coding sequence; the target sequence, optionally sequence-optimized; - at least one ribosome binding site (RBS); at least one expression product cleavage site; and - at least one tag, 27. The system of embodiment 26, comprising a linear DNA template having:

[0047] 28. The system of embodiment 1, wherein the nucleic acid synthesis template comprises an RNA template.

[0048] 29. The reaction mixture comprises the following components: - a quantity of ribosomes, and optionally a quantity of ribosomes from thermophilic bacteria; - a certain amount of ribonuclease inhibitors; - a certain amount of RNA polymerase; an amount of tRNA, and optionally an amount of tRNA from a thermophilic bacterium; buffer solutions; and 2. The system of embodiment 1, comprising one or more of: an amount of amino acids.

[0049] 30. The reaction mixture comprises the following components: -Tris-acetate; -Mg(OAc)2; -K + -Glutamate; -amino-acetate; -NaCl; -KCl; -MgCk; -DTT; -octyl-b-glycoside; -NAD; -NADP; -Sorbitol; -FADH; -CoA; -PLP; and -SAM.

[0050] 31. The system of any of embodiments 1 and 29, further comprising an energy source.

[0051] 32. In embodiments, the energy source comprises a quantity of nucleotide triphosphates (NTPs). 32 series.

[0052] 33. The nucleotide triphosphate is adenine triphosphate (ATP); guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP) 33. The system of embodiment 32, comprising one or more of the selected nucleotide triphosphates.

[0053] 34. In an embodiment, the energy source comprises an inorganic polyphosphate-derived energy regeneration system. 31, 32 and 33.

[0054] 35. The inorganic polyphosphate-derived energy regeneration system is a cellular adenosine triphosphate (ATP) energy regeneration system, - a certain amount of adenosyl kinase (Gst AdK) enzyme; - a certain amount of polyphosphate kinase (TaqPPK) enzyme; an amount of inorganic polyphosphate (PPi); and -Cells containing a certain amount of adenosine monophosphate (AMP), adenosine triphosphate (ATP) Including energy regeneration systems, -The AdK enzyme and PPK enzyme act synergistically to convert PPi and AMP into cellular ATP 35. The system of embodiment 34, which regenerates energy.

[0055] 36. The system of embodiment 1, wherein the bioreactor comprises a continuous flow bioreactor.

[0056] 37. A recombinant cell-free expression reaction mixture comprising: -Multiple initiation factors (IFs); -Multiple elongation factors (EFs); -Multiple release factors (RFs); - at least one ribosome recycling factor (RRF); - several aminoacyl-tRNA-synthetases (RS); and - a composition containing at least one methionyl-tRNA transformylase (MTF) Recombinant cell-free expression reaction mixture.

[0057] 38. The multiple initiation factors (IFs) include multiple initiation factors from thermophilic bacteria. System of state 37.

[0058] 39. The multiple initiation factors from thermophilic bacteria are IF1, IF2, IF3, or any of them. 39. The system of any one of embodiments 37 and 38, comprising a fragment or variant of any of

[0059] 40. The multiple initiation factors comprise the amino acid sequences SEQ ID NOs: 2, 4, 6, 70, 72, and 74. 37. The sequence of claim 36, wherein the sequence is selected from the group of sequences of the present invention, or sequences having at least 90% sequence identity. , 38 and 39.

[0060] 41. The multiple elongation factors (EFs) include multiple elongation factors from thermophilic bacteria. System of state 37.

[0061] 42. The multiple elongation factors from thermophilic bacteria are EF-G, EF-Tu, EF-Ts, and E 37 and 38, which comprise F-4, EF-P, or a fragment or variant of either thereof. and one of 41 systems.

[0062] 43. The multiple elongation factors are selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16, 76, 78, 80, 8 2, and 84, or a group of amino acid sequences having at least 90% sequence identity. 43. The system of any one of embodiments 37, 41 and 42, selected from the series:

[0063] 44. The multiple peptide release factors (RFs) are derived from thermophilic bacteria or Bacillus sp. 38. The system of embodiment 37, comprising a plurality of release factors derived from a Bacterium typhimurium sp.

[0064] 45. The multiple peptide release factors are RF1, RF2, and RF3, or any of them. 45. The system of any one of embodiments 37 and 44, comprising a fragment or variant of:

[0065] 46. ​​The multiple peptide terminator is an amino acid sequence consisting of SEQ ID NOs: 18, 20, 22, 86, and 88. or sequences having at least 90% sequence identity, Any one of states 37, 44 and 45.

[0066] 47. The ribosome recycling factor (RRF) is a ribosome recycling factor derived from thermophilic bacteria. 38. The system of embodiment 37, comprising a cycling factor.

[0067] 48. The ribosome recycling factor is a ribosome recycling factor derived from Geobacillus 48. The system of any one of embodiments 37 and 47, wherein the system is derived from

[0068] 49. The ribosome recycling factor has the amino acid sequence SEQ ID NO: 14 and 90 or at least and a ribosome recycling factor according to a sequence having 90% sequence identity with the A system of any one of forms 37, 47 and 48.

[0069] 50. The plurality of aminoacyl-tRNA-synthetases (RS) includes at least one an embodiment comprising multiple aminoacyl-tRNA-synthetases, one of which is from a thermophilic bacterium; System of state 37.

[0070] 51. Several aminoacyl-tRNA synthetases are known: AlaRS, ArgRS, and As nRS;AspRS;CysRS;GlnRS;GluRS;GlyRS;HisRS; IleRS;LeuRS;LysRS;MetRS;PheRS(a);PheRS(b ); ProRS; SerRS; ThrRS; TrpRS; TyrRS; and ValRS, or any fragment or variant thereof. Two systems.

[0071] 52. The aminoacyl-tRNA-synthetases are selected from the group consisting of SEQ ID NOs: 26, 28, 32 , 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, 98, 100, 102, 104, 106, 108 , 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 and 130, or a group of amino acid sequences having at least 90% sequence identity. 52. The system of any one of embodiments 37, 50 and 51, selected from the series:

[0072] 53. The methionyl-tRNA transformylase (MTF) is derived from a thermophilic bacterium. 38. The system of any one of embodiments 37, comprising a methionyl-tRNA transformylase.

[0073] 54. The methionyl-tRNA transformylase is derived from Geobacillus 54. The system of any one of embodiments 37 and 53, wherein the system is derived from Bacillus illus.

[0074] 55. Methionyl-tRNA transformylase has the amino acid sequence SEQ ID NO: 68 and 1 32, or a methionyl-tRNA tRNA tRNA sequence with at least 90% sequence identity. 55. The system of any one of embodiments 37, 53, and 54, comprising a phosphodiesterase.

[0075] 56. An isolated nucleotide comprising: SEQ ID NOs: 1, 3, 5, 69, 71, and 73; SEQ ID NOs: 7, 9, 11, 13, 15, 75, 77, 79, 81, and 83; SEQ ID NOs: 17, 19, 21, 85, and 87; SEQ ID NOs: 23 and 89; and SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 4 7, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 91, 93, 95 , 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 1 17, 119, 121, 123, 125, 127, 129, and 131. An isolated nucleotide comprising a selected nucleotide.

[0076] 57. At least one of the nucleotide sequences of embodiment 56, operably linked to a promoter. an expression vector comprising at least one

[0077] 58. A bacterium transformed with one of the expression vectors of embodiment 57.

[0078] 59. The transformed bacterium of embodiment 58, wherein the bacterium comprises E. coli. .

[0079] 60. A peptide comprising: SEQ ID NOs: 2, 4, 6, 70, 72, and 74; SEQ ID NOs: 8, 10, 12, 14, 16, 76, 78, 80, 82, and 84; -SEQ ID NOs: 18, 20, 22, 86, 88; SEQ ID NOs: 14 and 90; SEQ ID NOs: 26, 28, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, SEQ ID NO: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, and 130; and - consisting of SEQ ID NOs: 68 and 132, or any fragment or variant thereof A peptide comprising an amino acid sequence selected from the group:

[0080] 61. A cell-free expression system using at least one of the peptides of embodiment 60.

[0081] Further objects of the present technology are set forth in the detailed disclosure, drawings and claims set forth below. It will become clear from [Brief explanation of the drawings]

[0082] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate exemplary embodiments of the present invention. This application describes one or more embodiments of the present invention and, together with this application, provides a The drawings merely illustrate one or more preferred embodiments of the invention. It is for the purpose of clarification and is not to be construed as limiting the invention.

[0083] [Figure 1] FIG. 1 shows the results of an aminoacyl-tRNA synthetase kinetic activity assay for the following synthetase enzymes: AlaRS, ArgRS, AsnRS, AspRS, CysRS, GlnRS(Ec), GluRS, GlyRS, HisRS, IleRS, and a no-tRNA control. [Figure 2] FIG. 2 shows the results of an aminoacyl-tRNA synthetase kinetic activity assay for the following synthetase enzymes: LeuRS, LysRS, MetRS, PheRS, ProRS, SerRS, ThrRS, TrpRS, TyrRS, and ValRS, and a no-tRNA control. [Figure 3A] FIG. 3A shows the results of an aminoacyl-tRNA synthetase activity assay utilizing exemplary tRNAs from E. coli. [Figure 3B] FIG. 3B shows the results of an aminoacyl-tRNA synthetase activity assay utilizing tRNA from the exemplary thermophilic bacterium Geobacillus stearothermophilus. [Figure 4] FIG. 4 shows the production of green fluorescent protein (muGFP, SEQ ID NO: 134) cell-free expression product using the recombinant cell-free expression system described herein. [Figure 5] FIG. 5 is a diagram of a hollow fiber reactor for cell-free production and continuous exchange in one embodiment thereof. [Figure 6] 6A-6B are diagrams of a hollow fiber reactor for cell-free production and continuous exchange in one embodiment thereof. [Figure 7] In Figure 7, the pET151 / D-TOPO vector was used to select synthetic genes that add an N-terminal tag to the expressed protein. All genes expressed in this vector were reverse-translated from the protein sequence to DNA and codon-optimized for expression in E. coli. The N-terminal tag can be omitted from the specific sequences identified below. [Figure 8] In Figure 8, the pET24a(+) vector was used to select synthetic genes that add a C-terminal 6x His-tag to the expressed protein. All genes expressed in this vector were reverse-translated from the protein sequence to DNA and codon-optimized for expression in E. coli. The C-terminal tag can be omitted from the specific sequences identified below. [Figure 9] In Figure 9, the pNAT vector was designed and used to clone and / or synthesize selection genes, which add an N-terminal FLAG tag and / or a C-terminal 6X His tag to expressed proteins. All genes expressed in this vector were reverse-translated from the protein sequence to DNA and codon-optimized for expression in E. coli. Tags may be omitted from the specific sequences identified below. [Figure 10] In Figure 10, the pNAT2.0 vector was designed and used to clone and / or synthesize selected genes, which add an N- or C-terminal 6X His tag to expressed proteins. All genes expressed in this vector were reverse-translated from the protein sequence to DNA and codon-optimized for expression in E. coli. Tags may be omitted from the specific sequences identified below. [Figure 11] FIG. 11 shows SDS-PAGE results for the following purified aminoacyl-tRNA-synthetase (aaRS) enzymes: AlaRS, ArgRS, AsnRS, AspRS, CysRS, GlnRS(Ec), GluRS, GlyRS, HisRS, IleRS, and LeuRS. [Figure 12] FIG. 12 shows SDS-PAGE results for the following purified aminoacyl-tRNA-synthetase (aaRS) enzymes: LysRS, MetRS, PheBRS, ProRS, SerRS, ThrRS, TrpRS, TyrRS, ValRS, and purified methionyl-tRNA-transformylase MTF. [Figure 13]Figure 13 shows the SDS-PAGE results for the following purified translation factors: IF-1, IF-2, IF-3, EF-G, EF-Ts, EF-Tu, EF-P, RF-1, RF-2, RF-3 and RRF. [Figure 14] FIG. 14 shows the SDS-PAGE results for purified translation factor EF-4. [Figure 15] FIG. 15 shows the real-time production of a fluorescent protein (muGFP, SEQ ID NO: 134) product using the recombinant cell-free expression system described herein. [Figure 16] Figure 16 shows a Western blot using an anti-FLAG antibody of a cell-free protein expression reaction after reverse purification but without filtered ribosomes, demonstrating specific detection of protein cell-free expression products, particularly deGFP (de-Green Fluorescent Protein) (deGFP, SEQ ID NO: 135), using the recombinant cell-free expression system described herein. [Figure 17A] (A) shows the results of an AMP-producing activity assay of three distinct aminoacyl-tRNA-synthetases using exemplary tRNAs from E. coli. [Figure 17B] (B) shows the AMP standard curve. DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention is particularly directed to the preparation of, for example, polysaccharides in a cell-free environment that are suitable for direct delivery to a patient. It is suitable for on-demand production of therapeutic macromolecules such as peptides. The present invention will be primarily described and explained in relation to the production of therapeutic proteins. However, the present invention also provides a method for preparing a toxic protein, a radiolabeled amino acid, an unnatural amino acid, etc. The enzyme can also be used to produce any type of protein, including proteins. Furthermore, the present invention is particularly suitable for on-demand production of proteins using cell-free expression, Therefore, the present invention will be described primarily in the context of cell-free protein expression.

[0085] The present invention includes various aspects, which can be combined in various ways. The following description is provided to list components and describe some of the embodiments of the present invention. Although these components are listed with the first embodiment, they may be used in any manner and at any time. It is understood that any number of combinations may be used to produce further embodiments. The examples and preferred embodiments described are for the explicitly described systems, techniques and applications. Furthermore, the present specification should not be construed as limiting the present invention to the The component alone, and also all components in this application or any subsequent application. Any number of the disclosed components may be in any and all various permutations and combinations. All descriptions and claims of various embodiments, systems, techniques, methods, devices and applications, including It should be understood that the term "supports and encompasses" refers to the area of ​​the device.

[0086] The inventive technology described herein may include novel recombinant cell-free expression systems. In an embodiment, the present invention provides a method for identifying multiple core fragments that can contribute to in vitro expression activity. Exemplary core proteins may include: :

[0087] In one embodiment, the recombinant cell-free expression system comprises one or more initiation factors (IFs). The reaction mixture may include a reaction mixture containing a nucleotide sequence that is a nucleotide sequence that is a sequence of ... For example, IF1, IF2, and IF3 may be used as initiation factors in the reaction mixture. In some embodiments, for example, IF3 can be used with 30S and 50S subunits. promotes the dissociation of the ribosome into the target protein (i.e., a step generally required to initiate translation) In addition, in the step of forming the initiation complex, other than formylmethionyl-tRNA, IF2 prevents tRNA from being inserted into the P-site. A and transfers the formylmethionyl-tRNA to the P-site of the 30S subunit. IF1 enhances the function of IF2 and IF3, thereby forming the initiation complex. In the present invention, initiation factors derived from one or more bacteria can be used. may be preferred, and more preferably thermophilic bacteria, such as Bacillus Bacterial families of the family Eae and / or Geobacillus, e.g., Geobaci llus subterraneus, or Geobacillus stearoth The IF of the present invention may be obtained from one or more of the following: Exemplary amino acid sequences may be selected from the group consisting of: IF1 (SEQ ID NOs: 2 and 70) IF2 (SEQ ID NOs: 4 and 72) IF3 (SEQ ID NOs: 6 and 74)

[0088] In an embodiment of the invention, thus, one or more of the above amino acid sequences is Amino acid sequences according to SEQ ID NOs: 1 to 2, 4, 6, 69 to 70, 72 and 74, or Amino acids encoded by any one of the fragments or variants of the amino acid sequence It contains at least one IF comprising or consisting of the amino acid sequence. wherein the coding region of at least one of the IFs according to the present invention is The fragments or variants thereof of the proteins selected are typically preferably those disclosed herein. Each naturally occurring whole amino acid sequence according to SEQ ID NOs: 1-2, 4, 6, 69-70, 72 and 74 The amino acid sequence of the long protein or its variants and at least 5%, 10%, 20%, 3 0%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or is 99%, preferably at least 70%, more preferably at least 80%, and even more preferably Preferably at least 85%, even more preferably at least 90%, and most preferably or may comprise an amino acid sequence having at least 95% or even 97% sequence identity.

[0089] In the present invention, one or more bacteria, more preferably, for example, E. coli and / or In the present invention, it may be preferable to use an initiation factor isolated from the bacterium. Alternatively, exemplary nucleotide sequences for multiple IFs may be selected from the group consisting of: : IF1 (SEQ ID NOs: 1 and 69) IF2 (SEQ ID NOs: 3 and 71) IF3 (SEQ ID NOs: 5 and 73)

[0090] In particular, the nucleotide sequence may be a sequence encoding one or more bacterial or other proteins, e.g., yeast. The codons may be optimized for expression in a protein expression system. For example, in this embodiment, Exemplary nucleotide sequences, SEQ ID NOs: 1, 3 and 5, are expressed in E. coli. It has been codon-optimized for expression in

[0091] In an embodiment of the invention, thus, one or more of the above nucleotide sequences is a nucleotide sequence according to SEQ ID NO: 1, 3, 5, 69, 71 and 73 or a fragment thereof; or a nucleotide sequence encoded by a variant thereof The present invention relates to a method for producing a nucleic acid sequence comprising the steps of: In the present context, the coding region of at least one of the IFs according to the present invention is The fragments or variants thereof of the proteins provided herein are typically and preferably Each naturally occurring full-length transcript is shown in accordance with SEQ ID NOs: 1, 3, 5, 69, 71, and 73. The nucleotide sequence of the protein or its variants is at least 5%, 10%, 20%, 3 0%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or is 99%, preferably at least 70%, more preferably at least 80%, and even more preferably Preferably at least 85%, even more preferably at least 90%, and most preferably or may contain nucleotide sequences having at least 95% or even 97% sequence identity. do.

[0092] In one embodiment, the recombinant cell-free expression system comprises a reaction mixture having one or more elongation factors. Elongation factors, such as EF-Tu, can be of two types: GTP and EF-Tu of the GTP type can be classified into the GDP type and the GDP type. EF-Tu binds to and transfers it to the A-site of the ribosome. EF-Tu is released from the ribosome. When this happens, GTP is hydrolyzed to GDP. Another elongation factor, EF-Ts, reacts with GDP-tied Another elongation factor, E, binds to EF-Tu and promotes its conversion to the GTP type. FG is a translocation molecule that occurs after peptide bond formation during peptide chain elongation. In the present invention, the present invention is directed to the production of bacteria, more preferably thermophilic bacteria. It is preferred to use EF from bacteria, for example from the Bacillaceae family and / or the family of bacteria of the Geobacillaceae family, e.g. Geobacillus s ubterraneus, or Geobacillus stearothermoph ilus et al., for example. The amino acid sequence may be selected from the group consisting of: EF-G (SEQ ID NOs: 8 and 76) EF-Tu (SEQ ID NOs: 10 and 78) EF-Ts (SEQ ID NOs: 12 and 80) EF-4 (SEQ ID NOs: 14 and 82) EF-P (SEQ ID NOs: 16 and 84)

[0093] In an embodiment of the invention, thus, one or more of the above amino acid sequences is Amino acids according to SEQ ID NOs: 8, 10, 12, 14, 16, 76, 78, 80, 82 and 84 or a fragment or variant of any one of these amino acid sequences. At least one EF containing or consisting of the encoded amino acid sequence. In this context, at least one coding region of one or more EFs according to the invention Fragments or variants thereof of the protein encoded thereby are typically and preferably SEQ ID NOs: 8, 10, 12, 14, 16, 76, 78, 80, 82 and 84 disclosed herein and a sequence of the amino acid sequence of each naturally occurring full-length protein or a variant thereof according to At least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 8 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 5%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably At least 80%, even more preferably at least 85%, even more preferably Sequence identity of at least 90%, and most preferably at least 95% or even 97% It may comprise an amino acid sequence having the following structure:

[0094] In the present invention, one or more bacteria, more preferably, for example, E. coli and / or It may be preferable to use EF isolated from the bacterium. Exemplary nucleotide sequences for the multiple EFs may be selected from the group consisting of: EF-G (SEQ ID NOs: 7 and 75) EF-Tu (SEQ ID NOs: 9 and 77) EF-Ts (SEQ ID NOs: 11 and 79) EF-4 (SEQ ID NOs: 13 and 81) EF-P (SEQ ID NOs: 15 and 83)

[0095] In particular, the nucleotide sequence may be a sequence encoding one or more bacterial or other proteins, e.g., yeast. The codons may be optimized for expression in a protein expression system. For example, in this embodiment, Exemplary nucleotide sequences, SEQ ID NOs: 7, 9, 11, 13 and 15, are sequences derived from Escherichia coli (E. It has been codon-optimized for expression in E. coli.

[0096] In an embodiment of the invention, thus, one or more of the above nucleotide sequences are nucleotides according to SEQ ID NOs: 7, 9, 11, 13, 15, 75, 77, 79 and 83 or comprising a nucleotide sequence encoded by the sequence or a fragment or variant thereof At least one code encoding at least one EF consisting of the nucleotide sequence In this context, at least one code region of one or more EFs according to the invention Fragments of proteins encoded by the code regions or variants thereof are typically preferred. Preferably, SEQ ID NOs: 7, 9, 11, 13, 15, 75, 77, 79 and 8 disclosed herein. 3, the nucleotide sequence of each naturally occurring full-length protein or a variant thereof and at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably Preferably at least 80%, even more preferably at least 85%, even more preferably or at least 90%, and most preferably at least 95% or 97% It may contain nucleotide sequences that have identity.

[0097] In one embodiment, the recombinant cell-free expression system comprises one or more peptide release factors (RFs). The RF may comprise a reaction mixture having a nucleotide sequence that is a sequence of a protein synthesized, a translated peptide, or a sequence of a protein synthesized, a nucleotide sequence that is a sequence of a protein synthesized, ... involved in the recycling of the ribosome for chain release and subsequent initiation of mRNA translation. When a protein is synthesized in a reaction system that does not contain a release factor, the reaction is terminated. It stops before the codon and therefore consists of a stable ribosome, peptide, and mRNA. The ternary complex (polysome presentation) can be easily formed. or UGA), when located in the A-site of the ribosome, release factors RF1 and RF2 Enters the A-site and dissociates the peptide chain from the peptidyl-tRNA in the P-site RF1 recognizes the stop codons UAA and UAG and promotes the transcription of ribosomal proteins. On the other hand, RF2 recognizes UAA and UGA. Another release factor, RF3, recognizes RF1 and RF2. After dissociation of the peptide chain by ribosomal transport, RF1 and RF2 are released from the ribosome.

[0098] In the present invention, R from bacteria, more preferably from thermophilic bacteria, It is preferred to use F, for example Bacillaceae and / or Ge Bacterial family of the Ocacillaceae family, e.g., Geobacillus subter raneus, or Geobacillus stearothermophilus etc. An exemplary amino acid sequence for one or more RFs of the invention is , may be selected from the group consisting of: RF1 (SEQ ID NOs: 18 and 86) RF2 (SEQ ID NOs: 20 and 88) RF3 (SEQ ID NO: 22)

[0099] In an embodiment of the invention, thus, one or more of the above amino acid sequences is Amino acid sequences according to SEQ ID NOs: 18, 20, 22, 86, and 88 or these amino acid sequences The amino acid sequence encoded by any one of the fragments or variants of In this context, the present invention Proteins encoded by at least one coding region of one or more RFs according to the invention The fragment of a protein or variant thereof is typically preferably the fragment of SEQ ID NO: 1 disclosed herein. 8, 20, 22, 86, and 88. The amino acid sequence or its variants are at least 5%, 10%, 20%, 30%, 40%, 50%, %, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably is at least 70%, more preferably at least 80%, and even more preferably at least At least 85%, even more preferably at least 90%, and most preferably at least 95% % or even 97% sequence identity.

[0100] In the present invention, one or more bacteria, more preferably, for example, E. coli and / or It may be preferable to use RF isolated from the bacterium. Exemplary nucleotide sequences for the multiple RFs may be selected from the group consisting of: RF1 (SEQ ID NOs: 17 and 85) RF2 (SEQ ID NOs: 19 and 87) RF3 (SEQ ID NO: 21)

[0101] In particular, the nucleotide sequence may be a sequence encoding one or more bacterial or other proteins, e.g., yeast. The codons may be optimized for expression in a protein expression system. For example, in this embodiment, Exemplary nucleotide sequences, SEQ ID NOs: 17, 19 and 21, are sequences derived from E. coli ) has been codon-optimized for expression in

[0102] In an embodiment of the invention, thus, one or more of the above nucleotide sequences also include nucleotide sequences according to SEQ ID NOs: 17, 19, 21, 85, and 87 or fragments thereof. or a variant thereof, or a nucleotide sequence encoded by the At least one coding region encoding at least one RF consisting of a sequence of In this context, the coding region of at least one of the RFs according to the present invention is The encoded protein fragment or variant thereof is typically preferably a fragment of the protein described herein. The respective naturally occurring full-length sequences according to the disclosed SEQ ID NOs: 17, 19, 21, 85, and 87 At least 5%, 10%, 20% of the nucleotide sequence of the protein or a variant thereof, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably at least 80%, even better More preferably at least 85%, even more preferably at least 90%, and most preferably or nucleotide sequences having at least 95% or 97% sequence identity. obtain.

[0103] In one embodiment, the recombinant cell-free expression system comprises a P-site following protein synthesis. Release of the remaining tRNA and subsequent lysis of the ribosome for protein synthesis Contains one or more ribosome recycling factors (RRFs) that promote cyclin In the present invention, the reaction mixture may contain a bacterial, more preferably It is preferable to use RRF from thermophilic bacteria, for example, Bacillus Bacterial families of the family Eae and / or Geobacillus, e.g., Geobaci llus subterraneus, or Geobacillus stearoth The RRFs of the present invention are obtained from, for example, E. coli ... Exemplary amino acid sequences may be selected from the group consisting of: RRF (SEQ ID NOs: 24 and 90)

[0104] In an embodiment of the invention, thus, one or more of the above amino acid sequences is Amino acid sequences according to SEQ ID NOs: 23 and 90 or any one of these amino acid sequences or an amino acid sequence encoded by a fragment or variant of In this context, one or more RRFs according to the present invention comprise at least one RRF consisting of the sequence a fragment of a protein encoded by at least one coding region of a plurality of RRFs; or The variants are typically preferably in accordance with SEQ ID NOs: 23 and 90 disclosed herein. The amino acid sequence of each naturally occurring full-length protein or a variant thereof is at least All are 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 85% ,86%,87%,88%,89%,90%,91%,92%,93%,94%,95% , 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably At least 80%, even more preferably at least 85%, even more preferably at least and most preferably at least 95% or even 97% sequence identity. It may comprise an amino acid sequence that

[0105] In the present invention, one or more bacteria, more preferably, for example, E. coli and / or It may be preferable to use RRF isolated from the bacterium. Exemplary nucleotide sequences for multiple RRFs may be selected from the group consisting of: R: RRF (SEQ ID NOs: 23 and 89)

[0106] In particular, the nucleotide sequence may be a sequence encoding one or more bacterial or other proteins, e.g., yeast. The codons may be optimized for expression in a protein expression system. For example, in this embodiment, is an exemplary nucleotide sequence, SEQ ID NO: 23 is a sequence encoding the nucleotide sequence of ... It has been codon-optimized for

[0107] In an embodiment of the invention, thus, one or more of the above nucleotide sequences are derived from nucleotide sequences according to SEQ ID NOs: 23 and 89 or fragments or variants thereof. At least one nucleotide sequence comprising or consisting of a nucleotide sequence encoded by In this context, the present invention A protein encoded by at least one coding region of one or more RFs according to or variants thereof are typically preferably SEQ ID NO: 23, as disclosed herein, and 89, the nucleotide sequence of each naturally occurring full-length protein or its variant. riant and at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% ,80%,85%,86%,87%,88%,89%,90%,91%,92%,93% , 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 70% , more preferably at least 80%, even more preferably at least 85%, even more preferably More preferably at least 90%, and most preferably at least 95% or even 97%. The fragment may include a nucleotide sequence having sequence identity with the fragment.

[0108] In one embodiment, the recombinant cell-free expression system comprises one or more aminoacyl-tRNAs The reaction mixture may include an aminoacyl-tRNA synthetase (RS) enzyme. tRNA is a tRNA by which an amino acid is covalently attached to tRNA in the presence of ATP. In the present invention, the enzyme is used to synthesize aminoacyl-tRNA. It is preferred to use an aminoacyl-tRNA synthetase derived from a nucleotide sequence such as Bacillus subtilis. from the bacterial families of the family Illaceae and / or Geobacillus, or More specifically, G. stearothermophilus or Geobacil It is obtained from the species Lus stearothermophilus. An embodiment is an aminoacyl-tRNP from a non-thermophilic organism such as E. coli. A synthetase enzyme, which may be derived from a thermophilic organism, may be used to synthesize aminoacyl- Used in conjunction with tRNA synthetase enzymes. Exemplary nucleotide and amino acid sequences are selected from the group consisting of: AlaRS (SEQ ID NO: 26 and SEQ ID NO: 92) ArgRS (SEQ ID NO: 28 and SEQ ID NO: 94) AsnRS (SEQ ID NO: 30 and SEQ ID NO: 96) AspRS (SEQ ID NO: 32 and SEQ ID NO: 98) CysRS (SEQ ID NO: 34 and SEQ ID NO: 100) GlnRS(Ec) (SEQ ID NO: 36) GluRS (SEQ ID NO: 38 and SEQ ID NO: 102) GlyRS (SEQ ID NO: 40 and SEQ ID NO: 104) HisRS (SEQ ID NO: 42 and SEQ ID NO: 106) IleRS (SEQ ID NO: 44 and SEQ ID NO: 108) LeuRS (SEQ ID NO: 46 and SEQ ID NO: 110) LysRS (SEQ ID NO: 48 and SEQ ID NO: 112) MetRS (SEQ ID NO: 50 and SEQ ID NO: 114) PheRS(a) (SEQ ID NO: 52 and SEQ ID NO: 116) PheRS(b) (SEQ ID NO: 54 and SEQ ID NO: 118) ProRS (SEQ ID NO: 56 and SEQ ID NO: 120) SerRS (SEQ ID NO: 58 and SEQ ID NO: 122) ThrRS (SEQ ID NO: 60 and SEQ ID NO: 124) TrpRS (SEQ ID NO: 62 and SEQ ID NO: 126) TyrRS (SEQ ID NO: 64 and SEQ ID NO: 128) ValRS (SEQ ID NO: 66 and SEQ ID NO: 130)

[0109] In an embodiment of the invention, thus, one or more of the above amino acid sequences is SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 92, 94, 96, 98, 1 00, 102, 104, 106, 108, 110, 112, 114, 116, 118, 1 20, 122, 134, 126, 128 and 130 or the amino acid sequences according to these amino acids The amino acid sequence encoded by any one of the fragments or variants of the amino acid sequence In this context, the RS comprises at least one RS comprising or consisting of the amino acid sequence. a tag encoded by at least one coding region of one or more RSs according to the invention; Protein fragments or variants thereof typically have the sequence numbers preferably as disclosed herein. No. 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 134, 126, 128, and 130. and at least 5%, 10%, 20%, 30%, or 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 %, preferably at least 70%, more preferably at least 80%, even more preferably or at least 85%, even more preferably at least 90%, and most preferably at least It may comprise an amino acid sequence having at least 95% or even 97% sequence identity.

[0110] In the present invention, one or more bacteria, more preferably, for example, E. coli and / or In the present invention, it may be preferable to use RS isolated from the bacterium. Exemplary nucleotide sequences for the RSs may be selected from the group consisting of: AlaRS (SEQ ID NO: 25 and SEQ ID NO: 91) ArgRS (SEQ ID NO: 27 and SEQ ID NO: 93) AsnRS (SEQ ID NO: 29 and SEQ ID NO: 95) AspRS (SEQ ID NO: 31 and SEQ ID NO: 97) CysRS (SEQ ID NO: 33 and SEQ ID NO: 99) GlnRS(Ec) (SEQ ID NO: 35) GluRS (SEQ ID NO: 37 and SEQ ID NO: 101) GlyRS (SEQ ID NO: 39 and SEQ ID NO: 103) HisRS (SEQ ID NO: 41 and SEQ ID NO: 105) IleRS (SEQ ID NO: 43 and SEQ ID NO: 107) LeuRS (SEQ ID NO: 45 and SEQ ID NO: 109) LysRS (SEQ ID NO: 47 and SEQ ID NO: 111) MetRS (SEQ ID NO: 49 and SEQ ID NO: 113) PheRS(a) (SEQ ID NO: 51 and SEQ ID NO: 115) PheRS(b) (SEQ ID NO: 53 and SEQ ID NO: 117) ProRS (SEQ ID NO: 55 and SEQ ID NO: 119) SerRS (SEQ ID NO: 57 and SEQ ID NO: 121) ThrRS (SEQ ID NO: 59 and SEQ ID NO: 123) TrpRS (SEQ ID NO: 61 and SEQ ID NO: 125) TyrRS (SEQ ID NO: 63 and SEQ ID NO: 127) ValRS (SEQ ID NO: 65 and SEQ ID NO: 129)

[0111] In particular, the nucleotide sequence may be a sequence encoding one or more bacterial or other proteins, e.g., yeast. The codons may be optimized for expression in a protein expression system. For example, in this embodiment, are exemplary nucleotide sequences, SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, and and 65 are codon-optimized for expression in E. coli.

[0112] In an embodiment of the invention, thus, one or more of the above nucleotide sequences are SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 4 7, 49, 51, 53, 55, 57, 59, 61, 63, 65, 91, 93, 95, 97 , 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, and 129 or the nucleotide sequences thereof a fragment or variant of a nucleotide sequence encoding the nucleotide sequence of the nucleotide sequence at least one coding region encoding at least one RS consisting of a nucleotide sequence In this context, the coding region of at least one of the RSs according to the invention Fragments of proteins encoded by the present invention are typically and preferably fragments of the proteins encoded by the present invention. SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 disclosed in the specification; 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 91, 93, 9 5, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, and 129, respectively. The nucleotide sequence of the full-length protein occurring in %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99%, preferably at least 70%, more preferably at least 80% %, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or even 97% sequence identity. It may contain a sequence of

[0113] In one embodiment, the recombinant cell-free expression system comprises a methionyl-tRNA transforming The reaction mixture may contain N-formylmethionine, which is a methionine-binding protein. The amino group at the end of the amino acid has a formyl group attached to it, and the amino acid is then used in prokaryotic protein synthesis systems. This formyl group is converted to methionyl-t by MTF. MTF is an N-formyltetrahydrochloride that binds to methionine in RNA. The formyl group in folic acid is transferred to the N-terminus of the methionyl-tRNA corresponding to the initiation codon, This gives formylmethionyl-tRNA. The added formyl group is It is recognized by IF2 and functions as a start signal for protein synthesis. In this case, MTF from bacteria, more preferably from thermophilic bacteria, is used. are preferred, for example Bacillaceae and / or Geobaci Bacterial family of the family Illus, e.g., Geobacillus subterraneu s, or Geobacillus stearothermophilus, etc. Exemplary amino acid sequences for one or more MTFs of the present invention are as follows: may be selected from the group consisting of: MTF (SEQ ID NOs: 68 and 132)

[0114] In an embodiment of the invention, thus, one or more of the above amino acid sequences is Amino acid sequences according to SEQ ID NOs: 68 and 132 or any one of these amino acid sequences or an amino acid sequence encoded by a fragment or variant thereof In this context, one or more MTFs according to the invention comprise at least one MTF consisting of the sequence A fragment of a protein encoded by at least one coding region of one or more MTFs or Variants of the present invention are typically preferably in accordance with SEQ ID NOs: 68 and 132 disclosed herein. The amino acid sequence of each naturally occurring full-length protein or a variant thereof is at least All are 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 85% ,86%,87%,88%,89%,90%,91%,92%,93%,94%,95% , 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably At least 80%, even more preferably at least 85%, even more preferably at least and most preferably at least 95% or even 97% sequence identity. It may comprise an amino acid sequence that

[0115] In the present invention, one or more bacteria, more preferably, for example, E. coli and / or In the present invention, it may be preferable to use MTF isolated from the bacterium. Exemplary nucleotide sequences for multiple MTFs may be selected from the group consisting of: R: MTF (SEQ ID NOs: 67 and 131)

[0116] In particular, the nucleotide sequence may be a sequence encoding one or more bacterial or other proteins, e.g., yeast. The codons may be optimized for expression in a protein expression system. For example, in this embodiment, is an exemplary nucleotide sequence, SEQ ID NO: 67 is a sequence encoding the nucleotide sequence for expression in E. coli. It has been codon-optimized for

[0117] In an embodiment of the invention, thus, one or more of the above nucleotide sequences is a nucleotide sequence according to SEQ ID NOs: 67 and 131 or a fragment or variant thereof At least one of the nucleotide sequences encoding the In this context, the present invention also includes at least one coding region encoding one or more MTFs. A protein encoded by at least one coding region of one or more MTFs according to the invention. Protein fragments or variants thereof typically comprise a fragment of a protein, preferably one of the SEQ ID NOs: 67, and 131, respectively, the nucleotide sequences of the full-length naturally occurring proteins has at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% ,70%,80%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least more preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, Even more preferably at least 90%, and most preferably at least 95% or 97% The nucleotide sequence may contain nucleotide sequences with at least 10% sequence identity.

[0118] In one embodiment, the recombinant cell-free expression system comprises a reaction mixture having a quantity of ribosomes. Ribosomes are the particles in which peptides are synthesized. It binds to mRNA and attaches aminoacyl-tRNA to the A-site and formylmethionyl-tRNA to the ATP-site. Ligating RNA or peptidyl-tRNA to the P-site, thereby forming a peptide bond In the present invention, any ribosome can be used regardless of its origin. However, in a preferred embodiment, the ribosomes are expressed in a recombinant cell-free expression system. For use, the enzyme may be isolated from a thermophilic bacterium, and preferably from a bacterium such as Bacillus subtilis. Bacterial families of the family Geobacillaceae and / or Geobacillus, e.g., Geobacil lus subterraneus, or Geobacillus stearothe It can be isolated from cell lysates of thermophilic bacteria, such as from R. rhamophilus.

[0119] In one embodiment, recombinant cell-free expression systems can be used to express DNA sequences that occur in a variety of organisms. A quantity of RNA polymerase, an enzyme that transcribes RNA, or a fragment or barrier thereof In one preferred embodiment, the reaction mixture may include a mixture of: The present invention includes, for example, a T7 RNA polymerase according to the amino acid sequence SEQ ID NO: 136. T7 RNA polymerase binds to a specific DNA sequence called the T7 promoter. Derived from the T7 phage, an enzyme that binds and then transcribes downstream DNA sequences into RNA In the present invention, in addition to T7 RNA polymerase, various RNA polymerases are Available.

[0120] In one embodiment, the recombinant cell-free expression system comprises an amount of a ribonuclease inhibitor. The reaction mixture may include a ribonuclease (RNase) enzyme that degrades the oligonucleotides of RNA. Ribonuclease inhibitors are known in the art and are Therefore, the type and amount of ribonuclease inhibitor to be included in the recombinant cell-free expression system should be Non-limiting examples of ribonuclease inhibitors include mammalian ribonuclease inhibitors. Ribonuclease inhibitor proteins of the substance [e.g., porcine ribonuclease inhibitor and human Ribonuclease inhibitors (e.g., human placental ribonuclease inhibitor and recombinant human ribonuclease inhibitor) nuclease inhibitors)], aurintricarboxylic acid (ATA) and its salts [e.g., aurintricarboxylic acid (ATA) triammonium phosphate tricarboxylate (aluminon)], adenosine 5'-pyrophosphate ( adenosine 5'-pyrophosphate), 2'-cytidine monophosphate 5'-diphosphoadenosine 3'-phosphate (5'-diphosp hoadenosine 3'-phosphate) (ppA-3'-p), 5'-di 5'-diphosphoadenosine 2'-phosphate phosphate) (ppA-2'-p), leucine, oligovinylsulfonic acid (oli govinysulfonic acid), poly(aspartic acid), tyrosine-glucan Amino acid polymer, adenosine 3'-phosphate 5'-phospho-2'-deoxyuridine 3'-pyrophosphate P' → 5'-enyl ester Stereo(5'-phospho-2'-deoxyuridine 3'-pyroph phosphate P'→5'-ester) (pdUppAp) and its analogs , derivatives, and salts.

[0121] In one embodiment, the recombinant cell-free expression system is typically a plasmid synthesis template or a vector. A template encoding the target sequence in the form of a synthetic expression (or LST) template, e.g., m A quantity of amino acids, polynucleotides, such as RNA or DNA templates, and inorganic polyphosphates Other compounds and sequences identified in the '121 application related to acid salt energy regeneration systems; and preferably may be necessary to energetically drive an in vitro expression reaction. The reaction mixture may include a bound AdK / PPK energy regeneration system.

[0122] As shown generally in FIG. 8 of the '121 application (incorporated herein by reference), another In a preferred embodiment, isolated and purified Gst AdK (as herein incorporated by reference) is used. SEQ ID NO:8 of the '121 application, incorporated herein by reference) and / or TaqPPK (incorporated herein by reference). SEQ ID NO: 11 of the '121 application, which is incorporated herein by reference, describes this inorganic polyphosphate containing an amount of inorganic polyphosphate. In one embodiment, the amount of inorganic polyphosphate is optimally In this preferred embodiment, the optimum polyphosphate concentration range may be The polyphosphate concentration range is generally determined to be within the range of inorganic polyphosphate (P) that maintains an equilibrium state for reaction stability. In this preferred embodiment, the optimal polyphosphate concentration is defined as the concentration of The concentration range can be approximately 0.2-2 mg / ml of PPi.

[0123] As described above, PPK synthesizes ADP from polyphosphate and AMP. In this preferred embodiment, the combined Gst AdK and PPK It converts two ADPs into one ATP and one adenosine monophosphate (AMP) By doing so, adenosine diphosphate (ADP) can be removed from the system. [ka]

[0124] This reaction is fast enough to drive the equilibrium reaction of PPK toward the production of ADP. It can be. [ka]

[0125] In this system, the presence of higher concentrations of AMP inhibits the TaqPPK reaction toward ADP. can be further driven.

[0126] In a preferred embodiment, the production of macromolecules using the recombinant cell-free system of the present invention comprises: This can be achieved in a bioreactor system. The "microreactor" is configured to maintain an environment conducive to the production of macromolecules in vitro. The bioreactor can be any form of enclosed device. Depending on the reaction solution, it may be configured to operate batchwise, continuously, or semi-continuously. 14 of the application (which is incorporated herein by reference), in this embodiment The present invention may further include a cell-free culture device. In one preferred embodiment, the cell culture device In a preferred embodiment, the invention can be adapted to culture thermophilic bacteria. The fermentation vessel is removable and can be autoclaved independently. Furthermore, this cell-free culture device can be used not only for aerobic culture with organisms but also for Furthermore, cell-free expression bioreactors and All cell-free culture devices are adaptable to various cell cultures, such as microalgae and plant cells. .

[0127] In one embodiment, the present invention provides a continuous exchange or flow bioreactor (1). In this preferred embodiment, the continuous exchange production device in continuous flow operations to process biologicals, vaccines, proteins, enzymes, In vitro transcription, in vitro translation, and in vitro biosynthesis of biosimilars and enzymes Multiple fibers and hollow fibers as exchange media for biosynthesis or chemical modification of small molecules using The system may include a bioreactor.

[0128] Generally, referring to FIG. 5, a continuous flow bioreactor device is a continuous flow operation. In vitro transcription and immunoassay of biologics, proteins, enzymes, and biosimilars in In vitro translation and in vitro biosynthesis, as well as enzymatic biosynthesis or chemical modification of small molecules One or more hollow fibers (2) as an exchange medium for the hollow fiber-based bioreactor In this embodiment, the continuous transfer of the substrate as described herein may include The feed can be introduced into the device and the inner and outer compartments of the hollow fiber reactor (2) This can further involve the removal of reaction products via a concentration gradient between the inner Biological materials and biologically modified organisms that can be isolated from the "flow-through" solution of the compartments. Allows for long run and batch independent production of materials.

[0129] The operation of an exemplary hollow fiber reactor (2) is described as shown in Figures 5A and 5B. In this embodiment, the feed solution is pushed through the inner compartment (3) of the reactor. , the permeability of the fiber allows the substrates for mRNA synthesis (nucleotides), usually proteins (amino acids), substrates (for in vitro biosynthesis or chemical modification of compounds), and ribonucleotides. ATP and G (via nucleotide kinases, e.g., NDPK) for the operation of the ATPase The ATP regeneration system, as incorporated herein by reference from the '121 application, provides a continuous The outer compartment (4) allows for continuous exchange of in vitro transcription and in vitro Contains enzymes and factors that drive translation and in vitro biosynthetic reactions. Proteins, enzymes, and larger biological products can be isolated and purified in a closed loop system as shown in Figure 5B. This closed loop system prevents and minimizes the risk of potential product contamination, spillage, or exposure. and / or reduce the amount of processing required and production capacity for any type of biological product. Reduced laboratory footprint. Linearly increasing reactor volume allows for It is adaptable from research-scale biosynthesis to industrial-scale manufacturing, thereby accelerating development efforts and This reduces the costs associated with scaling processes and development timelines.

[0130] As used herein, recombinant cell-free expression in vitro refers to expression in biological extracts and / or The present invention relates to cell-free synthesis of polypeptides in a reaction mixture or solution containing defined cell-free reaction components. The reaction mixture contains a template or a target molecule for macromolecule production, e.g., DNA, mRNA, etc. is a genetic template; e.g., amino acids, nucleotides, etc. a monomer for the macromolecule to be synthesized, which is a cofactor, an enzyme, and other reagents required for synthesis, such as ribosomes, tRNA, polymerase, transcription factors, etc. Recombinant cell-free synthesis reactions and / or cellular adenosine triphosphate (ATP) enzymes may be used. The components of the energy regeneration system, which are incorporated herein by reference, are a batch continuous flow It can be implemented / applied as a continuous or semi-continuous flow.

[0131] Some of the target proteins that can be expressed by the present invention include vaccines, eukaryotic peptides, and the like. Tides, prokaryotic peptides, bacterial-associated peptides, fungal-associated peptides, yeast-associated, human-associated peptides, plant-related peptides, toxic peptides, vasoactive intestinal peptides, vasopressin Peptides, novel or engineered peptides, virus-related peptides, bacteriophages Phage-related proteins, hormones, antibodies, cell receptors, cell regulatory proteins, and the above These may include, but are not limited to, fragments of any of the listed polypeptides.

[0132] The present invention involves the production of genetically modified organisms and involves recombinant DNA technology. Therefore, the following definitions are provided to aid in the description of the present invention.

[0133] As used herein, the terms "isolated," "purified," or "biologically pure" The term "non-food" refers to the term that is normally associated with the material in its original state or when it is produced. It refers to a material that is substantially or essentially free of the components that accompany it. For the preparation of pharmaceuticals, purity and homogeneity may be assessed by polyacrylamide gel electrophoresis or high performance liquid chromatography. The nuclei are the predominant species present in the preparation and are determined using analytical chemistry techniques such as raphy. The acid or specific bacterium is substantially purified. In an exemplary embodiment, "purified" The term "single band" refers to a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. Typically, an isolated nucleic acid or protein is a protein that is expressed in a range of ways. The lower end of the purity range for this component is about 60%, about 70%, or about 80%. and the upper end of the range of purity is about 70%, about 80%, about 90%, or greater than about 90%. .

[0134] In a preferred embodiment, the result of the cell-free expression system is a peptide, for example. In this embodiment, the target Protein solation or purification, wherein the target protein is e.g. Precipitation with organic solvents, for example precipitation with methanol, ethanol or acetone, with organic or inorganic salts such as trichloroacetic acid (TCA) or ammonium sulfate precipitation precipitation, e.g., non-ionic polymer precipitation such as polyethylene glycol (PEG) precipitation; pH precipitation, temperature precipitation, immunoprecipitation, e.g. adsorption, ion exchange, affinity and gel exclusion precipitation Chromatography, chromatofocusing, isoelectric focusing, high performance liquid chromatography Chromatographic separation, such as high performance liquid chromatography (HPLC), gel electrophoresis, dialysis, and microfiltration, at least partially separated from at least one other component in the reaction mixture.

[0135] As used herein, the term "active" refers to a peptide that binds to the full-length (complete) protein. or a part thereof. Functional activity includes catalytic or enzymatic activity, antigenicity, etc. (ability to bind or compete with the polypeptide for binding to anti-polypeptide antibodies), immunological The ability to bind to a receptor or ligand for the polypeptide, including its affinity, ability to form multimers, and specificity for the receptor or ligand for the polypeptide. Preferably, the produced protein activity of at least 100% of the initial activity at temperatures between about 0°C and 30°C for at least 3 days. Maintain 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95% or higher do.

[0136] As used herein, the term "nucleic acid" refers to a ribonucleotide or deoxyribonucleic acid. Typically, a "nucleic acid" polymer is in either single-stranded or double-stranded form. Although they occur in a single chain, they are also known to form structures containing three or more chains. are known nucleotides, including naturally occurring nucleic acid polymers as well as synthetic, natural, and non-natural nucleotides. Nucleic acids containing analogs or modified backbone residues or linkages, which are similar to the reference nucleic acid They have similar binding properties and are metabolized in a manner similar to the reference nucleotide. Exemplary analogs are: These include phosphorothioates, phosphoramidates, methylphosphonates, chiral methyl These include phosphonic acids, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). "DNA," "RNA," "polynucleotide," "polynucleotide" "nucleotide sequence," "oligonucleotide," "nucleotide," "nucleic acid," "nucleic acid molecule" The terms "nucleic acid sequence," "nucleic acid fragment," and "isolated nucleic acid fragment" are used herein to distinguish between the terms "nucleic acid sequence," "nucleic acid fragment," and "isolated nucleic acid fragment." For nucleic acids, sizes are expressed in either kilobases (kb) or base pairs (bp). Estimation is typically performed by agarose or acrylamide gel electrophoresis. Derived from sequenced nucleic acids or from published DNA sequences. Sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are from gel electrophoresis, from sequenced proteins, from the amino acid sequence of origin, or is predicted from the published protein sequence.

[0137] As used herein, the term "target protein" generally refers to a protein having about five or more amino acids. Polypeptides refer to any peptide or protein in which bacteria can produce Produced in a host cell, such as a bacterial cell, e.g., E. coli. It may be homologous to a bacterial protein or yeast protein, or preferably They may be exogenous, meaning they are heterologous, i.e., foreign, to the bacterium. Preferably, mammalian polypeptides, viral, bacterial, and / or human polypeptides are used. Bacterial and engineered polypeptides are used.

[0138] As is known in the art, different organisms use different methods to produce polypeptides. These "codon usage" choices depend on the particular host cell system. The nucleic acid molecules encoding the proteins and chimeras of the invention are It can be used in the design of

[0139] All nucleotide sequences described in this invention may be modified for expression in a particular organism. Codon optimization can be performed for the gene of interest or for increased yield. Improves protein expression by increasing gene translation efficiency. Gene functionality was also increased by optimizing codon usage within custom-designed genes. In a codon optimization embodiment, low frequency codons in a species can be It can be substituted with a frequent codon, for example, for leucine, the rare codon UUA can be substituted with the frequent codon The codon can be replaced by the codon CUG. Codon optimization can increase mRNA stability, Therefore, the rate of protein translation or protein folding may be altered. Codon optimization can customize transcriptional and translational regulation, or ribosome binding sites. may be modified or mRNA degradation sites may be stabilized.

[0140] Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants thereof (e.g., For example, degenerate codon substitutions), complementary (or complementary) sequences, and reverse complementary sequences. It implicitly includes not only the (generic complement sequence) but also the explicit sequence. In particular, degenerate codon substitutions include substitutions of one or more selected (or all) codons. The third position of the amino acid sequence is replaced with a mixed-base and / or deoxyinosine residue. This can be achieved by generating (e.g., Batzer et al., Nucle ic Acid Res.19:5081(1991);Ohtsuka et al. , J. Biol. Chem. 260:2605-2608 (1985); and Rosso lini et al.,Mol.Cell.Probes 8:91-98(1994 )). The degeneracy of nucleotide codons that code for amino acids In addition to the natural sequence, the amino acid sequence also results in the production of chemically equivalent amino acids at a given site. Polynucleotides that result in the formation of polypeptides that do not affect the functional properties of the encoded polypeptide. Modifications in amino acids are well known in the art. "Conservative amino acid substitutions" are defined as Such substitutions are predicted to have the least interference with the properties of the polypeptide. The targeted amino acid substitutions substantially preserve the structure and function of the reference protein. The codon for the amino acid alanine, a hydrophobic amino acid, is e.g., glycine. Another residue that is less hydrophobic, such as valine, leucine, or isoleucine. Codons encoding more hydrophobic residues may be substituted. Charged residues to other amino acids, e.g., aspartic acid to glutamic acid, etc. or substitution of one positively charged residue for another amino acid, e.g., arginine or Changes resulting in substitutions such as lysine for histidine also produce functionally equivalent proteins. Conservative amino acid substitutions can be expected to produce proteins or polypeptides. Conservative amino acid substitutions are generally known to those skilled in the art. or alpha-helical conformation of the polypeptide in the substituted region. (b) the structure of the backbone, (b) the charge or hydrophobicity of the molecule at the site of substitution, and / or (c) the size of the side chain. Part, maintain.

[0141] Homology (e.g., percent homology, sequence identity + sequence similarity) is calculated by pairwise sequence alignment. The homology can be determined using any homology comparison software that calculates the homology. When used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to "Identity" includes reference to residues in two sequences that are the same when aligned. When percentage sequence identity is used in the context of proteins, residue positions that do not match are often Conservative amino acid substitutions are used to identify amino acid residues that have similar chemical properties (e.g., The functional properties of the molecule are affected by substitution of other amino acid residues with different properties (e.g., charge or hydrophobicity). It is recognized that the quality of the sequence remains unchanged. If the sequences differ by conservative substitutions, The percent identity may be adjusted upwards to reflect the conservative nature of the substitutions. Sequences that differ in their sequence are said to have "sequence similarity" or "similarity." Means for this are well known to those skilled in the art. Typically, this means that there is a mismatch rather than a complete mismatch. Conservative substitutions are scored as a fraction, thereby increasing the percentage of sequence identity. Therefore, for example, a matching amino acid will receive a score of 1, and a non-conservative substitution will receive a score of 2. Conservative substitutions are given a score between 0 and 1, with a score of 0 being given. Coring is performed using, for example, the algorithm of Henikoff S and Henikoff JG. It is calculated according to the following formula: [Amino acid substitution matr ices from protein blocks.Proc.Natl.Acad. Sci.USA1992,89(22):10915-9].

[0142] According to certain embodiments, the homologous sequence is a sequence (nucleic acid or amino acid) At least 60%, 65%, 70%, 75%, 80%, 85%, 90% of the total amino acid sequence %, 95%, 99%, or 50% to 99% identical homologues of SEQ ID NOs: 1 to 22 Sequences may be included in certain embodiments of the present invention.

[0143] As used herein, the term "primer" refers to a point at which DNA synthesis can be initiated under suitable conditions. The conditions include the oligonucleotides in a suitable buffer solution. and at a suitable temperature, four different nucleoside triphosphates and reagents for elongation (e.g., a primer complementary to a nucleic acid strand in the presence of a DNA polymerase or reverse transcriptase - those that induce the synthesis of extension products.

[0144] Preferably, the primer is a single-stranded DNA. Typically, the length of the nucleus is about 6 to about 225, including the middle region, depending on the intended use of the nucleus. nucleotides, such as 15-35 nucleotides, 18-75 nucleotides, and 25-150 nucleotides Short primer molecules generally have a sufficiently stable bond with the template. A lower temperature is required to form the complex. The primer binds to the correct position of the template nucleic acid. It need not mirror the sequence, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for amplification of a given target sequence is well known in the art. , as described in the references cited herein.

[0145] As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides. "DNA polymerase" catalyzes the polymerization of deoxyribonucleotides. NA polymerases include, inter alia, those derived from, for example, Pyrococcus furiosus (P fu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase "RNA polymerase" catalyzes the polymerization of ribonucleotides. The aforementioned examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases are also included in the DNA polymerase family. Reverse transcriptase activates the viral polymerase encoded by retroviruses. RNA polymerase ("RNP") is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerases ("APs") include, inter alia, T3 RNA polymerase, T7 RNA polymerase, A polymerase, SP6 RNA polymerase and E. coli RNA polymerase The aforementioned examples of RNA polymerases also include DNA-dependent RNA polymerases. The polymerase activity of any of the above enzymes is known as a polymerase. This can be determined by means well known in the art.

[0146] As used herein, "reaction mixture" or "cell-free reaction mixture" refers to a The term "recombinant cell-free reaction mixture" or "recombinant cell-free reaction mixture" refers to a given The term "reaction mixture" refers to a solution containing the reagents necessary to carry out the reaction of a cell-free expression system. The "product" or "reaction solution" is typically a crude or partially purified extract, (e.g., bacterial a nucleotide translation template (e.g., from a plant cell, microalgae, fungus, or mammalian cell), and It contains a reaction buffer suitable for promoting cell-free protein synthesis from the translation template. In one embodiment, the CF reaction mixture can include an exogenous RNA translation template. In other embodiments, the CF reaction mixture contains a primer for a DNA-dependent RNA polymerase. DNA encoding an open reading frame operably linked to a promoter element In these other embodiments, the CF reaction mixture may also include: DNA that directs transcription of an RNA translation template encoding an open reading frame In these other embodiments, further dependent RNA polymerases may be included. NTPs and divalent cation cofactors can be included in the CF reaction mixture. A reaction mixture is considered complete if it contains all the reagents necessary to enable the reaction. If it contains only a subset of the required reagents, it is called incomplete. The components are always stored as separate solutions, each containing a subset of the total components. This may be for convenience, shelf life, or to allow application-dependent adjustment of ingredient concentrations. This is because the reaction components are combined to form a complete reaction mixture prior to reaction. It will be understood by those skilled in the art that the reaction components may be packaged separately for commercial sale. It is understood that useful commercial kits may contain any subset of the reaction components of the present invention. It will be understood by those skilled in the art that some components in the reaction mixture may be present. While utilized in some embodiments, it is not required for cell-free expression product production. It will be understood that the term "cell-free expression product" refers to any product produced through a cell-free expression system. It can be any biological product.

[0147] The terms "about" or "approximately" are used to describe, for example, a described concentration, length, molecular weight, pH, time frame, It means that a temperature, pressure, volume, etc., is within a statistically significant range of values. The value or range is typically within 20%, more typically within 10%, of a given value or range. It can be within a single order of magnitude, and even more typically within 5%. The permissible variation encompassed by "about" or "approximately" is indicative of the specific system under test. "Contains," "has," "includes," and "contains" The term "things" should be construed as open-ended (i.e., (i.e., "including but not limited to").

[0148] The recitation of ranges of values ​​herein simply refers to each separate value that falls within that range. are intended to serve as a shorthand way of referring to each individually, and are used throughout this specification. Unless otherwise indicated, the range is inclusive of both endpoint boundaries and each distinct value is an integer. Each of these is incorporated herein by reference as if individually recited herein. This is what is done.

[0149] For example, when used in reference to a cell, or a nucleic acid, protein, or vector, The terms "recombinant" or "genetically modified" refer to a cell, organism, nucleic acid, protein, or The vector is capable of transferring a heterologous nucleic acid or protein or a native nucleic acid or protein. that the cell has been modified by altering a protein, or that the cell has been so modified That is, for example, a recombinant cell is derived from a cell that is naturally (non-recombinantly) It is possible to express genes not found in the normal (e.g., wild-type) form of the gene, or to express genes in another way. Abnormally expressed, over-expressed, under-expressed, or not expressed at all in the The gene can be expressed.

[0150] As used herein, the terms "transformed" or "genetically modified" refer to the process by which a cell The term refers to the transfer of one or more nucleic acid molecules into a microorganism. The microorganism is capable of stably replicating the nucleic acid molecule. A bacterium or cell or organism is "transformed" when it becomes As used herein, "transformed" or "genetically modified" means The term "genetically modified" refers to a nucleic acid molecule that has been engineered to be a nucleic acid molecule of a cell or organism, e.g., a bacterium. This includes all technologies that can be introduced into

[0151] As used herein, the term "promoter" can be upstream from the start of transcription, or D, which may be involved in the recognition and binding of RNA polymerase and other proteins that initiate transcription A promoter is a region of a gene that allows a coding sequence to function for expression in a cell. The promoter can be operably linked to a coding sequence for expression in a cell. It may be operably linked to a nucleotide sequence encoding a signal sequence to which it can bind.

[0152] The term "operably linked" when used in reference to regulatory and coding sequences means that the It is meant that a regulatory sequence influences the expression of a linked coding sequence. " or "regulatory element" refers to a gene that regulates transcription, RNA processing or stability, or related refers to a nucleotide sequence that influences the timing and level / amount of translation of a code sequence Regulatory sequences include promoters; translation leader sequences; introns; enhancers; It contains a loop structure, a repressor or binding sequence, a termination sequence, a polyadenylation recognition sequence, etc. Certain regulatory sequences may be located upstream and downstream of the coding sequence to which they are operably linked. Also, certain regulatory sequences operably linked to a coding sequence may be located downstream or / and downstream. It may be located on the associated complementary strand of a double-stranded nucleic acid molecule.

[0153] As used herein, the term "genome" refers to the chromosomal DNA found in the nucleus of a cell. It also refers to organelle DNA found within the intracellular compartments of cells. The term "genome" in this specification refers to both the chromosome and the plasmids in a bacterial cell. In this embodiment, the DNA molecule is integrated into the genome of the bacterium. In these and further embodiments, The DNA molecule is integrated onto the chromosome as or in a stable plasmid. It can be either embedded or located.

[0154] The term "gene" or "sequence" refers to any gene product (e.g., a polypeptide or is operably linked to an appropriate regulatory sequence capable of regulating the expression of functional RNA A gene is a coding region (open reading frame, ORF) ), as well as the code that precedes (upstream) and follows (downstream) the Intervening sequences (i.e., introns) between regions (i.e., exons) of DNA This includes untranslated regulatory regions (e.g., promoters, enhancers, repressors, etc.). As used herein, the term "structural gene" refers to a gene that is followed by the amino acid sequence of a specific polypeptide. It is intended to mean a DNA sequence that is transcribed into mRNA that is translated into a specific sequence. .

[0155] As used herein, "expression" or "expression of a coding sequence" (e.g., a gene or transgene) refers to The term "gene" thereby refers to a nucleic acid transcription unit (e.g., including genomic DNA or cDNA). The encoded information in the cells (including the nucleus) is converted into functional, non-functional, or structural parts of the cell. Gene expression is the process by which genes are expressed, often including the synthesis of proteins. For example, by exposing a cell, tissue, or organism to an agent that increases or decreases gene expression. Gene expression can also be affected by changes in the pathway from DNA to RNA to protein. Regulation of gene expression can occur at any point in the body, including transcription, translation, and RNA export. through regulation of the transport and processing of proteins, and the degradation of intermediate molecules such as mRNA; or After that, activation, deactivation, and compartmentalization of specific protein molecules are performed. through mentalization, or decomposition, or a combination thereof Gene expression was measured by Northern blot, RT-PCR, and Western blot. or in vitro, in situ or in vivo protein activity assays, RNA levels or protein levels can be measured by any method known in the art, including, but not limited to, It can be measured at the protein level.

[0156] The term "vector" refers to a vector that carries DNA, RNA, protein, or polypeptide. refers to any means by which a polypeptide to be introduced into a host can be introduced. The peptides, proteins and polypeptides may be therapeutic or prophylactic in nature. It may be codable or antigenic; regulatable in nature, etc. Various types of vectors, including viruses, plasmids, bacteriophages, cosmids, and bacteria There is a tar.

[0157] An "expression vector" is a nucleic acid that is replicable in a selected host cell or organism. Expression vectors may be replicable as autonomous structures or alternatively may be whole or multi-organisms. Partially, it can be integrated into the host cell chromosome or into the organelle nucleic acid, or It is a shuttle that delivers foreign DNA into cells, where it replicates along with the host cell's genome. That is, expression vectors can be, for example, plasmids, viruses, artificial chromosomes, etc. a nucleic acid fragment capable of replicating in a selected host cell, organelle or organism; and for that purpose, a gene (including a gene of interest) on the expression vector is expressed in the cell, Polynucleotides that are transcribed and translated into polypeptides or proteins within a luganella or organism. any suitable construct known in the art containing an "expression cassette"; On the other hand, as described in the examples of the present specification, a "cassette" is a A polynucleotide containing a portion of an expression vector of the invention. The use of the cassette is It aids in the construction of expression vectors. Expression vectors can be, for example, plasmids, A replicon such as a gene, virus, chimeric virus or cosmid, which is an expression control It contains the desired polynucleotide sequence operably linked to the sequence.

[0158] The methods outlined herein in connection with protein expression in cell-free expression systems are The term "expression product" is used interchangeably when referring to a gene having more than about five amino acids. Polypeptides refer to any peptide or protein produced in a cell-free extract. For example, human proteins, plant proteins, viral proteins, yeast proteins, etc. The cell-free extract may be homologous to the organism from which it is derived or may be exogenous. may be present, meaning that they are heterologous, i.e., foreign, to the organism. In some embodiments, the term "derived from" refers to a substance extracted or derived from a bacterium. means expressed and isolated. For example, in one embodiment, the protein is , which may be derived from a thermophilic bacterium, which is endogenous to the thermophilic bacterium and from the bacterium Individual proteins or cells isolated from different bacteria or heterologously expressed in different bacteria It may refer to a protein that has been isolated as an extract.

[0159] "Cell-free extracts" or "lysates" are those derived from bacteria, thermophilic bacteria, thermotolerant bacteria, archaea, fungi, and fungi. Species including Micutes, fungi, algae, microalgae, plant cell cultures, and plant suspension cultures The nucleic acid may be derived from various organisms and / or cells.

[0160] As used herein, the singular forms "a," "and," and "the" are used interchangeably where the context requires. Unless otherwise specified, plurals are included, i.e., "a cell" and "a cell" are not included. and "a culture of" includes one or more such cells. includes descriptions of multiple cultures and equivalents thereof known to those skilled in the art, etc. All technical and scientific terms used herein are intended to be understood as meaning the principles of the present invention unless otherwise specified. It has the same meaning as commonly understood by a person skilled in the art to which it pertains.

[0161] The invention generally described herein will be more readily understood by reference to the following examples. It will be understood that the examples are provided merely for the purpose of illustrating certain aspects of embodiments of the present invention. Other techniques and methods may satisfy the claims, and other techniques and methods are also included. and methods may be employed without departing from the scope of the claimed invention. Those skilled in the art will be able to recognize from the above teachings and the examples below. As such, the examples are not intended to limit the invention. While shown and described herein with reference to preferred embodiments thereof, the scope of the appended claims is limited to the present invention. Descriptive variations in form and details may be made therein without departing from the scope of the invention encompassed therein. Those skilled in the art will understand that variations can be made.

[0162] The invention generally described herein will be more readily understood by reference to the following examples. It will be understood that the examples are provided merely for the purpose of illustrating certain aspects of embodiments of the present invention. Other techniques and methods may satisfy the claims, and other techniques and methods are also included. and methods may be employed without departing from the scope of the claimed invention. Those skilled in the art will be able to recognize from the above teachings and the examples below. As such, the examples are not intended to limit the invention. While shown and described herein with reference to preferred embodiments thereof, the scope of the appended claims is limited to the present invention. Descriptive variations in form and details may be made therein without departing from the scope of the invention encompassed therein. Those skilled in the art will understand that variations can be made. [Example]

[0163] Example 1: Protein synthesis and cloning for recombinant cell-free expression systems The present inventors have prepared multiple recombinant core proteins, preferably or multiple recombinant core proteins from selected thermophilic bacteria in synthetic and selected expression vectors. In this embodiment, the inventors cloned multiple core recombinant thermophilic initiating The factors (IFs) were cloned into synthetic and selectable expression vectors. The present inventors have synthesized and synthesized multiple core recombinant thermophilic elongation factors (EFs) in a selection expression vector. In this embodiment, the inventors cloned multiple core recombination termination factors ( RF) was cloned into a synthetic and selected expression vector. The inventors synthesized and synthesized at least one core recombinant ribosome recycling factor (RRF). and cloned into a selection expression vector. In this embodiment, the inventors Synthesis and selection of core recombinant aminoacyl-tRNA-synthetase (RS) expression vectors In this embodiment, the inventors cloned at least one core recombinant into Methionyl-tRNA transformylase (MTF) is synthesized and placed in a selectable expression vector. was cloned into

[0164] As outlined in Table 1, in a preferred embodiment, the inventors provide a composition of the present invention. Exemplary recombinants of at least 34 proteins that can be applied to recombinant cell-free expression systems At least one of the nucleotide and / or amino acid sequences forming the core protein mixture At least 12 different recombination factors, as well as at least 12 different nucleotide and / or amino acid sequences, are included. Two recombinant synthetases (SEQ ID NOs: 1 to 132) were synthesized, cloned, and transformed into Escherichia coli ( These core proteins were expressed in E. coli and purified. In the expression vector, for example, pET151 / D-TOPO (pET151) , pET24a(+), or pNAT.

[0165] The inventors have identified one or more of the 34 proteins as well as exemplary thermophilic Recombinant cell-free reaction mixtures incorporating selected ribosomes and tRNA isolated from bacteria Next, the inventors introduced a quantity of RNA polymerase into the recombinant cell-free reaction mixture. polymerases, particularly T7 RNA polymerase enzymes, as well as exemplary amino acids and As noted above, the present inventors have disclosed in PCT Application No. PCT / US2018 Inorganic polyphosphate energy compounds specified in the claims of / 012121 ('121 application) - Further producing a recombinant cell-free reaction mixture incorporating one or more of the components of the regeneration system. did.

[0166] Example 2: Generation of an exemplary recombinant cell-free reaction mixture In one embodiment, the inventors have discovered an in vitro method for detecting leukemia cells selected from the group consisting of: A recombinant cell-free reaction mixture capable of transcription and translation was generated: - a reaction mixture of at least 33 thermophilic core proteins identified in Table 1; - one core protein from E. coli identified in Table 1; -tRNA from thermophiles; - a quantity of ribosomes isolated from selected thermophilic organisms; - a certain amount of amino acids; - a certain amount of nucleotide triphosphates (NTPs), such as ATP, CTP, GTP, TTP, etc. ); an amount of reaction buffer; and -The claims, drawings, sequences, and specifications of the '121 application, which are incorporated herein by reference. Energy regeneration or energy recovery from inorganic polyphosphates as specified in the document. One or more components of the system.

[0167] Example 3: Activity of recombinant aminoacyl-tRNA-synthetases The present inventors have investigated the activity of each of the purified aminoacyl-tRNA-synthetases (RS). Generally, the aminoacyl-tRNA synthetase reaction proceeds in the following two steps: It is legal: Step 1: Activation of amino acid + ATP => aminoacyl-AMP + PPi Step 2: Transfer aminoacyl-AMP + tRNA => aminoacyl Leu-tRNA+AMP

[0168] The resulting PPi was analyzed using the EnzCheck pyrophosphate kit (EnzCheck pyrophosphate kit). This can be measured using a phosphate kit. The present inventors have used a commercially available pyrophosphate assay kit (EnzCheck Pyrophosphate Assay Seikit (Pyrophosphate Assay Kit), Molecular Probes, E- Kinetic assays were performed using the ELISA kit (6654, incorporated herein by reference). This commercially available assay indirectly and spectrophotometrically measures the enzymatic production of pyrophosphate. Each RS reaction was set up in a total volume of 30 μl with the following final concentrations as shown in Table 2: 12.5 50 μl of RS reaction mixture was used for pyrophosphate assay as shown in Table 3. The pyrophosphate assay was set up in a 96-well plate and 3 The plate was automatically read at 2-minute intervals on a plate reader set to read absorbance at 60 nm. These kinetic measurements provide insight into the activity and functionality of all RS proteins. This was used as a qualitative first test.

[0169] The assay was performed according to the manufacturer's instructions, and the change in absorbance over time was measured using the R As shown in Figures 1 and 2, each RS showed good activity. (without tRNA as a control), and inorganic pyrophosphate inhibits the conversion of ATP to ADP+Pi. Pi is generated by hydrolysis and can be indirectly measured using the EnzCheck assay kit. Even if the change in absorbance is small, it is possible to detect the Data are based on published reports of RS and A provided by manufacturer guidelines. For clarity, the graphs shown for other enzyme kinetics for TP use are comparable. In both Figures 1 and 2, only 10 RSs are plotted in each graph. They result from the same experiment.

[0170] AMP obtained from the aminoacyl-tRNA-synthetase reaction is called AMP-Glo( The present inventors have used commercially available AMP detection kits. (AMP-Glo™ Assay, Promega V5012, incorporated herein by reference) The assay was performed using a commercially available assay (incorporated herein by reference). The enzymatic production of AMP is indirectly measured by the enzyme. The included standards are used to compare the amount of AMP produced. This assay is a quantitative endpoint measurement assay and can be used for calculations. Each RS reaction was set up in a total volume of 100 μL with the final concentrations shown in Table 4. The subsequent AMP detection assay was performed according to the manufacturer's instructions. Duplicate runs were performed and the AMP produced was calculated using a standard curve (Figure 17B). Figure 17A shows three distinct tRNA sequences utilizing exemplary tRNAs from E. coli. The results of the AMP-producing activity assay of the aminoacyl-tRNA-synthetase of The quasi-AMP curve is provided in Figure 17B.

[0171] Example 4: Confirmation of the activity of recombinant aminoacyl-tRNA-synthetase As further confirmation of the activity of each of the cloned RSs, the present inventors have used commercially available kits. Malachite Green Phosphate Assay Kit (Cayman) Malachite glycol was extracted using a malachite extract (British Patent No. 10009325, incorporated herein by reference). A pyrophosphate assay was performed. The pyrophosphate produced was complexed with malachite green. This leads to a color change that can be measured as absorption. The resulting standard can be used to calibrate and calculate the amount of PPi produced. The assay is a quantitative endpoint measurement assay. All reactions were performed according to the manufacturer's instructions. The results were analyzed according to the standard curve (shown as a small inlet on the graph). The calculation was performed using the following formula:

[0172] The final concentrations per RS ​​reaction were contained in a total volume of 150 μl as shown in Table 4 below. An exemplary tRNA from E. coli was used in this assay. As shown, the graph shows the control without reaction buffer (no ATP) and the control with one of the RSs. Compared with different amino acids (AsnRS+Arg), all RSs showed good activity. Each RS was used at the same molar concentration and incubated for 60 minutes before being analyzed using the kit. Each bar represents the concentration of PPi relative to the background / blank measurement. The values ​​are corrected and represent the average of duplicate measurements. The same assay was performed on a variety of strains, e.g., Geobacillus subterraneus, or Ge Geobacillus such as obacillus stearothermophilus The tRNA from the thermophile Lus was reproduced generally as described above.

[0173] Example 5: Recombinant Cell-Free Expression of Exemplary Proteins The present inventors have demonstrated the expression of two exemplary GFP peptides (sequences) in the recombinant cell-free expression system of the present invention. As specified in Table 6, the control and template The recombinant cell-free expression mixtures of the recombinant core proteins identified in Table 6 below were generated. The isolation of the protein is shown in Figures 11 to 14. As shown in Figure 4, the recombinant cell-free expression system The template DNA was transcribed and the resulting mRNA was analyzed as shown by the bands in Figure 4. As further shown in Figure 15, the inventors have synthesized the nucleotide sequences described herein. Production of a fluorescent protein (muGFP, SEQ ID NO: 134) using a recombinant cell-free expression system As further shown in Figure 16, the inventors have demonstrated real-time production of Fluorescent protein (deGFP, SEQ ID NO: 135) production using a recombinant cell-free expression system Furthermore, the present inventors have demonstrated the production of a protein from the produced GFP peptide through reverse purification. We have demonstrated the removal of translational components in a recombinant cell-free expression system. As specifically shown in Figure 16, Western blot analysis of cell-free protein expression reactions using anti-FLAG antibody after reverse purification was carried out. table [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] TIFF2025131577000010.tif212170 [Table 7]

[0174] References The following references are incorporated herein by reference in their entirety: [1] Carlson, Erik D. et al. “Cell-Free Prote in Synthesis:Applications Come of Age.” Biotechnology advances 30.5(2012):1185-1 194.PMC.Web.1 Jan.2018. [2] Lloyd, A. J., Thomann, H. U., Ibba, M., & Soi l,D.(1995).A broadly applicable continuo us spectrophotometric assay for measurin g aminoacyl-tRNA synthetase activity.Nuc leic acids research,23(15),2886-2892.

[0175] Sequence Listing SEQ ID NO: 1 DNA IF-1 - GbIF-1-EcOpt Bacillaceae (codon optimized for E. coli) ATGGCCAAAGATGATGTGATTGAAGTTGAAGGCACCGTTA TTGAAACCCTGCCGAATGCAATGTTTCGTGTTGAACTGGA AAATGGTCATACCGTTCTGGCACATGTTAGCGGTAAAATT CGCATGCACTTTATTCGTATTCTGCCTGGTGATCGTGTTA CCGTTGAACTGAGCCCGTACGATCTGACCCGTGGTCGTAT TACCTATCGTTATAAATGA SEQ ID NO: 2 amino acid IF-1-GbIF-1-EcOpt Bacillaceae MAKDDVIEVEGTVIETLPNAMFRVELENGHTVLAHVSGKI RMHFIRILPGDRVTVELSPYDLTRGRITYRYK SEQ ID NO: 3 DNA IF-2-GsIF-2-EcOpt Geobacillus stearothermophilus (E. col Codon optimization for i) ATGAGCAAAATGCGCGTTTATGAGTACGCCAAAAAACAGA ATGTTCCGAGCAAAGATGTGATCCACAAACTGAAAGAAAT GAACATCGAAGTGAACAACCATATGGCAATGCTGGAAGCA GATGTTGTTGAAAAACTGGATCATCAGTATCGTCCGAATA CCGGCAAAAAAGAAGAAAAAAAAGCCGAGAAGAAAACCGA GAAACCGAAACGTCCGACACCAGCAAAAGCAGCAGATTTT GCAGATGAAGAAATCTTCGATGATAGCAAAGAAGCAGCCA AAATGAAACCGGCAAAGAAAAAAGGTGCACCGAAAGGTAA AGAAACCAAAAAAACCGAAGCACAGCAGCAAGAGAAAAAA CTGCTGCAGGCAGCGAAAAAGAAAGGCAAAGGTCCGGCAA AAGGGAAAAAACAGGCAGCACCGGCAGCCAAACAGGCACC GCAGCCTGCGAAAAAAGAAAAAGAACTGCCGAAAAAAATC ACCTTTGAAGGTAGCCTGACCGTTGCAGAACTGGCAAAAA AACTGGGTCGTGAACCGAGCGAAATTATCAAAAAACTGTT TATGCTGGGTGTGATGGCCACCATTAATCAGGATCTGGAT AAAGATGCCATTGAACTGATTTGCAGCGATTATGGTGTTG AGGTTGAAGAAAAAGTGACCATCGATGAAACCAACTTTGA AGCCATTGAAATTGTTGATGCACCGGAAGATCTGGTTGAA CGTCCGCCTGTTGTTACCATTATGGGTCATGTTGATCATG GTAAAACCACACTGCTGGATGCAATTCGTCATAGCAAAGT TACCGAACAAGAAGCAGGCGGTATTACACAGCATATTGGT GCATATCAGGTTACCGTGAACGATAAGAAAATCACGTTTC TGGATACACCGGGTCATGAAGCATTTACCACCATGCGTGC ACGTGGTGCACAGGTGACCGATATTGTTATTCTGGTTGTT GCAGCAGATGATGGCGTTATGCCGCAGACCGTTGAAGCAA TTAATCATGCAAAAGCCGCAAACGTTCCGATTATTGTTGC CATCAACAAAATCGATAAACCGGAAGCAAATCCGGATCGT GTTATGCAAGAACTGATGGAATATAATCTGGTTCCGGAAG AATGGGGTGGTGATACCATTTTTTGTAAACTGAGCGCCAA AACCAAAGAAGGTCTGGACCATCTGCTGGAAATGATTCTG CTGGTTAGCGAAATGGAAGAACTGAAAGCCAATCCGAATC GTCGTGCAGTTGGCACCGTTATTGAAGCCAAACTGGACAA AGGTCGTGGTCCGGTTGCGACCCTGCTGATTCAGGCAGGC ACCCTGCGTGTTGGTGATCCGATTGTTGTGGGCACCACCT ATGGTCGTGTTCGTGCAATGGTTAATGATAGCGGTCGTCG TGTTAAAGAAGCAACCCCGAGCATGCCGGTTGAAATTACC GGTCTGCATGAAGTTCCGCAGGCAGGCGATCGTTTTATGG TTTTTGAAGATGAGAAAAAGGCACGCCAGATTGCCGAAGC ACGTGCACAGCGTCAGCTGCAAGAACAGCGTAGCGTTAAA ACCCGTGTTAGCCTGGATGACCTGTTTGAGCAGATTAAAC AGGGTGAAATGAAAGAGCTGAACCTGATTGTTAAAGCCGA TGTTCAGGGTAGCGTTGAAGCCCTGGTTGCAGCACTGCAG AAAATTGATGTTGAAGGTGTTCGCGTGAAAATTATCCATG CAGCCGTTGGTGCAATTACCGAAAGCGATATTAGCCTGGC AACCGCAAGCAATGCAATTGTGATTGGTTTTAATGTTCGT CCGGATGCAAATGCAAAACGTGCAGCAGAAAGTGAAAAAG TGGATATTCGTCTGCACCGCATTATCTATAACGTGATCGA AGAAATTGAGGCAGCCATGAAAGGTATGCTGGATCCGGAA TATGAAGAGAAAGTTATTGGTCAGGCAGAAGTTCGTCAGA CCTTTAAAGTTAGCAAAGTGGGTACAATTGCCGGTTGTTA TGTTACCGATGGTAAAATTACCCGTGATAGTAAAGTTCGT CTGATTCGTCAGGGTATTGTTGTGTATGAAGGTGAAATTG ATAGCCTGAAACGCTATAAAGATGATGTTCGTGAAGTTGC CCAGGGTTATGAATGTGGTCTGACCATTAAAAACTTCAAC GACATTAAAGAGGGCGACGTTATCGAAGCCTATATCATGC AAGAAGTTGCACGCGCATAA sequence number 4 amino acid IF-2-GsIF-2-EcOpt Geobacillus stearothermophilus MSKMRVYEYAKKQNVPSKDVIHKLKEMNIEVNNHMAMLEA DVVEKLDHQYRPNTGKKEEKKAEKKTEKPKRPTPAKAADF ADEEIFDDSKEAAKMKPAKKKGAPKGKETKKTEAQQQEKK LLQAAKKKGKGPAKGKKQAAPAAKQAPQPAKKEKELPKKI TFEGSLTVAELAKKLGREPSEIIKKLFMLGVMATINQDLD KDAIELICSDYGVEVEEKVTIDETNFEAIEIVDAPEDLVE RPPVVTIMGHVDHGKTTLLDAIRHSKVTEQEAGGITQHIG AYQVTVNDKKITFLDTPGHEAFTTMRARGAQVTDIVILVV AADDGVMPQTVEAINHAKAANVPIIVAINKIDKPEANPDR VMQELMEYNLVPEEWGGDTIFCKLSAKTTKEGLDHLLEMIL LVSEMEELKANPNRRAVGTVIEAKLDKGRGGPVATLLIQAG TLRVGDPIVVGTTYGRVRAMVNDSGRRVKEATPSMPVEIT GLHEVPQAGDRFMVFEDEKKARQIAEARAQRQLQEQRSVK TRVSLDDLFEQIKQGEMKELNLIVKADVQGSVEALVAALQ KIDVEGVRVKIIHAAVGAITESDISLATASNAIIGFNVR PDANAKRAAESEKVDIRLHRIIYNVIEEIEAAMKGMLDPE YEEKVIGQAEVRQTFKVSKVGTIAGCYVTDGKITRDSKVR LIRQGIVVYEGEIDSLKRYKDDVREVAQGYECGLTIKNFN DIKEGDVIEAYIMQEVARA SEQ ID NO:5 DNA IF-3-GbIF-3-EcOpt Geobacillus (codon optimized for E. coli) ATGATCAGCAAGGACTTTATCATCAATGAGCAGATTCGTG CACGTGAAGTTCGTCTGATTGATCAGAATGGTGAACAGCT GGGTATCAAAAGCAAACAAGAAGCACTGGAAATTGCAGCA CGTCGTAATCTGGATCTGGTTCTGGTGGCACCGAATGCAA AACCGCCTGTTTGTCGTATTATGGATTATGGCAAATTTCG CTTCGAGCAGCAGAAAAAAGAAAAAGAGGCACGCAAAAAG CAGAAAGTGATCAATGTTAAAGAAGTGCGTCTGAGCCCGA CCATTGAAGAACATGATTTTAACACCAAACTGCGCAACGC ACGCAAATTTCTGGAAAAAGGTGATAAAGTGAAAGCCACC ATTCGTTTTAAAGGTCGTGCAATCACCCATAAAGAAATTG GTCAGCGTGTTCTGGATCGTTTTAGCGAAGCATGTGCAGA TATTGCAGTTGTTGAAACCGCACCGAAAATGGATGGTCGT AATATGTTTCTGGTGCTGGCTCCGAAAAACGACAACAAAT AA SEQ ID NO:6 amino acid IF-3-GbIF-3-EcOpt Geobacillus MISKDFIINEQIRAREVRLIDQNGEQLGIKSKQEALEIAA RRNLDLVLVAPNAKPPVCRIMDYGKFRFEQQKKEKEARKK QKVINVKEVRLSPTIEEHDFNTKLRNARKFLEKGDKVKAT IRFKGRAITHKEIGQRVLDRFSEACADIAVVETAPKMDGR NMFLVLAPKNDNK SEQ ID NO:7 DNA EF-G-GsEF-G-EcOpt Geobacillus (codon optimized for E. coli) ATGGCACGTGAATTCAGCCTGGAAAAAACCCGTAATATTG GTATTATGGCCCATATCGATGCAGGTAAAACCACCACCAC CGAACGTATTCTGTTTTATACCGGTCGTGTGCATAAAATT GGTGAAGTTCATGAAGGTGCAGCAACCATGGATTGGATGG AACAAGAACAAGAGCGTGGTATTACCATTACCAGCGCAGC CACCACCGCACAGTGGAAAGGTCATCGTATTAACATTATT GATACACCGGGTCACGTTGATTTTACCGTTGAAGTTGAAC GTAGCCTGCGTGTTCTGGATGGTGCAATTACCGTGCTGGA TGCACAGAGCGGTGTTGAACCGCAGACCGAAACCGTTTGG CGTCAGGCAACCACCTATGGTGTTCCGCGTATTGTTTTTG TGAACAAGATGGATAAAATCGGTGCCGATTTCCTGTATAG CGTTAAAACCCTGCATGATCGTCTGCAGGCAAATGCACAT CCGGTTCAGCTGCCGATTGGTGCAGAAGATCAGTTTAGCG GTATTATTGATCTGGTTGAAATGTGCGCCTATCACTATCA TGATGAACTGGGCAAAAAACATCGAACGCATTGATATTCCG GAAGAATATCGTGATATGCCGAAGAGTATCACAACAAAC TGATTGAAGCAGTTGCAGAACTGGATGAAGAACTGATGAT GAAATATCTGGAAGGCGAAGAAATTACCGCAGAGGAACTG AAAGCAGCAATTCGTAAAGCAACCATTAGCGTGGAATTTT TTCCGGTTTTTTGTGGTAGCGCCTTCAAAACAAAGGTGT GCAGCTGCTGCTGGATGGCGTTGTTGATTATCTGCCGAGT CCGGTGGATATTCCTGCAATTCGTGGTGTTGTTCCGGATA CCGAAGAAAGTTACACGCGAAGCAAGTGATGATGCACCC GTTTGCAGCACTGGCCTTTAAAATCATGACCGATCCGTAT GTTGGTAAGCTGACCTTTTATTCGTGTTTATAGCGGCACCC TGGATAGCGGTAGCTATGTTTATGAATACCACCAAAGGTAA ACGTGAACGTATTGGTCGTCTGCTGCAGATGCATGCAAAT CATCGTCAAGAAATCAGCAAAGTTTATGCCGGTGATATTG CAGCAGCAGTTGGTCTGAAAGATACCACAACCGGTGATAC CCTGTGTGATGAAAAACAATCGGTGATTCTGGAAAGCATG CAGTTTCCGGAACCGGTTATTAGCGTTGCAATTGAACCGA AAAGCAAAGCCGATCAGGATAAAATGAGCCAGGCACTGCA GAAACTGCAAGAAGAGGATCCGACCTTTCGTGCACATACC GATCCGGAAACCGGTCAGACCATTATTAGTGGTATGGTG AACTGCATCTGGATATCATTGTTGATCGTATGCGTCGCGA ATTTAAAGTTGAAGCAAATGTTGGTGCACCGCAGGTTGCA TATCGTGAACCTTTCGTAAAAGCGCACAGGTTGAAGGCA AATTTATCCGTCAGAGTGGTGGTCGTGGTCAGTATGGTCA TGTTTGGATTGAATTTTCACCGAACGAACGCGGTAAAGGC TTTGAATTTGAAAATGCAATTGTTGGTGGTGTGGTGCCGA AAGAATATGTTCCGGCAGTTCAGGCAGGTCTGGAAGAGGC AATGCAGAATGGTGTTCTGGCAGGTTATCCGGTTGTTGAT ATTAAAGCCAAACTGTTCGATGGCAGCTATCACGATGTTG ATAGCAGCGAAATGGCATTCAAAATTGCAGCAAGCCTGGC ACTGAAAAATGCCGCAACCAAATGTGATCCTGTTCTGCTG GAACCGATTATGAAAGTGGAAGTTGTTATCCCTGAGGAAT ATCTGGGTGATATTATGGGCGATATTACCAGCCGTCGTGG TCGCATTGAAGGTATGGAAGCACGTGGTAATGCCCAGGTT GTTCGTGCAATGGTTCCGCTGGCAGAAATGTTTGGTTATG CAACCAGCCTGCGTAGCAATACCCAAGGTCGTGGCACCTT TAGCATGGTTTTTGATCATTATGAAGAGGTGCCCAAAAAC ATTGCCGATGAGATCATCAAAAAAAACAAGGGCGAATAA SEQ ID NO:8 amino acid EF-G-GsEF-G-EcOpt Geobacillus MAREFSLEKTRNIGIMAHIDAGKTTTTERILFYTGRVHKI GEVHEGAATMDWMEQEQERGITITSAATTAQWKGHRINII DTPGHVDFTVEVERSLRVLDGAITVLDAQSGVEPQTETVW RQATTYGVPRIVFVNKMDKIGADFLYSVKTLHDRLQANAH PVQLPIGAEDQFSGIIDLVEMMCAYHYHDELGKNIERIDIP EEYRDMAEEYHNKLIEAVAELDEELMMKYLEGEEITAEEL KAAIRKATISVEFFPVFCGSAFKNKGVQLLLDGVVDYLPS PVDIPAIRGVVPDTEEEVTREASDDAPFAALAFKIMTDPY VGKLTFIRVYSGTLDSGSYVMNTTKGKRERIGRLLQMHAN HRQEISKVYAGDIAAAVGLKDTTTGDTLCDEKHPVILESM QFPEPVISVAIEPKSKADQDKMSQALQKLQEEDPTFRAHT DPETGQTIISGMGELHLDIIVDRMREFKVEANVGAPQVA YRETFRKSAQVEGKFIRQSGGRGQYGHVWIEFSPNERGKG FEFENAIVGGVVPKEYVPAVQAGLEEAMQNGVLAGYPVVD IKAKLFDGSYHDVDSSEMAFKIAASLAKNAATKCDPVLL EPIMKVEVVIPEEYLGDIMGDITSRRGRIEGMEARGNAQV VRAMVPLAEMFGYATSLRSNTQGRGTFSMVFDHYEEVPKN IADEIIKKNKGE sequence number 9 DNA EF-Tu-GsEF-Tu-EcOpt Geobacterium ATGGCCAAAGCCAAATTGAACGTACCAAACCGCATGTTA ATATTGGCACCATTGGTCATGTTGATCATGGTAAAACCAC ACTGACCGCAGCAATTACCACCGTTCTGGCAAAACAGGGT AAAGCCGAAGCAAAAGCATATGATCAGATTGATGCAGCAC CGGAAGAACGTGAACGTGGTATTACCATTAGCACCGCACA TGTTGAATATGAAACCGATGCACGTCATTATGCCCATGTT GATTGTCCGGGTCATGCAGATTATGTGAAAAAATAGATTA CCGGTGCAGCACAGATGGATGGTGCAATTCTGGTTGTTAG CGCAGCAGATGGTCCGATGCCGCAGACACGTGAACATATT CTGCTGAGCCGTCAGGTTGGTGTTCCGTATATTGTTGTGT TTCTGAACAAATGCGATATGGTGGATGATGAAGAACTGCT GGAACTGGTTGAAATGGAAGTTCGTGATCTGCTGTCCGAA TATGATTTTCCGGGTGATGAAGTTCCGGTTATTAAAGGTA GCGCACTGAAAGCACTGGAAGGTGATCCGCAGTGGGAAGA AAAAATCATTGAACTGATGAATGCCGTGGATGAGTATATT CCGACACCGCAGCGTGAAGTTGATAAACCGTTTATGATGC CGATCGAAGATGTGTTTAGCATTACCGGTCGTGGCACCGT TGCAACCGGTCGCGTTGAACGTGGCACCCTGAAAGTTGGT GATCCGGTTGAAATTATTGGTCTGAGTGATGAACCGAAAA CCACCACCGTTACCGGTGTTGAAATGTTTCGTAAACTGTT AGATCAGGCCGAAGCCGGTGATAATATTGGTGCACTGCTG CGTGGTGTTTCACGTGATGAGGTGGAACGTGGTCAGGTTC TGGCGAAACCTGGTAGCATTACACCGCATACCAAATTCAA AGCACAGGTTTATGTTCTGACCAAAGAAGAAGGCGGTCGT CATACCCCGTTTTTTAGCAATTATCGTCCGCAGTTTTATT TCCGTACCACCGATGTTACCGGTATTATTACCCTGCCGGA AGGTGTGGAAATGGTTATGCCTGGTGATAACGTTGAAATG ACCGTGGAACTGATTGCACCGATTGCAATTGAAGAAGGCA CCAAATTTAGCATTCGTGAAGGTGGTCGTACCGTTGGTGC AGGTAGCGTTAGCGAAATTATCGAATAA SEQ ID NO: 10 Amino acid EF-Tu-GsEF-Tu-EcOpt Geobacillus MAKAKFERTKPHVNIGTIGHVDHGKTTLTAAITTVLAKQG KAEAKAYDQIDAAPEERERGITISTAHVEYETDARHYAHV DCPGHADYVKNMITGAAQMDGAILVVSAADGPMPQTREHI LLSRQVGVPYIVVFLNKCDMVDDEELLELVEMEVRDLLSE YDFPGDEVPVIKGSALKALEGDPQWEEKIIELMNAVDEYI PTPQREVDKPFMMPIEDVFSITGRGTVATGRVERGTLKVG DPVEIIGLSDEPKTTTVTGVEMFRKLLDQAEAGDNIGALL RGVSRDEVERGQVLAKPGSITPHTKFKAQVYVLTKEEGGR HTPFFSNYRPQFYFRTTDVTGIITLPEGVEMVMPGDNVEM TVELIAPIAIEEGTKFSIREGGRTVGAGSVSEIIE sequence number 11 DNA EF-Ts-GsEF-Ts-EcOpt Geobacterium ATGGCAATTACCGCACAGATGGTTAAAGAACTGCGTGAAA AAACCGGTGCAGGTATGATGGATTGTAAAAAGCACTGAC CGAAACCAATGGCGATATGGAAAAAGCAATTGATTGGCTG CGCGAAAAGGTATTGCAAAAGCAGCAAAAAAAGCCGATC GTATTGCAGCAGAAGGTATGGCATATATTGCAGTTGAAGG TAATACCGCAGTTATCCTGGAAGTTAATAGCGAAACCGAT TTTGTGGCAAAAAACGAAGCATTTCAGACCCTGGTGAAAG AGCTGGCAGCACATCTGCTGAAACAGAAACCGGCAAGCCT GGATGAAGCACTGGGTCAGACCATGGATAATGGTAGCACC GTTCAGGATTATATCAATGAAGCCATTGCCAAAATCGGCG AAAAAATCACCCTGCGTCGTTTTGCAGTTGTTAATAAAGC AGATGGTGAAACCTTTGGTGCCTATCTGCATATGGGTGGT CGTATTGGTGTTCTGACCCTGCTGGCAGGTAATGCAAGCG AAGATGTTGCAAAAGATGTGGCAATGCATATTGCAGCCCT GCATCCGAAATATGTTAGCCGTGATGATGTTCCGCAAGAA GAAATTGCACACGAACGTGAAGTTCTGAAACAGCAGGCAC TGAATGAAGGCAAACCGGAAAAAATTGTGGAAAAGATGGT TGAAGGTCGCCTGAACAAATTCTATGAAGATGTTTGTCTG CTGGAACAGGCCTTTGTTAAAAATCCGGATGTTACCGTTC GTCAGTATGTTGAAAGCAATGGTGCCACCGTTAAACAGTT TATTCGTTATGAAGTTGGTGAGGGCTTAGAAAAACGCCAG GATAATTTTGCCGAAGAAGTTATGAGCCAGGTTCGCAAAC AGTAA SEQ ID NO: 12 Amino acid EF-Ts-GsEF-Ts-EcOpt Geobacillus MAITAQMVKELREKTGAGMMDCKKALTETNGDMEKAIDWL REKGIAKAAKKADRIAAEGMAYIAVEGNTAVILEVNSETD FVAKNEAFQTLVKELAAHLLKQKPASLDEALGQTMDNGST VQDYINEAIAKIGEKITLRRFAVVNKADGETFGAYLHMGG RIGVLTLLAGNASEDVAKDVAMHIAALHPKYVSRDDVPQE EIAHEREVLKQQALNEGKPEKIVEKMVEGRLNKFYEDVCL LEQAFVKNPDVTVRQYVESNGATVKQFIRYEVGEGLEKRQ DNFAEEVMSQVRKQ SEQ ID NO: 13 DNA EF-4-GsEF-4-EcOpt Geobacillus (codon optimized for E. coli) ATGAACCGTGAGGAACGTCTGAAACGTCAGGAGCGTATTC GTAACTTCAGCATCATTGCGCACATCGACCACGGTAAAAG CACCCTGGCGGATCGTATCCTGGAGAAAACCGGTGCGCTG AGCGAGCGTGAACTGCGTGAACAGACCCTGGACATGATGG ATCTGGAGCGTGAACGTGGTATCACCATTAAGCTGAACGC GGTGCAACTGACCTATAAGGCGAAAAACGGCGAGGAATAC ATCTTCCACCTGATTGACACCCCGGGCCACGTGGATTTTA CCTATGAAGTTAGCCGTAGCCTGGCGGCGTGCGAAGGTGC GATTCTGGTGGTTGATGCGGCGCAGGGTATTGAGGCGCAA ACCCTGGCGAACGTGTACCTGGCGATTGACAACAACCTGG AAATCCTGCCGGTTATCAACAAAATTGATCTGCCGAGCGC GGAGCCGGAACGTGTGCGTCAGGAGATCGAAGACGTTATT GGTCTGGATGCGAGCGAGGCGGTGCTGGCGAGCGCGAAGG TTGGTATCGGCATTGAGGAAATCCTGGAGCAAATTGTGGA AAAAATTCCGGCGCCGAGCGGTGACCCGGATGCGCCGCTG AAGGCGCTGATCTTTGACAGCCTGTACGATCCGTATCGTG GCGTGGTTGCGTACGTGCGTATTGTTGACGGTACCGTTAA GCCGGGCCAGCGTATCAAAATGATGAGCACCGGCAAGGAG TTCGAAGTGACCGAGGTGGGCGTTTTTACCCCGAAGCAAA AAATCGTTGACGAACTGACCGTGGGTGATGTTGGCTATCT GACCGCGAGCATTAAGAACGTGAAAGATACCCGTGTTGGT GACACCATTACCGATGCGGAGCGTCCGGCGGCGGAACCGC TGCCGGGTTACCGTAAACTGAACCCGATGGTTTTCTGCGG CATGTATCCGATCGACACCGCGCGTTACAACGATCTGCGT GAGGCGCTGGAAAAGCTGCAGCTGAACGACGCGGCGCTGC ACTTCGAGCCGGAAACCAGCCAAGCGCTGGGTTTCGGCTT TCGTTGCGGTTTTCTGGGCCTGCTGCACATGGAGATCATT CAGGAACGTATCGAGCGTGAATTTCACATCGATCTGATTA CCACCGCGCCGAGCGTGGTTTATAAAGTGCACCTGACCGA CGGTACCGAGGTGAGCGTTGATAACCCGACCAACATGCCG GACCCGCAAAAAATCGATCGTATTGAGGAACCGTATGTGA AGGCGACCATTATGGTTCCGAACGACTACGTGGGCCCGGT TATGGAACTGTGCCAGGGTAAACGTGGCACCTTCGTGGAC ATGCAATACCTGGATGAGAAGCGTGTTATGCTGATCTATG ACATTCCGCTGAGCGAAATCGTTTACGACTTCTTTGATGC GCTGAAGAGCAACACCAAAGGTTACGCGAGCTTTGATTAT GAGCTGATTGGCTACCGTCCGAGCAACCTGGTGAAAATGG ACATCCTGCTGAACGGTGAAAAGATTGATGCGCTGAGCTT CATCGTTCACCGTGAGGCGGCGTATGAACGTGGCAAAGTG ATTGTTGAGAAGCTGAAAGACCTGATCCCGCGTCAGCAAT TTGAAGTGCCGGTTCAGGCGGCGATTGGTAACAAAATCAT TGCGCGTAGCACCATCAAGGCGCTGCGTAAAAACGTGCTG GCGAAGTGCTACGGTGGCGATGTTAGCCGTAAGCGTAAAC TGCTGGAGAAGCAGAAAGAAGGTAAGAAACGTATGAAACA GATTGGTAGCGTTGAGGTGCCGCAAGAAGCGTTCATGGCG GTGCTGAAGATCGACGATCAAAAGAAA SEQ ID NO: 14 amino acid EF-4-GsEF-4-EcOpt Geobacillus MNREERLKRQERIRNFSIIAHIDHGKSTLADRILEKTGAL SERELREQTLDMMDLERERGITIKLNAVQLTYKAKNGEEY IFHLIDTPGHVDFTYEVSRSLAACEGAILVVDAAQGIEAQ TLANVYLAIDNNLEILPVINKIDLPSAEPERVRQEIEDVI GLDASEAVLASAKVGIGIEEILEQIVEKIPAPSGDPDAPL KALIFDSLYDPYRGVVAYVRIVDGTVKPGQRIKMMSTGKE FEVTEVGVFTPKQKIVDELTVGDVGYLTASIKNVKDTRVG DTITDAERPAAEPLPGYRKLNPMVFCGMYPIDTARYNDLR EALEKLQLNDAALHFEPETSQALGFGFRCGFLGLLHMEII QERIEREFHIDLITTAPSVVYKVHLTDGTEVSVDNPTNMP DPQKIDRIEEPYVKATIMVPNDYVGPVMELCQGKRGTFVD MQYLDEKRVMLIYDIPLSEIVYDFFDALKSNTKGYASFDY ELIGYRPSNLVKMDILLNGEKIDALSFIVHREAAYERGKV IVEKLKDLIPRQQFEVPVQAAIGNKIIARSTIKALRKNVL AKCYGGDVSRKRKLLEKQKEGKKRMKQIGSVEVPQEAFMA VLKIDDQKK SEQ ID NO: 15 DNA EF-P-GsEF-P-EcOpt Geobacterium ATGATCAGCGTGAACGACTTCCGTACCGGTCTGACCATCG AAGTTGATGGCGAGATTTGGCGTGTGCTGGAATTCCAGCA CGTTAAGCCGGGTAAAGGCGCGGCGTTTGTGCGTAGCAAG CTGCGTAACCTGCGTACCGGTGCGATCCAAGAACGTACCT TCCGTGCGGGCGAGAAGGTGAACCGTGCGCAGATTGACAC CCGTAAAATGCAATACCTGTATGCGAACGGTGACCAGCAC GTTTTTATGGATATGGAGACCTACGAACAGATCGAGCTGC CGGCGAAACAAATTGAGTATGAACTGAAGTTCCTGAAAGA AAACATGGAAGTGTTTATCATGATGTACCAAGGTGAAACC ATCGGCATTGAGCTGCCGAACACCGTTGAGCTGAAGGTGG TTGAGACCGAACCGGGTTATTAAAGGTGATACCGCGAGCGG TGGCAGCAAGCCGCGAAACTGGAAACCGGCCTGGTGGTT CAGGTGCCGTTCTTTTGTTAACGAGGGTGACACCCTGATCA TTAACACGCGGATGGCACCTATGTTAGCCGTGCG sequence number 16 amino acid EF-P-GsEF-P-EcOpt Geobacillus MISVNDFRTGLTIEVDGEIWRVLEFQHVKPGKGAAFVRSK LRNLRTGAIQERTQUESTIONKVNRAQIDTRKMQYLYANGDQH VFMDMETYEQIELPAKQIEYELKFLKENMEVFIMMYQGET IGIELPNTVELKVVETEPGIKGDTASGGSKPAKLETGLVV QVPFFVNEGDTLIINTADGTYVSRA SEQ ID NO: 17 DNA RF-1 Title: GsRF-1-EcOpt Origin: Geobacillus stearothermophilus (codon-optimized for Escherichia coli) ATGTTTGATCGTCTGGAAGCAGTTGAACAGCGTTATGAAA AACTGAATGAACTGCTGATGGAACCGGATGTTATTAACGA TCCGAAAAAACTGCGCGATTATAGCAAAGAACAGGCAGAT CTGGAAGAAACCGTTCAGACCTATCGTGAGTATAAAAGCG TTCGTGAACAGCTGGCCGAAGCAAAAGCAATGCTGGAAGA GAAACTGGAACCTGAACTGCGTGAAATGGTGAAAGAAGAA ATTGGCGAACTGGAAGAACGTGAAGAAGCACTGGTTGAGA AACTGAAAGTTCTGCTGCTGCCGAAAGATCCGAATGATGA AAAAAACGTGATCATGGAAATTCGTGCAGCAGCCGGTGGC GAAGAAGCAGCACTGTTTGCCGGTGATCTGTATCGTATGT ATACCCGTTATGCAGAAAGCCAAGGTTGGAAAACCGAAGT TATTGAAGCAAGCCCGACCGGTTTAGGTGGTTATAAAGAA ATCATCTTCATGATCAATGGCAAGGGTGCATACAGCAAAC​ TGAAATTTGAAAATGGTGCACATCGTGTTCAGCGTGTTCC GGAAACCGAAAGCGGTGGTCGTATTCATACCAGCACCGCA ACCGTTGCATGTCTGCCGGAAATGGAAGAAATCGAAGTGG AAATCAACGAGAAAGATATTCGCGTTGATACCTTTGCAAG CAGCGGTCCTGGTGGTCAGAGCGTTAATACCACCATGAGC GCAGTTCGTCTGACCCATATTCCGACCGGTATTGTTGTTA CCTGTCAGGATGAAAAATCCCAGATCAAAAACAAAGAAAA AGCCATGAAAGTGCTGCGTGCCCGTATCTATGATAAATAT CAGCAAGAGGCACGTGCGGAATATGATCAGACCCGTAAAC AGGCAGTTGGCACCGGTGATCGTAGCGAACGTATTCGTAC CTATAACTTTCCGCAGAATCGTGTTACCGATCATCGTATT GGTCTGACCATTCAAAAACTGGATCAGGTTCTGGATGGTC ATCTGGATGAAATTATCGAAGCACTGATTCTGGATGACCA GGCAAAAAAGCTGGAACAGGCAAATGATGCAAGCTAA SEQ ID NO: 18 Amino acid RF-1-GsRF-1-EcOpt Geobacillus stearothermophilus MFDRLEAVEQRYEKLNELLMEPDVINDPKKLRDYSKEQAD LEETVQTYREYKSVREQLAEAKAMLEEKLEPELREMVKEE IGELEEREEALVEKLKVLLLPKDPNDEKNVIMEIRAAAGG EEAALFAGDLYRMYTRYAESQGWKTEVIEASPTGLGGYKE IIFMINGKGAYSKLKFENGAHRVQRVPETESGGRIHTSTA TVACLPEMEEIEVEINEKDIRVDTFASSGPGGQSVNTTMS AVRLTHIPTGIVVTCQDEKSQIKNKEKAMKVLRARIYDKY QQEARAEYDQTRKQAVGTGDRSERIRTYNFPQNRVTDHRI GLTIQKLDQVLDGHLDEIIEALILDDQAKKLEQANDAS SEQ ID NO: 19 DNA RF-2-GsRF-2-EcOpt Geobacillus stearothermophilus (E. col Codon optimization for i) ATGGCAGCACCGAATTTTTGGGATGATCAGAAAGCAGCAC AGGCAGTTATTAGCGAAGCAAATGCACTGAAAGATCTGGT GGAAGAATTTAGCAGCCTGGAAGAACGTTTTGATAATCTG GAAGTTACCTACGAACTGCTGAAAGAAGAACCGGACGACG AACTGCAGGCAGAACTGGTTGAAGAGGCAAAAAAACTGAT GAAAGATTTTAGCGAATTTGAACTGCAGCTGCTGCTGAAT GAACCGTATGATCAAAATAATGCCATCCTGGAACTGCATC CTGGTGCCGGTGGCACCGAAAGCCAGGATTGGGCAAGCAT GCTGCTGCGTATGTATACCCGTTGGGCAGAAAAAAAGGC TTTAAAGTTGAAACCCTGGATTATCTGCCTGGTGAAGAAG CAGGTATTAAAAGCGTTACCCTGCTGATTAAAGGCCATAA TGCATATGGTTATCTGAAAGCCGAAAAAGGTGTTCATCGT CTGGTTCGTATTAGCCCGTTTGATGCAAGCGGTCGTCGTC ATACCAGCTTGTTAGCTGTGAAGTTGTGCCGGAACTGGA TGATAACATTGAAATTGAAATTCGCCCTGAAGAACTGAAG ATTGATACCTATCGTAGCAGCGGTGCAGGCGGTCAGCATG TTAATACACCGATAGCGCAGTGCGTATTACCCATCTGCC GACCGGTATTGTTGTTACCTGTCAGAGCGAACGTAGCCAG ATTAAAAACCGTGAAAAGCCATGAATATGCTGAAAGCCA AACTGTACCAGAAGAAATTAGAAGAACAGCAGGCCGAGCT GGCCGAACTGCGTGGTGAACAGAAAGAAATTGGTTGGGGT AATCAGATTCGCAGCTATGTTTTTTCATCCGTACAGCCTGG TTAAAGATCATCGTACCAATGTTGAAGTTGGTAATGTTCA GGCCGTTATGGATGGTGAAATTGATGTTTTTATCGATGCA TACCTGCGTGCCAAACTGAAATAA sequence no. 20 amino acid RF-2-GsRF-2-EcOpt Geobacillus stearothermophilus MAAPNFWDDQKAAQAVISEANALKDLVEEFSSLEERFDNL EVTYELLKEEPDDELQAELVEEAKKLMKDFSEFELQLLLN EPYDQNNAILELHPGAGGTESQDWASMLLRMYTRWAEKKG FKVETLDYLPGEEAGIKSVTLLIKGHNAYGYLKAEKGVHR LVRISPFDASGRRHTSFVSCEVVPELDDNIEIEIRPEELK IDTYRSSGAGGQHVNTTTDSAVRITHLPTGIVVTCQSERSQ IKNREKAMNMLKAKLYQKKLEEQQAELAELRGEQKEIGWG NQIRSYVFHPYSLVKDHRTNVEVGNVQAVMDGEIDVFIDA YLRAKLK SEQ ID NO: 21 DNA RF-3- BX1-RF-3-EcOpt Bacillus sp. X1 (E. coli) optimization) ATGGGTAACGATTTCAAGAAAGAAGTGCTGAGCCGTCGTA CCTTTGCGATCATTAGCCATCCGGATGCGGGCAAGACCAC CCTGACCGAGAAACTGCTGCTGTTCGGTGGCGCGATCCGT GATGCGGGTACCGTTAAGGCGAAGAAAACCGGCAAATACG CGACCAGCGACTGGATGGAAATCGAGAAACAGCGTGGTAT TAGCGTGACCAGCAGCGTTATGCAATTCGATTACAACGGT TATCGTGTGAACATTCTGGACACCCCGGGCCACCAGGACT TTAGCGAAGATACCTATCGTACCCTGATGGCGGTGGACAG CGCGGTTATGATCATTGATAGCGCGAAGGGCATCGAGGAC CAAACCATTAAGCTGTTCAAAGTGTGCCGTATGCGTGGTA TCCCGATTTTCACCTTTATCAACAAGCTGGACCGTCAGGG CAAACAACCGCTGGAGCTGCTGGCGGAACTGGAGGAAGTT CTGGGTATCGAGAGCTACCCGATGAACTGGCCGATTGGTA TGGGCAAAGAATTTCTGGGCATCTATGATCGTTACTATAA CCGTATTGAGCAGTTCCGTGTGAACGAGGAAGAGCGTTTT ATCCCGCTGAACGAAGACGGTGAAATTGAGGGCAACCACA AGCTGGTTAGCAGCGGTCTGTACGAGCAGACCCTGGAAGA GATCATGCTGCTGAACGAGGCGGGTAACGAATTTAGCGCG GAGCGTGTGGCGGCGGGTCAACTGACCCCGGTTTTCTTTG GTAGCGCGCTGACCAACTTCGGCGTGCAGACCTTTCTGGA AACCTATCTGCAATTTGCTCCGCCGCCGAAGGCGCGTAAC AGCAGCATCGGCGAGATTGATCCGCTGAGCGAAGAGTTTA GCGGCTTCGTTTTTAAAATTCAGGCGAACATGAACCCGGC GCACCGTGACCGTATCGCGTTCGTGCGTATTTGCAGCGGC AAGTTTGAGCGTGGCATGAGCGTTAACCTGCCGCGTCTGG GCAAGCAGCTGAAACTGACCCAAAGCACCAGCTTCATGGC GGAAGAGCGTAACACCGTGGAAGAGGCGGTTAGCGGTGAC ATCATTGGCCTGTACGATACCGGTACCTATCAGATCGGCG ATACCCTGACCGTGGGCAAAAACGACTTCCAGTTTGAGCG TCTGCCGCAATTCACCCCGGAACTGTTTGTGCGTGTTAGC GCGAAGAACGTTATGCGTCAGAAGAGCTTTTACAAAGGTC TGCACCAGCTGGTGCAAGAAGGCGCGATTCAACTGTACAA GACCGTTAAAACCGATGAGTATCTGCTGGGTGCGGTGGGC CAGCTGCAATTCGAAGTTTTTGAGCACCGTATGAAGAACG AATATAACGCGGAAGTGCTGATGGAACGTCTGGGTAGCAA AATCGCGCGTTGGATTGAAAACGACGAGGTTGATGAAAAC CTGAGCAGCAGCCGTAGCCTGCTGGTGAAAGACCGTTACG ATCACTATGTTTTCCTGTTTGAGAACGACTTCGCGCTGCG TTGGTTTCAGGAAAAGAACCCGACCATCAAACTGTACAAC CCGATGGACCAACACGAT SEQ ID NO: 22 Amino acid RF-3 BX1-RF-3-EcOpt Bacillus sp. X1 MGNDFKKEVLSRRTFAIISHPDAGKTTLTEKLLLFGGAIR DAGTVKAKKTGKYATSDWMEIEKQRGISVTSSVMQFDYNG YRVNILDTPGHQDFSEDTYRTLMAVDSAVMIIDSAKGIED QTIKLFKVCRMRGIPIFTFINKLDRQGKQPLELLAELEEV LGIESYPMNWPIGMGKEFLGIYDRYYNRIEQFRVNEEERF IPLNEDGEIEGNHKLVSSGLYEQTLEEIMLLNEAGNEFSA ERVAAGQLTPVFFGSALTNFGVQTFLETYLQFAPPPKARN SSIGEIDPLSEEFSGFVFKIQANMNPAHRDRIAFVRICSG KFERGMSVNLPRLGKQLKLTQSTSFMAEERNTVEEAVSGD IIGLYDTGTYQIGDTLTVGKNDFQFERLPQFTPELFVRVS AKNVMRQKSFYKGLHQLVQEGAIQLYKTVKTDEYLLGAVG QLQFEVFEHRMKNEYNAEVLMERLGSKIARWIENDEVDEN LSSSRSLLVKDRYDHYVFLFENDFALRWFQEKNPTIKLYN PMDQHD SEQ ID NO: 23 DNA RRF-GbRRF-EcOpt Geobacillus (codon optimized for E. coli) ATGGCCAAACAGGTTATTCAGCAGGCCAAAGAAAAAATGG ATAAAGCCGTTCAGGCATTTACCCGTGAACTGGCAAGCAT TCGTGCAGGTCGTGCAAATGCAGGTCTGCTGGAAAAAGTT ACCGTTGATTATTATGGTGTTCCGACGCCGATTAATCAGC TGGCGAGCATTAGCGTTCCGGAAGCACGTCTGCTGGTGAT TCAGCCGTATGATAAAAGCGCAATCAAAGAGATGGAAAAA GCAATTCTGGCAAGCGATCTGGGTCTGACCCCGAGCAATG ATGGTAGCGTTATTCGTCTGGTTATTCCGCCTCTGACCGA AGAACGTCGTCGCGAACTGGCGAAACTGGTGAAAAAAATAC AGCGAAGATGCAAAAGTTGCCGTGCGTAATATTCGTCGTG ATGCAAATGATGAGCTGAAAAAAGCTGGAAAAGAATGGCGA AATTACCGAAGATGAACTGCGTAGCTATACCGATGAAGTT CAGAAACTGACCGATGATCATATCGCAAAAATTGACGCCA TCACCAAAGAGAAAGAAAAAGAAGTCATGGAAGTTTAA SEQ ID NO: 24 amino acid RRF GbRRF-EcOpt Geobacillus MAKQVIQQAKEKMDKAVQAFTRELASIRAGRANAGLLEKV TVDYYGVPTPINQLASISVPEARLLVIQPYDKSAIKEMEK AILASDLGLTPSNDGSVIIRLVIPPLTEERRRELAKLVKKY SEDAKVAVRNIRRDANDELKKLEKNGEITEDELRSYTDEV QKLTDDHIAKIDAITKEKEKEVMEV SEQ ID NO: 25 DNA AlaRS-GsAlaRS-EcOpt Geobacillus stearothermophilus (E. col Codon optimization for i) ATGAAAAACTGACCAGCGCACAGGTTCGTCGCATGTTTC TGGAATTTTTTCAAGAAAAAGGTCATGCCGTTGAACCGAG CGCAAGCCTGATTCCGGTTGATGATCCGAGCCTGCTGTGG ATTAATAGCGGTGTTGCAACCCTGAAAAAATACTTTGATG GTCGTATTGTTCCGGAAAATCCGCGTATTTGTAATGCCCA GAAAAGCATTCGTACCAACGATATTGAAAATGTGGGTAAA ACCGCACGCCATCACACCTTTTTTGAAATGCTGGGCAATT TTAGCATCGGCGATTATTTCAAACGTGAAGCAATTCATTG GGCCTGGGAATTTCTGACCAGTGATAAATGGATTGGTTTT GATCCGGAACGTCTGAGCGTTACCGTTCATCCGGAAGATG AAGAAGCATATAACATTTGGCGCAATGAAATTGGTCTGCC GGAAGAACGTATTATTCGTCTGGAAGGTAACTTTTGGGAT ATTGGTGAAGGTCCGAGCGGTCCGAATACCGAAATCTTTT ATGATCGTGGTGAAGCCTTTGGTAATGATCCGAATGATCC TGAACTGTATCCAGGTGGTGAAAATGATCGTTATCTGGAA GTTTGGAATCTGGTGTTTAGCCAGTTTAATCATAATCCGG ATGGCACCTATACACCGCTGCCGAAAAAAAACATTGATAC CGGCATGGGTTTAGAACGTATGTGTAGCATTCTGCAGGAT GTTCCGACCAATTTTGAAACCGACCTGTTTCTGCCGATTA TTCGTGCAACCGAGCAGATTGCCGGTGAACGTTATGGTGA AGATCCGGATAAAGATGTTGCCTTTAAAGTGATTGCCGAT CATATTCGCGCAGTTACCTTTGCAATTGGTGATGGTGCAC TGCCGAGCAATGAAGGTCGTGGTTATGTTCTGCGTCGTCT GCTGCGTCGTGCAGTTCGTTATGCAAAACATATTGGTATT GAACGTCCGTTCATGTATGAACTGGTTCCGGTTGTTGGTG AAATCATGCACGATTATTATCCCGAGGTTAAAGAGAAAGC CGATTTTATTGCACGTGTGATTCGTACCGAAGAAGAACGT TTTCACGAAACCCTGCATGAAGGTCTGGCAATTCTGGCAG AAGTTATTGAAAAAGCAAAAGAACAGGGTTCCGATGTTAT TCCGGGTGAAGAGGCATTTCGTCTGTATGATACCTATGGT TTTCCGATTGAACTGACCGAAGAATATGCAGCCGAAGCAG GTATGACCGTTGATCATGCAGGTTTTGAACGTGAAATGGA ACGTCAGCGTGAACGTGCCCGTGCAGCACGTCAGGATGTT GATAGTATGCAGGTTCAAGGTGGTGTTCTGGGTGATATTA AAGATGAAAGTCGCTTTGTGGGCTATGATGAGCTGGTTGC AGCAAGCACCGTTATTGCAATTGTTAAAGATGGTCGTCTG GTGGAAGAAGTTAAAGCAGGCGAAGAAGCACAGATTATTG TTGATGTTACCCCGTTTTATGCAGAAAGCGGTGGTCAGAT TGCAGATCAGGGTGTTTTTGAAAGCGAAACCGGCACCGCA GTTGTGAAAGATGTTCAGAAAGCACCGAATGGTCAGCATC TGCATGCAATTATTGTGGAACATGGCACCGTTAAAAAAGG TAGCCGTTATACCGCACGTGTTGATGAAGCAAAACGTATG CGTATTGTGAAAAATCATACCGCAACACATCTGCTGCATC AGGCACTGAAAGACGTTCTGGGTCGTCATGTTAATCAGGC AGGTAGCCTGGTTGCACCGGATCGTCTGCGTTTTGACTTT ACCCATTTTGGTCAGGTTAAACCCGAAGAACTGGAACGTA TTGAAGCGATTGTTAATGAGCAGATTTGGAAAAGCCTGCC GGTGGATATTTTCTATAAACCGCTGGAAGAGGCAAAAGCA ATGGGTGCAATGGCACTGTTTGGTGAAAAATATGGTGATA TTGTGCGTGTGGTTAAAGTGGGTGATTATAGCCTGGAACT GTGTGGTGGTTGTCATGTGCCGAATACCAGCGCCATTGGT CTGTTTAAAATCGTTAGCGAAAGCGGTATTGGTGCAGGCA CCCGTCGCATTGAAGCAGTTACCGGTGAAGCAGCATATCG TTTTATGAGCGAACAGCTGGCCATTCTGCAAGAAGCAGCA CAGAAACTGAAAACCAGTCCGAAAGAACTGAATGCACGTC TGGATGGCCTGTTTGCAGAACTGAAAGAATTAGAACGCGA AAATGAAAGCCTGGCAGCCCGTCTGGCACATATGGAAGCA GAACATCTGACCCGTCAGGTAAAAGATGTTAATGGTGTTC CGGTTCTGGCAGCAAAAGTTCAGGCAAATGATATGAATCA GCTGCGTGCCATGGCCGATGATCTGAAACAAAAACTGGGT ACAGCAGTTATTGTTCTGGCAAGCGCACAAGGTGGTAAAG TTCAGCTGATTGCAGCCGTTACAGATGACCTGGTAAAAAA AGGTTTTCATGCGGGTAAACTGGTTAAAGAAGTTGCAAGC CGTTGCGGTGGTGGTGGCGGTGGTCGTCCGGATCTGGCAC AGGCAGGCGGTAAAGATCCGAGCAAAGTTGGTGAAGCACT GGGTTATGTTGAAACCTGGGTTAAAAGCGTGAGCTAA SEQ ID NO: 26 Amino acid AlaRS-GsAlaRS-EcOpt Geobacillus stearothermophilus MKKLTSAQVRRMFLEFFQEKGHAVEPSASLIPVDDPSLLW INSGVATLKKYFDGRIVPENPRICNAQKSIRTNDIENVGK TARHHTFFEMLGNFSIGDYFKREAIHWAWEFLTSDKWIGF<00,02510>DPERLSVTVHPEDEEAYNIWRNEIGLPEERIIRLEGNFWD IGEGPSGPNTEIFYDRGEAFGNDPNDPELYPGGENDRYLE / / 这里原文中的 疑似应为 ,我按照正确的编号进行了翻译,你可根据实际情况调整 VWNLVFSQFNHNPDGTYTPLPKKNIDTGMGLERMCSILQD VPTNFETDLFLPIIRATEQIAGERYGEDPDKDVAFKVIAD HIRAVTFAIGDGALPSNEGRGYVLRRLLRRAVRYAKHIGI ERPFMYELVPVVGEIMHDYYPEVKEKADFIARVIRTEEER FHETLHEGLAILAEVIEKAKEQGSDVIPGEEAFRLYDTYG FPIELTEEYAAEAGMTVDHAGFEREMERQRERARAARQDV DSMQVQGGVLGDIKDESRFVGYDELVAASTVIAIVKDGRL VEEVKAGEEAQIIVDVTPFYAESGGQIADQGVFESETGTA VVKDVQKAPNGQHLHAIIVEHGTVKKGSRYTARVDEAKRM RIVKNHTATHLLHQALKDVLGRHVNQAGSLVAPDRLRFDF THFGQVKPEELERIEAIVNEQIWKSLPVDIFYKPLEEAKA MGAMALFGEKYGDIVRVVKVGDYSLELCGGCHVPNTSAIG LFKIVSESGIGAGTRRIEAVTGEAAYRFMSEQLAILQEAA QKLKTSPKELNARLDGLFAELKELERENESLAARLAHMEA EHLTRQVKDVNGVPVLAAKVQANDMNQLRAMADDLKQKLG TAVIVLASAQGGKVQLIAAVTDDLVKKGFHAGKLVKEVAS RCGGGGGGRPDLAQAGGKDPSKVGEALGYVETWVKSVS SEQ ID NO: 27 DNA ArgRS-GsArgRS-EcOpt Geobacillus (codon optimized for E. coli) ATGAATATTGTGGGCCAGATCAAAGAAAAAATGAAAGAAG AAATTCGTCAGGCAGCAGTTCGTGCAGGTCTGGCAAGCGC AGATGAACTGCCGGATGTTCTGCTGGAAGTTCCGCGTGAT AAAGCACATGGTGATTATAGCACCAATATTGCAATGCAGC TGGCACGTATTGCAAAAAAACCGCCTCGTGCAATTGCCGA AGCAATTGTTGGTCAGCTGGATCGTGAACGTATGAGCGTT GCCCGTATTGAAATTGCAGGTCCGGGTTTTATCAACTTCT ATATGGATAATCGTTACCTGACCGCAGTTGTTCCGGCAAT TCTGCAGGCAGGTCAGGCATATGGTGAAAGTAATGTTGGT AATGGTGAGAAAGTCCAGGTTGAATTTGTTAGCGCAAATC CGACCGGTGATCTGCATCTGGGTCATGCACGTGGTGCAGC AGTTGGTGATAGCCTGTGTAATATTCTGGCAAAAGCAGGT TTTGATGTGACCCGTGAATACTATATTAATGATGCAGGCA AGCAGATCTACAATCTGGCCAAAAGCGTTGAAGCACGTTA TTTTCAGGCACTGGGTGTTGATATGCCGCTGCCGGAAGAT GGTTATTATGGTGATGATATTGTGGAAATCGGCAAAAAAC TGGCCGAAGAATATGGTGATCGTTTCGTTGAAATGGAAGA AGAGGAACGTCTGGCATTTTTTCGTGATTATGGTCTGCGT TATGAGCTGGAAAAAATCAAAAAAGATCTGGCCGATTTTC GCGTTCCGTTTGATGTTTGGTATAGCGAAACCAGCCTGTA TGAAAGCGGTAAAATTGATGAAGCACTGAGCACCCTGCGT GAACGTGGTTATATCTATGAACAGGATGGTGCAACCTGGT TTCGTAGCACCGCATTTGGAGATGATAAAGATCGTGTTCT GATTAAACAGGACGGCACCTATACCTATCTGCTGCCGGAT ATTGCATATCATCAGGATAAACTGCGTCGCGGTTTTAAGA AACTGATTAACATTTGGGGTGCCGATCATCATGGTTATAT TCCTCGCATGAAAGCAGCAATTGCAGCACTGGGTTATGAT CCGGAAGCACTGGAAGTTGAAATTATTCAGATGGTGAATC TGTATCAGAATGGCGAACGTGTGAAAATGAGCAAACGTAC CGGTAAAGCAGTTACCATGCGTGAACTGATGGAAGAGGTT GGTGTTGATGCAGTTCGTTATTTCTTTGCAATGCGTAGCG GTGATACCCATCTGGATTTTGATATGGATCTGGCAGTTAG CCAGAGCAATGAAAATCCGGTTTATTATGTTCAGTATGCC CATGCGCGTGTTAGCAGCATTCTGCGTCAGGCGGAAGAAC AGCATATTAGCTATGATGGTGATCTGGCACTGCATCATCT GGTTGAAACCGAAAAAGAAATTGAGCTGCTGAAAGTGCTG GGTGATTTTCCGGATGTTGTTGCAGAAGCAGCACTGAAAC GTATGCCGCATCGTGTTACCGCATATGCATTTGACCTGGC CAGCGCACTGCATAGCTTTTATAACGCCGAAAAAAGTTCTG GATCTGGACAACATCGAAAAAACCAAAGCACGTCTGGCCC TGGTTAAAGCCGTTCAGATTACACTGCAGAATGCACTGGC CCTGATTGGTGTGAGCGCACCGGAACAAATGTAA SEQ ID NO: 28 amino acid ArgRS-GsArgRS-EcOpt Geobacillus MNIVGQIKEKMKEEIRQAAVRAGLASADELPDVLLEVPRD KAHGDYSTNIAMQLARIAKKPPRAIAEAIVGQLDRERMSV ARIEIAGPGFINFYMDNRYLTAVVPAILQAGQAYGESNVG NGEKVQVEFVSANPTGDLHLGHARGAAVGDSLCNILAKAG FDVTREYYINDAGKQIYNLAKSVEARYFQALGVDMPLPED GYYGDDIVEIGKKLAEEYGDRFVEMEEEERLAFFRDYGLR YELEKIKKDLADFRVPFDVWYSETSLYESGKIDEALSTLR ERGYIYEQDGATWFRSTAFGDDKDRVLIKQDGTYTYLLPD IAYHQDKLRRGFKKLINIWGADHHGYIPRMKAAIAALGYD PEALEVEIIQMVNLYQNGERVKMSKRTGKAVTMRELMEEV GVDAVRYFFAMRSGDTHLDFDMDLAVSQSNENPVYYVQYA HARVSSILRQAEEQHISYDGDLALHHLVETEKEIELLKVL GDFPDVVAEAALKRMPHRVVTAYAFDLASALHSFYNAEKVL DLDNIEKTKARLALVKAVQITLQNALALIGVSAPEQM SEQ ID NO: 29 DNA AsnRS-GsAsnRS-EcOpt Geobacillus (codon optimized for E. coli) ATGGATGTGAGCATTATTGGTGGTAATCAGTGTGTTAAAA CCACCACCATTGCCGAAGTTAATCAGTATGTTGGTCAGCA GGTTACCATTGGTGCATGGCTGGCAAATAAACGTAGCAGC GGTAAAATTGTTTTTCTGCAGCTGCGTGATGGCACCGGTT TTATTCAGGGTGTTGTTGAAAAAGCCAATGTTAGCGAAGA GGTTTTTCAGCGTGCAAAAACCCTGACACAAGAAACCAGC CTGTATGTGACCGGCACCGTTCGTATTGATGAACGTAGCC CGTTTGGTTATGAACTGAGCGTTGCCGATCTGCAGGTTAT TCAAGAAGCAGTTGATTATCCGATTACGCCGAAAGAACAT GGTGTTGAATTTCTGATGGATCATCGTCATCTGTGGCTGC GTAGCCGTCGTCAGCATGCAATTATGAAAATTCGCAACGA AATTATCCGTGCCACCTATGAATTTTTCAACGATCGTGGT TTTGTGAAAGTGGATGCACCGATTCTGACCGGTAGCGCAC CGGAAGGCACCACCGAACTGTTTCATACCAAATATTTCGA TGAGGATGCATATCTGAGCCAGAGCGGTCAGCTGTATATG GAAGCAGCAGCAATGGCACTGGGTAAAGTTTTTAGCTTTG GTCCGACCTTTCGTGCCGAAAAAAGCAAAACCCGTCGCCA TCTGATTGAATTTTGGATGGTTGAACCGGAAATGGCCTTT TATGAATTTGAAGATAATCTGCGCCTGCAAGAGGAATATG TTAGCTATCTGGTTCAGAGCGTTCTGGAACGTTGTCGTCT GGAACTGGGTCGCCTGGGTCGTGATGTTAGCAAACTGGAA TTAGTTAAACCGCCTTTTCCGCGTCTGACCTATGATGAAG CAATTAAACTGCTGCATGAAAAAAGGCCTGACCGATATTGA ATGGGGTGATGATTTTGGTGCACCGCATGAAACCGCAATT GCAGAAAGCTTTGATAAACCGGTGTTTATCACCCATTATC CGACCAGCCTGAAACCGTTTTATATGCAGCCGGATCCGAA TCGTCCGGATGTTGTTCTGTGTGCAGATCTGATTGCTCCG GAAGGTTATGGTGAAATTATTGGCGGTAGCGAACGCATCC ATGATTATGAGCTGCTGAAACGTCGCCTGGAAGAACATCA TCTGCCGCTGGAAGCATATGAATGGTATCTGGATCTGCGT AAATATGGTAGCTTCCGCATAGCGGTTTTGGTCTGGGTT TAGAACGTACCGTTGCATGGATTTGCGGTGTTGAACATGT GCGTGAAACCATTCCGTTTCCACGTCTGCTGAATCGTCTG TATCCGTAA sequence number 30 amino acid AsnRS-GsAsnRS-EcOpt Geobacillus MDVSIIGGNQCVKTTTIAEVNQYVGQQVTIGAWLANKRSS GKIVFLQLRDGTGFIQGVVEKANVSEEVFQRAKTLTQETS LYVTGTVRIDERSPFGYELSVADLQVIQEAVDYPITPKEH GVEFLMDHRHLWLRSRRQHAIMKIRNEIIRATYEFFNDRG FVKVDAPILTGSAPEGTTELFHTKYFDEDAYLSQSGQLYM EAAMALGKVFSFGPTFRAEKSKTRRHLIEFMVEPEMAF YEFEDNLRLQEEYVSYLVQSVLERCRLELGRLGRDVSKLE LVKPPFPRLTYDEAIKLLHEKGLTDIEWGDDFGAPHETAI AESFDKPVFITHYPTSLKPFYMQPDPNRPDVVLCADLIAP EGYGEIIGGSERIHDYELLKRRLEEHHLPLEAYEWYLDLR KYGSVPHSGFGLGLERTVAWICGVEHVRETIPFPRLLNRL YP SEQ ID NO: 31 DNA AspRS-GsAspRS-EcOpt Geobacillus (codon optimized for E. coli) ATGGAACGCACCTATTATTGTGGTGAAGTTCCGGAAAACCG CAGTTGGTGAACGTGTTGTTCTGAAAGGTTGGGTTCAGAA ACGTCGTGATTTAGGTGGTCTGATTTTTATCGATCTGCGT GATCGTACCGGTATTGTTCAGGTTGTTGCAAGTCCGGATG TTAGCGCAGAAGCACTGGCAGCAGCAGAACGTGTTCGTAG CGAATATGTTCTGAGCGTTGAAGGCACCGTTGTTGCCCGT GCACCGGAAACAGTTAATCCGAATATTGCAACCGGTCGCA TTGAAATTCAGGCAGAACGTATTGAAATTATCAACGAAGC AAAAACCCCTCCGTTTAGCATTAGTGATGATACCGATGCA GCCGAAGATGTTCGTCTGAAATATCGTTATCTGGATCTGC GTCGTCCGGTTATGTTTCAGACCCTGGCACTGCGTCATAA AATCACCAAAACCGTTCGTGATTTTCTGGATAGCGAACGC TTTCTGGAAATTGAAACCCCGATGCTGACCAAAAGCACAC CGGAAGGTGCACGTGATTATCTGGTTCCGAGCCGTGTTCA TCCGGGTGAATTTTATGCACTGCCGCAGAGTCCGCAGATC TTTAAACAGCTGCTGATGGTTGGTGGTGTGGAACGTTATT ATCAGATTGCACGTTGTTTTCGTGATGAGGACCTGCGTGC AGATCGTCAGCCGGAATTTACCCAGATTGATATTGAAATG AGCTTCATCGAGCAAGAGGATATCATTGATCTGACCGAAC GTATGATGGCAGCAGTTGTTAAAGCAGCAAAAGGTATTGA TATTCCGCGTCCGTTTCCGCGTATTACCTATGATGAAGCA ATGAGCTGTTATGGTAGCGATAAACCGGATATTCGTTTTG GTCTGGAACTGGTTGATGTGAGCGAAATTGTTCGTGATAG CGCATTTCAGGTTTTTGCGCGTGCAGTTAAAGAAGGTGGT CAGGTTAAAGCAATTAATGCAAAAGGTGCAGCACCGCGTT ATAGCCGTAAAGATATTGATGCACTGGGCGAATTTGCAGG TCGTTATGGTGCCAAAGGTCTGGCATGGCTGAAAGCAGAA GGTGAAGAACTGAAAGGTCCGATTGCAAAATTCTTTACCG ATGAAGAACAGGCAGCCCTGCGTCGTGCACTGGCCGTTGA AGATGGTGACCTGCTGCTGTTTGTTGCAGATGAAAAAGCA ATTGTTGCAGCAGCACTGGGTGCGCTGCGTCTGAAACTGG GTAAAGAACTGGGTCTGATTGATGAAGCCAAACTGGCATT TCTGTGGGTTACCGATTGGCCTCTGCTGGAATACGATGAA GAGGAAGGTCGCTATTACGCAGCACATCATCCGTTTACCA TGCCGGTGCGTGATGATATCCCGCTGCTGGAAACCAATCC GAGCGCAGTTCGTGCACAGGCATATGATCTGGTTCTGAAT GGTTATGAATTAGGTGGTGGTAGCCTGCGTATTTTTGAAC GTGATGTGCAAGAAAAAATGTTTCGTGCCCTGGGTTTTAG CGAAGAAGAAGCACGTCGTCAGTTTGGTTTTCTGTTAGAA GCATTTGAATATGGCACCCCTCCGCATGGTGGTATTGCAC TGGGTTTAGATCGTCTGGTTATGCTGCTGGCAGGTCGTAC CAATCTGCGCGATACCATTGCATTTCCGAAAACCGCCAGC GCAAGCTGTCTGCTGACCGAAGCACCGGGTCCTGTTAGCG ACAAACAGCTGGAAGAACTGCATCTGGCAGTTGTTCTGCC GGAAAATGAATAA SEQ ID NO: 32 amino acid AspRS-GsAspRS-EcOpt Geobacillus MERTYYCGEVPETAVGERVVLKGWVQKRRDLGGLIFIDLR DRTGIVQVVASPDVSAEALAAAERVRSEYVLSVEGTVVAR APETVNPNIATGRIEIQAERIEIINEAKTPPFSISDDTDA AEDVRLKYRYLDLRRPVMFQTLALRHKITKTVRDFLDSER FLEIETPMLTKSTPEGARDYLVPSRVHPGEFYALPQSPQI FKQLLMVGGVERYYQIARCFRDEDLRADRQPEFTQIDIEM SFIEQEDIIDLTERMMAAVVKAAKGIDIPRPFPRITYDEA MSCYGSDKPDIRFGLELVDVSEIVRDSAFQVFARAVKEGG QVKAINAKGAAPRYSRKDIDALGEFAGRYGAKGLAWLKAE GEELKGPIAKFFTDEEQAALRRALAVEDGDLLLFVADEKA IVAAALGALRLKLGKELGLIDEAKLAFLWVTDWPLLEYDE EEGRYYAAHHPFTMPVRDDIPLLETNPSAVRAQAYDLVLN GYELGGGSLRIFERDVQEKMFRALGFSEEEARRQFGFLLE AFEYGTPPHGGIALGLDRLVMLLAGRTNLRDTIAFPKTAS ASCLLTEAPGPVSDKQLEELHLAVVLPENE SEQ ID NO: 33 DNA CysRS-GsCysRS-EcOpt Geobacillus (codon optimized for E. coli) ATGAGCAGCATTCGTCTGTATAATACCCTGACGCGTAAAA AAGAACCGTTTGAACCGCTGGAACCGAACAAAGTTAAAAT GTATGTTTGTGGTCCGACCGTGTATAACTATATTCATATT GGTAATGCCCGTGCAGCCATTGTGTTTGATACCATTCGTC GTTATCTGGAATTTCGCGGTTATGATGTTACCTATGTGAG CAATTTTACCGACGTGGATGACAAACTGATTAAAGCAGCA CGTGAACTGGGTGAAAGCGTTCCGGCAATTGCAGAACGTT TTATTGAAGCCTATTTCGAAGATATTCAGGCCCTGGGTTG TAAAAAAGCAGATATTCATCCGCGTGTGACCGAAAATATC GATACCATTATTGAATTTATCCAGGCGCTGATCGATAAAG GCTATGCATATGAAGTTGATGGCGACGTTTATTATCGTAC CCGTAAATTTCGCGAATATGGCAAACTGAGCCATCAGAGC ATTGATGAACTGCAGGCAGGCGCACGTATTGAAATTGGTG AAAAAAAAGATGATCCGCTGGATTTTGCACTGTGGAAAGC AGCAAAAAGGTGAAATTTGTTGGGATAGCCCGTGGGGT AAAGGTCGTCCTGGTTGGCATATTGAATGTAGCGCAATGG CACGTAAATATCTGGGTGATACGATTGATATTCATGCCGG TGGTCAGGATCTGACCTTTCCGCATCATGAAAATGAAATT GCACAGAGCGAAGCACTGACCGGTAAACCGTTTGCCAAAT ATTGGCTGCATAATGGCTATCTGAACATCAACAACGAA AATGAGCAAAAGCCTGGGTAATTTTGTTCTGGTGCATGAT ATTATTCGCGAGATTGATCCGCAGGTTCTGCGCTTTTTTA TGCTGAGCGTTCATTATCGTCATCCGATCAATTATAGCGA AGAACTGCTGGAAAGCGCACGTCGTGGTCTGGAACGTCTG AAAACCGCATATAGCAATCTGCAGCACCGTCTGCAGGCAA GCACCAATCTGACCGATAATGATGAAGAATGGGTTAGCCG TATTGCCGATTCGTGCAAGCTTTATTCGTGAAATGGAT GATGATTTTAACACCGCCAATGGTATTGCCGTTCTGTTTG AACTGGCAAAACAGGCAAATCTGTATCTGCAAGAAAAAAC CACCTCCGAAAAAGTGATTCATGCATTTCTGCGTGAATTT GAACAGCTGGCAGATGTTCTGGGTCTGACCCTGAAACAGG ATGAGCTGCTGGATGAAGAAATTGAAGCCCTGATTCAGAA ACGTAATGAAGCCCGTAAAAATCGTGATTTTGCCCTGGCA GATCGTATTCGTGATGAATTACGTGCGAAAAACATCATCC TGGAAGATACACCGCAGGGCACCCGTTGGAAACGTGGTTA A SEQ ID NO: 34 amino acid CysRS-GsCysRS-EcOpt Geobacillus MSSIRLYNTLTRKKEPFEPLEPNKVKMYVCGPTVYNYIHI GNARAAIVFDTIRRYLEFRGYDVTYVSNFTDVDDKLIKAA RELGESVPAIAERFIEAYFEDIQALGCKKADIHPRVTENI DTIIEFIQALIDKGYAYEVDGDVYYRTRKFREYGKLSHQS IDELQAGARIEIGEKKDDPLDFALWKAAKEGEICWDSPWG KGRPGWHIECSAMARKYLGDTIDIHAGGQDLTFPHHENEI AQSEALTGKPFAKYWLHNGYLNINNEKMSKSLGNFVLVHD IIREIDPQVLRFFMLSVHYRHPINYSEELLESARGLERL KTAYSNLQHRLQASTNLTDNDEEWVSRIADIRASFIREMD DDFNTANGIAVLFELAKQANLYLQEKTTSEKVIHAFLREF EQLADVLGLTLKQDELLDEEIEALIQKRNEARKNRDFALA DRIRDELRAKNIILEDTPQGTRWKRG SEQ ID NO: 35 DNA GlnRS-EcGlnRS-EcOpt Escherichia coli ATGAGCGAAGCAGAAGCACGTCCGACCAACTTTATTCGTC AGATTATTGATGAAGATCTGGCCAGCGGTAAACATACCAC CGTTCATACCCGTTTTCCGCCTGAACCGAATGGTTATCTG CATATTGGTCATGCCAAAAGCATTTGCCTGAATTTTGGTA TTGCCCAGGATTATAAAGGTCAGTGCAATCTGCGTTTCGA TGATACCAATCCGGTGAAAGAAGATATCGAATACGTCGAG AGCATCAAAAATGATGTTGAATGGCTGGGTTTTCATTGGA GCGGTAATGTTCGTTATAGCAGCGATTATTTTGATCAGCT GCATGCCTATGCAATCGAACTGATTAACAAAGGTCTGGCC TATGTTGATGAACTGACACCGGAACAAATTCGTGAATATC GTGGTACACTGACCCAGCCTGGTAAAAATAGCCCGTATCG TGATCGTAGCGTTGAAGAAAATCTGGCCCTGTTTGAAAAA ATGCGTGCCGGTGGTTTTGAAGAAGGTAAAGCCTGTCTGC GTGCAAAAATTGATATGGCAAGCCCGTTTATTGTTATGCG TGATCCGGTTCTGTATCGCATCAAATTTGCAGAACATCAT CAGACCGGTAACAAATGGTGTATCTATCCGATGTATGATT TCACCCATTGCATTAGTGATGCCCTGGAAGGTATTACCCA TAGCCTGTGTACCCTGGAATTTCAGGATAATCGTCGTCTG TATGATTGGGTGTTAGACAATATCACCATTCCGGTGCATC CGCGTCAGTATGAATTTAGCCGTCTGAATCTGGAATACAC CGTTATGAGCAAACGTAAACTGAATCTGCTGGTGACCGAT AAACATGTTGAAGGTTGGGATGATCCGCGTATGCCGACCA TTAGCGGTCTGCGTCGTCGTGGTTATACCGCAGCAAGCAT CCGTGAATTTTGTAAACGTATTGGTGTGACCAAACAGGAT AACACCATTGAAATGGCCAGCCTGGAAAGCTGTATTCGCG AAGATCTGAATGAAAATGCACCGCGTGCAATGGCAGTTAT CGATCCGGTTAAACTGGTGATCGAAAATTATCAAGGTGAA GGTGAAATGGTGACCATGCCGAATCATCCGAATAAACCGG AAATGGGTAGCCGTCAGGTTCCGTTTAGCGGTGAAATTTG GATTGATCGTGCAGATTTTCGTGAAGAAGCCAACAAACAG TATAAACGTCTGGTTCTGGGTAAAGAAGTTCGTCTGCGTA ACGCCTATGTTATTAAAGCAGAACGTGTTGAAAAAGATGC CGAAGGCAATATTACCACCATTTTTTGTACCTATGACGCA GATACCCTGAGCAAAGATCCGGCAGATGGTCGTAAAGTTA AAGGTGTTATTCATTGGGTTAGCGCAGCACATGCACTGCC GGTTGAAATTCGCCTGTATGATCGTCTGTTTAGCGTTCCG AATCCGGGTGCAGCAGATGATTTTCTGAGCGTTATTAATC CGGAAAGCCTGGTTATTAAACAGGGTTTTGCCGAACCGAG CCTGAAAGATGCAGTTGCAGGTAAAGCATTTCAGTTTGAA CGCGAAGGTTATTTTTGTCTGGATAGCCGTCATAGCACCG CAGAAAAACCGGTGTTTAATCGTACCGTTGGTCTGCGTGA TACCTGGGCAAAAGTTGGTGAATAA SEQ ID NO: 36 amino acid GlnRS-EcGlnRS-EcOpt E. coli MSEAEARPTNFIRQIIDEDLASGKHTTVHTRFPPEPNGYL HIGHAKSICLNFGIAQDYKGQCNLRFDDTNPVKEDIEYVE SIKNDVEWLGFHWSGNVRYSSDYFDQLHAYAIELINKGLA YVDELTPEQIREYRGTLTQPGKNSPYRDRSVEENLALFEK MRAGGFEEGKACLRAKIDMASPFIVMRDPVLYRIKFAEHH QTGNKWCIYPMYDFTHCISDALEGITHSLCTLEFQDNRRL YDWVLDNITIPVHPRQYEFSRLNLEYTVMSKRKLNLLVTD KHVEGWDDPRMPTISGLRRRGYTAASIREFCKRIGVTKQD NTIEMASLESCIREDLNENAPRAMAVIDPVKLVIENYQGE GEMVTMPNHPNKPEMGSRQVPFSGEIWIDRADFREEANKQ YKRLVLGKEVRLRNAYVIKAERVEKDAEGNITTIFCTYDA DTLSKDPADGRKVKGVIHWVSAAHALPVEIRLYDRLFSVP NPGAADDFLSVINPESLVIKQGFAEPSLKDAVAGKAFQFE REGYFCLDSRHSTAEKPVFNRTVGLRDTWAKVGE sequence no. 37 DNA GluRS-GsGluRS-EcOpt Geobacterium ATGGCCAAAGAAGTTCCGGTTCGTTACGCACCGAGTCCGA CCGGTCATCTGCATATTGGTGGTGCACGTACCGCACTGTT TAATTACCTGTTTGCACGTCATCATGGTGGCAAAATGATT GTGCGTATTGAAGATACCGATATCGAACGTAATGTTGAAG GTGGTGAAAAAAGCCAGCTGGAAAATCTGAAATGGCTGGG CATTGATTATGATGAAAGCATTGATCAGGATGGTGGTTAT GGTCCGTATCGTCAGACCGAACGTCTGGATATTTATCGCA AATATGTGAACGAACTGCTGGAACAGGGTCATGCCTATAA ATGTTTTTGTACACCGGAAGAACTGGAACGTGAACGTGAA GCACAGCGTGCAGCAGGTATTGCAGCACCGCAGTATAGCG GTAAATGTCGTCATCTGACACCGGAACAGGTTGCCGAACT GGAAGCACAGGTAAACCGTATACCATTCGTCTGAAAGTT CCGGAAGGTAAAACCTATGAATTCTATGATCTGGTGCGTG GCAAAGTTGTGTTGAAAGCAAAGATGTTGGTGGCGATTG GGTTATTGTTAAAGCAAATGGTATTCCGACCTATAACTTT GCCGTTGTGATTGATGATCACCTGATGGAAATTTCACATG TGTTTCGTGGTGAAGAACATCTGAGCAATACCCCGAAACA GCTGATGGTGTATGAATATTTTGGTTGGGAACCGCCTCAG TTTGCACATCTGACCCTGATTGTTAATGAACAGCGTAAAA AACTGAGCAAACGCGACGAAAGCATTATTCAGTTTGTGAG CCAGTATAAAGAACTGGGTTATCTGCCGGAAGCCATGTTT AACTTTTTTGCACTGTTAGGTTGGTCACCGGAAGGTGAAG AAGAAATCTTTACCAAAGATGAACTGATCCGCATGTTTGA TGTTAGCCGTCTGAGCAAAAGCCCGAGTATGTTTGATACC AAAAAGCTGACCTGGATGAACAACCAGTACATCAAAAAAC TGGATCTGGATCGTCTGGTTGAACTGGCACTGCCGCATCT GGTTAAAGCAGGTCGTCTGCCTGCAGATATGACCGATGAG CAGCGTCAGTGGGCACGTGATCTGATTGCACTGTATCAAG AGCAGATGAGCTATGGTGCAGAAATTGTTCCGCTGAGCGA ACTGTTTTTCAAAGAAGAGATTGATTACGAGGATGAAGCA CGTCAGGTTCTGGCAGAAGAACAGGTTCCGGCAGTTCTGA GCACCTTTCTGGAAAGCGTTCGTGAGCTGGAACCGTTTAC CGCAGATGAAATTAAAGCAGCAATTAAAGCCGTTCAGAAA GCAACCGGTCAGAAAGGGAAAAAACTGTTTATGCCGATTC GTGCAGCCGTTACAGGTCAGACCCATGGTCCGGAACTGCC GTTTGCAATTCAGCTGCTGGGTAAAGAAAAAGTGATTGAA CGCCTGGAACGCGCACTGCAAGAAAAATTCTAA SEQ ID NO: 38 amino acid GluRS-GsGluRS-EcOpt Geobacillus MAKEVRVRYAPSPTGHLHIGGARTALFNYLFARHHGGKMI VRIEDTDIERNVEGGEKSQLENLKWLGIDYDESIDQDGGY GPYRQTERLDIYRKYVNELLEQGHAYKCFCTPEELERERE AQRAAGIAAPQYSGKCRHLTPEQVAELEAQGKPYTIRLKV PEGKTYEFYDLVRGKVVFESKDVGGDWVIVKANGIPTYNF AVVIDDHLMEISHVFRGEEHLSNTPKQLMVYEYFGWEPPQ FAHLTLIVNEQRKKLSKRDESIIQFVSQYKELGYLPEAMF NFFALLGWSPEGEEEIFTKDELIRMFDVSRLSKSPSMFDT KKLTWMNNQYIKKLDLDRLVELALPHLVKAGRLPADMTDE QRQWARDLIALYQEQMSYGAEIVPLSELFFKEEIDYEDEA RQVLAEEQVPAVLSTFLESVRELEPFTADEIKAAIKAVQK ATGQKGKKLFMPIRAAVTGQTHGPELPFAIQLLGKEKVIE RLERALQEKF SEQ ID NO: 39 DNA GlyRS-GsGlyRS-EcOpt Geobacterium ATGGCAGTTACCATGGAAGAAATTGTTGCACATGCAAAAC ATCGTGGTTTTGTTTTCCGGGTAGCGAAATTTATGGTGG TCTGGCAAATACCTGGGATTATGGTCCGCTGGGTGTTGAA CTGAAAAAATAACATTAAACGTGCCTGGTGGAAAAAATTCG TTCAAGAAAGCCCGTATAATGTTGGTCTGGATGCAGCAAT TCTGATGAATCCGCGTACCTGGGAAGCAAGCGGTCATCTG GGTAACTTTAATGATCCGATGGTTGATTGCAAACAGTGTA AAGCACGTCATCGTGCAGATAAACTGATTGAAAAAGCCCT GGAAGAAAAAGGCATTGAGATGATTGTTGATGGTCTGCCG CTGGCAAAAATGGATGAACTGATTAAAGAATATGATATCG CCTGTCCGGAATGTGGTAGCCGTGATTTTACCAATGTTCG TCAGTTTAACCTGATGTTCAAAACCTATCAGGGTGTTACC GAAAGCAGCGCCAATGAAATTTATCTGCGTCCGGAAACCG CACAGGGTATTTTTTGTTAATTTCAAAAATGTGCAGCGCAC CATGCGTAAAAAACTGCCGTTTGGTATTGCACAGATTGGC AAAAGCTTTCGCAACGAAATTACCCCTGGTAATTTTACCT TTCGCACCCGTGAATTTGAGCAGATGGAACTGGAATTTTT CTGTAAACCGGGTGAAGAACTGCAGTGGCTGGAATATTGG AAACAGTTTTGTAAAGAATGGCTGCTGAGCCTGGGTATGA AAGAAGATAATATTCGTCTGCGTGATCATGCCAAAGAAGA ACTGAGCCATTATAGCAATGCAACCACCGATATCGAATAT CATTTTCCGTTTGGTTGGGGTGAACTGTGGGGTATTGCAA GCCGTACCGATTATGATCTGAAACGCCATATGGAATATAG CGGTGAAGATTTCCATTACCTGGATCAAGAAACCAACGAA CGTTATATTCCGTATTGTATTGAACCGAGTCTGGGTGCAG ATCGTGTTACCCTGGCATTTATGATTGATGCCTATGATGA AGAGGAACTTGAAGATGGTACAACCCGTACCGTGATGCAT CTGCATCCGGCACTGGCACCGTATAAAGCAGCAGTGCTGC CGTTAAGCAAAAACTGGCAGATGGTGCACGTCGTATTTA TGAGGAACTGGCAAAACACTTCATGGTGGATTATGATGAA ACCGGTAGTATTGGTAAACGTTATCGTCGTCAGGATGAAA TTGGCACCCGTTTTGTTATTACCTATGATTTTGAAAGCGA ACAGGATGGTCAGGTTACCGTTCGTGATCGTGATACAATG GAACAGGTTCGTCTGCCGATTGGCGAACTGAAAGCATTTC TGGAAGAGAAAATCGCCTTCTAA sequence number 40 amino acid GlyRS-GsGlyRS-EcOpt Geobacillus MAVTMEEIVAHAKHRGFVFPGSEIYGGLANTWDYGPLGVE LKNNIKRAWWKKFVQESPYNVGLDAAILMNPRTWEASGHL GNFNDPMVDCKQCKARHRADKLIEKALEEKGIEMIVDGLP LAKMDELIKEYDIACPECGSRDFTNVRQFNLMFKTYQGVT ESSANEIYLRPETAQGIFVNFKNVQRTMRKKLPFGIAQIG KSFRNEITPGNFTFRTREFE Q MELEFFCKPGEEL Q WLEYW K Q FCKEWLLSLGMKEDNIRLRDHAKEELSHYSNATTDIEY HFPFGWGELWGIASRTDYDLKRHMEYSGEDFHYLDQETNE RYIPYCIEPSLGADRVTLAFMIDAYDEEELEDGTTRTVMH LHPALAPYKAAVLPLSKKLADGARRIYEELAKHFMVDYDE TGSIGKRYRRQDEIGTPFCITYDFESEQDGQVTVRDRDTM EQVRLPIGELKAFLEEKIAF SEQ ID NO: 41 DNA HisRS-GsHisRS-EcOpt Geobacillus (codon optimized for E. coli) ATGGCATTTCAGATTCCGCGTGGCACCCAGGATGTTCTGC CTGGTGATACCGAAAAATGGCAGTATGTTGAACATGTTGC ACGTAATCTGTGTAGCCGTTATGGTTATCGTGAAATTCGT ACCCCGATTTTTGAACACACCGAACTGTTTCTGCGTGGTG TGGGTGATACCACCGATATTGTTCAGAAAGAAATGTATAC CTTCGAGGATAAAGGTGGTCGTGCACTGACCCTGCGTCCG GAAGGCACCGCACCGGTTGTTCGTGCATTTGTGGAACATA AACTGTATGGTAGTCCGCATCAGCCGCTGAAACTGTATTA TTCAGGTCCGATGTTTCGTTATGAACGTCCTGAAGCAGGT CGTTTTCGTCAGTTTGTTCAGTTTGGTGTTGAAGCACTGG GTAGCAGCGATCCGGCAATTGATGCAGAAGTTATGGCACT GGCAATGCATATTTATGAAGCCCTGGGTCTGAAACGTATT CGTCTGGTGATTAATAGCCTGGGTGATCTGGATAGCCGTC GTGCACATCGTGAAGCGCTGGTTCGTCATTTTAGCAGCCG TATTCATGAACTGTGTCCGGATTGTCAGACCCGTCTGCAT ACCAATCCGCTGCGTATTCTGGATTGTAAAAAAGATCGTG ATCATGAGCTGATGGCAACCGCACCGAGCATCCTGGATTA TCTGAATGAAGATAGCCGTGCCTATTTCGAGAAAGTGAAA CAGTATCTGACCAATCTGGGTATTCCGTTTGTTATTGATA GTCGTCTGGTTCGTGGTCTGGATTATTACAATCATACCAC CTTTGAAATCATGAGCGAAGCCGAAGGTTTTGGTGCAGCA GCAACCCTGTGTGGTGGTGGTCGTTATAATGGTCTGGTTC AAGAAATTGGTGGTCCGGAAACACCTGGTATTGGTTTTGC ACTGAGCATTGAACGTCTGCTGGCAGCACTGGATGCCGAA GGTGTTGAACTGCCGGTTGAAAGTGGCCTGGATTGTTATG TTGTTGCAGTTGGTGAACGTGCAAAAGATGAAGCAGTGCG TCTGGTTTATGCCCTGCGTCGTAGCGGTCTGCGTGTTGAT CAGGATTACCTGGGTCGTAAACTGAAAGCACAGCTGAAAG CAGCAGATCGTCTGGGTGCAAGCTTTGTTGCAATTATTGG TGATGAGGAACTGGAACGTCAAGAAGCAGCAGTTAAACAT ATGGCAAGCGGTGAACAGACCAATGTTCCGCTGGGTGAAC TGGCACATTTTCTGCATGAACGTATTGGCAAAGAAGAATA A SEQ ID NO: 42 amino acid HisRS-GsHisRS-EcOpt Geobacillus MAFQIPRGTQDVLPGDTEKWQYVEHVARNLCSRYGYREIR TPIFEHTELFLRGVGDTTDIVQKEMYTFEDKGGRALTLRP EGTAPVVRAFVEHKLYGSPHQPLKLYYSGPMFRYERPEAG RFRQFVQFGVEALGSSDPAIDAEVMALAMHIYEALGLKRI RLVINSLGDLDSRRAHREALVRHFSSRIHELCPDCQTRLH TNPLRILDCKKDRDHELMATAPSILDYLNEDSRAYFEKVK QYLTNLGIPFVIDSRLVRGLDYYNHTTFEIMSEAEGFGAA ATLCGGGRYNGLVQEIGGPETPGIGFALSIERLLAALDAE GVELPVESGLDCYVVAVGERAKDEAVRLVYALRRSGLRVD QDYLGRKLKAQLKAADRLGASFVAIIGDEELERQEAAVKH MASGEQTNVPLGELAHFLHERIGKEE SEQ ID NO: 43 DNA IleRS-GsIleRS-EcOpt Geobacillus stearothermophilus (E. coli (codon optimized for i)) ATGGACTACAAAGAAACCCTGCTGATGCCGCAGACCGAAT TTCCGATGCGTGGTAATCTGCCGAAACGTGAACCGGAAAT GCAGAAAAAATGGGAAGAGATGGATATCTACCGCAAAGTT CAAGAACGTACCAAAGGTCGTCCGCTGTTTGTTCTGCATG ATGGTCCGCCTTATGCAAATGGTGATATTCATATGGGTCA TGCCCTGAACAAAATCCTGAAAGATATTATCGTGCGCTAT AAGAGCATGAATGGTTATTGTGCACCGTATGTTCCAGGTT GGGATACCCATGGTCTGCCGATTGAAACCGCACTGGCAAA ACAGGGTGTTGATCGTAAAAGCATGAGCGTTGCAGAATTT CGTAAACGTTGTGAACAGTATGCCTATGAGCAGATTGATA ATCAGCGTCGTCAGTTTAAACGTCTGGGTGTTCGTGGTGA TTGGGATAATCCGTATATTACCCTGAAACCGGAATATGAA GCACAGCAGATTAAAGTGTTTGGCGAGATGGCAAAAAAAG GCCTGATCTATAAAGGTCTGAAACCTGTTTATTGGAGCCC GAGCAGCGAAAGTGCACTGGCAGAAGCAGAAATTGAGTAT AAAGATAAACGCTCCCCGAGCATTTATGTTGCCTTTCCGG TTAAAGATGGTAAAGGTGTTCTGGAAGGTGATGAACGTAT TGTGATTTGGACCACCACACCGTGGACCATTCCGGCAAAT CTGGCAATTGCAGTTCATCCGGATCTGGATTATCATGTTG TTGATGTTAGCGGTAAACGTTATGTTGTTGCAGCAGCACT GGCCGAAAGCGTTGCAAAAGAAATTGGTTGGGATGCATGG TCAGTTGTGAAAACCGTTAAAGGTAAAGAACTGGAATATG TGGTTGCGAAACACCCGTTTTATGAACGTGATAGCCTGGT TGTTTGTGGTGAACATGTGACCACCGATGCAGGCACCGGT TGTGTTCATACCGCACCTGGTCATGGTGAAGATGATTTTC TGGTTGGTCAGAAATATGGCCTGCCGGTTCTGTGTCCGGT GGATGAACGTGGTTATATGACCGAAGAAGCACCGGGTTTT GAAGGTATGTTTTATGAGGATGCCAACAAAGCGATTACGC AGAAACTGGAAGAAGTTGGCGCACTGCTGAAACTGGGTTT TATTACCCATAGCTATCCGCATGATTGGCGTACCAAACAG CCGACCATTTTTCGTGCAACCACACAGTGGTTTGCAAGCA TTGATAAAATTCGCAATGAACTGCTGCAGGCCATCAAAGA AACAAAATGGATCCCGGAATGGGGTGAAATTCGCATTCAT AACATGGTTCGTGATCGCGGTGATTGGTGTATTAGCCGTC AGCGTGCATGGGGTGTTCCGATTCCGGTGTTTTATGGTGA AAATGGTGAACCGATTATCACCGATGAAACCATTGAACAT GTTAGCAACCTGTTTCGTCAGTATGGTAGCAATGTTTGGT TTGAACGTGAAGCAAAAGATCTGCTGCCGGAAGGTTTTAC CCATCCGAGCAGCCCGAATGGTATTTTTACAAAAGAAACC GATATCATGGACGTGTGGTTTGATAGCGGTAGCAGCCATC AGGCAGTTCTGGTGGAACGTGATGATCTGATGCGTCCGGC AGATCTGTATCTGGAAGGCAGCGATCAGTATCGTGGTTGG TTTAATAGCAGCCTGAGCACCGCAGTTGCAGTGACCGGTA AAGCACCGTATAAAGGTGTGCTGAGCCATGGTTTTGTGCT GGATGGTGAAGGTCGTAAAATGAGCAAAAGCCTGGGTAAT GTTGTTGTTCCTGCAAAAGTTATGGAACAGTTTGGTGCAG ATATTCTGCGTCTGTGGGTTGCCAGCGTTGATTATCAGGC AGATGTTCGTATTAGCGATCATATTCTGAAACAGGTGAGC GAAGTGTATCGCAAAATTCGTAATACCTTTCGCTTTATGC TGGGTAACCTGTTTGATTTTGATCCGAATCAGAATGCAGT TCCGATTGGTGAACTGGGTGAAGTTGATCGTTATATGCTG GCCAAACTGAATAAACTGATCGCCAAAGTGAAAAAAGCCT ATGATAGCTACGATTTCGCAGCCGTTTATCATGAAATGAA CCATTTTTGTACCGTTGAACTGAGCGCCTTTTATCTGGAT ATGGCAAAAGATATCCTGTATATCGAAGCAGCAGATAGCC GTGCACGTCGTGCAGTTCAGACCGTTCTGTATGAAACCGT TGTTGCACTGGCGAAACTGATTGCACCGATTCTGCCGCAT ACCGCAGATGAAGTTTGGGAACATATTCCGAATCGTCGTG AAAATGTGGAAAGCGTTCAGCTGACCGATATGCCGGAACC GATTGCAATTGATGGCGAAGAGGCACTGCTGGCAAAATGG GATGCCTTTATGGATGTTCGTGATGATATGCTGAAAGCAC TGGAAAATGCCCGTAACGAAAAAGTGATTGGTAAAAGCCT GACCGCAAGCGTTATTGTTTATCCGAAAGATGAAGCACGT AAACTGCTGGCGAGCCTGGATGCCGATCTGCGTCAGCTGC TGATTGTTAGCGCATTTAGCATTGCAGATGAACCGTATGA TGCTGCCCCTGCAGAAGCCGAACGTCTGGATCATGTTGCC GTTCTGGTTCGTCCTGCCGAAGGTGAAACCTGCGAACGTT GTTGGACCGTTACACCGGCAGTTGGTCAGGATCCGAGCCA TCCGACCTTTTGTCCGCGTTGTGCACATATTGTTAACGAA CATTATAGCGCCTAA SEQ ID NO: 44 Amino acid IleRS - GsIleRS - EcOpt Geobacillus stearothermophilus MDYKETLLMPQTEFPMRGNLPKREPEMQKKWEEMDIYRKV QERTKGRPLFVLHDGPPYANGDIHMGHALNKILKDIIVRY KSMNGYCAPYVPGWDTHGLPIETALAKQGVDRKSMSVAEF RKRCEQYAYEQIDNQRRQFKRLGVRGDWDNPYITLKPEYE AQQIKVFGEMAKKGLIYKGLKPVYWSPSSESALAEAEIEY KDKRSPSIYVAFPVKDGKGVLEGDERIVIWTTTPWTIPAN LAIAVHPDLDYHVVDVSGKRYVVAAALAESVAKEIGWDAW SVVKTVKGKELEYVVAKHPFYERDSLVVCGEHVTTDAGTG CVHTAPGHGEDDFLVGQKYGLPVLCPVDERGYMTEEAPGF EGMFYEDANKAITQKLEEVGALLKLGFITHSYPHDWRTKQ PTIFRATTQWFASIDKIRNELLQAIKETKWIPEWGEIRIH NMVRDRGDWCISRQRAWGVPIPVFYGENGEPIITDETIEH VSNLFRQYGSNVWFEREAKDLLPEGFTHPSSPNGIFTKET DIMDVWFDSGSSHQAVLVERDDLMRPADLYLEGSDQYRGW FNSSLSTAVAVTGKAPYKGVLSHGFVLDGEGRKMSKSLGN VVVPAKVMEQFGADILRLWVASVDYQADVRISDHILKQVS EVYRKIRNTFRFMLGNLFDFDPNQNAVPIGELGEVDRYML AKLNKLIAKVKKAYDSYDFAAVYHEMNHFCTVELSAFYLD MAKDILYIEAADSRARRAVQTVLYETVVALAKLIAPILPH TADEVWEHIPNRRENVESVQLTDMPEPIAIDGEEALLAKW DAFMDVRDDMLKALENARNEKVIGKSLTASVIVYPKDEAR KLLASLDADLRQLLIVSAFSIADEPYDAAPAEAERLDHVA VLVRPAEGETCERCWTVTPAVGQDPSHPTFCPRCAHIVNE HYSA Sequence number 45 DNA LeuRS-GsLeuRS-EcOpt Geobacillus stearothermophilus (Escherichia coli (E.col codon optimization for i)) ATGAGCTTTAACCACCGTGAAATCGAACAGAAATGGCAGG ATTATTGGGAGAAGAATAAAACCTTTCGTACACCGGATGA TGATGACAAACCGAAATTCTATGTGCTGGATATGTTTCCG TATCCGAGCGGTGCAGGTCTGCATGTTGGTCATCCGGAAG GTTATACCGCAACCGATATTCTGGCACGTATGAAACGTAT GCAGGGTTATAATGTTCTGCATCCGATGGGTTGGGATGCA TTTGGTCTGCCTGCAGAACAGTATGCACTGGATACCGGTA ATGATCCGGCAGAATTTACCCAGAAAAACATCGATAACTT TCGTCGCCAGATTAAAAGCCTGGGTTTTAGCTATGATTGG GATCGTGAAATCAATACCACCGATCCGAATTATTACAAAT GGACCCAGTGGATCTTCCTGAAACTGTATGAAAAAGGTCT GGCCTATATGGATGAAGTTCCGGTTAATTGGTGTCCGGCA CTGGGCACCGTTCTGGCAAATGAAGAAGTTATTAACGGTC GTAGCGAACGTGGTGGCCATCCGGTTATTCGTAAACCGAT GCGTCAGTGGATGCTGAAAATTACCGCATATGCAGATCGT CTGCTGGAAGATCTGGAAGAATTAGATTGGCCTGAAAGCA TCAAAGAAATGCAGCGTAATTGGATTGGTCGTAGTGAAGG TGCAGAAATTGAATTTGCAGTTGATGGTCACGATGAAACC TTTACCGTTTTTACCACACGTCCGGATACACTGTTTGGTG CAACCTATACCGTGCTGGCACCGGAACATCCGCTGGTTGA AAAAATCACCACTCCGGAACAGAAACCTGCCGTTGATGCA TATCTGAAAGAAATTCAGAGCAAAAGCGATCTGGAACGTA CCGATCTGGCCAAAGAAAAAACCGGTGTGTTTACCGGTGC ATATGCCATTCATCCTGTTACCGGTGATCGCCTGCCGATT TGGATTGCAGATTATGTTCTGATGAGCTATGGTACAGGTG CAATTATGGCAGTTCCGGCACATGATGAACGTGATTATGA ATTCGCCAAAAAATTCCATCTGCCGATGAAAGAAGTTGTT GCAGGCGGTAATATTGAGAAAGAAGCATATACAGGCGACG GCGAACATATTAACAGCGAATTTCTGAATGGCCTGAATAA ACAAGAGGCCATCGATAAAATGATTGCCTGGCTGGAAGAA CATGGTAAAGGTCGTAAAAAAGTTAGCTATCGTCTGCGTG ATTGGCTGTTTAGCCGTCAGCGTTATTGGGGTGAACCGAT TCCGATTATTCATTGGGAAGATGGCACCATGACACCGGTT CCGGAAGAAGAACTGCCGCTGGTTCTGCCGAAAACCGATG AAATTCGTCCGAGCGGCACCGGTGAAAGTCCGCTGGCAAA TATTGAAGAATGGGTTAATGTTGTGGATCCGAAAACGGGT AAAAAAGGTCGTCGCGAAACCAATACCATGCCGCAGTGGG CAGGTAGCTGTTGGTATTATCTGCGTTATATTGATCCGCA CAACGATAAACAGCTGGCAGATCCGGAAAAACTGAAAAAA TGGCTGCCGGTTGATGTGTATATTGGTGGTGCCGAACATG CAGTGCTGCATCTGCTGTATGCACGTTTTTGGCATAAATT TCTGTATGACCTGGGTATTGTTCCGACCAAAGAACCGTTT CAGAAACTGTTTAATCAGGGTATGATTCTGGGCGAGAACA ACGAAAAAATGAGCAAAAGTAAAGGCAATGTGGTGAACCC GGATGATATTATTGAAAGCCATGGTGCAGATACCCTGCGT CTGTATGAGATGTTTATGGGTCCGCTGGAAGCAAGCATTG CATGGTCAACCAAAGGCCTGGATGGTGCACGTCGTTTTCT GGATCGTGTTTGGCGTCTGTTTGTTACCGAAAATGGTGAA CTGAATCCGAACATTGTTGATGAACCGGCAAATGATACCC TGGAACGCATTTATCATCAGACCGTTAAAAAAGTGACCGA GGATTATGAAGCCCTGCGTTTTAATACCGCAATTAGCCAG CTGATGGTGTTTATTAACGAAGCCTATAAAGCCGAGCAGA TGAAAAAAGAATATATGGAAGGCTTCGTGAAACTGCTGAG TCCGGTTTGTCCGCATATTGGTGAAGAACTGTGGCAGAAA CTGGGTCATACCGATACCATTGCATATGAACCGTGGCCGA CCTATGATGAAACCAAACTGGTTGAAGATGTGGTGGAAAT TGTTGTGCAGATTAATGGTAAAGTGCGTAGTCGCCTGCAT GTGCCTGTTGATCTGCCTAAAGAAGCCTTAGAAGAACGCG CACTGGCGGATGAAAAGATTAAAGAACAGCTGGAAGGTAA AACCGTGCGTAAAGTTATTGCCGTTCCGGGTAAACTGGTT AATATTGTTGCCAACTAA SEQ ID NO: 46 Amino acid LeuRS-GsLeuRS-EcOpt Geobacillus stearothermophilus MSFNHREIEQKWQDYWEKNKTFRTPDDDKPKFYVLDMFP YPSGAGLHVGHPEGYTATDILARMKRMQGYNVLHPMGWDA FGLPAEQYALDTGNDPAEFTQKNIDNFRRQIKSLGFSYDW DREINTTDPNYKWTQWIFLKLYEKGLAYMDEVPVNWCPA LGTVLANEEVINGRSERGGHPVIRKPMRQWMLKITAYADR LLEDLEELDWPESIKEMQRNWIGRSEGAEIEFAVDGHDET FTVFTTRPDTLFGATYTVLAPEHPLVEKITTPEQKPAVDA YLKEIQSKSDLERTDLAKEKTGVFTGAYAIHPVTGDRLPI WIADYVLMSYGTGAIMAVPAHDERDYEFAKKFHLPMKEVV AGGNIEKEAYTGDGEHINSEFLNGLNKQEAIDKMIAWLEE HGKGRKKVSYRLRDWLFSRQRYWGEPIPIIHWEDGTMTPV PEEELPLVLPKTDEIRPSGTGESPLANIEEWVNVVDPKTG KKGRRETNTMPQWAGSCWYYLRYIDPHNDKQLADPEKLKK WLPVDVYIGGAEHAVLHLLYYARFWHKFLYDLGIVPTKEPF QKLFNQGMILGENNEKMSKSKGNVVNPDDIIESHGADTLR LYEMFMGPLEASIAWSTKGLDGARRFLDRVWRLFVTENGE LNPNIVDEPANDTLERIYHQTVKKVTEDYEALRFNTAISQ LMVFINEAYKAEQMKKEYMEGFVKLLSPVCPHIGEELWQK LGHTDTIAYEPWPTYDETKLVEDVVEIVVQINGKVRSRLH VPVDLPKEALEERALADEKIKEQLEGKTVRKVIAVPGKLV NIVAN Accession number 47 DNA LysRS-GsLysRS-EcOpt Geobacillus stearothermophilus(Escherichia coli (E.col codon-optimized for i)) ATGAGCCATGAAGAACTGAATGATCAGCTGCGTGTTCGTC GTGAAAAACTGAAAAAAATCGAAGAACTGGGCGTTGATCC GTTTGGTAAACGTTTTGAACGTACCCATAAAGCCCAAGAA CTGTTTGAACTGTATGGTGATCTGAGCAAAGAGGAACTGG AAGAAAAACAAATTGAAGTTGCAGTTGCCGGTCGCATTAT GACCAAACGTGGTAAAGGTAAAGCAGGCTTTGCACATATT CAGGATGTTACCGGTCAGATTCAGATTTATGTGCGTCAGG ATGATGTTGGTGAACAGCAGTATGAACTGTTCAAAATTAG CGATCTGGGTGATATTGTTGGTGTTCGTGGCACCATGTTT AAAACCAAAGTGGGTGAACTGAGCATTAAAGTGAGCAGCT ATGAATTTCTGACCAAAGCACTGCGTCCGCTGCCGGAAAA ATATCATGGTCTGAAAGATATTGAACAGCGTTATCGTCAG CGCTATCTGGATCTGATTATGAATCCGGAAAGCAAAAAAA CCTTTATTACCCGCTCACTGATTATCCAGAGCATGCGTCG TTATCTGGATAGCCGTGGATATCTGGAAGTTGAAACCCCG ATGATGCATGCCGTTGCCGGTGGTGCAGCAGCACGTCCGT TTATTACACATCATAATGCACTGGATATGACCCTGTATAT GCGTATTGCAATTGAACTGCATCTGAAACGTCTGATTGTT GGCGGTCTGGAAAAAGTGTATGAAATTGGTCGTGTGTTTC GCAATGAAGGTATTAGCACCCGTCATAATCCGGAATTTAC CATGCTGGAACTGTACGAAGCATATGCCGATTTTCACGAT ATTATGGAACTGACCGAAAACCTGATTGCCCATATTGCAA CCGAAGTTCTGGGCACCACCAAAATTCAGTATGATGAACA TGTTGTTGACCTGACACCGGAATGGCGTCGTCTGCATATG GTTGATGCAATTAAAGAATATGTCGGCGTGGATTTTTGGC GTCAGATGAGTGATGAAGAAGCACGCGAACTGGCAAAAGA ACATGGTGTGGAAGTTGCACCGCATATGACCTTTGGCCAT ATTGTGAACGAATTCTTTGAGCAGAAAGTGGAAAGCCATC TGATTCAGCCGACCTTTATCTATGGTCATCCGGTTGAAAT TAGTCCGCTGGCCAAAAAAAACCCGGATGATCCTCGTTTT ACCGATCGTTTTGAGCTGTTTATTGTGGGTCGTGAACATG CAAATGCCTTTACCGAACTGAACGATCCGATTGATCAGCG TCAGCGTTTTGAAGCACAGCTGAAAGAACGTGAACAGGGT AATGATGAAGCACACGAAATGGATGAAGATTTTCTGGAAG CACTGGAATATGGTATGCCTCCGACCGGTGGTTTTAGGTAT TGGTGTTGATCGTCTGGTTATGCTGCTGACCAATAGTCCG AGCATTCGTGATGTTCTGCTGTTTCCGCAGATGCGTCATA AAAAA sequence no. 48 amino acid LysRS-GsLysRS-EcOpt Geobacillus stearothermophilus MSHEELNDQLRVRREKLKKIEELGVDPFGKRFERTHKAQE LFELYGDLSKEELEEKQIEVAVAGRIMTKRGKGKAGFAHI QDVTGQIQIYVRQDDVGEQQYELFKISDLGDIVGVRGTMF KTKVGELSIKVSSYEFLTKALRPLPPEKYHGLKDIEQRYRQ RYLDLIMNPESKKTFITRSLIIQSMRRYLDSRGYLEVETP MMHAVAGGAAARPFITHHNALDMTLYMRIAIELHLKRLIV GGLEKVYEIGRVFRNEGISTRHNPEFTMLELYEAYADFHD IMELTENLIAHIATEVLGTTKIQYDEHVVDLTPEWRRLHM VDAIKEYVGVDFWRQMSDEEARELAKEHGVEVAPHMTFGH IVNEFFEQKVESHLIQPTFIYGHPVEISPLAKKNPDDPRF TDRFELFIVGREHANAFTELNDPIDQRQRFEAQLKEREQG NDEAHEMDEDFLEALEYGMPPTGGLGIGVDRLVMLLTNSP SIRDVLLFPQMRHK Sequence number 49 DNA MetRS-GsMetRS-EcOpt Geobacillus stearothermophilus (codon optimized for Escherichia coli (E. coli)) ) ATGGAAAAAAAGACCTTCTATCTGACCACGCCGATCTATT ATCCGAGCGATCGTCTGCATATTGGTCATGCATATACCAC CGTTGCCGGTGATGCAATGGCACGTTATAAACGTATGCGT GGTTATGATGTTATGTATCTGACCGGCACCGATGAACATG GTCAGAAAATTCAGCGTAAAGCCGAAGAAAAAGGTGTTAC ACCGCAGCAGTATGTTGATGAAATTGTTGCAGGTATTCAA GAACTGTGGAAAAAACTGGATATCAGCTATGATGATTTCA TCCGTACCACACAAGAACGCCATAAAAAAGTTGTTGAGCA GATTTTTACCCGTCTGGTTGAACAGGGTGATATTTATCTG GGTGAATATGAAGGTTGGTATTGTACCCCGTGTGAAAGCT TTTATACCGAACGTCAGCTGGTTGATGGTAATTGTCCGGA TTGTGGTCGTCCGGTTGAAAAAGTTAAAGAGGAAAGCTAT TTTTTCCGCATGAGCAAATATGTTGATCGCCTGCTGCAGT ATTATGAAGAAAACCCGGATTTCATTCAGCCGGAAAGCCG TAAAAATGAGATGATTAACAACTTTATCAAACCTGGCCTG GAAGATCTGGCAGTTAGCCGTACCACCTTTGATTGGGGTA TTAAAGTTCCGGGTAATCCGAAACATGTGATCTATGTTTG GATTGATGCACTGGCCAACTATATTACCGCATTAGGTTAT GGCACCGATAACGATGAAAAATTCCGTAAATATTGGCCTG CCGATGTTCATCTGGTTGGTAAAGAAATTGTTCGCTTCCA TACCATTTATTGGCCGATTATGCTGATGGCACTGGGTCTG CCGCTGCCGAAAAAAGTTTTTGGTCATGGTTGGCTGCTGA TGAAAGATGGTAAAATGAGCAAAAGCAAAGGCAATGTTGT TGATCCGGTTACACTGATTGATCGTTATGGTCTGGATGCA CTGCGTTATTATCTGCTGCGTGAAGTTCCGTTTGGTGCAG ATGGTGTTTTTACACCGGAAGGTTTTATTGAGCGCATCAA TTATGATCTGGCAAATGATCTGGGTAATCTGCTGCATCGT ACCGTTGCAATGATCGAAAAATACTTTGATGGTGTGATTC CGCCTTATCGTGGTCCGAAAACACCGTTTGATCAAGAGCT GGTTCAGACCGCACGTGAAGTTGTTCGTCAGTATGAAGAG GCAATGGAAGGTATGGAATTTAGCGTTGCACTGGCAGCAG TTTGGCAGCTGATTAGTCGTACCAATAAATACATTGATGA AACCCAGCCGTGGGTGTTAGCAAAAGATGAACAGAAACGT GATGAACTGGCAGCCGTTATGACCCATCTGGCAGAAAGCC TGCGTCATACCGCAGTTCTGCTGCAGCCGTTTCTGACCCG CACACCGGAACGTATGCTGGCACAGCTGGGTATTACCGAT CATAGCCTGAAAGAATGGGATAGCCTGTATGATTTTGGTC TGATTCCGGAAGGCACCAAAGTTCAGAAAGGTGAACCGCT GTTTCCGCGTCTGGATATTGAAGCAGAAGTGGAATATATC AAAGCCCATATGCAAGGTGGTAAACCGGCAGCCGAACCGG TTAAAGAAGAAAAAAAAGCAGCCGAAGCAGCGGAAATTAG CATCGATGAATTTGCAAAAGTTGATCTGCGTGTTGCCGAA GTTATTCATGCAGAACGTATGAAAAACGCCGATAAACTGC TGAAACTGCAGCTGGATTTAGGTGGTGAAAAACGTCAGGT TATTAGCGGTATTGCCGAATTCTATAAACCGGAAGAACTG GTGGGTAAAAAAGTGATTTGTGTGGCAAATCTGAAACCGG CAAAACTGCGTGGTGAATGGTCTGAAGGCATGATTCTGGC AGGCGGTAGCGGTGGTGAATTTAGCCTGGCAACCGTTGAT CAGCATGTTCCGAATGGTACGAAAATCAAATAA SEQ ID NO: 50 Amino acid MetRS-GsMetRS-EcOpt Geobacillus stearothermophilus MEKKTFYLTTPIYYPSDRLHIGHAYTTVAGDAMARYKRMR GYDVMYLTGTDEHGQKIQRKAEEKGVTPQQYVDEIVAGIQ ELWKKLDISYDDFIRTTQERHKKVVEQIFTRLVEQGDIYL GEYEGWYCTPCESFYTERQLVDGNCPDCGRPVEKVKEESY FFRMSKYVDRLLQYYEENPDFIQPESRKNEMINNFIKPGL EDLAVSRTTFDWGIKVPGNPKHVIYVWIDALANYITALGY GTDNDEKFRKYWPADVHLVGKEIVRFHTIYWPIMLMALGL PLPKKVFGHGWLLMKDGKMSKSKGNVVDPVTLIDRYGLDA LRYYLLREVPFGADGVFTPEGFIERINYDLANDLGNLLHR TVAMIEKYFDGVIPPYRGPKTPFDQELVQTAREVVRQYEE AMEGMEFSVALAAVWQLISRTNKYIDETQPWVLAKDEQKR DELAAVMTHLAESLRHTAVLLQPFLTRTPERMLAQLGITD HSLKEWDSLYDFGLIPEGTKVQKGEPLFPRLDIEAEVEYI KAHMQGGKPAAEPVKEEKKAAEAAEISIDEFAKVDLRVAE VIHAERMKNADKLLKLQLDLGGEKRQVISGIAEFYKPEEL VGKKVICVANLKPAKLRGEWSEGMILAGGSGGEFSLATVD QHVPNGTKIK SEQ ID NO:51 DNA Phe-aRS-GsPhe-aRS-EcOpt Geobacillus (codon optimized for E. coli) ATGAAAGAACGCCTGTATGAACTGAAACGTCAGGCACTGG AACAAATTGGTCAGGCACGTGATCTGCGTATGCTGAATGA TGTTCGTGTTGCATATCTGGGTAAAAAAGGTCCGATTACC GAAGTTCTGCGTGGTATGGGTGCACTGCCTCCGGAAGAAC GTCCGAAAATTGGTGCACTGGCAAATGAAGTTCGTGAAGC AATTCAGCAGGCCCTGGAAGCAAAACAGGCAAAACTTGAA CAAGAAGAAGTGGAACGTAAACTGGCAGCCGAAGCAATTG ATGTTACCCTGCCTGGTCGTCCGGTTAGCCTGGGTAATCC GCATCCGCTGACACGTGTTATTGAAGAAATTGAGGACCTG TTTATTGGCATGGGTTATACCGTTGCAGAAGGTCCGGAAG TTGAAACCGATTATTACAATTTTGAAGCCCTGAATCTGCC GAAAGGTCATCCGGCACGCGATATGCAGGATAGCTTTTAT ATCACCGAAGAAATTCTGCTGCGTACCCATACCTCACCGA TGCAGGCACGTACCATGGAAAAACATCGTGGTCGTGGTCC GGTTAAAATCATTTGTCCGGGTAAAGTTTATCGTCGCGAT ACCGATGATGCAACCCATAGCCATCAGTTTACACAGATTG AAGGTCTGGTTGTGGATCGTAATATTCGTATGAGCGATCT GAAAGGCACCCTGCGTGAATTTGCCCGTAAACTGTTTGGT GAAGGTCGTGATATTCGTTTTCGTCCGAGCTTTTTTCCGT TTACCGAACCGAGCGTTGAAGTTGATGTTAGCTGTTTTCG TTGTGAAGGCCGTGGTTGCGGTGTTGTTAAAGGCACCGGT TGGATTGAAATTTTAGGTGCAGGTATGGTTCATCCGAATG TTCTGGAAATGGCAGGTTTTGATAGTAAAACCTATACCGG TTTTGCATTCGGTATGGGTCCTGAACGTATTGCAATGCTG AAATATGGCATTGATGATATCCGCCACTTCTATCAGAATG ATCTGCGCTTTCTGCGTCAGTTTCTGCGTGTTTAA sequence number 52 amino acid Phe-aRS-GsPhe-aRS-EcOpt Geobacillus MKERLYELKRQALEQIGQARDLRMLNDVRVAYLGKKGPIT EVLRGMGALPPEERPKIGALANEVREAIQQALEAKQAKLE QEEVERKLAAEAIDVTLPGRPVSLGNPHPLTRVIEEIEDL FIGMGYTVAEGPEVETDYYNFEALNLPKGHPARDMQDSFY ITEEILLRTHTSPMQARTMEKHRGRGPVKIICPGKVYRRD TDDATHSHQFTQIEGLVVDRNIRMSDLKTGTLREFARKLFG EGRDIRFRPSFFPFTEPSVEVDVSCFRCEGRGCGVCKGTG WIEILGAGMVHPNVLEMAGFDSKTYTGFAFGMGPERIAML KYGIDDIRHFYQNDLRFLRQFLRV sequence number 53 DNA Phe-bRS-GsPhe-bRS-EcOpt Geobacillus stearothermophilus (Escherichia coli (E.coli) codon-optimized for (i)) ) ATGCTGGTTAGCTATCGTTGGCTGGGTGAATATGTTGATC TGACCGGTATTACCGCAAAAGAACTGGCAGAACGTATTAC CAAAAGCGGTATTGAAGTTGAACGTGTTGAAGCACTGGAT CGTGGTATGAATGGTGTTGTTATTGGTCATGTTCTGGAAT GTGAACCGCATCCGAATGCAGATAAACTGCGTAAATGTCT GGTTGATTTAGGTGAAGGTGAACCGGTGCGTATTATTTGT GGTGCACCGAATGTTGCAAAAGGTCAGAAAGTTGCAGTTG CCAAAGTTGGTGCAGTTCTGCCTGGTAACTTTAAAATCAA ACGTGCAAAACTGCGTGGCGAAGAAAGCAATGGTATGATT TGTAGCCTGCAAGAACTGGGTGTTGAAACCAAAGTTGTTC CGAAAGAATATGCCGATGGCA TTTTT G TTTTTCC GAG T GA T G C A CC GG TT GG T G CC GA T [[ID=四十七]]G[[ID=四十八]] C [[ID=四十九]]A[[ID=五十]] CT [[ID=五十一]]GGAA[[ID=五十二]] T [[ID=五十三]]GG[[ID=五十四]] CT [[ID=五十五]]GGG[[ID=五十六]] TCT [[ID=五十七]]G[[ID=五十八]] [[ID=五十九]] C [[ID=六十]]A[[ID=六十一]] T [[ID=六十二]]GA[[ID=六十三]] T [[ID=六十四]]GAAGTTCTGGAACTGGCACTGACCCCGAATCGTG[[ID=六十五]] CAGATTGTCTGAGCATGATTGGTGTTGCCTATGAAGTTGC AGCAATTCTGGGTCGTGATGTTAAACTGCCGGAAGCAGCA GTTAAAGAAAATAGCGAACATGTGCACGAATATATCAGCG TTCGTGTGGAAGCACCGGAAGATAATCCGCTGTATGCAGG TCGTATTGTTAAAAATGTTCGTATTGGTCCGAGTCCGCTG TGGATGCAGGCACGTCTGATGGCAGCAGGTATTCGTCCGC ATAATAATGTTGTTGACATCACCAACTATATCCTGCTGGA ATATGGTCAGCCGCTGCATGCATTTGATTATGATCGTCTG GGTAGCAAAGAAATTGTTGTTCGTCGTGCAAAAGCCGGTG AAACCATTATTACCCTGGATGATGTTGAACGTAAACTGAC CGAAAATCATCTGGTGATTACCAATGGTCGCGAACCGGTT GCACTGGCAGGCGTTATGGGTGGTGCCAATAGCGAAGTTC GTGATGATACCACCACCGTTTTTATTGAAGCAGCCTATTT CACCAGTCCGGTTATTCGTCAGGCCGTTAAAGATCATGGT CTGCGTAGCGAAGCGAGCACCCGTTTTGAAAAAGGTATTG ATCCGGCACGTACCAAAGAGGCCCTGGATCGCGCAGCAGC ACTGATGAGCGAATATGCAGGCGGTGAAGTTGTTGGTGGT ATTGTTGAAGCCAGCGTTTGGCGTCAGGATCCGGTTGTTG TTACCGTTACACTGGAACGCATTAATGGTGTTCTGGGCAC CGCAATGACCAAAGAAGAAGTGGCTGCCATTCTGAGCAAT CTGCAGTTTCCGTTTACCGAAGATAATGGCACCTTTACCA TTCATGTTCCGAGCCGTCGTCGTGATATTGCAATTGAAGA AGATATTATTGAAGAGGCAGCCCGTCTGTATGGTTATGAT CGCCTGCCTGCAACACTGCCGGTTGCCGAAGCAAAACCTG GTGGTCTGACACCGCATCAGGCAAAACGTCGTCGCGTTCG TCGTTATCTGGAAGGCACCGGTCTGTTTCAGGCAATTACC TATAGCCTGACCTCACCGGATAAAGCAACCCGCTTTGCCC TGGAAACCGCAGAACCGATTCGTCTGGCACTGCCGATGAG TGAAGAACGTAGCGTTCTGCGTCAGAGCCTGATTCCGCAT CTGCTGGAAGCCGCAAGCTATAATCGTGCACGTCAGGTTG AAGATGTTGCCCTGTATGAAATTGGTAGCGTTTATCTGAG CAAAGGTGAACATGTACAGCCTGCAGAAAAAGAACGTTTA GCCGGTGTGCTGACAGGTCTGTGGCATGCACATCTGTGGC AGGGTGAAAAAAAAGCCGTTGATTTTTATGTGGCCAAAGG TATTCTGGATGGTCTGTTTGATCTGCTGGGTTTAGCAGCA CGTATTGAATATAAACCGGCAAAACGCGCTGATCTGCATC CGGGTCGTACCGCAGATATTGTGCTGGATGGCCGTGTGAT TGGTTTTGTTGGTCAGCTGCATCCTGCAGTTCAGAAAGAG TATGATCTGAAAGAAACCTATGTGTTTGAGCTGGCCCTGA CCGATCTGCTGAATGCAGAAAGCGAAGCAATTCGTTATGA ACCTATTCCGCGTTTTCCGAGCGTTGTGCGCGACATTGCA CTGGTTGTTGATGAAAATGTTGAAGCGGGTGCACTGAAAC AGGCAATCGAAGAAGCAGGTAAACCGCTGGTTAAAGATGT TAGCCTGTTCGATGTTTATAAAGGCGATCGTCTGCCGGAT GGTAAAAAAAGTCTGGCATTTAGCCTGCGTTATTATGATC CGGAACGCACCCTGACAGATGAAGAGGTTGCAGCAGTGCA TGAACGTGTGCTGGCAGCAGTTGAAAAACAGTTTGGTGCC GTGCTGCGTGGTTAA sequence number 54 amino acid Phe-bRS-GsPhe-bRS-EcOpt Geobacillus stearothermophilus MLVSYRWLGEYVDLTGITAKELAERITKSGIEVERVEALD RGMNGVVIGHVLECEPHPNADKLRKCLVDLGEGEPVRIIC GAPNVAKGQKVAVAKVGAVLPGNFKIKRAKLRGEESNGMI CSLQELGVETKVVPKEYADGIFVFPSDAPVGADALEWLGL HDEVLELALTPNRADCLSMIGWAYEVAAILGRDVKLPEAA VKENSEHVHEYISVRVEAPEDNPLYAGRIVKNVRIGPSPL WMQARLMAAGIRPHNNVVDITNYILLEYGQPLHAFDYDRL GSKEIVVRRAKAGETIITLDDVERKLTENHLVITNGREPV ALAGVMGGANSEVRDDTTTVFIEAAYFTSPVIRQAVKDHG LRSEASTRFEKGIDPARTKEALDRAAALMSEYAGGEVVGG IVEASVWRQDPVVVTVTLERINGVLGTAMTKEEVAAILSN LQFPFTEDNGTFTIHVPSRRRDIAIEEDIIEEAARLYGYD RLPATLPVAEAKPGGLTPHQAKRRRVRRYLEGTGLFQAIT YSLTSPDKATRFALETAEPIRLALPMSEERSVLRQSLIPH LLEAASYNRARQVEDVALYEIGSVYLSKGEHVQPAEKERL AGVLTGLWHAHLWQGEKKAVDFYVAKGILDGLFDLLGLAA RIEYKPAKRADLHPGRTADIVLDGRVIGFVGQLHPAVQKE YDLKETYVFELALTDLLNAESEAIRYEPIPRFPSVVRDIA LVVDENVEAGALKQAIEEAGKPLVKDVSLFDVYKGDRLPD GKKSLAFSLRYYDPERTLTDEEVAAVHERVLAAVEKQFGA VLRG SEQ ID NO: 55 DNA ProRS-GsProRS-EcOpt Geobacillus stearothermophilus (E. col Codon optimization for i) ATGCGTCAGAGCCAGGCATTTATTCCGACACTGCGTGAAG TTCCGGCAGATGCAGAAGTTAAAAGCCATCAGCTGCTGCT GCGTGCAGGTTTTATTCGTCAGAGCGCAAGCGGTGTTTAT ACCTTTCTGCCGCTGGGTCAGCGTGTGCTGCAGAAAGTTG AAGCAATTATTCGCGAAGAAATGAATCGTATTGGTGCCAT GGAACTGTTTATGCCTGCACTGCAGCCTGCAGAACTGTGG CAGCAGAGCGGTCGTTGGTATAGCTATGGTCCGGAACTGA TGCGTCTGAAAGATCGTCATGAACGTGATTTTGCACTGGG TCCGACACATGAAGAGATGATTACCGCAATTGTTCGTGAT GAGGTGAAAACCTATAAACGTCTGCCTCTGGTTCTGTATC AGATCCAGACCAAATTCCGTGATGAAAAACGTCCGCGTTT TGGTCTGTTACGTGGTCGTGAATTTATGATGAAAGATGCC TATAGCTTCCATACCAGCAAAGAAAGCCTGGATGAAACCT ACAACAATATGTATGAAGCCTACGCCAACATTTTTCGTCG TTGCGGTCTGAATTTTCGTGCAGTTATTGCAGATAGCGGT GCAATTGGTGGTAAAGATACCCACGAATTCATGGTTCTGA GCGATATTGGTGAAGATACCATTGCATATAGTGATGCAAG CGATTATGCAGCCAATATTGAAATGGCACCGGTTGTTGCA ACCTATGAAAAAAGTGATGAACCTCCGGCAGAACTGAAGA AAGTTGCCACACCGGGTCAGAAAACCATTGCCGAAGTTGC AAGCCATCTGCAAATTAGTCCGGAACGTTGTATTAAAAGC CTGCTGTTTAATGTGGATGGTCGTTATGTTCTGGTGCTGG TTCGTGGTGATCATGAAGCAAATGAAGTGAAAGTGAAAAA TGTGCTGGATGCCACCGTTGTTGAACTGGCAAAACCGGAA GAAACCGAACGTGTTATGAATGCACCGATTGGTAGCCTGG GTCCTATTGGTGTTAGCGAAGATGTTACCGTTATTGCCGA TCATGCAGTTGCAGCAATTGTTAATGGTGTTTGTGGTGCC AATGAAGAGGGCTATCATTACATTGGTGTGAATCCGGGTC GCGATTTTGCAGTTAGCCAGTATGCCGATCTGCGTTTTGT TAAAGAAGGTGATCCGAGTCCGGATGGTAAAGGCACCATT CGTTTTGCACGTGGTATTGAAGTTGGCCATGTTTTTAAAC TGGGCACCAAATATAGCGAAGCCATGAATGCAGTTTATCT GGATGAGAATGGTCAGACCCAGACAATGATTATGGGTTGT TATGGTATTGGCGTTAGCCGTCTGGTTGCAGCCATTGCAG AACAGTTTGCCGATGAACATGGTCTGGTTTGGCCTGCAAG CGTTGCACCGTTTCATATTCATCTGCTGACCGCAAATGCC AAATCAGATGAACAGCGTGCACTGGCCGAAGAATGGTATG AAAAACTGGGTCAAGCAGGTTTTGAAGTGCTGTATGATGA TCGTCCAGAACGTGCCGGTGTTAAATTTGCCGATAGCGAT CTGATTGGTATTCCGCTGCGTGTTACCGTGGGTAAACGTG CAGGCGAAGGTGTTGTTGAAGTTAAAGTTCGTAAAACCGG TGAAACCTTTGATGTTCCGGTTAGCGAACTGGTTGATACC GCACGTCGTCTGCTGCAGAGCTAA SEQ ID NO: 56 Amino acid ProRS-GsProRS-EcOpt Geobacillus stearothermophilus MRQSQAFIPTLREVPADAEVKSHQLLLRAGFIRQSASGVY TFLPLGQRVLQKVEAIIREEMNRIGAMELFMPALQPAELW QQSGRWYSYGPELMRLKDRHERDFALGPTHEEMITAIVRD EVKTYKRLPLVLYQIQTKFRDEKRPRFGLLRGREFMMKDA YSFHTSKESLDETYNNMYEAYANIFRRCGLNFRAVIADSG AIGGKDTHEFMVLSDIGEDTIAYSDASDYAANIEMAPVVA TYEKSDEPPAELKKVATPGQKTIAEVASHLQISPERCIKS LLFNVDGRYVLVLVRGDHEANEVKVKNVLDATVVELAKPE ETERVMNAPIGSLGPIGVSEDVTVIADHAVAAIVNGVCGA NEEGYHYIGVNPGRDFAVSQYADLRFVKEGDPSPDGKGTI RFARGIEVGHVFKLGTKYSEAMNAVYLDENGQTQTMIMGC YGIGVSRLVAAIAEQFADEHGLVWPASVAPFHIHLLTANA KSDEQRALAEEWYEKLGQAGFEVLYDDRPERAGVKFADSD LIGIPLRVTVGKRAGEGVVEVKVRKTGETFDVPVSELVDT ARRLLQS Sequence number 57 DNA SerRS-GsSerRS-EcOpt Geobacillus stearothermophilus(Escherichia coli (E.col codon-optimized for i) ATGCTGGATGTGAAAATTCTGCGTACCCAGTTTGAAGAGG TGAAAGAAAAACTGATGCAGCGTGGTGGTGATCTGACCAA TATTGATCGTTTTGAACAGCTGGATAAAGATCGTCGTCGT CTGATTGCAGAAGTTGAAGAACTGAAAAGCAAACGCAATG ATGTTAGCCAGCAGATTGCAGTTCTGAAACGCGAAAAAAA AGATGCAGAACCGCTGATTGCACAGATGCGTGAAGTTGGT GATCGTATTAAACGTATGGATGAGCAGATTCGTCAGCTGG AAGCAGAACTGGATGATCTGCTGCTGAGCATTCCGAATGT TCCGCATGAAAGCGTTCCGATTGGCCAGAGCGAAGAAGAT AACGTTGAAGTTCGTCGTTGGGGTGAACCGCGTAGCTTTA GCTTTGAACCGAAACCGCATTGGGAAATTGCAGATCGTCT GGGTCTGCTGGATTTTGAACGTGCAGCAAAAGTTGCAGGT AGCCGTTTTGTTTTCTATAAAGGTCTGGGTGCACGTCTGG AACGTGCACTGATTAACTTTATGCTGGATATTCACCTGGA TGAGTTTGGCTATGAAGAAGTTCTGCCTCCGTATCTGGTT AATCGTGCAAGCATGATTGGCACCGGTCAGCTGCCGAAAT TTGCAGAAGATGCATTTCATCTGGATAGCGAGGATTATTT TCTGATTCCGACCGCAGAAGTTCCGGTTACCAATCTGCAT CGTGATGAAATTCTGGCAGCAGATGACCTGCCGATCTATT ATGCAGCATATAGCGCATGTTTTCGTGCAGAAGCAGGTAG CGCAGGTCGTGATACCCGTGGTCTGATTCGCCAGCATCAG TTCAATAAAGTTGAACTGGTGAAATTCGTGAAGCCGGAAG ATAGCTATGATGAACTGGAAAAGCTGACCCGTCAGGCAGA AACCATTCTGCAGCGTCTGGGCCTGCCGTATCGTGTTGTT GCACTGTGTACCGGTGATCTGGGTTTTAGCGTTGCAAAAA CCTATGATATTGAAGTTTGGCTGCCGAGCTATGGCACCTA TCGTGAAATTAGCAGCTGTAGCAATTTTGAAGCATTTCAG GCACGTCGTGCCAATATTCGTTTTCGTCGTGATCCGAAAG CAAAACCGGAATATGTTCATACCCTGAATGGTAGCGGTCT GGCAATTGGTCGTACCGTTGCAGCAATTCTGGAAAATTAT CAGCAAGAAGATGGCAGCGTTATTGTTCCGGAAGCACTGC GTCCGTATATGGGCAATCGTGATGTTATTCGTTAA SEQ ID NO:58 amino acid SerRS-GsSerRS-EcOpt Geobacillus stearothermophilus MLDVKILRTQFEEVKEKLMQRGGDLTNIDRFEQLDKDRRR LIAEVEELKSKRNDVSQQIAVLKREKKDAEPLIAQMREVG DRIKRMDEQIRQLEAELDDLLLSIPNVPHESVPIGQSEED NVEVRRWGEPRSFSFEPKPHWEIADRLGLLDFERAAKVAG SRFVFYKGLGARLERALINFMLDIHLDEFGYEEVLPPYLV NRASMIGTGQLPKFAEDAFHLDSEDYFLIPTAEVPVTNLH RDEILAADDLPIYYAAYSACFRAEAGSAGRDTRGLIRQHQ FNKVELVKFVKPEDSYDELEKLTRQAETILQRLGLPYRVV ALCTGDLGFSVAKTYDIEVWLPSYGTYREISSCSNFEAFQ ARRANIRFRRDPKAKPEYVHTLNGSGLAIGRTVAAILENY QQEDGSVIVPEALRPYMGNRDVIR SEQ ID NO:59 DNA ThrRS-GsThrRS-EcOpt Geobacillus (codon optimized for E. coli) ATGCCGGATGTTATTCGTATTACCTTTCCGGATGGTGCCG AAAAAAGAATTTCCGAAAGGCACCACCACCGAAGATGTTGC AGCAAGCATTAGTCCGGGTCTGAAAAAAAAAGGCAATTGCG GGTAAACTGAATGGTCGTTTTGTTGATCTGCGTACACCGC TGCATGAAGATGGTGAACTGGTGATTATTACCCAGGATAT GCCGGAAGCACTGGATATTCTGCGTCATAGCACCGCACAT CTGATGGCACAGGCAATTAAACGTCTGTATGGCAATGTGA AATTAGGTGTTGGTCCGGTGATTGAAAACGGCTTCTATTA TGATATCGACATGGAACATAAACTGACACCGGATGATCTG CCGAAAATTGAAGCAGAAATGCGCAAAATCGTGAAAGAGA ACCTGGATATTGTTCGCAAAGAAGTTAGTCGCGAAGAGGC AATTCGCCTGTATGAAGAAATTGGTGATGAACTGAAACTG GAACTGATTGCAGATATTCCGGAAGGTGAACCGATTAGCA TTTATGAACAGGGCGAATTTTTTGATCTGTGCCGTGGTGT TCATGTTCCGAGCACCGGTAAAATCAAAGAATTTAAACTG CTGAGCATCAGCGGTGCATATTGGCGTGGTGATAGCAATA ACAAAATGCTGCAGCGTATTTATGGCACCGCGTTTTTCAA AAAAGAAGATCTGGATCGTTATCTGCGTCTGCTGGAAGAA GCAAAAGAACGCGATCATCGTAAACTGGGTAAAGAGCTGG AACTGTTTACCACCAGTCAGCAGGTTGGTCAGGGTCTGCC GCTGTGGCTGCCGAAAGGTGCAACCATTCGTCGTATTATT GAACGCTATATCGTGGATAAAGAAGTTGCACTGGGTTACG ATCATGTTTATACACCGGTTCTGGGTAGCGTTGAACTGTA TAAAACCAGCGGTCATTGGGATCACTACAAAGAAAATATG TTTCCGCCTATGGAAATGGACAATGAAGAACTGGTTCTGC GTCCGATGAATTGTCCGCATCACATGATGATCTATAAAAG CAAACTGCACAGCTATCGTGAACTGCCGATTCGTATTGCA GAACTGGGCACCATGCATCGTTATGAAATGAGCGGTGCAC TGACCGGTCTGCAGCGTGTTCGTGGTATGACCCTGAATGA TGCACATATCTTTGTTCGTCCGGATCAGATCAAAGATGAA TTCAAACGTGTGGTGAACCTGATCCTGGAAGTGTATAAAG ATTTTGGCATCGAAGAATACAGCTTCCGTCTGAGTTATCG TGATCCGCATGATAAAGAAAAATACTATGATGACGATGAA ATGTGGGAAAAAGCACAGCGTATGCTGCGTGAAGCAATGG ATGAATTAGGTCTGGATTATTATGAAGCCGAAGGTGAAGC AGCCTTTTATGGTCCGAAACTGGATGTTCAGGTTCGTACC GCACTGGGAAAAGATGAAACCCTGAGCACCGTTCAGCTGG ATTTTCTGCTGCCGGAACGTTTCGATCTGACCTATATTGG TGAAGATGGCAAACCGCATCGTCCGGTTGTTATTCATCGT GGTGTTGTTAGCACCATGGAACGTTTTGTGGCATTTCTGA TCGAAGAGTATAAAGGTGCATTCCGACCTGGCTGGCACC GGTTCAGGTTAAAGTTATTCCGGTTAGTCCGGAAGCGCAC CTGGATTATGCATATGATGTTCAGCGTACCCTGAAAGAAC GTGGTTTTCGTGTTGAAGTTGAATGAACGCGACGAAAAAAAT CGGCTATAAAATCCGTGAAGCACAGATGCAGAAAATCCCG TATATGCTGGTTGTTGGTGATAAAGAGGTTAGCGAACGCG CAGTTAATGTTCGTCGTTATGGTGAAAAAGAAAGCCGTAC CATGGGCCTTGATGAATTTATGGCCCTGCTGGCAGATGAT GTTCGTGAAAAACGTACCCGTCTGGGCAAAGCACAGTAA sequence no.60 amino acid ThrRS-GsThrRS-EcOpt Geobacillus MPDVIRITFPDGAEKEFPKGTTTEDVAASISPGLKKAIA GKLNGRFVDLRTPLHEDGELVIITQDMPEALDILRHSTAH LMAQAIKRLYGNVKLWGPVIENGFYYDIDMEHKLTPDDL PKIEAEMRKIVKENLDIVRKEVSREEAIRLYEEIGDELKL ELIADIPEGEPISIYEQGEFFDLCRGVHVPSTGKIKEFKL LSISGAYWRGDSNNKMLQRIYGTAFFKKEDLDRYLRLLEE AKERDHRKLGKELELFTSQQVGQGLPLWLPKGATIRRII ERYIVDKEVALGYDHVYTPVLGSVELYKTSGHWDHYKENM FPPMEMDNEELVLRPMNCPHHMMIYKSKLHSYRELPIRIA ELGTMHRYEMSGALTGLQRVRGMTLNDAHIFVRPDQIKDE FKRVVNLILEVYKDFGIEEYSFRLSYRDPHDKEKYYDDDE MWEKAQRMLREAMDELGLDYYEAEGEAAFYGPKLDVQVRA LGKDETLSTVQLDFLLPERFDLTYIGEDGKPHRPVVIHRG VVSTMERFVAFLIEEYKGAFPTWLAPVQVKVIPVSPEAHL DYAYDVQRTLKERGFRVEVDERDEKIGYKIREAQMQKIPY MLVVGDKEVSERAVNVRRYGEKESRTMGLDEFMALLADDV REKRTRLGKAQ SEQ ID NO: 61 DNA TrpRS-GsTrpRS-EcOpt Geobacillus stearothermophilus (E. col Codon optimization for i) ATGAAAACCATCTTTAGCGGTATTCAGCCGAGCGGTGTTA TTACCCTGGGTAACTATATTGGTGCACTGCGTCAGTTTAT TGAACTGCAGCATGAATATAACTGCTATTTCTGCATTGTT GATCAGCATGCAATTACCGTTTGGCAGGATCCGCATGAAC TGCGCCAGAATATTCGTCGTCTGGCAGCACTGTATCTGGC AGTTGGTATTGATCCGACACAGGCAACCCTGTTTATTCAG AGCGAAGTTCCGGCACATGCACAGGCAGCATGGATGCTGC AATGTATTGTTTATATTGGCGAACTGGAACGCATGACCCA GTTTAAAGAAAAAAGCGCAGGTAAAGAAGCAGTTAGCGCA GGTCTGCTGACCTATCCGCCTCTGATGGCAGCCGATATTC TGCTGTATAACACCGATATTGTTCCGGTTGGTGATGATCA GAAACAGCATATCGAACTGACCCGTGATCTGGCAGAACGT TTTAACAAACGTTATGGTGAGCTGTTTACCATTCCGGAAG CACGTATTCCGAAAGTTGGTGCACGTATTATGAGCCTGGT GGATCCGACCAAAAAAATGAGCAAAAGCGATCCGAATCCG AAAGCCTATATTACACTGCTGGATGATGCAAAAACCATCG AGAAAAAAATCAAAAGTGCCGTGACCGATAGCGAAGGCAC CATTCGTTATGATAAAGAAGCCAAACCGGGTATTAGCAAC CTGCTGAACATTTATAGCACCCTGAGCGGTCAGAGCATTG AAGAATTAGAACGTAAATATGAAGGCAAAGGCTACGGTGT TTTTAAAGCAGATCTGGCACAGGTTGTTATTGAAACCCTG CGTCCGATTCAAGAACGTTATCATCATTGGATGGAAAGCG AAGAACTGGATCGTGTTCTGGATGAAGGTGCAGAAAAAGC AAATCGTGTTGCAAGCGAAATGGTGCGTAAAATGGAACAG GCAATGGGTCTGGGTCGTCGTCGTTAA[[ID=3??]] SEQ ID NO: 62 Amino acid TrpRS-GsTrpRS-EcOpt Geobacillus stearothermophilus MKTIFSGIQPSGVITLGNYIGALRQFIELQHEYNCYFCIV DQHAITVWQDPHELRQNIRRLAALYLAVGIDPTQATLFIQ SEVPAHAQAAWMLQCIVYIGELERMTQFKEKSAGKEAVSA GLLTYPPLMAADILLYNTDIVPVGDDQKQHIELTRDLAER FNKRYGELFTIPEARIPKVGARIMSLVDPTKKMSKSDPNP KAYITLLDDAKTIEKKIKSAVTDSEGTIRYDKEAKPGISN LLNIYSTLSGQSIEELERKYEGKGYGVFKADLAQVVIETL RPIQERYHHWMESEELDRVLDEGAEKANRVASEMVRKMEQ AMGLGRRR SEQ ID NO: 63 DNA TyrRS-GsTyrRS-EcOpt Geobacillus stearothermophilus (E. coli codon optimized for i) ATGGATCTGCTGGCAGAACTGCAGTGGCGTGGTCTGGTGA ATCAGACCACCGATGAAGATGGTCTGCGTGAACTGCTGAA AGAAGAACGCGTTACCCTGTATTGTGGTTTTGATCCGACC GCAGATAGCCTGCATATTGGTAATCTGGCAGCAATTCTGA CCCTGCGTCGTTTTCAGCAGGCAGGTCATCAGCCGATTGC ACTGGTTGGTGGTGCAACCGGTCTGATTGGTGATCCGAGC GGTAAAAAAAGCGAACGTACCCTGAATGCAAAAGAAACCG TTGAAGCATGGTCAGCACGTATTCAAGAACAGCTGAGCCG TTTTCTGGATTTTGAAGCACATGGTAATCCGGCAAAAATC AAGAACAACTATGATTGGATTGGTCCGCTGGATGTTATTA CCTTTCTGCGTGATGTTGGCAAACATTTCAGCGTGAATTA TATGATGGCCAAAGAAAGCGTTCAGAGCCGTATTGAAACC GGTATTAGCTTTACCGAATTCAGCTATATGATGCTGCAGG CCTATGATTTTCTGCGTCTGTATGAAACCGAAGGTTGTCG TCTGCAGATTGGTGGTAGCGATCAGTGGGGCAATATTACC GCAGGTCTGGAACTGATTCGTAAAACCAAAGGTGAAGCAC GTGCATTTGGTCTGACCATTCCGCTGGTTACCAAAGCAGA TGGTACAAAATTTGGTAAAACCGAAAGCGGCACCATTTGG CTGGATAAAGAAAAAACCAGTCCGTATGAGTTCTACCAGT TTTGGATTAATACCGATGATCGTGATGTGATCCGCTACCT GAAATACTTTACATTTCTGAGCAAAGAAGAGATCGAAGCC TTTGAACAAGAACTGCGTGAAGCACCGGAAAAACGTGCAG CACAGAAAGCACTGGCAGAAGAAGTTACCAAACTGGTTCA TGGTGAAGAAGCACTGCGTCAGGCAGTTCGTATTAGCGAA GCACTGTTTAGCGGTGATATTGGCAACCTGACCGCAGCAG AAATTGAACAGGGTTTTAAAGATGTTCCGAGCTTTGTTCA TGAAGGTGGTGATGTGCCGCTGGTCGAACTGCTGGTTAGC GCAGGTATTAGCCCGAGCAAACGTCAGGCACGTGAAGATA TTCAGAATGGTGCCATTTATGTGAATGGTGAACGTCTGCA GGATGTTGGTGCGATTCTGACAGCAGAACATCGTCTGGAA GGTCGTTTTACCGTTATTCGTCGTGGCAAAAAAAAGTATT ACCTGATTCGCTATGCCTAA SEQ ID NO: 64 Amino acid TyrRS - GsTyrRS - EcOpt Geobacillus stearothermophilus MDLLAELQWRGLVNQTTDEDGLRELLKEERVTLYCGFDPT ADSLHIGNLAAILTLRRFQQAGHQPIALVGGATGLIGDPS GKKSERTLNAKETVEAWSARIQEQLSRFLDFEAHGNPAKI KNNYDWIGPLDVITFLRDVGKHFSVNYMMAKESVQSRIET GISFTEFSYMMLQAYDFLRLYETEGCRLQIGGSDQWGNIT AGLELIRKTKGEARAFGLTIPLVTKADGTKFGKTESGTIW<00??3784>LDKEKTSPYEFYQFWINTDDRDVIRYLKYFTFLSKEEIEA FEQELREAPEKRAAQKALAEEVTKLVHGEEALRQAVRISE ALFSGDIGNLTAAEIEQGFKDVPSFVHEGGDVPLVELLVS AGISPSKRQAREDIQNGAIYVNGERLQDVGAILTAEHRLE GRFTVIRRGKKYYLIRYA sequence number 65 DNA ValRS-GsValRS-EcOpt Geobacterium ATGGCACAGCATGAAGTTAGCATGCCTCCGAAATATGATC ATCGTGCAGTTGAAGCAGGTCGTTATGAATGGTGGCTGAA AGGTAAATTCTTTGAAGCAACCGGTGATCCGAATAAACGT CCGTTTACCATTGTTATTCCGCCTCCGAATGTGACCGGTA AACTGCATCTGGGTCATGCATGGGATACCACACTGCAGGA TATTATCACCCGTATGAAACGTATGCAGGGTTATGATGTT CTGTGGCTGCCTGGTATGGATCATGCAGGTATTGCAACCC AGGCAAAAGTTGAAGAAAAACTGCGTCAGCAGGGTCTGAG CCGTTATGATCTGGGTCGTGAAAAAATTCTGGAAGAAACC TGGAAATGGAAAGAAGAATACGCAGGTCATATTCGTAGCC AGTGGGCAAAATTAGGTCTGGGTTTAGATTATACCCGTGA ACGTTTTCACCTGGATGAAGGTCTGAGCAAAGCAGTTCGT GAAGTTTTTGTTAGCCTGTATCGTAAAGGTCTGATTTATC GCGGTGAGTATATCATTAATTGGGACCCTGTTACCAAAAC CGCACTGAGCGATATTGAAGTGGTTTACAAAGAAGTTAAA GGCGCACTGTATCATCTGCGTTATCCGCTGGCAGATGGTA GCGGTTGTATTGAAGTTGCAACCACACGTCCGGAAACCAT GCTGGGTGATACCGCAGTTGCAGTTCATCCTGATGATGAA CGTTATAAACATCTGATCGGCAAAATGGTGAAACTGCCGA TTGTTGGTCGCGAAATTCCGATTATTGCAGATGAATATGT GGACATGGAATTTGGTAGTGGTGCCGTGAAAATTACACCG GCACATGATCCGAACGATTTTGAAATTGGTAATCGCCATA ATCTGCCTCGTATTCTGGTGATGAATGAAGATGGCACCAT GAATGAAAATGCCATGCAGTATCAAGGTCTGGATCGTTTT GAATGCCGTAAACAAATTGTTCGCGATCTGCAAGAACAGG GTGTTCTGTTTAAAATCGAAGAACATGTGCATAGCGTTGG TCATAGCGAACGTAGCGGTGCAGTTATTGAACCGTATCTG AGCACCCAGTGGTTTGTTAAAATGAAACCGCTGGCCGAAG CAGCAATTAAACTGCAGCAGACCGATGGTAAAGTTCAGTT TGTGCCGGAACGCTTTGAAAAAACCTATCTGCATTGGCTG GAAAACATTCGTGATTGGTGTATTAGCCGTCAGCTGTGGT GGGGTCATCGTATTCCGGCATGGTATCATAAAGAAACCGG TGAAATTTATGTGGATCACGAACCGCCTAAAGATATCGAA AATTGGGAACAAGATCCGGATGTTCTGGATACCTGGTTTA GCAGCGCACTGTGGCCGTTTAGCACCATGGGTTGGCCTGA TGTTGAAAGTCCGGATTATAAACGTTATTATCCGACCGAT GTGCTGGTTACCGGTTATGATATTATCTTTTTTTGGGTGA GCCGCATGATTTTTCAAGGCCTGGAATTTACCGGCAAACG CCCTTTTAAAGATGTTCTGATTCATGGTCTGGTGCGTGAT GCACAGGGTCGTAAAATGAGCAAAAGCTTAGGTAATGGTG TTGATCCGATGGATGTGATTGATCAGTATGGTGCAGATGC ACTGCGTTATTTTCTGGCAACCGGTAGCAGCCCTGGTCAG GATCTGCGTTTTAGCACCGAAAAAGTGGAAGCAACGTGGA ATTTTGCCAACAAAATTTGGAATGCAAGCCGTTTTGCACT GATGAACATGGGTGGTATGACCTATGAAGAACTGGATCTG AGCGGTGAAAAAACAGTTGCGGATCATTGGATTCTGACCC GTCTGAATGAAACCATTGATACCGTTACCAAACTGGCCGA AAAATATGAATTTGGTGAAGCCGGTCGTACCCTGTATAAC TTTATTTGGGATGATCTGTGCGATTGGTATATCGAAATGG CAAAACTGCCGCTGTATGGTGATGATGAGGCAGCAAAAAA AACAACCCGTAGCGTTCTGGCATATGTGCTGGATAATACC ATGCGCCTGCTGCATCCGTTTATGCCGTTTATTACCGAAG AAATTTGGCAGAATCTGCCGCATGAAGGTGAAAGCATTAC CGTTGCACCGTGGCCTCAGGTTCGTCCGGAACTGAGCAAT GAAGAGGCAGCGGAAGAAATGCGTATGCTGGTTGATATTA TTCGTGCCGTTCGTAATGTTCGTGCCGAAGTTAATACCCC TCCGAGCAAACCGATTGCACTGTATATCAAAGTTAAAGAC GAACAGGTTCGTGCAGCCCTGATGAAAAATCGTGCATATC TGGAACGTTTTTGCAATCCGAGCGAACTGCTGATTGATAC CAATGTTCCTGCACCGGATAAAGCAATGACCGCAGTGGTG ACCGGTGCAGAACTGATTATGCCGCTGGAAGGCCTGATTA ACATTGAAGAAGAAATTAAACGCCTGGAAAAAGAACTTGA TAAATGGAACAAAGAGGTGGAACGCGTCGAAAAAAAACTG GCAAATGAAGGTTTTCTGGCCAAAGCACCAGCGCATGTTG<X TGGAAGAAGAACGTCGTAAACGTCAGGATTACATGGAAAA ACGTGAAGCAGTTAAAGCACGTCTGGCCGAACTGAAACGT TAA Sequence number 66 Amino acid ValRS - GsValRS - EcOpt Geobacillus MAQHEVSMPPKYDHRAVEAGRYEWWLKGKFFEATGDPNKR PFTIVIPPPNVTGKLHLGHAWDTTLQDIITRMKRMQGYDV LWLPGMDHAGIATQAKVEEKLRQQGLSRYDLGREKFLEET WKWKEEYAGHIRSQWAKLGLGLDYTRERFTLDEGLSKAVR EVFVSLYRKGLIYRGEYIINWDPVTKTALSDIEVVYKEVK GALYHLRYPLADGSGCIEVATTRPETMLGDTAVAVHPDDE RYKHLIGKMVKLPIVGREIPIIADEYVDMEFGSGAVKITP AHDPNDFEIGNRHNLPRILVMNEDGTMNENAMQYQGLDRF ECRKQIVRDLQEQGVLFKIEEHVHSVGHSERSGAVIEPYL STQWFVKMKPLAEAAIKLQQTDGKVQFVPERFEKTYLHWL ENIRDWCISRQLWWGHRIPAWYHKETGEIYVDHEPPKDIE NWEQDPDVLDTWFSSALWPFSTMGWPDVESPDYKRYYPTD VLVTGYDIIFFWVSRMIFQGLEFTGKRPFKDVLIHGLVRD AQGRKMSKSLGNGVDPMDVIDQYGADALRYFLATGSSPGQ DLRFSTEKVEATWNFANKIWNASRFALMNMGGMTYEELDL SGEKTVADHWILTRLNETIDTVTKLAEKYEFGEAGRTLYN FIWDDLCDWYIEMAKLPLYGDDEAAKKTTRSVLAYVLDNT MRLLHPFMPFITEEIWQNLPHEGESITVAPWPQVRPELSN EEAAEEMRMLVDIIRAVRNVRAEVNTPPSKPIALYIKVKD EQVRAALMKNRAYLERFCNPSELLIDTNVPAPDKAMTAVV TGAELIMPLEGLINIEEEIKRLEKELDKWNKEVERVEKKL ANEGFLAKAPAHVVEEERRKRQDYMEKREAVKARLAELKR Sequence number 67 DNA MTF-GsMTF-EcOpt Geobacillus stearothermophilus (Escherichia coli (E.col codon optimization for i) ATGACCAACATTGTGTTTATGGGCACACCGGATTTTGCAG TTCCGATTCTGCGTCAGCTGCTGCATGATGGTTATCGTGT TGCAGCAGTTGTTACCCAGCCGGATAAACCGAAAGGTCGT AAACGTGAACCTGTTCCGCCTCCGGTTAAAGTTGAAGCAG AACGTCGTGGTATTCCGGTTCTGCAGCCGACCAAAATTCG TGAACCGGAACAGTATGAACAGGTGCTGGCATTTGCACCG GATCTGATTGTTACCGCAGCATTTGGTCAGATTCTGCCGA AAGCACTGCTGGATGCACCGAAATATGGTTGCATTAATGT TCATGCAAGCCTGCTGCCGGAACTGCGTGGTGGTGCACCG ATTCATTATGCAATTTGGCAGGGTAAAACCAAAACCGGTG TTACCATTATGTATATGGTTGAACGTCTGGATGCCGGTGA TATGCTGGCACAGGTTGAAGTGCCGATTGCAGAAACCGAT ACCGTTGGCACCCTGCATGATAAACTGAGCGCAGCGGGTG CAAAACTGCTGAGCGAAACCCTGCCGCTGCTGCTGGAAGG CAATATTACACCGGTTCCGCAGGATGAAGAAAAAGCAACC TATGCACCTAATATTCGTCGTGAACAAGAACGTATTGATT GGACCCAGCCTGGTGAAGCCATTTATAACCATATTCGTGC CTTTCATCCGTGGCCTGTTACCTATACCACACAGGATGGT CATATTTGGAAAGTTTGGTGGGGTGAAAAAGTTCCTGCAC CGCGTAGCGCACCGCCTGGCACCATTCTGGCACTGGAAGA AAATGGTATTGTTGTTGCAACCGGTAATGAAACCGCAATT CGTATTACCGAACTGCAGCCTGCAGGTAAAAAACGTATGG CAGCCGGTGAATTTCTGCGTGGCGCAGGTAGCCGTCTGGC AGTTGGTATGAAACTGGGTGAAGATCATGAACGTACCTAA SEQ ID NO: 68 Amino acid MTF-GsMTF-EcOpt Geobacillus stearothermophilus MTNIVFMGTPDFAVPILRQLLHDGYRVAAVVTQPDKPKGR KREPVPPPVKVEAERRGIPVLQPTKIREPEQYEQVLAFAP DLIVTAAFGQILPKALLDAPKYGCINVHASLLPELRGGAP IHYAIWQGKTKTGVTIMYMVERLDAGDMLAQVEVPIAETD TVGTLHDKLSAAGAKLLSETLPLLLEGNITPVPQDEEKAT YAPNIRREQERIDWTQPGEAIYNHIRAFHPWPVTYTTQDG HIWKVWWGEKVPAPRSAPPGTILALEENGIVVATGNETAI RITELQPAGKKRMAAGEFLRGAGSRLAVGMKLGEDHERT Sequence number 69 DNA IF-1-GsuIF-1 Geobacillus subterraneus DSM 13552 (91A1 ) ATGTTACTCATTCGAAGGAGGGAGAGCCGCTCGATGGCAA AAGACGATGTAATTGAAGTGGAAGGCACCGTCATTGAAAC ATTGCCAAATGCGATGTTTCGTGTAGAATTAGAAAATGGG CACACAGTATTGGCCCATGTGTCCGGCAAAATCCGTATGC ACTTCATCCGCATTTTGCCTGGCGATAAAGTGACGGTGGA GTTGTCGCCGTATGATTTAACGCGTGGACGGATTACGTAT CGATATAAA Sequence number 70 Amino acid IF-1-GsuIF-1[[ID=3-3]] Geobacillus subterraneus DSM 13552 (91A1 ) MLLIRRRESRSMAKDDVIEVEGTVIETLPNAMFRVELENG HTVLAHVSGKIRMHFIRILPGDKVTVELSPYDLTRGRITY RYK Sequence number 71 DNA IF-2-GsuIF-2 Geobacillus subterraneus DSM 13552 (91A1 ) ATGGTGTCCCGCTTTGCAAAGTGCCGGACCGGTATACGCT CGGCGGCGCGATCGGCAAAGACGCCCGCGTCGTTGTCGCC GTCACCGACGAAGGGTTCGCGCGCCAATTGCAAACGATGC TCGACTGATCTTTATGGGGGTGAATGTATGTCGAAAATGC GTGTGTACGAATACGCCAAAAAACATAATGTGCCAAGCAA GGACGTTATTCATAAATTGAAAGAAATGAATATTGAAGTG AACAACCATATGACTATGCTCGAAGCCGATGTCGTCGAAA AGCTCGATCATCAATACCGCGTGAACTCAGAGAAAAAAGC GGAAAAGAAAACGGAGAAACCGAAGCGGCCGACGCCGGCG AAAGCCGCCGATTTTGCCGACGAGGAAATGTTTGAGGACA AGAAAGAAACGGCAAAGACGAAGCCGGCGAAGAAAAAGGG AGCAGTGAAAGGAAAGGAAACGAAAAAAACAGAAGCACAG CAGCAAGAAAAGAAACTGTTCCAAGCGGCGAAGAAAAAAG GAAAAGGACCGATGAAAGGCAAAAAACAAGCTGCCCCAGC CTCAAAGCAGGCGCAGCAGCCGGCGAAAAAAGAAAAAGAG CTCCCGAAAAAAATTACGTTCGAAGGTTCGCTCACGGTAG CCGAATTGGCGAAAAAACTTGGCCGCGAGCCGTCGGAAAT CATTAAAAAACTGTTTATGCTCGGCGTCATGGCGACGATT AACCAAGATTTAGACAAAGATGCGATCGAGCTCATTTGCT CTGATTACGGAGTTGAAGTCGAAGAAAAAGTGACGATCGA TGAAACGAATTTTGAAACGATCGAAATTGTCGATGCACCG GAAGATTTGGTGGAACGGCCGCCGGTCGTCACGATTATGG GGCACGTTGACCACGGGAAAACAACGCTGCTTGACGCAAT CCGCCACTCGAAAGTGACCGAGCAAGAGGCGGGCGGTATT ACACAGCATATCGGTGCTTATCAAGTCACGGTCAACGGCA AGAAAATTACGTTCCTCGATACGCCGGGGCATGAAGCGTT TACGACGATGCGGGCGCGCGGTGCGCAAGTGACGGATATC GTCATCCTTGTTGTTGCTGCTGATGATGGGGTCATGCCGC AGACGGTCGAGGCGATTAACCACGCCAAAGCGGCGAACGT ACCGATTATCGTCGCCATTAACAAAATGGATAAGCCGGAA GCAAACCCGGATCGCGTTATGCAAGAGTTGATGGAGTACA ACCTCGTTCCGGAAGAATGGGGTGGCGATACGATTTTCTG CAAGCTGTCGGCGAAAACCCAAGACGGTATTGACCATCTG TTGGAAATGATTTTGCTTGTCAGCGAAATGGAAGAACTAA AAGCGAACCCGAACCGCCGCGCGCTCGGTACGGTGATCGA AGCGAAGCTCGATAAAGGGCGCGGTCCGGTAGCGACGTTG CTCGTCCAAGCCGGTACGCTAAAAGTCGGTGATCCGATTG TTGTCGGAACAACGTACGGACGCGTGCGCGCGATGGTCAA TGACAGCGGTCGGCGTGTCAAAGAAGCGGGTCCGTCGATG CCGGTCGAAATCACAGGGCTTCATGATGTGCCGCAAGCCG GGGACCGCTTTATGGTATTTGAAGATGAGAAGAAAGCGCG ACAAATCGGAGAAGCGCGGGCACAGCGGCAGCTGCAAGAG CAGCGGAGCGTGAAAACGCGCGTCAGCTTGGACGATTTGT TTGAACAAATTAAGCAAGGTGAAATGAAAGAGCTGAACTT GATCGTTAAGGCCGACGTCCAAGGATCGGTCGAAGCGCTT GTCGCCGCCTTGCAAAAAATCGATATCGAAGGCGTGCGTG TGAAAATTATCCACGCGGCGGTCGGCGCCATTACGGAGTC AGACATCTTGTTGGCAACGACCTCGAACGCGATCGTCATC GGTTTTAACGTCCGTCCGGACACCAATGCGAAGCGGGCTG CCGAATCAGAAAACGTCGACATCCGCCTCCACCGCATTAT TTACAATGTCATCGAAGAAATTGAAGCGGCGATGAAAGGG ATGCTCGACCCAGAATATGAAGAAAAAGTGATCGGTCAGG CGGAAGTGCGGCAAACGTTCAAAGTGTCGAAAGTCGGCAC GATCGCCGGGTGCTACGTCACCGACGGCAAAATTACCCGC GACAGCAAAGTGCGCCTTATCCGTCAAGGCATCGTCGTGT ACGAAGGCGAAATCGACTCGCTCAAACGGTATAAAGATGA TGTGCGTGAGGTGGCGCAAGGATACGAATGCGGCGTGACC ATCAAAAACTTCCAACGATATTAAAGAAGGGGACGTCATCG AGGCGTACATCATGCAGGAAGTGGCTCGCGCA sequence number 72 amino acid IF-2-GsuIF-2 Geobacillus subterraneus DSM 13552 (91A1 ) MVSRFAKCRTGIRSAARSAKTPASLSPSPTKGSRANCKRC STDLYGGECMSKMRVYEYAKKHNVPSKDVIHKLKEMNIEV NNHMTMLEADVVEKLDHQYRVNSEKKAEKKTEKPKRTPPA KAADFADEEMFEDKKETAKTKPAKKKGAVKGKETKKTEAQ QQEKKLFQAAKKKGKGPMKGKKQAAPASKQAQQPACKAGE LPKKITEFGSLTVAELAKKLGREPSEIIKKLFMLGVMATI NQDLDKDAIELICSDYGVEVEEKVTIDETNFETIEIVDAP EDLVERPPVVTIMGHVDHGKTTLLDAIRHSKVTEQEAGGI TQHIGAYQVTVNGKKITFLDTPGHEAFTTMRARGAQVTDI VILVVAADDGVMPQTVEAINHAKAANVPIIVAINKMDKPE ANPDRVMQELMEYNLVPEEWGGDTIFCKLSAKTQDGIDHL LEMILLVSEMEELKANPNRRALGTVIEKLDKGRGPVATL LVQAGTLKVGDPIVVGTTYGRVRAMVNDSGRRVKEAGPSM PVEITGLHDVPQAGDRFMVFEDEKKARQIGEARAQRQLQE QRSVKTRVSLDDLFEQIKQGEMKELNLIVKADVQGSVEAL VAALQKIDIEGVRVKIIHAAVGAITESDILLATTSNAIVI GFNVRPDTNAKRAAESENVDIRLHRIIYNVIEEIEAAMKG MLDPEYEEKVIGQAEVRQTFKVSKVGTIAGCYVTDGKITR DSKVRLIRQGIVVYEGEIDSLKRYKDDVREVAQGYECGVT IKNFNDIKEGDVIEAYIMQEVARA SEQ ID NO: 73 DNA IF-3-GsuIF-3 Geobacillus subterraneus DSM 13552 (91A1 ) ATGGACTACGGCAAATTCCGCTTTGAGCAGCAAAAGAAAG AAAAAGAAGCGCGCAAAAAGCAAAAGGTGATCAACATTAA AGAGGTGCGCCTCAGCCCGACAATTGAGGAACACGACTTT AATACGAAACTACGCAATGCGCGCAAGTTTTTAGAAAAAG GCGATAAAGTGAAGGCGACGATCCGCTTTAAAGGGCGGGC GATCACCCATAAAGAAATCGGGCAGCGCGTCCTTGACCGC TTCTCGGAAGCATGCGCTGATATCGCGGTCGTCGAAACGG CGCCGAAATTGGAAGGGCGCAACATGTTTTTAGTGCTGGC ACCGAAAAATGACAACAAG SEQ ID NO: 74 Amino acid IF-3-GsuIF-3 Geobacillus subterraneus DSM 13552 (91A1 ) MDYGKFRFEQQKKEKEARKKQKVINIKEVRLSPTIEEHDF NTKLRNARKFLEKGDKVKATIRFKGRAITHKEIGQRVLDR FSEACADIAVVETAPKLEGRNMFLVLAPKNDNK sequence no. 75 DNA EF-G-GsuEF-G Geobacillus subterraneus DSM 13552 (91A1 ) ATGGCAAGAGAGTTCTCCTTAGAAAAACACTCGTAACATAG GAATCATGGCGCACATTGACGCCGGAAAAACGACGACGAC GGAACGAATCCTGTTCTACACAGGCCGCGTTCATAAAATC GGGGAAACGCATGAAGGCTCAGCTACGATGGACTGGATGG AACAAGAGCAAGAGCGCGGGATTACGATTACGTCGGCGGC GACAACGGCGCAATGGAAAGGCCATCGCATCAACATCATC GACACGCCAGGGCACGTCGACTTCACGGTTGAGGTTGAAC GTTCGTTGCGCGTGTTGGACGGAGCCATTACAGTTCTTGA CGCCCAATCTGGTGTAGAACCGCAAACGGAAACAGTTTGG CGTCAAGCGACTACATATGGTGTTCCGCGGATTGTATTCG TCAACAAAATGGACAAAATCGGTGCGGACTTCTTGTATGC GGTAAAAACGCTCCATGACCGCTTACAAGCGAATGCCTAC CCGGTGCAGTTGCCGATCGGCGCTGAAGACCAATTCACCG GCATTATTGACCTCGTGGAAATGTGTGCATACCATTACCA CGACGACCTTGGCAAAAACATCGAACGCATCGAAATTCCG GAAGACTACCGCGATTTAGCGGAAGAATATCATGGCAAGC TCATTGAGGCTGTTGCGGAACTCGATGAAGAGCTGATGAT GAAATATTTAGAAGGAGAAGAAATTACGAAAGAAGAGCTG AAAGCCGCAATCCGTAAGGCGACGATCAACGTTGAATTCT ATCCAGTCTTCTGCGGTTCAGCTTTTAAAAACAAAGGTGT TCAGCTGCTTCTTGACGGGGTTGTCGACTACTTGCCGTCT CCGTTAGATATCCCGGCGATTCGCGGTATCATTCCGGATA CGGAAGAAGAAGTGGCTCGCGAAGCACGCGATGACGCTCC GTTCTCCGCGTTGGCATTCAAAATTATGACTGACCCGTAC GTTGGGAAGTTGACGTTCTTCCGCGTCTACTCCGGAACGC TTGATTCCGGTTCTTACGTCATGAACTCAACGAAACGGAA GCGTGAACGGATCGGTCGCTTGCTGCAAATGCATGCGAAC CACCGTCAAGAAATTTCGACAGTCTATGCCGGTGATATTG CGGCAGCAGTAGGTTTAAAAGAAACAACGACCGGCGATAC TCTATGTGATGAGAAAAATCTTGTCATCTTAGAGTCGATG CAATTCCCAGAGCCGGTTATCTCGGTGGCGATCGAACCGA AATCGAAAGCCGACCAAGATAAGATGGGTCAAGCATTGCA AAAACTGCAAGAGGAAGACCCGACATTCCGTGCGCATACC GATCCGGAAACAGGACAAACGATCATTTCCGGGATGGGCG AGCTGCACTTGGACATTATCGTCGACCGGATGCGTCGCGA ATTCAAAGTCGAGGCGAACGTTGGTGCACCGCAAGTTGCT TACCGTGAAACGTTCCGTCAATCGGCTCAAGTCGAAGGGA AATTTATTCGCCAGTCCGGTGGTCGTGGTCAGTACGGTCA CGTTTGGATCGAATTCACACCGAACGAACGCGGTAAAGGC TTTGAATTTGAAAATGCGATCGTCGGTGGGGTCGTTCCGA AAGAGTACGTGCCGGCTGTTCAAGCTGGATTGGAAGAAGC GATGCAAAACGGTGTCTTAGCTGGCTACCCGGTTGTTGAC ATCAAAGCGAAACTGTTTGATGGATCGTACCATGATGTCG ACTCGAGTGAGATGGCGTTCAAAATTGCTGCTTCGATGGC GTTGAAAAACGCGGCAGCGAAGTGTGAACCGGTTCTGCTT GAACCGATCATGAAAGTAGAAGTCGTCATCCCTGAAGAAT ACCTCGGCGACATTATGGGTGACATCACATCCCGCCGCGG TCGCGTCGAAGGGATGGAAGCGCGCGGAAACGCCCAAGTT GTTCGTGCAATGGTGCCGCTGGCCGAAATGTTCGGTTATG CAACATCGCTCCGTTCGAACACGCAAGGGCGTGGAACGTT CTCGATGGTATTTGACCATTCGAAGAAGTTCCGAAAAAC ATCGCCGATGAAATTATCAAAAAAAAATAAAGGCGAA sequence no. 76 amino acid EF-G-GsuEF-G Geobacillus subterraneus DSM 13552 (91A1 ) MAREFSLENTRNIGAMIHIDAGKTTTTERILFYTGRVHKI GETHEGSATMDWMEQEQERGITITSAATTAQWKGHRINII DTPGHVDFTVEVERSLRVLDGAITVLDAQSGVEPQTETVW RQATTYGVPRIVFVNKMDKIGADFLYAVKTLHDRLQANAY PVQLPIGAEDQFTGIIDLVEMCAYHYHDDLGKNIERIEIP EDYRDLAEEYHGKLIEAVAELDEELMMKYLEGEEITKEEL KAAIRKATINVEFYPVFCGSAFKNKGVQLLLDGVVDYLPS PLDIPAIRGIIPDTEEEVAREARDDAPFSALAFKIMTDPY VGKLTFFRVYSGTLDSGSYVMNSTKRKRERIGRLLQMHAN HRQIISTVYAGDIAAAVGLKETTGDTLCDEKNLVILESM QFPEPVISVAIEPKSKADQDKMGQALQKLQEEDPTFRAHT DPETGQTIISGMGELHLDIIVDRMRREFKVEANVGAPQVA YRETFRQSAQVEGKFIRQSGGRGQYGHVWIEFTPNERGKG FEFENAIVGGVVPKEYVPAVQAGLEEAMQNGVLAGYPVVD IKAKLFDGSYHDVDSSEMAFKIAASMALKNAAAKCEPVLL EPIMKVEVVIPEEYLGDIMGDITSRRGRVEGMEARGNAQV VRAMVPLAEMFGYATSLRSNTQGRGTFSMVFDHYEEVPKN IADEIIKKNKGE sequence no. 77 DNA EF-Tu-GsuEF-Tu Geobacillus subterraneus DSM 13552 (91A1 ) ATGGCTAAAGCGAAATTTGAGCGTACGAAACCGCACGTCA ACATTGGCACGATCGGCCACGTTGACCATGGGAAAACGAC GTTGACAGCTGCGATCACGACAGTTCTTGCGAAACAAGGT AAAGCAGAAGCGAGAGCGTACGACCAAATCGACGCTGCTC CGGAAGAGCGTGAACGCGGAATCACGATTTCGACGGCTCA CGTTGAGTATGAACAGAAAACCGTCACTATGCGCCACGTT GACTGCCCGGGCCACGCTGACTACGTGAAAAACATGATCA CGGGCGCAGCGCAAATGGACGGCGCGATCCTTGTTGTATC GGCTGCTGACGGTCCGATGCCGCAAACTCGCGAACACATT CTTCTTTCCCGCCAAGTCGGTGTTCCGTACATCGTTGTTT TCTTGAACAAATGCGACATGGTGGACGACGAAGAATTGCT TGAACTCGTTGAAATGGAAGTTCGCGATCTTCTTTCTGAA TATGACTTCCCGGGCGACGAAGTGCCGGTTATCAAAGGTT CGGCATTAAAAGCGCTCGAAGGCGATGCACAATGGGAAGA AAAAATCGTTGAACTGATGAAGCGGTTGACGAGTACATC CCAACTCGCAACGTGAAGTAGCAAACCGTTCATGATGC CGGTTGAGGACGTCTTCTCGATCACGGGTCGTGGGTACGGT TGCAACGGGCCGTGTTGAGCGCGGTACGTTAAAAGTTGGT GACCCGGTTGAAATCATCGGTCTTTCGGACGAGCCGAAAT CGACGACTGTTACGGGTGTAGAAATGTTCCGTAAGCTTCT CGACCAAGCAGAAGCTGGTGACAACATCGGTGCGCTTCTC CGCGGTGTATCCGCGTGACGAAGTTGAGCGCGGTCAAGTAT TGGCGAAACCGGGCTCGATCACGCCACACACGAAATTTAA AGCACAAGTTTACGTTCTGACGAAAGAAGAGCGGACGC CATACTCCGTTCTTTCTCGAACTACCGTCCGCATTCTACT TCCGTACAACGGACGTAACGGGCATCATCACGCTTCCAGA AGGCGTTGAAATGGTTATGCCTGGCGACAACGTTGAAATG ACGGTTGAACTGATCGCTCCGATCGCGATCGAAAAGGTA CGAAATTCTCGATCCGTGAAGGCGGCCGCACGGTTGGTGC TGGTTCCGTATCGGAAATCATTGAG SEQ ID NO:78 amino acid EF-Tu-GsuEF-Tu Geobacillus subterraneus DSM 13552 (91A1 ) MAKAKFERTKPHVNIGTIGHVDHGKTTLTAAITTVLAKQG KAEARAYDQIDAAPEERERGITISTAHVEYETENRHYAHV DCPGHADYVKNMITGAAQMDGAILVVSAADGPMPQTREHI LLSRQVGVPYIVVFLNKCDMVDDEELLELVEMEVRDLLSE YDFPGDEVPVIKGSALKALEGDAQWEEKIVELMNAVDEYI PTPQREVDKPFMMPVEDVFSITGRGTVATGRVERGTLKVG DPVEIIGLSDEPKSTTVTGVEMFRKLLDQAEAGDNIGALL RGVSRDEVERGQVLAKPGSITPHTKFKAQVYVLTKEEGGR HTPFFSNYRPQFYFRTTDVTGIITLPEGVEMVMPGDNVEM TVELIAPIAIEEGTKFSIREGGRTVGAGSVSEIIE SEQ ID NO:79 DNA EF-Ts-GsuEF-Ts Geobacillus subterraneus DSM 13552 (91A1 ) ATGGCGATTACAGCACAAATGGTAAAAGAGCTGCGCGAAA AAACGGGCGCAGGCATGATGGACTGCAAAAAAGCGCTCAC CGAAACGAACGGTGACATGGAAAAAGCGATCGACTGGCTG CGTGAAAAAAGGAATTGCTAAAAGCAGCGAAAAAAGCAGATC GCATCGCAGCGGAAGGAATGACATACATCGCGACGGAAGG CAATGCGGCTGTCATTTTGGAAGTAAACTCGGAAACGGAC TTCGTTGCCAAAAACGAAGCGTTCCAAACGCTCGTTAAGG AGCTGGCTGCACATCTGCTGAAACAAAAGCCAGCCACGCT TGATGAAGCGCTCGGACAAACGATGAGCAGTGGTTCCACT GTTCAAGATTACATTAACGAAGCAGTTGCTAAAATCGGTG AAAAAATTACGCTCCCGCCGCTTTGCTGTTGTCAACAAAGC GGATGATGAAACGTTTGGCGCGTACTTGCACATGGGCGGG CGCATCGGCGTATTAACATATTAGCCGGCAACGCAACTG AAGAGGTCGCTAAAGATGTGGCGATGCATATTGCTGCGCT CCATCCGAAATACGTTTCGCGCGATGAAGTGCCGCAAGAA GAGATTGCCGCGGAACGTGAAGTGTTGAACAACAAGCGT TGAACGAAGGTAAGCCGGAAAACATCGTTGAAAAAATGGT TGAAGGCCGTCTGAAAAAGTTTTACGAAGATGTTTGCCTG CTTGAGCAAGCGTTCGTGAAAACCCGGATGTGACGGTAC GCCAATACGTCGAATCGAGGCGGAGCAACCGTGAAGCAGTT CATCCGCTACGAAGTTGGTGAAGGGCTCGAAAAACGTCAA GATAATTTCGCTGAAGAAGTCATGAGCCAAGTAAGAAAC AA sequence no. 80 amino acid EF-Ts-GsuEF-Ts Geobacillus subterraneus DSM 13552 (91A1 ) MAITAQMVKELREKTGAGMMDCKKALTETNGDMEKAIDWL REKGIAKAAKKADRIAAEGMTYIATEGNAAVILEVNSETD FVAKNEAFQTLVKELAAHLLKQKPATLDEALGQTMSSGST VQDYINEAVAKIGEKITLRRFAFVNKADDETFGAYLHMGG RIGVLTLLAGNATEEVAKDVAMHIAALHPKYVSRDEVPQE EIAREREVLKQQALNEGKPENIVEKMVEGRLKKFYEDVCL LEQAFVKNPDVTVRQYVESSGATVKQFIRYEVGEGLEQRQ DNFAEEVMSQVRKQ sequence no. 81 DNA EF-4-GsuEF-4 Geobacillus subterraneus DSM 13552 (91A1 ) ATGAACCGGGAAGAACGGTTGAAACGGCAGGAACGGATTC GCAACTTTTCGATTATCGCTCACATTGACCACGGAAAATC GACGCTTGCGGACCGCATTTTAGAAAAACAGGTGCGCTG TCGGAGCGCGAGTTGCGCGAGCAGACGCTCGATATGATGG AGCTCGAGCGCGAGCGCGGCATCACGATCAAATTGAATGC GGTCCAGTTGACATATAAAGCGAAAAACGGGGAAGAGTAT ATTTTCCATTTGATCGATACGCCGGGCCACGTCGATTTTA CGTATGAAGTGTCGCGCAGCTTGGCTGCTTGCGAAGGAGC GATCTTAGTCGTCGATGCGGCGCAAGGCATTGAAGCGCAG ACGCTCGCAAACGTGTATTTGGCCATTGACAACAATTTAG AAATTTTACCAGTCATTAATAAAATCGATTTGCCAAGCGC CGAGCCGGAGCGTGTCCGCCAAGAAATCGAAGACGTCATT GGCCTCGATGCCTCTGAAGCGGTGCTCGCCTCCGCGAAAG TCGGCATCGGCGTCGAGGACATTTTAGAACAAATCGTGGA AAAAATTCCTGCTCCGTCAGGCGATCCGGACGCGCCGTTG AAGGCGCTCATTTTTGATTCACTTTATGACCCGTACCGCG GCGTTGTCGCCTACGTCCGTATCGTCGATGGAACGGTTAA GCCGGGCCAGCGCATTAAAATGATGTCGACCGGCAAAGAG TTTGAAGTGACCGAAGTCGGCGTGTTTACACCAAAACCAA AAGTTGTCGACGAACTGATGGTCGGTGATGTCGGCTATTT AACTGCGTCGATCAAAAACGTACAAGATACGCGCGTCGGC GATACGATTACCGATGCCGAACGGCCGGCTGCTGAGCCAC TCCCTGGCTACCGGAAGCTCAATCCGATGGTGTTTTGCGG CATGTACCCGATCGACACGGCGCGCTACAACGACTTGCGC GAAGCGTTAGAAAAAGCTGCAGCTCAACGATGCGGCGCTTC ACTTTGAACCGGAAACGTCGCAGGCGCTCGGGTTTGGCTT TCGTTGCGGGTTTCTCGGCTTGCTTCATATGGAGATTATC CAAGAGCGGATTGAACGTGAATTTCATATCGATTTAATTA CAACGGCGCCGAGCGTTGTCTACAAAGTATATTTAACGGA CGGAACGGAAGTCGATGTCGACAACCCGACGAACATGCCG GATCCGCAAAAAATCGACCGCATCGAAGAGCCGTATGTAA AAGCGACGATTATGGTGCCGAACGACTACGTCGGACCGGT GATGGAGCTGTGCCAAGGAAAGCGTGGCACGTTCGTTGAC ATGCAATATTTAGATGAAAGCGGGTCATGTTGATTTACG ATATTCCGCTGTCGGAAATCGTGTATGACTTTTTCGATGC GTTAAAGTCGAACACGAAAGGGTATGCGTCGTTTGACTAT GAATTGATCGGTTACCGGCCGTCCAATCTTGTCAAAATGG ATATTTTGTTGAATGGCGAAAAAATTGACGCTTTATCGTT TATTGTTCACCGCGATTCGGCTTATGAGCGCGGCAAAGTG ATCGTCGAGAAGCTGAAAGATTTAATTCCACGCCAACAGT TTGAAGTGCCTGTGCAGGCGGCGATCGGCAATAAGATCAT CGCCCGTTCGACGATCAAGGCGCTGCGTAAAAACGTGCTC GCCAAATGTTACGGCGGCGACGTGTCGCGGAAACGGAAAC TGCTTGAGAAAAAAAAAGAAGGAAAGAAACGGATGAAACA AATCGGTTCGGTCGAAGTGCCGCAGGAAGCGTTTATGGCT GTCTTGAAAATCGACGACCAGAAAAAAA sequence no. 82 amino acid EF-4-GsuEF-4 Geobacillus subterraneus DSM 13552 (91A1 ) MNREERLKRQERIRNFSIIAHIDHGKSTLADRILEKTGAL SERELREQTLDMELELERERGITIKLNAVQLTYKAKNGEEY IFHLIDTPGHVDFTYEVSRSLAACEGAILVVDAAQGIEAQ TLANVYLAIDNNLEILPVINKIDLPSAEPERVRQEIEDVI GLDASEAVLASAKVGIGVEDILEQIVEKIPAPSGDPDAPL KALIFDSLYDPYRGVVAYVRIVDGTVKPGQRIKMMSTGKE FEVTEVGVFTPKPKVVDELMVGDVGYLTASIKNVQDTRVG DTITDAERPAAEPLPGYRKLNPMVFCGMYPIDTARYNDLR EALEKLQNLDAALHFEPETSQALGFGFRCGFLGLLHMEII QERIEREFHIDLITTAPSVVYKVYLTDGTEVDVDNPTNMP DPQKIDRIEEPYVKATIMVPNDYVGPVMELCQGKRGTFVD MQYLDEKRVMLIYDIPLSEIVYDFFFDALKSNTKGYASFDY ELIGYRPSNLVKMDILLLNGEKIDALSFIVHRDSAYERGKV IVEKLKDLIPRQQFEPVVQAAIGNKIIARSTIKALRKNVL AKCYGGDVSRKRKLLEKQKEGKKRMKQIGSVEVPQEAFMA VLKIDDQKK sequence no. 83 DNA EF-P-GsuEF-P Geobacillus subterraneus DSM 13552 (91A1 ) ATGATTTCAGTGAACGATTTTCGCACAGGGCTTACGATTG AGGTCGACGGCGAGATTTGGCGCGTCCTTGAGTTCCAGCA TGTTAAGCCGGGCAAAGGGGCGGCGTTCGTCCGTTCGAAG CTGCGCAACTTGCGTACCGGCGCCATTCAAGAGCGGACGT TCCGCGCTGGCGAAAAAGTAAACCGGGCACAAATTGATAC GCGCAAAATGCAATATTATACGCTAACGGCGACTTGCAT GTCTTTATGGATATGGAAACATACGAACAAATCGAGCTGC CAGCGAAACAAATTGAGTATGAGCTGAAGTTCTTAAAAAGA AAACATGGAAGTATTTATCATGATGTATCAAGGCGAAACG ATCGGTGTTGAGCTGCCGAACACCGTCGAGTTGAAAGTCG TTGAAACAGAGCCGGGCATCAAAGGTGACACGGCTTCCGG CGGTTCGAAGCCGGCCAAGCTCGAAACCGGTCTTGTCGTT CAAGTGCCGTTTTTCGTCAATGAAGGCGACACGCTCATCA TTAACACGGCTGACGGTACGTACGTTTCGCGGGCA sequence no. 84 amino acid EF-P-GsuEF-P Geobacillus subterraneus DSM 13552 (91A1 ) MISVNDFRTGLTIEVDGEIWRVLEFQHVKPGKGAAFVRSK LRNLRTGAIQERTQUESTIONKVNRAQIDTRKMQYLYANGDLH VFMDMETYEQIELPAKQIEYELKFLKENMEVFIMMYQGET IGVELPNTVELKVVETEPGIKGDTASGGSKPAKLETGLVV QVPFFVNEGDTLIINTADGTYVSRA sequence no. 85 DNA RF-1-GsuRF-1 Geobacillus subterraneus DSM 13552 (91A1 ) ATGGATCCAGCCGTTATCAACGACCCGAAAAGTTGCGCG ATTATTCGAAAGAGCAGGCTGATTTGACTGAAACGGTGCA AACGTACCGTGAATACAAGTCCGTTCGCAGTCAGCTCGCG GAAGCGAAGGCTATGCTGGAAGAAAAACTTGAGCCAGAGC TGCGCGAGATGGTGAAAGAGGAAATTGATGAGCTCGAAGA ACGGGAAGAGGCCTCGTTGAGAAGTTGAAAGTGTTGCTT TTGCCGAAAGATCCGAATGATGAGAAAAACGTCATTATGG AAATTCGTGCCGCCGCCGGTGGCGAGAAGCCGCGCTGTT TGCCGGCGACTTGTACCGGATGTATACGCGCTATGCGGAG TCGCAAGGGTGGAAAACGGAAGTGATCGAAGCAAGCCCAA CAGGTCTTGGCGGCTATAAAGAAATCTCTTTATGGTCAA TGGGAAAGGGCGTATTCGAAGCTGAAGTTTGAAAACGGC GCTCATCGCGTCCAACGCGTCCCGGAAACGGAATCAGGCG GACGCATCCATACATCGACGGCAACGGTCGCCTGCTTGCC GGAAATGGAAGAAGTCGAAGTCGAAATTCATGAAAAAAGAC ATTCGCGTCGATACGTACGCCTCGAGCGGGGCCAGGGGGAC AAAGCGTGAACACGACGATGTCAGCCGTACGCCTCACCCA TATTCCGACCGGCATTGTCGTTACTTGCCAAGACGAAAAA TCGCAAATTAAAAACAAAGAAAAGCGATGAAAGTGTTGC GCGCCCGCATTTACGACAAATACCAGCAAGAAGCGCGCGC CGAGTATGACCAAACCGGTAAGCCAAGCAGTCGGCACCGGC GATCGCTCAGAGCGCATCCGCACGTACAACTTCCCGCAAA ACCGCGTCACTGACCACCGTATCGGGTTGACGATTCAAAA GCTTGACCTCGTGTTAGACGGGCAGCTCGATGAAATTATC GAGGCGCTCATTTTAGACGACCAGTCGAAAAAACTGGAGC AAGCGAACGATGCGTCG sequence number 86 amino acid RF-1-GsuRF-1 Geobacillus subterraneus DSM 13552 (91A1 ) MDPAVINDPKKLRDYSKEQADLTETVQTYREYKSVRSQLA EAKAMLEEKLEPELREMVKEEIDELEEREEEALVEKLKVLL LPKDPNDEKNVIMEIRAAAGGEEAALFAGDLYRMYTRYAE SQGWKTEVIEASPTGLGGYKEIIFMVNGKGAYSKLKFENG AHRVQRVPETESGGRIHTSTATVACLPEMEEVEVEIHEKD IRVDTYASSGPGGQSVNTTMSAVRLTHIPTGIVVTCQDEK SQIKNKEKAMKVLRARIYDKYQQEARAEYDQTRKQAVGTG DRSERIRTYNFPQNRVTDHRIGLTIQKLDLVLDGQLDEII EALILDDQSKKLEQANDAS sequence number87 DNA RF-2-Gsu-RF2 Geobacillus subterraneus DSM 13552 (91A1 ) ATGGCCGCGCCCGGCTTTTGGGATGACCAGAAAGCGGCGC AGGCGATCATTTCCGAAGCGAATGCGCTCAAGGAATTAGT CGGCGAGTTTGAATCGCTCGCGGAACGGTTCGACAACTTG GAAGTGACGTATGAGTTGTTGAAAGAGGAGCCGGATGACG AGCTGCAGGCTGAACTTGTGGAAGAAGCGAAAAAATTGAC GAAAGACTTCAGCCAGTTTGAGCTGCAGCTGTTGCTCAAC GAGCCGTACGACCAAAATAACGCGATTTTGGAGCTTCATC CGGGTGCGGGCGGCACGGAATCGCAAGACTGGGCGTCGAT GCTGTTGCGCATGTACACGCGCTGGGCGGAGAAAAAAGGA TTTAAAGTCGAAACACTGGATTATCTCCCAGGCGAGGAAG CCGGGGTGAAAAGCGTCACCTTGCTTATCAAGGGACATAA TGCATACGGCTACTTAAAGGCGGAAAAAGGGGTACACCGG CTTGTGCGCATCTCCCCGTTTGACGCCTCAGGCCGCCGCC ATACGTCGTTCGTGTCATGCGAAGTCGTGCCGGAGATGGA CGATAACATTGAGATTGAGATCCGTCCGGAAGAGCTGAAA ATCGACACGTACCGCTCAAGCGGTGCGGGCGGGCAGCACG TCAACACGACCGACTCCGCGGTGCGCATCACCCACTTGCC GACCGGCATTGTCGTTACGTGCCAATCGGAGCGGTCGCAA ATTAAAAACCGCGAAAAAGCGATGAATATGTTAAAAGCGA AGCTGTATCAAAAGAAAATGGAGGAACAGCAAGCTGAACT CGCCGAGCTGCGCGGCGAGCAAAAAGAAATCGGCTGGGGC AGCCAAATCCGCTCCTACGTCTTCCATCCGTATTCGCTTG TCAAAGACCATCGGACGAATGTGGAGGTCGGCAACGTGCA AGCGGTGATGGATGGGGAAATCGATGTGTTCATTGACGCG TATTTGCGCGCGAAATTGAAG SEQ ID NO: 88 amino acid RF-2-GsuRF-2 Geobacillus subterraneus DSM 13552 (91A1 ) MAAPGFWDDQKAAQAIISEANALKELVGEFESLAERFDNL EVTYELLKEEPDDELQAELVEEAKKLTKDFSQFELQLLLN EPYDQNNAILELHPGAGGTESQDWASMLLRMYTRWAEKKG FKVETLDYLPGEEAGVKSVTLLIKGHNAYGYLKAEKGVHR LVRISPFDASGRRHTSFVSCEVVPEMDDNIEIEIRPEELK IDTYRSSGAGGQHVNTTTDSAVRITHLPTGIVVTCQSERSQ IKNREKAMNMLKAKLYQKKMEEQQAELAELRGEQKEIGWG SQIRSYVFHPYSLVKDHRTNVEVGNVQAVMDGEIDVFIDA YLRAKLK SEQ ID NO:89 DNA RRF-GsuRRF Geobacillus subterraneus DSM 13552 (91A1 ) ATGGCAAAGCAAGTGATCCAACAGGCGAAAGAAAAAATGG ATAAAGCTGTGCAAGCGTTCAGCCGCGAGTTGCGGACCGT CCGTGCCGGTCGGGCGAACGCGGGGTTGCTTGAGAAAGTA ACCGTTGACTATTACGGTGTCGCAACGCCGATCAACCAGC TCGCTACGATCAGCGTGCCGGAAGCGCGTATGCTTGTCAT TCAGCCGTATGACAAATCGGTCATTAAAGAAATGGAAAAA GCGATTTTAGCGTCGGACTTAGGAGTGACGCCGTCGAATG ACGGATCGGTTATCCGCCTTGTCATTCCGCCGCTTACTGA AGAACGTCGCCGTGAACTGGCGAAGCTCGTCAAAAAAATAT TCGGAAGAAGCGAAAGTTGCGGTGCGCAACATCCGTCGCG ATGCAAACGATGAGCTGAAAAAACTCGAGAAAAATAGCGA GATTACGGAAGATGAGCTGCGCAGCTATACCGACGAAGTG CAAAAGCTGACCGACAGCCATATCGCCAAAATTGACGCCA TCACAAAAGAGAAAGAAAAAGAAGTGATGGAAGTA sequence number 90 amino acid RRF-GsuRRF Geobacillus subterraneus DSM 13552 (91A1 ) MAKQVIQQAKEKMDKAVQAFSRELATVRAGRANAGLLEKV TVDYYGVATPINQLATISVPEARMLVIQPYDKSVIKEMEK AILASDLGVTPSNDGSVIRLVIPPLTEERRRELAKLVKKY SEEAKVAVRNIRRDANDELKKLEKNSEITEDELRSYTDEV QKLTDSHIAKIDAITKEKEKEVMEV sequence number 91 DNA AlaRS-GsuAlaRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAGAGTTTTTTTATATAAAAGACCAAAGGGGAGGATTG TTATGAAAAAGTTAACATCTGCCGAAGTGCGGCGTATGTT TTTGCAGTTTTTCCAAGAAAAAGGCCATGCGGTCGAGCCG AGCGCTTCGCTCATTCCTGTCGATGACCCGTCGTTATTAT GGATCAACAGCGGTGTCGCGACGCTGAAAAAATATTTTGA TGGCCGTATCATCCCGGACAACCCGCGCATTTGCAATGCG CAAAAATCGATCCGCACAAACGACATCGAAAATGTCGGGA AAACGGCTCGCCACCATACGTTTTTTGAAATGCTCGGCAA CTTTTCGATCGGCGATTATTTCAAGCGTGAAGCGATTCAT TGGGCATGGGAGTTTTTAACAAGTGAAAAGTGGATTGGTT TTGATCCAGAGCGGTTGTCAGTCACTGTTCATCCGGAAGA CGAAGAGGCGTATAACATTTGGCGCAACGAGATCGGTCTT CCTGAAGAGCGGATTATTCGTTTAGAAGGAAACTTCTGGG ATATCGGTGAAGGCCCGAGCGGTCCGAACACGGAAATTTT TTATGACCGCGGTGAAGCGTTCGGCAACGATCCAAACGAT CCAGAACTGTATCCAGGCGGGGAAAATGACCGCTACTTAG AAGTATGGAATCTCGTCTTTTCACAGTTCAACCATAACCC GGACGGCACGTACACGCCGCTGCCGAAGAAAAACATCGAT ACCGGCATGGGCTTAGAGCGGATGTGCTCGATTTTGCAAG ATGTACCGACGAACTTTGAAACTGATTTGTTCATGCCGAT CATCCGCGCGACTGAGCAGATCGCGGGTGAGCAATACGGC AAAGATCCGAATAAAGACGTTGCTTTTAAGGTCATCGCTG ACCATATTCGTGCCGTGACGTTTGCGGTCGGCGACGGGGC GCTGCCGTCGAACGAAGGACGAGGCTATGTATTGCGCCGC CTGCTTCGCCGCGCTGTGCGCTATGCGAAACAAATCGGCA TTGACCGTCCATTTATGTATGAGCTTGTTCCGGTTGTCGG TGAAATTATGCAAGACTATTATCCGGAAGTGAAAGAAAAA GCCGATTTCATCGCCCGCGTCATTCGGACGGAAGAAGAGC GGTTCCACGAAACGCTTCATGAAGGGCTCGCCATTTTGGC AGAAGTGATGGAAAAGGCGAAAAAACAAGGAAGCACCGTC ATTCCAGGAGAAGAGGCGTTCCGCTTGTACGATACGTACG GCTTCCCGCTCGAGCTGACGGAAGAATATGCTGCTGAAGC GGGCATGTCGGTCGATCACGCCGGTTTTGAGCGCGAGATG GAGCGCCAGCGCGAACGGGCCCGTGCCGCTCGCCAAGATG TCGATTCGATGCAAGTGCAAGGCGGGGTGCTCGGCGACAT TAAAGACGAAAGCCGTTTTGTCGGCTACGATGAGCTCGTC GTTTCTTCGACGGTCATTGCCATCATTAAAGACGGACAGC TCGTGGAGGAAGTCGGGACTGGCGAGGAAGCACAAATCAT CGTTGATGTGACGCCGTTTTACGCCGAAAGCGGCGGACAA ATCGCTGACCAAGGTGTGTTTGAAGGCGAAACGGGAACAG CGGTCGTCAAAGATGTGCAAAAAGCACCGAACGGTCAGCA CCTCCATTCGATTGTCGTCGAACGCGGTGCGGTGAAAAAA GGCGATCGCTATACGGCGCGCGTCGATGAAGTGAAGCGGT CGCAAATCGTGAAAAACCATACGGCGACCCACTTGCTTCA TCAAGCGTTAAAAGACGTTCTTGGCCGCCATGTCAACCAG GCCGGATCACTCGTTGCCCCGGATCGGCTTCGCTTTGACT TTACTCATTTCGGGCAAGTGAAGCCTGATGAGCTCGAGCG CATTGAGGCGATCGTCAATGAACAAATTTGGAAGAGTATT CCGGTCGACATTTTTTACAAACCGCTCGAGGAAGCAAAAG CGATGGGGGCGATGGCGCTGTTTGGTGAAAAATACGGCGA TATCGTCCGCGTTGTTAAAGTTGGCGACTACAGCTTAGAG TTGTGCGGCGGCTGCCATGTGCCGAATACAGCGGCCATTG GGTTGTTTAAAATCGTCTCCGAGTCCGGCATCGGTGCCGG CACGCGCCGGATTGAAGCGGTGACTGGGGAAGCGGCATAC CGCTTTATGAGCGAACAGCTTGCTCTGTTGCAAGAAGCGG CGCAAAAGCTGAAAACGAGCCCGAGAGAGCTGAATGCCCG CCTTGATGGGCTGTTTGCCGAACTGCGCCAACTGCAGCGC GAAAATGAGTCGCTTGCTGCCCGTCTCGCCCATATGGAGG CGGAACACCTCACCCGTCAAGTGAAAGAGGTGGGCGGTGT GCCGGTATTAGCCGCAAAAGTGCAGGCGAACGACATGAAC CAATTGCGGGCGATGGCTGATGACTTGAAGCAAAAACTAG GGACGGCGGTCATCGTGTTAGCGGCCGTGCAAGGTGGCAA AGTCCAATTGATTGCTGCGGTGACTGATGACTTAGTGAAA AAAGGATACCACGCCGGCAAACTCGTCAAAGAAGTGGCTT CACGTTGCGGCGGCGGAGGCGGCGGACGTCCTGATATGGC GCAGGCCGGTGGGAAGGACGCGAACAAAGTCGGCGAAGCG CTCGATTATGTCGAAACATGGGTCAAATCCATTTCC SEQ ID NO: 92 Amino acid AlaRS - GsuAlaRS Geobacillus subterraneus DSM 13552 (91A1 ) MRVFLYKRPKGRIVMKKLTSAEVRRMFLQFFQEKGHAVEP SASLIPVDDPSLLWINSGVATLKKYFDGRIIPDNPRICNA QKSIRTNDIENVGKTARHHTFFEMLGNFSIGDYFKREAIH WAWEFLTSEKWIGFDPERLSVTVHPEDEEAYNIWRNEIGL PEERIIRLEGNFWDIGEGPSGPNTEIFYDRGEAFGNDPND PELYPGGENDRYLEVWNLVFSQFNHNPDGTYTPLPKKNID TGMGLERMCSILQDVPTNFETDLFMPIIRATEQIAGEQYG KDPNKDVAFKVIADHIRAVTFAVGDGALPSNEGRGYVLRR LLRRAVRYAKQIGIDRPFMYELVVPVGEIMQDYYPEVKEK ADFIARVIRTEEERFETHELHGLAILAEVMEKAKKQGSTV IPGEEAFRLYDTYGFPLELTEEYAAEAGMSVDHAGFEREM ERQRERARAARQDVDSMQVQGGVLGDIKDESRFVGYDELV VSSTVIAIIKDGQLVEEVGTGEEAQIIVDVTPFYAESGGQ IADQGVFEGETGTAVVKDVQKAPNGQHLHSIVVERGAVKK GDRYTARVDEVKRSQIVKNHTATHLLHQALKDVLGRHVNQ AGSLVAPDRLRFDFTHFGQVKPDELERIEAIVNEQIWKSI PVDIFYKPLEEAKAMGAMALFGEKYGDIVRVVKVGDYSLE LCGGCHVPNTAAIGLFKIVSEGSIGAGTRRIEAVTGEAAY RFMSEQLALLQEAAQKLKTSPRELNARLDGLFAELRQLQR ENESLAARLAHMEAEHLTRQVKEVGGVPVLAAKVQANDMN QLRAMADDLKQKLGTAVIVLAAVQGGKVQLIAAVTDDLVK KGYHAGKLVKEVASRCGGGGGRPDMAQAGGKDANKVGEA LDYVETWVKSIS sequence no. 93 DNA ArgRS-GsuArgRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAACATTGTCGGACAAATGAAAGAACAGCTGAAAGAGG AAATTCGCCAGGCGGTGGGAAAAGCCGGGCTGGTGGCGGC TGAGGAGCTGCCAGAAGTATTGCTTGAGGTGCCGCGCGAA AAGGCTCATGGCGATTATTCGACGAATATCGCCATGCAGC TCGCCCGCATCGCGAAAAAGCCACCGCGGGCAATCGCCGA AGCCATCGTTGAAAAGTTTGACGCCGAGCGTGTTTCGGTG GCGCGCATCGAGGTAGCCGGCCCAGGGTTTATTAACTTTT ACATGGACAATCGCTATTTGACAGCGGTTGTGCCGGCGAT TTTGCAAGCGGGCCAAGCGTATGGCGAGTCGAATGTCGGC AAAGGGGAAAAAGTGCAAGTCGAGTTCGTCTCGGCTAACC CGACCGGCAACTTGCATTTAGGTCATGCTCGCGGTGCGGC GGTTGGCGATTCACTTAGCAATATTTTGGCGAAAGCCGGA TTCGATGTGACGCGTGAATATTACATTAATGATGCCGGCA AACAAATTTATAACTTGGCGAAATCAGTCGAAGCCCGCTA TTTCCAAGCGCTCGGTACCGATATGCCGCTGCCGGAGGAC GGCTATTACGGTGACGACATCGTGGAAATCGGCAAAAAGC TCGCCGATGAATATGGCGATCGGTTCGTCCATGTGGACGA AGAAGAACGACTCGCCTTTTTCCGCGAATACGGCCTCCGT TATGAGCTCGACAAAATTAAAAACGATTTGGCTGCCTTCC GCGTTCCATTTGACGTTTGGTATTCGGAAACATCGCTTTA TGAGAGCGGCAAAATCGATGAGGCGCTCTCAACGCTGCGT GAGCGCGGTTACATTTACGAACAGGACGGAGCCACATGGT TTCGTTCGACGGCGTTTGGCGATGACAAAGACCGTGTGTT AATCAAGCAAGACGGAACGTATACGTATTTGCTTCCGGAC ATCGCTTACCATCAAGATAAGCTGCGGCGTGGGTTCACGA AGCTAATCAACGTCTGGGGAGCGGATCATCATGGCTACAT CCCGCGCATGAAAGCGGCGATCGCTGCGCTCGGCTACGAT CCAGAAGCGCTCGAGGTCGAAATTATCCAAATGGTGAACT TATACCAAAACGGCGAGCGCGTCAAAATGAGCAAACGTAC TGGCAAAGCGGTGACGATGCGCGAGCTGATGGAAGAAGTC GGCGTCGATGCTGTCCGCTACTTCTTCGCTATGCGTTCGG GCGATACGCATCTCGATTTTGATATGGACTTGGCTGTTGC CCAGTCGAATGAAAACCCGGTCTACTATGTCCAATATGCA CATGCCCGCGTCTCAAGCATTCTCCGTCAAGCAAAAGAGC ATCAACTGTCGTATGAAGGCGACGTCGATCTTCATCATCT CGTGGAAACAGAAAAAGAAATCGAGCTGCTCAAAGCGCTT GGCGACTTCCCGGACGTTGTCGCTGAGGCGGCCTTGAAAC GGATGCCACATCGCGTCACCGCCTATGCGTTTGATTTGGC GTCGGCGCTCCACAGCTTTTACAATGCGGAAAAAGTGCTT GACCTAGACCAGATCGAAAAAACGAAAGCTCGTCTCGCGC TTGTCAAGGCGGTGCAAATCACGCTGCAAAACGCTCTAGC GTTAATCGGCGTCTCAGCGCCGGAACAAATG SEQ ID NO: 94 Amino acid ArgRS - GsuArgRS Geobacillus subterraneus DSM 13552 (91A1 ) MNIVGQMKEQLKEEIRQAVGKAGLVAAEELPEVLLEVPRE KAHGDYSTNIAMQLARIAKKPPRAIAEAIVEKFDAERVSV ARIEVAGPGFINFYMDNRYLTAVVPAILQAGQAYGESNVG KGEKVQVEFVSANPTGNLHLGHARGAAVGDSLSNILAKAG FDVTREYYINDAGKQIYNLAKSVEARYFQALGTDMPLPED GYYGDDIVEIGKKLADEYGDRFVHVDEEERLAFFREYGLR YELDKIKNDLAAFRVPFDVWYSETSLYESGKIDEALSTLR ERGYIYEQDGATWFRSTAFGDDKDRVLIKQDGTYTYLLPD IAYHQDKLRRGFTKLINVWGADHHGYIPRMKAAIAALGYD PEALEVEIIQMVNLYQNGERVKMSKRTGKAVTMRELMEEV GVDAVRYFFAMRSGDTHLDFDMDLAVAQSNENPVYYVQYA HARVSSILRQAKEHQLSYEGDVDLHHLVETEKEIELLKAL GDFPDVVAEAALKRMPHRVTAYAFDLASALHSFYNAEKVL DLDQIEKTKARLALVKAVQITLQNALALIGVSAPEQM sequence number 95 DNA AsnRS-GsuAsnRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGGACGTGTCGATTATTGGAGGGAATGTGTACGTGAAAA CGACGATTGCTGAAGTGAACCAATATGTAGGTCAAGAAGT CACGATCGGCGCTTGGTTGGCGAACAAGCGCTCGAGCGGA AAAATCGCCTTTTTACAGCTGCGTGATGGGACTGGCTTTA TTCAAGGTGTAGTTGAAAAAGCGAACGTCTCAGAAGAGGT ATTTCAACGTGCGAAAACGCTGACGCAAGAAACGTCGCTC TATGTGACCGGCACGGTGCGCGTCGACGAGCGTTCACCGT TCGGTTATGAGCTTTCGGTGACGAACATACAGGTCATCAA TGAAGCGGTCGATTATCCGATTACGCCAAAAGAACACGGT GTCGAGTTTTTAATGGATCATCGTCACCTTTGGCTTCGTT CGCGGCGCCAACATGCGATCATGAAAATCCGCAACGAATT GATCCGTGCGACGTATGAGTTTTTTAACGAACGTGGCTTC GTCAAAGTCGATGCGCCGATTTTGACTGGCAGCGCACCGG AAGGAACGACCGAGCTGTTCCATACGAAGTATTTTGACGA GGATGCCTATTTATCGCAAAGCGGCCAGCTATATATGGAA GCAGCAGCCATGGCGCTCGGTAAAGTGTTTTCGTTCGGTC CGACATTCCGTGCCGAAAAGTCGAAAACCGCCGCCATTT GATCGAATTTTGGATGATCGAGCCTGAAATGGCGTTTTAAC GAATTTGAAGACAATTTGCGGCTGCAAGAAGGTATGTCT CTTATCTCGTACAGTCGGTGCTTAGCCGTTGCCAACTTGA GCTCGGGCGCCTTGGACGCGACGTCACCAAGCTTGAGCTT GTCAAGCCGCCGTTCCGCGTCTAACGTATGACGAAGCGA TCAAGCTGCTGCATGACAAAGGGTTTACCGATATCGAATG GGGCGATGACTTCGGTGCGCCGCATGAGACAGCCATCGCT GAAAGCTTCGACAAGCCGGTGTTTATCACTCACTACCCGA CGTCGTTAAAAGCCGTTTTATATGCAGCCAGATCCGAACCG TCCGGACGTCGTGCTATGTGCTGATTTAATCGCGCCGGAG GGATACGGGGAGATTATCGGCGGTTCCGAGCGCATTCATG ATTATGAGCTGCTCAAGCAGCGTCTCGAGGAGCATCATTT GCCGCTTGAAGCATATGAATGGTATTTAGATTTGCGCAAA TACGGTTCCGTGCGCACTCCGGATTCGGGCTCGGCCTCCG AGCGAACGGTTGCTTGGATTTGCGGCGTTGAGCATGTACG CGAGACGATCCCGTTTCCGCGGTTGCTCAACCGTCTATAC CCG SEQ ID NO:96 amino acid AsnRS-GsuAsnRS Geobacillus subterraneus DSM 13552 (91A1 ) MDVSIIGGNVYVKTTIAEVNQYVGQEVTIGAWLANKRSSG KIAFLQLRDGTGFIQGVVEKANVSEEVFQRAKTLTQETSL YVTGTVRVDERSPFGYELSVTNIQVINEAVDYPITPKEHG VEFLMDHRHLWLRSRRQHAIMKIRNELIRATYEFFNERGF VKVDAPILTGSAPEGTTELFHTKYFDEDAYLSQSGQLYME AAAMALGKVFSFGPTFRAEKSKTRRHLIEFWMIEPEMAFY EFEDNLRLQEEYVSYLVQSVLSRCQLELGRLGRDVTKLEL VKPPFPRLTYDEAIKLLHDKGFTDIEWGDDFGAPHETAIA ESFDKPVFITHYPTSLKPFYMQPDPNRPDVVLCADLIAPE GYGEIIGGSERIHDYELLKQRLEEHHLPLEAYEWYLDLRK YGSVPHSGFGLGLERTVAWICGVEHVRETIPFPRLLNRLY P SEQ ID NO:97 DNA AspRS-GsuAspRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGTTTCAAACACTTGAGCTTCGTCATAAAGTGGCGAAGG CGGTGCGCAACTTTTTAGACGGCGAACGCTTTTTAGAAGT GGAGACGCCAATGTTGACGAAAAGCACACCGGAAGGGGCG CGCGATTATTTAGTGCCAAGCCGCGTTCATCCGGGGGAAT TTTACGCCTTGCCGCAGTCGCCGCAAATTTTTAAGCAGCT TTTGATGGTCGGCGGTTTTGAACGCTATTACCAAATCACT CGTTGCTTCCGCGATGAAGATTTGCGCGCTGACCGCCAGC CAGAGTTTACGCAAATTGACATTGAAATGTCGTTTGTCGA CCAAGAAGACATCATCGATTTAACCGAACGGATGATGGCG GCGGTCGTCAAAGCAACTAAAGGGATTGACATTCCGCGCC CATTTCCACGCATCACGTATGACGAAGCGATGAGCCGTTA CGGTTCCGATAAGCCGGACGTACGTTTTGGCCTTGAGCTT GTCGATGTGTCGGAAGCGGTCCGCGGCTCCGCGTTTCAAG TGTTCGCCCGCGCCGTTGAGCAAGGTGGTCAAGTGAAGGC AATCAACGTAAAAGGAGCGGCGAGCCGTTATTCGCGTAAA GACATTGACGCGTTAGCGGAGTTTGCCGGCCGCTACGGAG CGAAAGGGCTCGCTTGGTTAAAAGTTGAAGGCGGGGAGCT GAAAGGGCCGATCGCCAAGTTTTTCGTCGATGATGAGCAA ACAGCGCTGCGCCAGCTGCTTGCTGCCGAAGATGGGGATT TGCTGTTGTTTGTTGCTGACGAGAAGGCGATTGTCGCGGC GGCTCTTGGTGCGTTGCGGTTAAAGCTCGGCAAAGAGCTT GGCTTGATCGATGAAACGAAGCTCGCTTTTTTATGGGTAA CAGATTGGCCGCTTTTAGAGTACGACGAAGAAGAAGGCCG ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​MFQTLELRHKVAKAVRNFLDGERFLEVETPMLTKSTPEGA RDYLVPSRVHPGEFYALPQSPQIFKQLLMVGGFERYYQIT RCFRDEDLRADRQPEFTQIDIEMSFVDQEDIIDLTERMMA AVVKATKGIDIPRPFPRITYDEAMSRYGSDKPDVRFGLEL VDVSEAVRGSAFQVFARAVEQGGQVKAINVKGAASRYSRK DIDALAEFAGRYGAKGLAWLKVEGGELKGPIAKFFVDDEQ TALRQLLAAEDGDLLLFVADEKAIVAAAALGALRLKLGKEL GLIDETKLAFLWVTDWPLLEYDEEEGRYYAAAHHPFTMPVR DDIPLLETNPGAVRAQAYDLVLNGYELGGGSLRIFERDVQ EKMFRALGFDQEEARQFGFLLEAFEYGTPPHGGIALGLD RLVMLLAGRTNLRDTIAFPKTASASCLLTEAPGPVSEKQL KELHLAVVLPDQQ sequence no. 99 DNA CysRS-GsuCysRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAAAGGAAGAGCGAATATGAGCAGTATCCGACTTTATA ATACGTTGACGCGAAAAAAGGAAACGTTTGAGCCGCTCGA ACCGAACAAAGTGAAAATGTATGTATGTGGCCCGACGGTC TATAATTATATTCATATCGGCAATGCTCGCGCCGCTATCG TCTTTGATACGATCCGCCGTTATTTAGAGTTCCGCGGTTA TGATGTGACGTATGTATCCAACTTTACTGATGTCGACGAC AAGCTAATCAGGGCGGCCCGCGAGCTTGGTGAGAGCGTGC CGGCGATCGCCGAGCGGTTTATTGAGGCGTATTTTGAGGA CATTGAGGCGCTCGGCTGCAAAAAAGCAGATATCCATCCG CGCGTGACGGAAAATATCGAAACGATTATCGAATTCATTC AAGCGCTCATTGACAAAGGCTATGCGTACGAAGTCGATGG TGACGTATACTATCGGACGCGCAAGTTTGATGGCTACGGC AAATTGTCGCATCAGTCGATCGATGAGCTACAAGCGGGGG CGCGCATCGAAGTTGGGGAAAAGAAAGATGATCCACTCGA TTTTGCTCTTTGGAAAGCAGCGAAAGAAGGAGAGATTTCT TGGGACAGCCCATGGGGGAAAGGGCGGCCCGGCTGGCATA TCGAATGTTCAGCGATGGCGCGCAAATATTTAGGAGATAC GATCGACATTCATGCTGGCGGCCAAGACTTAACGTTTCCA CACCATGAAAACGAAATTGCCCAATCGGAAGCACTGACCG GCAAACCGTTTGCGAAATATTGGCTGCACAATGGGTATTT AAATATTAACAATGAAAAAATGTCCAAGTCGCTTGGCAAC TTTGTACTTGTTCACGATATCATCCGGCAGATTGACCCAC AAGTGTTGCGTTTCTTTATGCTGTCGGTGCACTATCGCCA CCCGATCAACTATAGCGAGGAGCTGCTTGAGAGCGCTCGG CGTGGTCTCGAACGCTTGAGGACAGCATACGGTAATTTGC AGCACCGGCTTGGGGCGAGCACGAACTTAACCGATAACGA CGGCGAGTGGCTTTCGCGCCTCGCGGATATCCGCGCCTCG TTCATTCGTGAAATGGACGATGATTTCAACACAGCAAACG GCATTGCGGTCTTGTTCGAGCTCGCCAAACAAGCGAACTT GTATTTGCAGGAGAAAACGACATCCGAGAATGTCATTCAC GCGTTTTTGCGCGAATTTGAGCAGCTGATGGATGTACTCG GCCTTACTTTGAAACAAGAGGAGTTGCTTGACGAAGAAAT TGAGGCGCTGATCCGCCAGCGCAATGAAGCGCGGAAAAAT CGTGACTTTGCCTTAGCCGACCGCATCCGCGACGAGTTGA AAGCAAAAAATATCATTTTGGAAGATACGCCGCAAGGGAC GAGATGGAAACGGGGATCG SEQ ID NO: 100 Amino acid CysRS-GsuCysRS Geobacillus subterraneus DSM 13552 (91A1 ) MKGRANMSSIRLYNTLTRKKETFEPLEPNKVKMYVCGPTV YNYIHIGNARAAIVFDTIRRYLEFRGYDVTYVSNFTDVDD KLIRAARELGESVPAIAERFIEAYFEDIEALGCKKADIHP RVTENIETIIEFIQALIDKGYAYEVDGDVYYRTRKFDGYG KLSHQSIDELQAGARIEVGEKKDDDPLDFALWKAAKEGEIS WDSPWGKGRPGWHIECSAMARKYLGDTIDIHAGGQDLTFP HHENEIAQSEALTGKPFAKYWLHNGYLNINNEKMSKSLGN FVLVHDIIRQIDPQVLRFFMLSVHYRHPINISEELLESAR RGLERLRTAYGNLQHRLGASTNLDTNDGEWLSRLADIRAS FIREMDDDFNTANGIAVLFELAKQANLYLQEKTTSENVIH AFLREFEQLMDVLGLTLKQEELLDEEIEALIRQRNEARKN RDFALADRIRDELKAKNIILEDTPQGTRWKRGS sequence number 101 DNA GluRS-GsuGluRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGGAATTGGAGGTTTGGACGATGGCAAAAAACGTGCGCG TGCGCTATGCGCCGAGCCCGACTGGCCATTTGCATATCGG TGGGGCACGGACAGCGCTGTTTAACTATTTGTTTGCCCGC CATTACGGCGGAAAAAATGATCGTCCGCATCGAAGATACGG ATATTGAACGGAACGTTGAAGGCGGCGAAAGAGTCGCAGCT TGAAAACTTAAAATGGCTTGGCATCGATTATGACGAATCG ATTGATAAGGACGGCGGATATGGGCCGTATCGTCAGACGG AACGGCTCGATATCTATCGGAAGTATGTGAACGAGCTGCT TGAACAAGGGCATGCGTATAAATGTTTTTGTACACCGGAA GAGCTCGAGCGGGAACGTGAGGAGCAACGGGCGGCAGGTA TTGCTGCTCCGCAATACAGCGGCAAATGCCGCCATTTAAC GCCGGAGCAAGTTGCCGAGCTTGAAGCACAAGGAAAACCG TATACGATCCGCTTGAAAGTGCCGGAAGGGAAAACGTATG AAGTAGATGATTTAGTGCGCGGTAAAGTGACGTTTGAATC GAAAGACATCGGCGATTGGGTCATTGTGAAGGCGAACGGT ATTCCGACGTACAACTTTGCCGTTGTCATTGATGACCATT TGATGGAAATCAGCCATGTGTTCCGCGGTGAGGAGCATTT ATCCAACACGCCGAAACAGCTAATGGTGTACGAATATTTC GGTTGGGAGCCACCGCAATTCGCCCATATGACATTGATTG TCAACGAGCAGCGGAAAAAGCTATCCAAGCGCGATGAATC GATTATCCAGTTCGTGTCGCAATATAAAGAGCTCGGCTAT TTGCCGGAGGCGATGTTCAACTTTTTCGCCCTTCTTGGCT GGTCGCCGGAAGGAGAAGAAGAAATTTTTACGAAGGACGA GCTCATCCGCATTTTTGATGTCGCCCGGCTGTCGAAATCG CCGTCGATGTTTGATACGAAAAAGCTGACATGGATGAACA ACCAATATATCAAAAAGCTGGATCTCGACAGGCTTGTCGA GCTGGCGTTGCCGCATTTAGTGAAAGCCGGACGCCTGCCG GCAGATATGAGTGATGAGCAGCGGCAATGGGCACGCGATT TGATTGCCTTGTACCAAGAGCAAATGAGCTACGGTGCGGA GATCGTTTCGCTGTCCGAGCTGTTCTTTAAAGAAGAAGTC GAATACGAAGACGAAGCCCGCCAAGTGCTCGCCGAAGAAC AAGTACCGGATGTGCTCTCCGCCTTTTTGGCGAATGTGCG TGAGCTTGAGCCGTTTACGGCGGATGAGATTAAAGCAGCG ATCAAAGCAGTGCAAAAATCGACAGGGCAAAAAGGCAAGA AGCTGTTTATGCCGATTCGCGCCGCAGTGACTGGGCAAAC ACACGGACCGGAACTGCCGTTTGCCATCCAACTGCTTGGC AAACAAAAGGTGATTGAACGGCTCGAACGGGCACTGCATG AAAAATTT SEQ ID NO: 102 Amino acid GluRS - GsuGluRS Geobacillus subterraneus DSM 13552 (91A1 ) MELEVWTMAKNVRVRYAPSPTGHLHIGGARTALFNYLFAR HYGGKMIVRIEDTDIERNVEGGEESQLENLKWLGIDYDES IDKDGGYGPYRQTERLDIYRKYVNELLEQGHAYKCFCTPE ELEREREEQRAAGIAAPQYSGKCRHLTPEQVAELEAQGKP YTIRLKVPEGKTYEVDDLVRGKVTFESKDIGDWVIVKANG IPTYNFAVVIDDHLMEISHVFRGEEHLSNTPKQLMVYEYF GWEPPQFAHMTLIVNEQRKKLSKRDESIIQFVSQYKELGY LPEAMFNFFALLGWSPEGEEEIFTKDELIRIFDVARLSKS PSMFDTKKLTWMNNQYIKKLDLDRLVELALPHLVKAGRLP ADMSDEQRQWARDLIALYQEQMSYGAEIVSLSELFFKEEV EYEDEARQVLAEEQVPDVLSAFLANVRELEPFTADEIKAA IKAVQKSTGQKGKKLFMPIRAAVTGQTHGPELPFAIQLLG KQKVIERLERALHEKF sequence number 103 DNA GlyRS-GsuGlyRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGGAGGAGGATGATGACATGGCTGCAACAATGGAAGAAA TCGTTGCCCACGCCAAGCATCGCGGCTTCGTGTTTCCGGG GTCGGAAATTTACGGTGGGCTGGCGAACACATGGGATTAC GGTCCGCTCGGTGTCGAGCTGAAAAATAACATTAAACGGG CGTGGTGGAAAAAGTTCGTCCAAGAATCGCCACACAATGT CGGTTTGGACGCTGCCATTTTAATGAACCCAAAAACGTGG GAAGCATCCGGCCATTTAGGCAACTTCACGATCCGATGG TCGACTGCAAACAGTGTAAAGCGCGTCATCGCGCCGACAA GCTGATTGAGCAGGCACTTGAAGAAAAAAGGAATTGAGATG GTCGTTGACGGTTTGCCGCTTGCCAAGATGGAAGAGCTTA TCCGTGAATACGACATCGCTTGTCCAGAATGCGGCAGTCG TGACTTTACGAACGTGCGTCAGTTTAATTTAATGTTCAAA ACATACCAAGGTGTCACCGAATCAAGCGCTAACGAAATTT ATTTGCGCCCGGAGACGGCCCAAGGTATTTTTGTCAACTT TAAAAACGTCCAGCGCACGATGCGCAAAAAAATTACCGTTT GGCATCGCGCAAATCGGAAAAAAGTTTCCGCAACGAAATTA CGCCAGGGAACTTTACGTTCGCACACGTGAATTTGAACA AATGGAGCTTGAGTTTTTCTGCAAACCGGGCGAAGAGCTG AAATGGTTCGACTACTGGAAACAATTTTGCAAGGAATGGC TGTTGTCGCTCGGCATGAACGAAGAACATATCCGCCTGCG CGACCATACGAAAGAAGAATTATCCCACTATAGTAATGCG ACGACTGATATCGAGTATCAGTTCCCGTTCGGCTGGGGCG AGCTCTGGGGTATTGCGTCGCGCACCGATTACGACTTAAA ACAGCATATGGAACACTCCGGTGAGGATTTCCATTATCTT GACCAAGAAACGAATGAGCGCTACATCCCGTACTGCATTG AGCCGTCGCTCGGTGCCGACCGTGTCACGCTCGCGTTTAT GATTGACGCCTATGACGAGGAAGAGCTCGAAGACGGCACG ACCCGGACAGTTATGCATTGCATCCAGCGCTTGCGCCGT ACAAAGCAGCTGTCTTGCCGTTATCGAAAAAGCTGGGTGA CGGAGCGCGCCGAATTTATGAAGAGCTCGCGAAGCATTTC ATGGTCGACTACGAATGAAACAGGTTCGATTGGCAAGCGGT ATCGTCGTCAAGATGAAATCGGCACGCCGTTTTGTATCAC GTACGACTTTGAGTCCGAGCAAGATGGCCAAGTAACCGTT CGTGACCGTGACACGATGGAACAAGTGCGGTTGCCGATTG GGGAGCTCAAAGCCTTTTTTGGATAAAAAAATTGCCTTT sequence number 104 amino acid GlyRS-GsuGlyRS Geobacillus subterraneus DSM 13552 (91A1 ) MEEDDDMAATMEEIVAHAKHRGFVFPGSEIYGGLANTWDY GPLGVELKNNIKRAWWKKFVQESPHNVGLDAAILMNPKTW EAGSHLGNFNDPMVDCKQCKARHRADKLIEQALEEKGIEM VVDGLPLAKMEELIREYDIACPECGSRDFTNVRQFNLMFK TYQGVTESSANEIYLRPETAQGIFVNFKNVQRTMRKKLPF GIAQIGKSFRNEITPGNFTRTREFEQMELEFFCKPGEEL KWFDYWKQFCKEWLLSLGMNEEHIRLRDHTKEELSHYSNA TTDIEYQFPFGWGELWGIASRTDYDLKQHMEHSGEDFHYL DQETNERYIPYCIEPSLGADRVTLAFMIDAYDEEELEDGT TRTVMHLHPALAPYKAAVLPLSKKLDGGARRIYEELAKHF MVDYDETGSIGKRYRRQDEIGTPFCITYDFESEQDGQVTV RDRDTMEQVRLPIGELKAFLDKKIAF sequence number 105 DNA HisRS-GsuHisRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGGCTTTTCAAATTCCAAGAGGGACACAAGATTTATTAC CGGGTGAAACGGAAAAATGGCAATATGTCGAACAAGTGGC CCGCGACCTGTGTAGACGGTACGCTATGAAGAAATACGG ACGCCGATTTTTGAACATACGGAGCTGTTTTTACGTGGCG TTGGTGATACGACCGATATCGTCCAAAAAGAGATGTACAC GTTTGAAGACAAAGGGGGCCGTGCGTTGACGCTCCGTCCG GAAGGAACCGCACCGGTCGTGCGGGCGTTCGTCGAGCATA AGCTGTACGGCAGCCCGAATCAGCCGGTCAAGTTGTATTA TGCGGGACCAATGTTCCGTTATGAGCGGCCGGAAGCCGGA CGGTTCCGCCAATTCGTCCAGTTTGGTGTTGAGGCAATTG GCAGCAGTGATCCGGCGATTGACGCCGAGGTGATGGCGTT AGCGATGCATATTTATAAGGCGCTTGGTTTAAAACACATC CGGCTCGTAATCAACAGTTTAGGCGATGTAGACAGCCGCC GGGCGCATCGCGAAGCGCTTGTCCGCCATTTTTCTGACCG CATTCATGAACTGTGCCCGGACTGTCAGGCGCGGCTTGAG ACGAATCCGCTCCGCATTCTCGATTGTAAAAAGGACCGCG ATCATGAACTGATGGCGTCAGCACCGTCGATTTTAGACTA TTTGAATGACGAATCGCGCGCGTATTTTGAGAAGGTGAAG CAATATTTAACGATGCTTGACATCCCGTTTGTCATTGACT CGCGGCTCGTGCGCGGCCTCGATTATTACAACCATACGAC GTTTGAAATTATGAGCGAGGCTGAAGGATTCGGCGCAGCG GCGACTCTTTGCGGCGGCGGACGCTATAACGGGCTTGTGC AAGAAATTGGCGGCCCGGAAACGCCTGGCATCGGCTTTGC GTTAAGCATTGAACGGCTGCTGGCGGCGCTTGAAGCGGAA GGGATTGAACTGCCGATCCATCGAGGAATCGATTGCTATG TTGTCGCTGTCGGTGAGCGGGCAAAAGATGAAACTGTCCG CCTCGTTTACGAATTGCGCCGTGCCGGCCTGCGTGTGGAG CAAGACTATTTAGGTCGAAAAATGAAGGCACAGCTGAAGG CAGCTGACCGTCTTGGCGCATCATTCGTTGCCATCATCGG CGACGAGGAGCTGGAAAAACAGACAGCAGCTGTGAAACAC ATGGCGAGCGGCGAGCAAACTGATGTGCCGCTTGGAGAGT TGGCGTCCTTTTTAATAGAACGAACAAAACGGGAGGAG SEQ ID NO: 106 amino acid HisRS-GsuHisRS Geobacillus subterraneus DSM 13552 (91A1 ) MAFQIPRGTQDLLPGETEKWQYVEQVARDLCRRYGYEEIR TPIFEHTELFLRGVGDTTDIVQKEMYTFEDKGGRALTLRP EGTAPVVRAFVEHKLYGSPNQPVKLYYAGPMFRYERPEAG RFRQFVQFGVEAIGSSDPAIDAEVMALAMHIYKALGLKHI RLVINSLGDVDSRRAHREALVRHFSDRIHELCPDCQARLE TNPLRILDCKKDRDHELMASAPSILDYLNDESRAYFEKVK QYLTMLDIPFVIDSRLVRGLDYYNHTTFEIMSEAEGFGAA ATLCGGGRYNGLVQEIGGPETPGIGFALSIERLLAALEAE GIELPIHRGIDCYVVAVGERAKDETVRLVYELRRAGLRVE QDYLGRKMKAQLKAADRLGASFVAIIGDEELEKQTAAVKH MASGEQTDVPLGELASFLIERTKREE SEQ ID NO: 107 DNA IleRS-GsuIleRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGGACTACAAAGAGACGCTGCTCATGCCGCAAACGGAGT TCCCGATGCGTGGCAACTTGCCGAAGCGGGAGCCGGAAAT GCAAAAAAAATGGGAGGAAATGGACATTTACCGGAAAGTG CAGGAGCGGACGAAAGGACGGCCGCTGTTTGTGCTGCACG ACGGCCCGCCATACGCCAACGGTGATATTCATATGGGCCA TGCATTAAATAAAATTTTAAAAGATATTATCGTCCGCTAC AAGTCGATGAGCGGCTTTTGTGCGCCGTATGTGCCTGGCT GGGATACACATGGCTTACCGATTGAAACGGCACTGACGAA GCAAGGTGTCGACCGCAAATCGATGAGTGTCGCTGAGTTC CGCAAGCTGTGCGAACAATACGCGTATGAGCAAATCGACA ACCAGCGCCAACAGTTTAAACGGCTCGGGGTGCGGGGCGA TTGGGACAACCCGTACATTACGCTCAAGCCGGAATACGAA GCCCAGCAAATTAAAGTGTTCGGTGAAATGGCGAAAAAAG GGCTCATTTATAAAGGGCTGAAGCCGGTGTATTGGTCGCC GTCGAGCGAATCGGCGCTCGCCGAAGCGGAAATCGAATAT AAAGACAAACGGTCGCCGTCGATTTATGTCGCGTTCCCAG TTAAAGATGGTAAAGGTGTGCTTCAAGGGGATGAACGAAT CGTCATTTGGACGACGACACCGTGGACGATTCCAGCGAAC TTGGCGATCGCCGTTCACCCGGATTTGGACTACTATATTG TCGAAGCAAACGGGCAAAAATACGTTGTTGCTGCGGCCTT GGCGGAATCGGTAGCGAAAGAAGTCGGCTGGGAGGCATGG TCCGTCGTCAAAACGGTAAAAGGAAAAGAACTTGAGTACG TAGTCGCCAAACATCCGTTTTACGAGCGCGACTCGCTTGT CGTCTGCGGCGAGCACGTCACGACCGACGCCGGTACCGGC TGCGTTCATACGGCACCAGGACACGGGGAAGACGACTTTA TCGTCGGACAAAAATACGGGCTTCCGGTTCTTTGCCCGGT TGATGAGCGCGGCTATATGACAGAAGAAGCGCCTGGATTT GCAGGGATGTTTTACGACGAGGCGAACAAAGCGATTACAC AAAAGCTCGAGGAAGTTGGAGCGCTCCTTAAGCTCAGCTT CATTACCCACTCGTATCCGCATGATTGGCGGACGAAGCAA CCGACAATTTTCCGAGCGACGACACAATGGTTTGCCTCCA TTGATAAAATTCGTGATCAACTTCTTGATGCCATCAAGGA AACGAAATGGGTGCCAGAATGGGGAGAAATCCGCATCCAT AACATGGTGCGCGACCGCGGTGACTGGTGCATCTCCCGCC AACGCGCTTGGGGCGTGCCAATTCCGGTCTTTTACGGCGA AAACGGCGAGCCGATCATCACAGATGAGACGATCGAGCAC GTGTCAAACCTATTCCGCCAGTACGGCTCGAATGTTTGGT TTGAGCGTGAGGCGAAAGACTTATTGCCGGAAGGATTCAC CCATCCGTCCAGCCCGAACGGCCTCTTTACGAAAGAGACG GATATTATGGACGTCTGGTTTGACTCCGGTTCGTCGCATC AAGCCGTGCTTGTTGAACGCGATGACCTAGAGCGTCCGGC TGATTTATACTTAGAAGGATCTGACCAATATCGCGGCTGG TTTAACTCGTCGCTGTCTACAGCCGTTGCCGTCACCGGAA AAGCACCGTATAAAGGGGTGTTAAGCCATGGCTTCGTTTT AGACGGCGAAGGGCGAAAAATGAGCAAATCGCTCGGCAAC GTCGTCGTGCCGGCCAAAGTCATGGAACAGCTCGGTGCCG ACATTTTACGCCTTTGGGTCGCCTCGGTTGACTATCAGGC GGATGTACGCATTTCCGATAACATTTTAAAACAAGTGTCC GAAGTGTATCGGAAAATCCGCAATACGTTCCGCTTTATGC TCGGCAACTTGTTTGATTTTGACCCGAATCAAAACGCTGT GCCGGTTGGGGAGCTTGGCGAAGTCGATCGCTACATGTTA GCGAAATTAAATAAACTCATCGCTAAAGTGAAAAAGGCGT ATGACAGCTATGATTTTGCTGCTGTTTATCATGAGATGAA CCATTTCTGCACCGTCGAGTTAAGCGCATTTTATTTGGAT ATGGCGAAAGACATTTTGTACATCGAAGCGGCCGATTGTC GTGCCCGCCGTGCGGTGCAGACGGTGCTGTATGAAACGGT TGTCGCCTTGGCGAAGCTCATTGCGCCGATTTTGCCGCAC ACGGCCGATGAAGTGTGGGAGCATATCCCGAACCGGAAAG AGCAAGTGGAAAGCGTCCAGCTCACCGACATGCCGGAGTC AATGGCCATCGATGGTGAAGAAGCGCTGCTTGCGAAATGG GATGCGTTTATGGATGTACGAGATGACATTTTAAAAGCGC TCGAGAATGCGCGTAATGAAAAAGTGATCGGTAAGTCGCT CACGGCGAGCGTCACTGTTTACCCGAAAGACGAAGTGCGG GCGCTTTTGGCTTCGATCAACGAGGACTTGCGCCAACTTC TCATCGTTTCCGCGTTTTCGGTCGCCGATGAATCGTATGA CGCCGCGCCAGCCGAAGCAGAACGGCTCAACCATGTGGCC GTCATCGTTCGCCCGGCGGAAGGTGAGACGTGCGAACGTT GCTGGACGGTGACACCGGACGTCGGACGCGATGAGTCCCA CCCGACGCTTTGTCCGCGCTGCGCACATATTGTGAACGAA CATTATTCGGCA SEQ ID NO: 108 Amino acid IleRS - GsuIleRS<{0005077}>Geobacillus subterraneus DSM 13552 (91A1 ) MDYKETLLMPQTEFPMRGNLPKREPEMQKKWEEMDIYRKV QERTKGRPLFVLHDGPPYANGDIHMGHALNKILKDIIVRY KSMSGFCAPYVPGWDTHGLPIETALTKQGVDRKSMSVAEF RKLCEQYAYEQIDNQRQQFKRLGVRGDWDNPYITLKPEYE AQQIKVFGEMAKKGLIYKGLKPVYWSPSSESALAEAEIEY KDKRSPSIYVAFPVKDGKGVLQGDERIVIWTTTPWTIPAN LAIAVHPDLDYYIVEANGQKYVVAAALAESVAKEVGWEAW SVVKTVKGKELEYVVAKHPFYERDSLVVCGEHVTTDAGTG CVHTAPGHGEDDFIVGQKYGLPVLCPVDERGYMTEEAPGF AGMFYDEANKAITQKLEEVGALLKLSFITHSYPHDWRTKQ PTIFRATTQWFASIDKIRDQLLDAIKETKWVPEWGEIRIH NMVRDRGDWCISRQRAWGVPIPVFYGENGEPIITDETIEH VSNLFRQYGSNVWFEREAKDLLPEGFTHPSSPNGLFTKET DIMDVWFDSGSSHQAVLVERDDLERPADLYLEGSDQYRGW FNSSLSTAVAVTGKAPYKGVLSHGFVLDGEGRKMSKSLGN VVVPAKVMEQLGADILRLWVASVDYQADVRISDNILKQVS EVYRKIRNTFRFMLGNLFDFDPNQNAVPVGELGEVDRYML AKLNKLIAKVKKAYDSYDFAAVYHEMNHFCTVELSAFYLD MAKDILYIEAADCRARRAVQTVLYETVVALAKLIAPILPH TADEVWEHIPNRKEQVESVQLTDMPESMAIDGEEALLAKW DAFMDVRDDILKALENARNEKVIGKSLTASVTVYPKDEVR ALLASINEDLRQLLIVSAFSVADESYDAAPAEAERLNHVA VIVRPAEGETCERCWTVTPDVGRDESHPTLCPRCAHIVNE HYSA Sequence number 109 DNA LeuRS - GsuLeuRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAGGAGGAGTGCGACGATGAGTTTCAACCATCGCGAAA TTGAGAAAAAGTGGCAGGATTATTGGGAACAGCATAAAAC GTTCCGCACCCCGGATGAAAGCGATAAACCGAAGTTTTAC GTGTTGGATATGTTTCCGTATCCGTCTGGCGCTGGCTTGC ACGTCGGCCATCCGGAAGGGTATACGGCGACTGATATTTT GGCGCGCATGAAGCGGATGCAAGGGTACAATGTCCTTCAC CCGATGGGGTGGGACGCGTTCGGATTGCCGGCAGAACAAT ATGCGCTCGATACCGGCAACGACCCGGCCGAATTTACGCA AAAAAACATCGACAACTTCCGCCGGCAAATTAAGTCGCTT GGTTTTTCGTATGACTGGGATCGGGAAATTAACACGACTG ATCCGAACTATTACAAATGGACGCAATGGATTTTCTTGAA GCTGTATGAAAAAGGGCTCGCCTACATGGACGAAGTACCG GTCAACTGGTGTCCGGCGCTTGGCACCGTGCTGGCGAACG AAGAAGTCATCAACGGCCGGAGCGAGCGCGGTGGGCATCC GGTCATCCGCAAGCCAATGCGGCAATGGATGCTGAAAATT ACCGCCTATGCCGACCGGCTGCTCGAAGATTTGGAGGAGC TTGACTGGCCGGAAAGCATTAAAGAAATGCAACGCAACTG GATCGGCCGTTCGGAAGGAGCGGAAATTGAGTTTGCTGTC GACGGCCATGACGAGTCGTTCACGGTATTTACGACGCGGC CAGATACGCTGTTTGGCGCCACGTACGCAGTGTTGGCTCC GGAACATCCGCTTGTTGAGAAAATTACAACGCCGGAGCAA AAACCAGCCGTTGATGCTTACTTAAAAGAAGTGCAAAGCA AAAGCGACCTCGAGCGCACCGACTTGGCGAAAGAAAAAAC AGGCGTGTTCACTGGTGCGTACGCCATCCATCCAGTTACC GGCGACAAGCTGCCGATTTGGATCGCCGATTACGTGTTGA TGGGCTACGGCACTGGGGCGATCATGGCTGTACCGGCGCA TGATGAGCGCGACTACGAGTTTGCGAAAACATTCAACTTG CCGATCAAAGAAGTCGTTGCCGGCGGGAATGTCGAAAACG AGCCGTACACTGGCGACGGGGAGCACATCAACTCTGAGTT TTTGAACGGCTTGAACAAACAAGAAGCGATCGAAAAAATG ATCGCCTGGCTTGAAGAAAACGGAAAAGGACAAAAGAAAG TGTCGTACCGGCTGCGCGACTGGTTGTTTAGCCGCCAACG CTACTGGGGTGAGCCGATTCCGGTCATCCATTGGGAAGAT GGGACGATGACGACGGTGCCGGAAGAAGAATTGCCGCTTG TCTTGCCGAAAACGGATGAAATTAAACCGTCGGGAACGGG TGAATCGCCGCTCGCCAACATCGAAGAATGGGTCAATGTT GTCGATCCGAAAACCGGGAAAAAAGGGCGGCGTGAAACAA ACACGATGCCGCAATGGGCGGGAAGCTGCTGGTATTATTT GCGCTACATCGACCCGCATAACGACAAACAGCTCGCCGAT CCGGAAAAGTTGAAACAATGGCTGCCGGTTGACGTCTACA TCGGCGGGGCGGAGCATGCGGTCTTGCACTTGCTGTACGC TCGCTTCTGGCATAAAGTGTTGTACGACCTTGGCATCGTG CCGACGAAAGAGCCGTTCCAAAAGCTGTTTAACCAAGGGA TGATCTTAGGCGAAAACAATGAAAAAATGAGCAAATCGAA AGGCAATGTCGTCAACCCGGATGATATCGTCGAGAGCCAT GGCGCGGATACGTTGCGGCTGTATGAAATGTTTATGGGGC CGCTTGAAGCGTCGATCGCCTGGTCGACGAAAGGGCTTGA CGGAGCGCGCCGTTTCTTAGAGCGCGTCTGGCGTCTGTTT GTCACCGAAGATGGTCAACTGAACCCGAACATCGTTGACG AGCCAGCGAACGATACGCTCGAGCGCGTCTACCATCAAAC GGTGAAAAAAGTGACGGAAGACTACGAAGCGCTGCGCTTC AACACCGCCATTTCGCAGCTGATGGTGTTCATTAACGAAG CGTATAAAGCGGAGCAGATGAAAAAAGAATATATGGAAGG GTTCGTCAAGCTCTTATCGCCGGTTTGCCCGCATATTGGC GAAGAGCTCTGGCAAAAGCTCGGCCATACTGACACCATCG CCTATGAACCATGGCCGACATATGACGAAGCGAAACTCGT CGAAGATGTCGTTGAAATCGTGATCCAAATCAACGGCAAA GTGCGGGCGAAACTGAACGTGCCGGCGGACTTATCGAAAG AGGCGCTAGAAGAACGGGCGCTCGCCGATGAAAAAATTAA AGAGCAGCTTGCAGGGAAAACGGTGCGTAAGGTGATCACT GTCCCTGGTAAGCTCGTCAATATCGTCGCCAAC SEQ ID NO: 110 Amino acid LeuRS - GsuLeuRS Geobacillus subterraneus DSM 13552 (91A1 ) MRRSATMSFNHREIEKKWQDYWEQHKTFRTPDESDKPKFY s VLDMFPYPSGAGLHVGHPEGYTATDILARMKRMQGYNVLH PMGWDAFGLPAEQYALDTGNDPAEFTQKNIDNFRRQIKSL GFSYDWDREINTTDPNYYKWTQWIFLKLYEKGLAYMDEVP VNWCPALGTVLANEEVINGRSERGGHPVIRKPMRQWMLKI TAYADRLLEDLEELDWPESIKEMQRNWIGRSEGAEIEFAV DGHDESFTVFTTRPDTLFGATYAVLAPEHPLVEKITTPEQ KPAVDAYLKEVQSKSDLERTDLAKEKTGVFTGAYAIHPVT GDKLPIWIADYVLMGYGTGAIMAVPAHDERDYEFAKTFNL PIKEVVAGGNVENEPYTGDGEHINSEFLNGLNKQEAIEKM IAWLEENGKGQKKVSYRLRDWLFSRQRYWGEPIPVIHWED GTMTTVPEEELPLVLPKTDEIKPSGTGESPLANIEEWVNV VDPKTGKKGRRETNTMPQWAGSCWYYLRYIDPHNDKQLAD PEKLKQWLPVDVYIGGAEHAVLHLLYYARFWHKVLYDLGIV PTKEPFQKLFNQGMILGENNEKMSKSKGNVVNPDDIVESH GADTLRLYEMFMGPLEASIAWSTKGLDGARRFLERVWRLF VTEDGQLNPNIVDEPANDTLERVYHQTVKKVTEDYEALRF NTAISQLMVFINEAYKAEQMKKEYMEGFVKLLSPVCPHIG EELWQKLGHTDTTIAYEPWPTYDEAKLVEDVVEIVIQINGK VRAKLNVPADLSKEALEERALADEKIKEQLAGKTVRKVIT VPGKLVLIVAN sequence number 111 DNA LysRS-GsuLysRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAGCCATGAAGAATTGAACGACCAATTGCGTGTCCGCC GGGAAAAGTTAAAAAAAATCGAAGAGCTAGGTGTCGACCC GTTTGGCAAACGGTTCGAGCGCACGCATAAAGCAGAAGAG CTGTTTAAACTGTACGGCGATTTGTCCAAAGAAGAACTTG AAGATCAGCAAATTGAAGTCGCTGTCGCCGGCCGCATTAT GACGAAACGCGGTAAAGGAAAAGCAGGATTTGCTCACATT CAAGACGTCACAGGGCAAATTCAAATTTATGTCCGCCAAG ACGATGTCGGTGAACAGCAATATGAGCTGTTTAAAATCTC TGACCTTGGTGATATCGTCGGTGTGCGCGGCACTATGTTC AAAACAAAAGTCGGCGAGCTTTCCATCAAAGTGTCATCAT ATGAATTTTTAACAAAAGCATTGCGTCCATTGCCGGAAAA ATACCATGGTTTAAAGGACGTCGAACAACGTTACCGCCAA CGTTATCTCGACTTAACTATGAATCCGCAAAGTAAGCAGA CGTTTATCACCCGTAGTCTCATTATTCAATCGATGCGGCG TTATCTCGACAGCCAAGGTTATTTGGAAGTCGAAACACCG ATGATGCACGCCATAGCAGGTGGTGCGGCTGCACGTCCGT TTATTACGCACCATAATGCCCTTGATATGACACTTTATAT GCGAATCGCCATCGAACTCCATTTAAAACGGCTCATCGTC GGCGGTTTGGAAAAAGTGTATGAAATCGGACGCGTCTTCC GGAATGAGGGGATTTCCACCCGTCACAATCCGGAGTTTAC GATGCTTGAACTGTACGAGGCATATGCCGACTTCCGTGAC ATCATGAAATTGACAGAAAACTTAATTGCTCACATTGCCA CGGAAGTGCTTGGCACGACGAAAATTCAATACGGCGAACA TACCGTCGATTTAACGCCTGAATGGCGGCGACTTCATATG GTCGATGCGATTAAAGAATACGTCGGCGTTGATTTCTGGC GGCACATGGACGACGAGGAAGCGCGGGCGTTGGCGAAAGA ACATGGGGTCGAAATCGCCCCGCACATGACGTTTGGTCAT ATCGTCAATGAATTTTTTGAACAAAAAGTCGAGTCGCAAC TCATCCAACCGACGTTCATTTATGGCCACCCTGTCGAAAT TTCGCCGTTAGCTAAGAAAAACCCGGACGATCCACGCTTT ACCGATCGATTTGAGCTATTTATCGTTGGACGTGAACATG CGAACGCGTTTACGGAACTAAACGATCCGATCGACCAGCG CCAACGTTTCGAAGCACAGTTGAAAGAACGTGAACAAGGG AACGATGAAGCGCACGAAATGGACGAAGATTTCCTCGAAG CGCTCGAGTACGGTATGCCTCCAACAGGCGGACTCGGCAT CGGCGTTGACCGTCTAGTCATGCTCTTGACTAACTCTCCG TCCATTCGGGATGTGTTACTCTTCCCGCAAATGCGTCATA AA SEQ ID NO: 112 It should be noted that there seems to be a minor error in the original text where "配列番号" is misspelled as "配列番號" in ID=39. This has been corrected in the translation. Also, the "" tags are retained as they are part of the original text's formatting. If there are any further specific requirements or corrections related to this translation, please let me know. amino acid LysRS-GsuLysRS Geobacillus subterraneus DSM 13552 (91A1 ) MSHEELNDQLRVRREKLKKIEELGVDPFGKRFERTHKAEE LFKLYGDLSKEELEDQQIEVAVAGRIMTKRGKGKAGFAHI QDVTGQIQIYVRQDDVGEQQYELFKISDLGDIVGVRGTMF KTKVGELSIKVSSYEFLTKALRPLPPEKYHGLKDVEQRYRQ RYLDLTMNPQSKQTFITRSLIIQSMRRYLDSQGYLEVETP MMHAIAGGAAARPFITHHNALDMTLYMRIAIELHLKRLIV GGLEKVYEIGRVFRNEGISTRHNPEFTMLELYEAYADFRD IMKLTENLIAHIATEVLGTTKIQYGEHTVDLTPEWRRLHM VDAIKEYVGVDFWRHMDDEEARALAKEHGVEIAPHMTFGH IVNEFFEQKVESQLIQPTFIYGHPVEISPLAKKNPDDPRF TDRFELFIVGREHANAFTELNDPIDQRQRFEAQLKEREQG NDEAHEMDEDFLEALEYGMPPTGGLGIGVDRLVMLLTNSP SIRDVLLFPQMRHK sequence number 113 DNA MetRS-GsuMetRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGGAGAAAAAGACGTTTTATTTGACGACGCCGATTTATT ATCCGAGCGACAAATTGCACATCGGCCATGCTTATACAAC AGTGGCGGGGATACGCTAGCGCGCTATAAACGGATGCGC GGTTACGATGTTATGTATTTGACGGGAACCGATGAGCACG GGCAAAAAAATTCAACGCAAGGCGGAGGAAAAGGAGTAAC GCCGCAGCAATATGTCGATGAGATCGTCGCTGGCATTCAG GAGCTATGGAAAAAGCTCGACATTTCTTATGACGATTTCA TCCGTACAACGCAGGAGCGGCATAAAAAAGTAGTCGAAAAA GATTTTCGCGCGTCTTGTCGAACAAGGGGATATTATTTA GGTGAATATGAAGGATGGTATTGCACGCCATGCGAATCGT TTTACACTGAGCGACAGCTTGTCGACGGCAACTGCCCGGA CTGTGGTCGTCCGGTTGAAAAAGTGAAAGAGCAGTCGTAC TTTTTCCGAATGAGCAAATACGTCGACCGTTTGCTTCAAT ATTATGAGGAAATCCAGATTTCATCCAGCCGGAATCGCG GAAAAAACGAAATGATTAACAATTTTATTAAGCCGGGGCTT GAAGATTTAGCTGTGTCGCGGACGACGTTTGACTGGGGCA TTAAAGTGCCGGGCGATCCGAAACATGTCATTTACGTCTG GATTGACGCGCTTGCCAACTATATTACAGCGCTCGGTTAC GGCACGGACAATGATGAAAAGTTCCGCAAATATTGGCCGG CCGATGTCCATTTAGTCGGCAAGGAAATCATCCGCTTTCA TACGATTTATTGGCCGATTATGCTCATGGCGCTTGACTTG CCGCTGCCGAAAAAAGTATTCGGTCATGGCTGGCTGCTCA TGAAAGACGGGAAAATGTCGAAATCGAAAGGCAATGTCGT TGACCCGGTGACGTTGATCGATCGATACGGACTCGATGCG CTTCGTTATTATTTACTCAGGGAAGTGCCGTTCGGTTCTG ACGGCGTATTCACGCCGGAAGGATTTATTGAGCGCATCAA CTACGATTTAGCCAATGACCTAGGCAATTTATTGAATCGT ACAGTAGCGATGATTAAGAAATATTTTGATGGGGTGATTC CGCCGTACCGCGGTCCGAAAACGCCGTTTGACGAAGAGCT GGTACAAACGGCGCGTGAGGTGGTCCGTCAGTATGAGGAA GCGATGGAACGGATGGAGTTTTCCGTTGCCCTTGCTTCGG TTTGGCAACTGATTGGCCGGACGAACAAATACATTGATGA GACGCAGCCATGGGTATTGGCCAAAGATGAAAGCAAACGG GAAGAGCTTGCTTCTGTCATGACCCACCTAGCCGAGTCGC TCCGCCATACGGCAGTGCTGTTGCAGCCGTTTTTGACACG CACGCCAGAGCGCATTTTTGCCCAGCTCGGCATTGCCGAC CGTTCATTAAAAGAGTGGGATAGCTTGTACGAGTTCGGGC TCATTCCGGAAGGAACAAACGTGCAAAAAGGAGAACCACT GTTCCCGCGCCTTGATATTGAAGCGGAAGTCGAGTACATT AAGGCGCATATGCAAGGCGGCAAGCCGCGGTGGAACCCG TTAAAGAGGAGAAGCAAGCGGCTGAGACGGCCGAAATCTC AATTGATGAGTTTGCCAAAGTTGACTTGCGCGTTGCTGAA GTCGTGCATGCTGAACGGAATGAAAACGCCAATAAGCTGT TGAAGCTCCAACTTGATCTTGGCGGCGAGAAACGGCAAGT CATCTCTGGTATCGCTGAATTTTTACAAACCAGAGGAACTC ATCGGCAAAAAGGTCATTTGCGTCGCCAAATTTAAAACCGG CCAAACTGCGCGGTGAGTGGTCGGAAGGAATGATTTTGGC CGGCGGTAACGGCGGAGAGTTTTCACTGGCGACCGTCGAT CAACATGTGCCAAACGGAACAAAAATTAAA sequence number 114 amino acid MetRS-GsuMetRS Geobacillus subterraneus DSM 13552 (91A1 ) MEKKTFYLTTPIYYPSDKLHIGHAYTTVAGDTLARYKRMR GYDVMYLTGTDEHGQKIQRKAEEKGVTPQQYVDEIVAGIQ ELWKKLDISYDDFIRTQERHKKVVEKIFARLVEQGDIYL GEYEGWYCTPCESFYTERQLVDGNCPDCGRPVEKVKEQSY FFRMSKYVDRLLQYYEENPDFIQPESRKNEMINNFIKPGL EDLAVSRTTDFWGIKVPGDPKHVIYVWIDALANYITALGY GTDNDEKFRKYWPADVHLVGKEIIRFHTIYWPIMLMALDL PLPKKVFGHGWLLMKDGKMSKSKGNVVDPVTLIDRYGLDA LRYYLLREVPFGSDGVFTPEGFIERINYDLANDLGNLLNR TVAMIKKYFDGVIPPYRGPKTPFDEELVQTAREVVRQYEE AMERMEFSVALASVWQLIGRTNKYIDETQPWVLAKDESKR EELASVMTHLAESLRHTAVLLQPFLTRTPERIFAQLGIAD RSLKEWDSLYEFGLIPEGTNVQKGEPLFPRLDIEAEVEYI KAHMQGGKPAVEPVKEEKQAAETAEISIDEFAKVDLRVAE VVHAERMKNANKLLKLQLDLGGEKRQVISGIAEFYKPEEL IGKKVICVANLKPAKLRGEWSEGMILAGGNGEFSLATVD QHVPNGTKIK sequence number 115 DNA Phe-aRS-GsuPhe-aRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAGGGACGGGTTTTTTATTTTGTTAGAGGAGGATTG GCGTGAAAGAACGGTTGCATGAGCTTGAACGAGAAGCGCT TGAAAAAATTGAACAAGCTGGCGATTTAAAAGCGCTCAAC GATGTGCGTGTCGCCTATTTAGGAAAAAAGGGCCGATTA CCGAAGTGCTGCGCGGCATGGGAGCATTGCCGTCAGAAGA GCGTCCGAAAATTGGTGCGCTTGCCAATGAGGTAAGAGAG GCGATCCAAAAGGCGCTCGAAGCAAAACAAACGAAACTGG AAGAAGAAGAAGTCGAGCGGAAGTTGGCGGCTGAAGCGAT CGATGTGACGCTTCCGGGCCGTCCGGTGAAACTGGGGAAT CCTCATCCGCTGACGCGCGTCATCGAGGAAATTGAAGATT TGTTTATCGGCATGGGCTATACGGTCGCCGAAGGTCCGGA AGTCGAGACCGATTATTACAATTTTGAGGCGCTCAATTTG CCGAAAGGACACCCGGCCCGCGATATGCAAGATTCGTTTT ATATTACGGAAGAAATTCTGCTTCGCACCCACACGTCGCC GATGCAGGCACGGACGATGGAAAAACATCGCGGGCGCGGT CCGGTAAAAATCATTTGCCCGGGGAAAGTGTATCGCCGCG ATACCGATGATGCGACCCATTCACATCAGTTTACGCAAAT TGAAGGATTGGTTGTTGACCGCAACATCCGGATGAGCGAT TTAAAAGGGACGCTGCGCGAATTTGCCCGCAAGCTGTTCG GTGAAGGGCGCGACATCCGTTTTCGTCCGAGCTTTTTCCC GTTTACCGAGCCTTCAGTCGAGGTCGATGTGTCCTGCTTC CGCTGCGAAGGGCACGGCTGCAGCGTTTGCAAAGGTACGG GCTGGATTGAAATTTTAGGCGCTGGCATGGTGCACCCGAA CGTGCTTGAGATGGCCGGCTTTGATTCGAAAACGTATACC GGATTTGCGTTCGGCATGGGGCCGGAGCGGATCGCGATGT TGAAATACGGCATTGATGACATCCGCCATTTCTATCAGAA CGATCTTCGTTTCTTGCAACAATTTTTGCGTGTC sequence number 116 amino acid Phe-aRS-GsuPhe-aRS Geobacillus subterraneus DSM 13552 (91A1 ) MRDGFFYVRGGIGVKERLHELEREALEKIEQAGDLKALN DVRVAYLGKKGPITEVLRGMGALPSEERPKIGALANEVRE AIQKALEAKQTKLEEEEVERKLAAEAIDVTLPGRPVKLGN PHPLTRVIEEIEDLFIGMGYTVAEGPEVETDYYNFEALNL PKGHPARDMQDSFYITEEILLRTHTSPMQARTMEKHRGRG PVKIICPGKVYRRDTDDATHSHQFTQIEGLVVDRNIRMSD LKGTLREFARKLFGEGRDRIRFRPSFFPFTEPSVEVDVSCF RCEGHGCSVCKGTGWIEILGAGMVHPNVLEMAGFDSKTYT GFAFGMGPERIAMLKYGIDDIRHFYQNDLRFLQQFLRV sequence number 117 DNA Phe-bRS-GsuPhe-bRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGCTCGTTTCTTATCGTTGGCTAGGCGAATACGTCGATT TGACGGGCGTGACGGCGGAACAACTCGCTGATCGCATTAC AAAAAGCGGCATTGAAGTCGAGCGGGTTGAAGCGCTTGAG CGGGGAATGAAAGGAGTCGTCATCGGCCATGTGCTCGAAT GCGAGCCACACCCAAACGCCGATAAACTGCGGAAATGTCT TGTTGATCTTGGCGAAGGAGAGCCGGTGCAAATCATTTGC GGTGCCCCGAACGTCGCCAAGGGGCAAAAAGTTGCTGTAG CGAAAGTTGGAGCGAGACTGCCGGGCAATTTTAAAATCAA ACGGGCGAAGCTGCGCGGCGAAGAGTCGAACGGCATGATT TGCTCGCTCCAAGAACTCGGTGTTGAAACAAAAGTCGTGC CGAAAGAATACGCCGAAGGCATTTTCGTCTTCCCAAGCGA CGCGCCGGTCGGCGCTGATGCGCTTGAATGGCTCGGCTTG CACGATGAAGTGCTCGAACTCGCCTTGACGCCGAATCGCG CCGATTGCTTAAGCATGCTTGGCGTTGCCTACGAAGTCGC TGCGATTCTCGGCCGCGATGTGAAGTTGCCGGAAACGGCG GTGAACGAAAATGAAGAAAGCGTCCATGACTACATTTCTG TCCGTGTCGAGGCGCCGGAAGACAATCCGCTGTACGCCGG ACGGATCGTGAAAAACGTCCAAATCGGCCCGTCGCCGCTT TGGATGCAAGCGCGCTTGATGGCGGCCGGCATTCGTCCAC ACAACAATGTTGTCGATATCACCAACTACATTTTGCTTGA GTACGGCCAGCCGCTTCACGCGTTTGACTACGACCGTCTC GGTTCGAAGGAGATCGTCGTTCGTCGTGCCAAGGCGGGAG AAATGATCGTGACGCTTGACGATGTCGAGCGGAAGCTGAC TGAAGATCATCTCGTCATCACAAACGGCCGTGAGCCGGTC GCCTTAGCCGGTGTGATGGGCGGAGCGAACTCGGAAGTGC AGGATGACACGAAAACAGTGTTCATCGAAGCCGCGTATTT TACGAGCCCGGTCATCCGCCAGGCGGTGAAAGACCACGGG TTGCCGCAGCGAAGCGAGCACCCGGTTTGAAAAAGGGATTG ATCCGGCGCGGACGAAAGAAGCGCTCGAGCGCGCTGCTGC TTTGATGGCAGAATACGCCGGCGGCGAGGTCGTCAGCGGT ATCGTGGAAGCTAATACATGGAAAGAAGAGCCGGTTGTCG TAACGGTGGCGCTGGAACGCATCAACGGCGTCCTCCGGCAC AGCGATGACGAAAGAGGAAGTAGCTGGCATTCTTTCAAAC TTGCAATTCTCGTTTACGGAAGATAATGGAACGTTTACAA TCCATGTTCCATCGCGCCGCCGCGATATTACGATCGAAGA AGATATTATCGAGGAAGTCGCCCGTTTGTATGGCTACGAC CATTTGCCAGCGACTTTGCCGGTGGCCGAAGCAAAACCGG GCGAGTTGACACCGTACCAAGCGAAACGCCGCCGTGTCCG CCGCTATTTCGAAGGCGCGGGCTTGTTCCAGGCGATCACG TATTCGCTTACCAGTCCGGACAAAGCGACGCGGTTTGCTT TGGAGACAACCGAACCAGTCCGCTTGGCGTTGCCGATGAG TGAGGAGCGGAGCGTTCTCCGGCAAAGCTTGGTGCCGCAT TTGCTCGAAGCGGCGAGCTACAACCGTGCCCGCCAAGTTG AGAACGTCGCGCTATATGAAATCGGCTCTGTCTATTTGTC CAAGGGGGAAAATGTCCAACCGGCGGAAAAGAACGGCTC GCCGGCGTCATCACCGGTTTATGGCATGCCCACCTTTGGC AAGGAGAGAAAAAAGCAGCTGATTTCTATGTTGCAAAAGG CGTGCTTGACGGCTTGTTCGCCCTGCTTGGGCTGTCTGAT CGCATCAGCTACCGTCCGGCGAAGCGTGCTGATTTGCATC TGGGGCGGACAGCGGAGATTGTGCTTGACGGCAAAGAGAT CGGCTTTGTCGGCCAGCTCCATCCGGCTGTCAAAAAAGAG TACGATTTGAAAAAGGTATGTCTTTGAACTCGCCTTCG CTGAGCTACTGAATACAGAAGGCGAAACGATCCGTTACGA GTCGATTCCGCGCTTCCCGTCAGTCGTGCGCGACATCGCT TTAGTCGTCGACGACAATGTCGAAGCAGGTGCTCTCAAGC AGGCGATCGCCGAAGCGGGGAACCCGCTATTAAAGACGT GGCCCTCTTTGACGTCTATAAAGGCGACCGTCTGCCGGCC GGGAAAAAATCGCTCGCCTTCTCGCTCCGCTACTACGATC CGGAACGGACGCTCACTGATGAGGAAGTTACTGCCGTCCA TGAACGGGTTTTGGCAGGCGGTCGAGGAGCAGTTTGGCGCG GTGTTGCGCGGG sequence number 118 amino acid Phe-bRS-GsuPhe-bRS Geobacillus subterraneus DSM 13552 (91A1 ) MLVSYRWLGEYVDLTGVTAEQLADRITKSGIEVERVEALE RGMKGVVIGHVLECEPHPNADKLRKCLVDLGEGEPVQIIC GAPNVAKGQKVAVAKVGARLPGNFKIKRAKLRGEESNGMI CSLQELGVETKVVPKEYAEGIFVFPSDAPVGADALEWLGL HDEVLELALTPNRADCLSMLGVAYEVAAIlgRDVKLPETA VNENEESVHDYISVRVEAPEDNPLYAGRIVKNVQIGPSPL WMQARLMAAGIRPHNNVVDITNYILLEYGQPLHAFDYDRL GSKEIVVRRAKAGEMIVTLDDVERKLTEDHLVITNGREPV ALAGVMGGANSEVQDDTKTVFIEAAYFTSPVIRQAVKDHG LRSEASTRFEKGIDPARTKEALERAAALMAEYAGGEVVSG IVEANTWKEEPVVVTVALERINGVLGTAMTKEEVAGILSN LQFSFTEDNGTFTIHVPSRRRDITIEDIIEEVARLYGYD HLPATLPVAEAKPGELTPYQAKRRRVRRYFEGAGLFQAIT YSLTSPDKATRFALETTEPVRLALPMSEERSVLRQSLVPH LLEAASYNRARQVENVALYEIGSVYLSKGENVQPAEKERL AGVITGLWHAHLWQGEKKAADFYVAKGVLDGLFALLGLSD RICE PAKRADLHLGRTAEIVLDGKEIGFVGQLHPAVQKE YDLKETYVFELAFAELLNTEGETIRYESIPRFPSVVRDIA LVVDDNVEAGALKQAIAEAGNPLLKDVALFDVYKGDRLPA GKKSLAFSLRYYDPERTLTDEEVTAVHERVLAAVEEQFGA VLRG sequence number 119 DNA ProRS-GsuProRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGACATTCAAAAATTCTTCCTATAATGAAAGAGAAAAA CGAGGTGGCTATTGATGAGACAAAGTCAAGGGTTTATTCC GACATTGCGCGAAGTGCCGGCGGACGCGGAAGTGAAAAGC CATCAGCTCCTGTTGCGGCCGGCTTCGTCCGCCAAAGCG CAAGCGGCGTCTACACGTTTTTGCCGCTCGGGCAACGTGT TTTGCAAAAAGTGGAAGCGATTATTCGTGAGGAGAATGAAT CGCGCCGGAGCATTGGAGCTTCTCATGCCTGCTTTGCAGC CGGCTGAGCTTTGGCAGCAGTCCGGGCGCTGGTATTCGTA TGGACCGGAGCTCATGCGCCTGAAAGACCGTCACGAGCGC GATTTCGTTCTCGGACCGACACACGAAGAGATGATTACTA CGATCGTTCGCGATGAAGTGAAAACGTATAAGCGGCTGCC GCTTATCTTGTATCAAATTCAAACGAAATTCCGTGATGAA AAACGTCCGCGTTTCGGGCTGTTGCGCGGTCGCGAGTTCA TCATGAAAGATGCGTATTCATTCCACACATCGCAGGAAAG TTTGGACGAAACGTACAATAAAATGTATGAAGCGTACGCG AACATTTTCCGCCGCTGCGGCTTAAATTTCCGCGCTGTCA TTGCTGACTCCGGAGCGATGGGCGGCAAAGATACGCACGA GTTTATGGTGCTGTCTGATATTGGCGAGGATACGATCGCT TATTCCGATGCGTCCGACTATGCGGCCAACATTGAAATGG CACCGGTCGTCACTACGTATGAAAAAAGCAGTGAGCCGCT GGTGGAACTGAAAAAAGTGGCGACCCCGGAGCAAAAAACG ATTGCTGAAGTTGCTTCGTATTTGCAAGTAGCACCGGAAC GTTGCATTAAATCGCTTTTATTTAACGTTGATGGCCGCTA CGTGCTCGTTCTGGTGCGCGGCGATCATGAAGCGAATGAT GTGAAAGTGAAAAATGTGCTTGATGCGACTGTCGTGGAGC TGGCGACACCGGAAGAAACAGCACGAGTGATGAACTGCCC GGTTGGTTCGCTCGGCCCGATTGGCGTCAGCGAAGAGGTG ACGATTATCGCCGATCATGCTGTCGCGGCGATCGTAAACG GCGTCTGCGGCGCCAATGAGGAAGGATACCATTATACGGG TGTCAATCCAGACCGCGATTTTGCCGTCAGTCAATATGCG GATTTGCGTTTCGTCCAAGAAGGCGACCCTTCTCCGGATG GCAACGGGACGATCCGCTTCGCTCGTGGCATTGAAGTTGG ACATGTGTTTAAGCTCGGTACGAAATATAGCGAGGCGATG AACGCCGTTTACCTCGACGAAAATGGTCGGACACAGACGA TGATTATGGGTTGCTACGGCATTGGCGTCTCTAGGCTCGT TGCGGCGATCGCCGAGCAGTTCGCCGATGAGAACGGGCTT GTATGGCCGGTTTCGGTCGCACCGTTTCACGTTCATTTGC TGACGGCGAACGCGAAAAGCGATGAACAGCGCATGCTGGC TGAAGAGTGGTACGAAAAACTCGGACAGGCCGGATTTGAC GTGTTGTATGATGACCGTCCGGAACGGGCCGGGGTGAAGT TTGCCGACAGCGATTTGATCGGCATCCCGCTCCGCGTCAC CGTTGGCAAGCGGGCAAGTGAAGGTGTGGTCGAAGTAAAA GTTCGGAAAACAGGCGAGACGTTTGACGTGCCGGTCGGTG AGCTGATCGAAACAGTGCGCCGTCTTTTGCAAGGA sequence number 120 amino acid ProRS-GsuProRSt Geobacillus subterraneus DSM 13552 (91A1 ) MTFKNSSYNEREKTRWLLMRQSQGFIPTLREVPADAEVKS HQLLLRAGFVRQSASGVYTFLPLGQRVLQKVEAIIREEMN RAGALELLMPALQPAELWQQSGRWYSYGPELMRLKDRHER DFVLGPTHEEMITTIVRDEVKTYKRLPLILYQIQTKFRDE KRPRFGLLRGREFIMKDAYSFHTSQESLDETYNKMYEAYA NIFRRCGLNFRAVIADSGAMGGKDTHEFMVLSDIGEDTIA YSDASDYAANIEMAPVVTTYEKSEPLVELKKVATPEQKT IAEVASYLQVAPERCIKSLLFNVDGRYVLVLVRGDHEAND VKVKNVLDATVVELATPEETARVMNCPVGSLGPIGVSEEV TIIADHAVAAIVNGVCGANEEGHYHYTGVNPDRDFAVSQYA DLRFVQEGDPSPDGNGTIRFARGIEVGHVFKLGTKYSEAM NAVYLDENGRTQTMIMGCYGIGVSRLVAAIAEQFADENGL VWPVSVAPFHVHLLTANAKSDEQRMLAEEWYEKLGQAGFD VLYDDRPERAGVKFADSDLIGIPLRVTVGKRASEGVVEVK VRKTGETFDVPVGELIETVRRLLQG sequence number 121 DNA SerRS-GsuSerRS Geobacillus subterraneus DSM 13552 (91A1). ) ATGGTGGTAAGGAGGTAAAGCGAATGCTGGATGTGAAAT TACTACGCACCCAATTTCAAGAGGTGAAAGAAAAACTGCT GCAGCGCGGCGACGACTTGGCCAACATCGACCGGTTTGAG CAGCTTGATAAAGAGCGTCGTCGTTTGATCGCTCAGGTGG AGGATTAAAAAGCAAGCGCAATGAGGTGTCGCAACAAAT TGCTGTCTTAAAGCGTGAAAAAAAGGACGCCGAGTCGTTG ATCGTCGAAATGCGCGAAGTCGGCGACCGCATTAAACAAA TGGACGAGCAAATTCGCCAACTTGAAAGAGCTCGACAG CCTTCTGTTATCGATTCCGAATGTACCGCATGAGTCAGTG CCAGTCGGTCAGTCGGAAAGATAATGTCGAAGTGCGAA GATGGGGGGAACCGCGTTCGTTCTCGTTCGAACCGAAGCC ACATTGGGACATTGCTGACCAACTCGGTTTGCTCGATTTT GAGCGGGCTGCCAAAGTGGCAGGAAGTCGGTTTGTGTTTT ACAAAGGACTAGGGGCCTCGTCTTGAGCGGGCATTAATCAA CTTTATGCTCGACATCCATCTCGATGAATTTGGCTATCAA GAGGTGTTGCCGCCATACTTAGTGAACCGGGCGAGCATGA TCGGAACAGGGCAATTGCCAAAATTTGCGGAAGGCCGTT CCACTTGGACAATGAAGACTATTTTCTCATTCCAACAGCG GAAGTGCCTGTGACGAATTTGCATCGCGATGAAATTTTAA CGGCTGATGACTTGCCGCTTTACTATGCGGCTTACAGCGC GTGCTTCCGCGCCGAAGCTGGCTCGGCTGGCCGTGACACG CGGGGGCTCATCCGCCAGCACCAATTCAATAAAGTGGAGC TCGTCAAGTTCGTCAAGCCGGAGGATTCATATGACGAGTT GGAAAAATTGACGCACCAAGCCGAAACGATCCTGCAACGG CTCGGACTTCCGTATCGCGTCGTAGCCTTGTGTACAGGGG ATCTGGGATTTTCAGCGGCGAAGACGTATGATATTGAGGT GTGGCTGCCAAGCTATGGAACGTATCGGGAAATTTCGTCG TGCAGCAACTTTGAGGCGTTCCAGGCGCGCCGAGCTAATA TCCGCTTCCGTCGCGAGCCGAAAGCAAAGCCAGAATATGT GCATACGCTAAACGGTTCGGGGCTAGCCATCGGCCGCACG GTTGCTGCCATTTTGGAAAACTACCAACAAGAAGACGGAT CGGTCGTCATCCCGGAAGCGCTCCGTCCATATATGGGGAA TCGGGATGTCATTCGC SEQ ID NO: 122 Amino acid SerRS - GsuSerRS Geobacillus subterraneus DSM 13552 (91A1 ) MVDKEVKRMLDVKLLRTQFQEVKEKLQRGDDLANIDRFE QLDKERRRLIAQVEELKSKRNEVSQQIAVLKREKKDAESL IVEMREVGDRIKQMDEQIRQLEEELDSLLLSIPNVPHESV PVGQSEEDNVEVRRWGEPRSFSFEPKPHWDIADQLGLLDF ERAAKVAGSRFVFYKGLGARLERALINFMLDIHLDEFGYQ EVLPPYLVNRASMIGTGQLPKFAEDAFHLDNEDYFLIPTA EVPVTNLHRDEILTADDLPLYYAAYSACFRAEAGSAGRDT RGLIRQHQFNKVELVKFVKPEDSYDEELEKLTHQAETILQR LGLPYRVVALCTGDLGFSAAKTYDIEVWLPSYGTYREISS CSNFEAFQARRANIRFRREPKAKPEYVHTLNGSGLAIGRT VAAILENYQQEDGSVVIPEALRPYMGNRDVIR sequence number 123 DNA ThrRS-GsuThrRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGCCAGACGTTATTCGCATTACGTTCCCGGACGGGGCGA AAAAGGAGTTCCGAGCGGAACGTCAACTGAGGACATCGC TGCCTCGATCAGTCCGGGATTGAAGAAAAAGCGATTGCC GGGAAACTGAACGGCCGGTTTGTTGATTTACGCACGCCGC TTCAAGAAGACGGCGAGCTTGTCATTATTACCCAGGACAT GCCTGAGGCACTTGATATTTTGCGTCATAGCACCGCCCAT TTAATGGCGCAAGCGATCAAGCGGCTGTATGACAACGTCA AGCTTGGCGTCGGCCCGGTCATTGAAAACGGCTTCTACTA TGATATTGATATGGAACATAAGCTGACGCCGGATGATTTG CCGAAAATTGAGGCGGAAATGCGCAAAATCGTAAAGGAAA ATCTTGACGTTGTTCGCAAAGAGGTGAGCCGTGACGAGGC GATTCGCCTGTATGAAAAAATTGGTGATCACTTGAAACTG GAGCTCATCAACGATATTCCGGAAGGCGAGACGATTTCCA TTTACGAGCAAGGCGAGTTTTTCGATCTTTGTCGGGGTGT GCACGTGCCGTCGACCGGGAAAATCAAAGAGTTCAAGCTG CTCAGCATCTCGGGGGCCTACTGGCGCGGTGACAGCAACA ACAAAATGCTGCAGCGTATTTACGGTACGGCGTTTTTCAA AAAAGAAGATCTGGACCATTATTTGCAGTTGCTCGAAGAG GCGAAAGAGCGCGATCATCGCAAATTGGGCAAAGAGCTTG AGCTATTTACGACATCACAAAAAGTCGGACAAGGACTGCC GCTTTGGTTGCCGAAAGGGGCGACGATCCGTCGCTTGATT GAACGGTACATTGTCGATAAAGAAATCGCCCTTGGTTATG ATCATGTATATACGCCGGTGCTCGGCAGTGTGGAGCTGTA TAAAACCTCAGGACACTGGGACCATTATAAAGAAAACATG TTCCCACCGATGGAAATGGATAACGAAGAGCTCGTGCTGC GGCCGATGAACTGCCCGCACCATATGATGATTTATAAAAG CAAGCTTCATAGCTACCGTGAGCTGCCGATCCGCATCGCC GAGCTCGGCACGATGCATCGCTACGAAATGTCCGGGGCGC TTACTGGACTGCAGCGTGTCCGCGGCATGACGCTCAACGA CGCCCATATTTTCGTGCGCCCGGATCAAATTAAAGACGAG TTTAAGCGCGTCGTTAATTTGATTTTGGAAGTATACAAAG ACTTTGGGCTGGACGAATATTCGTTCCGCCTGTCGTACCG CGACCCACAAGATAAAGAAAAATATTACGACGACGACGAG ATGTGGGAAAAGGCGCAACGCATGCTGCGCGAGGCGATGG ATGAACTTGGCCTCGATTACTACGAAGCGGAAGGGGAAGC AGCGTTTTACGGACCGAAGCTCGATGTGCAAGTGCGCACG GCACTCGGCAAAGATGAGACGCTGTCGACTGTACAGCTTG ACTTCCTCTTGCCGGAGCGGTTTGACTTAACATATATCGG CGAAGATGGAAAACCGCACCGCCCGGTCGTCATCCACCGC GGCGTTGTTTCCACGATGGAACGGTTTGTCGCCTTCTTGA TCGAAGAATACAAAGGGGCATTTCCAACGTGGCTCGCCCC GGTGCAAGTGGAAGTCATCCCGGTATCGTCGGAAGCCCAT CTCGATTATGCGTATGAAGTGAAACAAGCGCTGCAAGTAA ACGGCTTCCGCGTCGAAGTCGACGAACGGGATGAAAAAAT CGGCTATAAAATCCGCGAAGCGCAAATGCAAAAAATTCCT TATATGCTCGTTGTCGGCGACAAAGAAGCGGCCGAGCGAG CGGTCAACGTCCGCCGCTACGGTGAAAAAGAAAGCGAGAC TGTGGCGCTTGACAAGTTTATCGCGATGCTAGAAGAAGAT GTGCGGCAAAAACGAGTGAAAAAACGA SEQ ID NO: 124 amino acid ThrRS-GsuThrRS Geobacillus subterraneus DSM 13552 (91A1 ) MPDVIRITFPDGAKKEFPSGTSTEDIAASISPGLKKKAIA GKLNGRFVDLRTPLQEDGELVIITQDMPEALDILRHSTAH LMAQAIKRLYDNVKLGVGPVIENGFYYDIDMEHKLTPDDL PKIEAEMRKIVKENLDVVRKEVSRDEAIRLYEKIGDHLKL ELINDIPEGETISIYEQGEFFDLCRGVHVPSTGKIKEFKL LSISGAYWRGDSNNKMLQRIYGTAFFKKEDLDHYLQLLEE AKERDHRKLGKELELFTTSQKVGQGLPLWLPKGATIRRLI ERYIVDKEIALGYDHVYTPVLGSVELYKTSGHWDHYKENM FPPMEMDNEELVLRPMNCPHHMMIYKSKLHSYRELPIRIA ELGTMHRYEMSGALTGLQRVRGMTLNDAHIFVRPDQIKDE FKRVVNLILEVYKDFGLDEYSFRLSYRDPQDKEKYYDDDE MWEKAQRMLREAMDELGLDYYEAEGEAAFYGPKLDVQVRT ALGKDETLSTVQLDFLLPERFDLTYIGEDGKPHRPVVIHR GVVSTMERFVAFLIEEYKGAFPTWLAPVQVEVIPVSSEAH LDYAYEVKQALQVNGFRVEVDERDEKIGYKIREAQMQKIP YMLVVGDKEAAERAVNVRRYGEKESETVALDKFIAMLEED VRQKRVKKR sequence number 125 DNA TrpRS-GsuTrpRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAAAACCATTTTTCTGGCATTCAGCCAAGCGGCGTCCA TTACCCTTGGCAACTACATTGGTGCGATGCGACAATTTGT CGAACTGCAGCATGAGTACAACTGCTATTTTTGCATTGTC GACCAACATGCCATTACTGTTCCGCAAAAATCCGAACGAAC TGCAACAAACATTCGCCGTCTCGCTGCCTTATATTGGC AGTCGGCATCGATCCTAAACAGGCGACGCTGTTCGTTCAA TCGGAGGTGCCGGCGCACGCCCAAGCGGCTTGGATGCTGC AATGCATCGTCTATATCGGCGAACTGGAGCGGATGACGCA GTTTAAAGACAAATCAGCCCGGTAAAAGGCGGTCAGTGCC GGGTTGCTCACGTATCCACCGCTTATGGCAGCCGACATTT TGCTTTACAACACGGACATTGTCCCAGTCGGCGAAGACCA AAAGCAGCACATCGAGCTGACGCGCGATTTAGCTGAGCGC TTCAACAAACGGTACGGCGAGCTGTTCACTATCCCGGAAG CGCGCATCCCGAAAATCGGCGCCCGCATTATGTCGCTTAC CGATCCGACGAAAAAAATGAGCAAATCTGACCCAAACCCG AAATCGTTTATTACGCTGCTTGACGACGCCAAAACGATTG AAAAGAAAATTAAAAGTGCTGTGACCGATTCAGAAGGAAC GATTCGCTATGACAAGGAAGCGAAACCGGGCATTTCGAAC TTGCTCAACATTTATTCGATTTTATCGGGTCAGCCGATTG ACGAACTTGAGCGGCAATACGAAGGAAAAGGATACGGGGT CTTTAAATCCGATTTGGCCCAAGTGGTCATTGAAACGCTC CAACCGATCCAAGAGCGGTATTATCATTGGCTCGAAAGTG AAGAGCTCGACCGCGTCCTAGACGAAGGGGCGGAAAAAGC GAACCGTGTCGCCTCGGAAATGGTGCGCAAAATGGAACAA GCCATGGGGCTTGGGCGGCGTCGG SEQ ID NO: 126 Amino acid TrpRS - GsTrpRS Geobacillus subterraneus DSM 13552 (91A1 ) MKTIFSGIQPSGVITLGNYIGAMRQFVELQHEYNCYFCIV DQHAITVPQNPNELQQNIRRLAALYLAVGIDPKQATLFVQ SEVPAHAQAAWMLQCIVYIGELERMTQFKDKSAGKEAVSA GLLTYPPLMAADILLYNTDIPVVGEDQKQHIELTRDLAER FNKRYGELFTIPEARIPKIGARIMSLTDPTKKMSKSDPNP KSFITLLDDAKTIEKKIKSAVTDSEGTIRYDKEAKPGGISN LLNIYSILSGQPIDELERQYEGKGYGVFKSDLAQVVIETL QPIQERYYHWLESEELDRVLDEGAEKANRVASEMVRKMEQ AMGLGRRR SEQ ID NO: 127 DNA TyrRS-GsuTyrRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAACCTGCTTGAAGAACTGCAATGGCGCGGACTTGTCA ATCAAACGACGGATGAGGATGGGCTTCGAAAGCTCCTGAA TGAGGAGAAGGTGACGCTTTATTGCGGGTTTGACCCGACA GCAGACAGCTTGCATATCGGCCATTTGGTCACGATCATGA CCTTGCGTCGTTTCCAACAGGCGGGGCATCAACCGATCGC CTTAGTCGGCGGCCACCGGGTTGATCGGCGATCCGAGT GGCAGAAAAAGCGAGCGCACGCTCAACGCCAAGGAGACGG TCGAGACGTGGAGCGCCCGAATCAAAGCGCAACTCGAGCG GTTTCTTGATTTTGAGGCTGAGAGCAATCCAGCGAAAATC AAAAACAACTACGACTGGATCGGGCCGCTTGATGTCATCT CGTTTTTGCGTGACATCGGCAAGCATTTCAGCGTCAATTA CATGCTTGCGAAAGAATCGGTGCAGTCGCGCATTGAAATG GGCATTTCGTTTACCGAGTTCAGCTATATGATGCTGCAGG CGTACGACTTCCTCAACTTGTACGAAACGGAAGGTTGCCG ACTACAAATCGGTGGCAGCGACCAATGGGGCAACATCACG GCGGGGCTTGAGCTCATCCGCAGAACGAAAGGTGAGGCGA AAGCATTTGGTTTGACGGTTCCGCTCGTGACGAAAGCCGA TGGGACGAAGTTCGGAAAAACGGAAAGCGGCGCGGTTTGG CTCGATCCGGAAAAAACGTCGCCGTATGAGTTTTACCAGT TCTGGATCAACACCGATGACCGCGATGTGATCCGTTACTT AAAATATTTCACGTTCTTGACAAAAGAAGAGATCGACGCG CTTGAACAAGAGCTGCGCGAAGCGCCGGAGAAGCGGGTGG CGCAAAAAACGCTTGCTTCCGAAGTGACGAAGCTCGTGCA TGGCGAAGAGGCGCTCAATCAAGCGATTCGTATTTCAGAA GCACTCTTTAGCGGCGACATTGCCGAACTGACGGCTGCGG AAATCGAGCAAGGGTTTAAAAACGTGCCGTCGTTTGTCCA TGAAGGAGGCGACGTCCCGCTCGTCGAGCTGCTCGTAGCT GCCGGCATCTCGCCATCGAAGCGGCAGGCGCGCGAAGATG TTCAAAACGGTGCGATTTATGTCAACGGCGAGCGCATCCA AGATGTCGGCGCTGTCTTAACGGCCGAACACCGTTTGGAA GGGCGGTTTACCGTGATCCGCCGCGGCAAGAAGAAGTATT ATTTAATCCGCTACGCT SEQ ID NO: 128 amino acid TyrRS-GsuTyrRS Geobacillus subterraneus DSM 13552 (91A1 ) MNLLEELQWRGLVNQTTDEDGLRKLLNEEKVTLYCGFDPT ADSLHIGHLVTIMTLRRFQQAGHQPIALVGGATGLIGDPS GRKSERTLNAKETVETWSARIKAQLERFLDFEAESNPAKI KNNYDWIGPLDVISFLRDIGKHFSVNYMLAKESVQSRIEM GISFTEFSYMMLQAYDFLNLYETEGCRLQIGGSDQWGNIT AGLELIRRTKGEAKAFGLTVPLVTKADGTKFGKTESGAVW LDPEKTSPYEFYQFWINTDDRDVIRYLKYFTFLTKEEIDA LEQELREAPEKRVAQKTLASEVTKLVHGEEALNQAIRISE ALFSGDIAELTAAEIEQGFKNVPSFVHEGGGDVPLVELLVA AGISPSKRQAREDVQNGAIYVNGERIQDVGAVLTAEHRLE GRFTVIRRGKKKYYLIRYA Accession number 129 DNA ValRS - GsuValRS Geobacillus subterraneus DSM 13552 (91A1 ) ATGAAAGGGGCTTTTTTGCTTGCCTATCGGACGGTTGATC CTGTAGGCAACACAGCCATTGTTTATCACATGAAGGAGGG AATAAAAGTGGCACAGCATGAAGTGTCGATGCCGCCAAAA TACGATCACCGCGCTGTTGAAGCGGGGCGCTATGACTGGT GGCTGAAAGGCAAGTTTTTTGAAACGACCGGCGATCCGGA CAAACAACCGTTTACGATCGTTATCCCACCGCCGAACGTC ACAGGCAAACTGCATTTGGGCCATGCGTGGGATACGACGC TGCAAGACATCATTACGCGCATGAAGCGGATGCAAGGGTA TGATGTCCTATGGCTTCCGGGTATGGACCATGCCGGCATC GCCACCCAGGCGAAAGTGGAAGAAAAATTGCGCCAACAAG GACTGTCCCGCTACGATTTAGGACGGGAAAAATTTTTGGA AGAAACGTGGAAATGGAAAGAAGAATATGCCGGCCATATC CGCAGCCAATGGGCAAAATTAGGGCTCGGCCTCGATTACA CGCGCGAGCGGTTTACGCTTGATGAAGGGCTGTCAAAAGC CGTACGCGAAGTGTTCGTCTCGCTTTACCGGAAAGGGCTC ATTTACCGCGGTGAATACATTATCAACTGGGATCCGGCGA CCAAACCGCCTTGTCCGACATCGAGGTCATTTACAAGGA AGTGAAAGGTGCGCTTTATCATTTGCGCTATCCGCTCGCT GACGGCTCGGGCTACATTGAAGTAGCGACAACCCGTCCAG AAACGATGCTCGGTGACAGGCCGTCGCGGTTCATCCGGA TGACGAGCGGTATAAACACTTGATCGGCAAGATGGTGAAA TTGCCAATCGTTGGCCGGGAAATTCCGATCATCGCTGATG AGTATGTCGATATGGAATTCGGTTCCGGCGCGGTAAAAAT TACACCGGCACACGATCCGAACGACTTTGAAGTTGGCAAC CGCCACAACTTGCCGCGCATTCTCGTCATGAACGAAGACG GTACAATGAACGAAAACGCATTGCAATATCAAGGGCTTGA CCGGTTTGAATGCCGGAAGCAAATCGTCCGTGATTTACAA GAGCAAGGCGTCCTCTTTAAATTGAGGAACACGTCCACT CGGTCGGGCACAGTGAACGGAGCGGCGCCGTTGTTGAACC GTATTTGTCGACACAATGGTTCGTAAAAATGAAGCCGCTC GCGGAAGCTGCCATCAAGATGCAGCAAACAGAAGGAAAAG TGCAATTTGTGCCGGAGCGGTTTGAAAAAACGTACTTGCA CTGGCTTGAGACATTCGCGACTGGTGCATTTCGCGTCAG CTTTGGTGGGGGCACCGCATTCCGGCGTGGTACCATAAAG AAACGGGTGAAATTTACGTCGACCACGAGCCGCCGGCAGA CATTGAAAATTGGGAGCAAGACCCGGATGTGCTTGATACA TGGTTCAGCTCGGCACTCTGGCCGTTCTCCACAATGGGGT GGCCGGATACGGAAGCGCCGGACTACAAGCGCTATTACCC GACCGATGTGCTTGTCACCGGCTATGACATCATTTTCTTC TGGGTGTCGCGCATGATTTTCCAAGGGCTTGAGTTCACTG GGAAGAGACCGTTTAAAGATGTGTTGATCCACGGCCTCGT CCGCGACGCTCAAGGAAGAAAAATGAGCAAGTCGCTCGGC AACGGTGTCGACCCGATGGATGTCATTGACCAATACGGCG CCGATGCGCTCCGCTACTTCCTAGCGACCGGTAGCTCGCC AGGACAAGATTTGCGCTTTAGCACGGAAAAAGTTGAGGCG ACGTGGAATTTTGCTAACAAAATTTGGAACGCTTCACGTT TCGCCTTAATGAACATGGGCGGCATGACATATGAGGAGCT CGATTTGAGCGGCGAAAAAACGGTCGCCGACCATTGGATT TTAACGCGCTTAAATGAAACGATCGACACGGTGACGAAGC TCGCCGACAAATACGAGTTTGGTGAAGTCGGTCGCACGTT GTACAACTTTATTTGGGACGATTTGTGCGACTGGTACATT GAAATGGCGAAGCTGCCGCTTTACGGCGATGATGAGACAG CGAAAAAGACGACGCGTTCAGTTTTAGCGTATGTGCTTGA CAATACGATGCGCTTGTTGCATCCATTCATGCCGTTCATT ACCGAGGAAATTTGGCAAAACTTGCCGCATGACGGCGAAT CGATTACCGTTGCCTCGTGGCCGCAAGTGCGTCCGGAGCT GTCAAACGAAGAAGCGGCGGAAGAAATGCGGATGCTCGTT GACATTATCCGCGCGGTCCGAAACGTTCGTGCCGAAGTCA ATACGCCGCCGAGCAAACCGATTGCGCTCTACATTAAGAC AAAAGACGAACAAGTGCGCGCAGCGCTTATGAAAAACCGC GCTTATCTCGAACGGTTCTGCAATCCGAGCGAATTGATCA TTGACACGGATGTTCCGGCGCCAGAAAAAGCGATGACTGC TGTCGTCACAGGGGCAGAGCTCATTTTGCCGCTTGAAGGA CTCATCAATATCGAAGAAGAAATCAAGCGGCTTGAGAAAG AGCTCGACAAATGGAACAAAGAAGTCGAGCGTGTCGAAAA GAAACTGGCGAACGAAGGCTTTTTGGCAAAAGCGCCGGCT CATGTCGTCGAGGAAGAGCGGCGCAAGCGGCAAGATTACA TCGAAAAACGCGAAGCAGTGAAAGCGCGTCTTGCCGAGTT GAAACGG SEQ ID NO: 130 Amino acid ValRS - GsuValRS Geobacillus subterraneus DSM 13552 (91A1 ) MKGAFLLAYRTVDPVGNTAIVYHMKEGIKVAQHEVSMPPK YDHRAVEAGRYDWWLKGKFFETTGDPDKQPFTIVIPPPNV TGKLHLGHAWDTTLQDIITRMKRMQGYDVLWLPGMDHAGI ATQAKVEEKLRQQGLSRYDLGREKFLEETWKWKEEYAGHI RSQWAKLGLGLDYTRERFTLDEGLSKAVREVFVSLYRKGL IYRGEYIINWDPATKTALSDIEVIYKEVKGALYHLRYPLA DGSGYIEVATTRPETMLGDTAVAVHPDDERYKHLIGKMVK LPIVGREIPIIADEYVDMEFGSGAVKITPAHDPNDFEVGN RHNLPRILVMNEDGTMNENALQYQGLDRFECRKQIVRDLQ EQGVLFKIEEHVHSVGHSERSGAVVEPYLSTQWFVKMKPL AEAAIKMQQTEGKVQFVPERFEKTYLHWLENIRDWCISRQ LWWGHRIPAWYHKETGEIYVDHEPPADIENWEQDPDVLDT WFSSALWPFSTMGWPDTEAPDYKRYYPTDVLVTGYDIIFF WVSRMIFQGLEFTGKRPFKDVLIHGLVRDAQGRKMSKSLG NGVDPMDVIDQYGADALRYFLATGSSPGQDLRFSTEKVEA TWNFANKIWNASRFALMNMGGMTYEELDLSGEKTVADHWI LTRLNETIDTVTKLADKYEFGEVGRTLYNFIWDDLCDWYI EMAKLPLYGDDETAKKTTRSVLAYVLDNTMRLLHPFMPFI TEEIWQNLPHDGESITVASWPQVRPELSNEEAAEEMRMLV DIIRAVRNVRAEVNTPSKPIALYIKTKDEQVRAALMKNR AYLERFCNPSELIIDTDVPAPEPAKMTAVVTGAELILPLEG LINIEEEIKRLEKELDKWNKEVERVEKKLANEGFLAKAPA HVVEEERRKRQDYIEKREAVKARLAELKR sequence number 131 DNA MTF-GsuMTF Geobacillus subterraneus DSM 13552 (91A1 ) ATGCTGATGACGAACATTGTCTTTATGGGAACGCCTGATT TTGCGGTGCCGGTTTTACGGCAGCTGCTTGATGACGGGTA TCGGGTTGTTGCCGTGTTACGCAGCCGGACAAGCCGAAA GGGCGAAAGCGCGAGCTTGTTCCGCCCCCCGTTAAGGTCG AGGCGCAAAAACAGCATCCCGGTATTGCAACCGACGAA AATTCGTGAACCGGAACAATACGAACAAGTGCTGGCGTTTT GCGCCTGACTTGATCGTGACCGCGGCATTTGGACAAATTT TGCCTAAGGCTCTGCTTGACGCTCCCAAATATGGCTGCAT TAATGTTCACGCCTCGCTTCTTCCCGAGCTGCGCGGCGGT GCGCCGATCCATTATGCCATTTGGCAAGGGAAAACGAAAA CAGGTGTCACGATTATGTATATGGCGGAAAGTTGGATGC CGGCGACATGTTGACGCAAGTCGAAGTGCCGATTGAAGAA ACCGATACCGTCGGCACACTGCATGATAAATTGAGCGCTG CCGGGGCTAAACTATTATCAGAAACGCTCCCGCTTTTATT GGAAGGTAACCTTGCGCCTATTCCCGCAAGAGGAAGAGAAA GCGACATATGCTCCGAATATCCGGGCGTGAACAAGAGCGGA TTGACTGGGCGCAGCCTGGTGAGGCGATTTACAACCATAT CCGTGCTTTTCATCCGTGGCCGGTTACGTATACGACATAC GACGGGAACGTTTGGAAAATCTGGTGGGGCGAAAAGTGC CGGCGCCAAGCTTAGCGTCGCCAGGCACGATTTTATCGCT TGAGGAAGACGGCATCGTCGTCGCCACCGGCAGTGAGACG GCCATTAAAATTACTGAATTGCAGCCGGCCGGCAAAAAGC GAATGGCGGCCAGCGAGTTTTGCGCGGTGCTGGCAGCCG GCTTGCGGTCGGCACGAAGCTAGGAGAGAACAATGAACGT ACG sequence number 132 amino acid MTF-GsuMTF Geobacillus subterraneus DSM 13552 (91A1 ) MLMTNIVFMGTPDFAVPVLRQLLDDGYRVVAVVTQPDKPK GRKRELVPPPVKVEAQKHGIPVLQPTKIREPEQYEQVLAF APDLIVTAAFGQILPKALLDAPKYCGINVHASLLPELRGG APIHYAIWQGKTKTGVTIMYMAEKLDAGDMLTQVEVPIEE TDTVGTLHDKLSAAGAKLLSETLPLLLEGNLAPIPQEEEK ATYAPNIRREQERIDWAQPGEAIYNHIRAFHPWPVTYTTY DGNVWKIWWGEKVPAPSLASPGTILSLEEDGIVVATGSET AIKITELQPAGKKRMAASEFLRGAGSRLAVGTKLGENNER T SEQ ID NO: 133 amino acid RF-1-Mut-GsRF-1-EcOpt Geobacillus stearothermophilus MFDRLEAVEQRYEKLNELLMEPDVINDPKKLRDYSKEQAD LGETVQTYREYKSVREQLAEAKAMLEEKLEPELREMVKEE IGELEEREEALVEKLKVLLLPKDPNDEKNVIMEIRAAAGG EEAALFAGDLYRMYTRYAESQGWKTEVIEASPTGLGGYKE IIFMINGKGAYSKLKFENGAHRVQRVPETESGGRIHTSTA TVACLPEMEEIEVEINEKDIRVDTFASSGPGGQSVNTTMS AVRLTHIPTGIVVTCQDEKSQIKNKEKAMKVLRARIYDKY QQEARAEYDQTRKQAVGTGDRSERIRTYNFPQNRVTDHRI GLTIQKLDQVPDGHLDEIIEALILDDQAKKLEQANDAS SEQ ID NO: 134 amino acid muGFP + His6 tag + C-tag Aequorea victoria MRGSHHHHHHGSSKGEELFTGVVPILVELDGDVNGHKFSV RGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVL CFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTR AEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHN VYITADKQKNGIKAYFKIRHNVEDGSVQLADHYQQNTPIG DGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEDVTAAGI THGMDELYKGSEPEA SEQ ID NO: 135 amino acid deGFP Aequorea victoria MELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFF KSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGI SEQ ID NO: 136 amino acid T7 RNA polymerase T7 Bacteriophage MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALE HESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPK MIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIK TTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAK HFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEA WSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQD SETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTG ITGGGYWANGRRPLAVRTHSKALMRYEDVYMPEVYKAI NIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREEL PMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEF MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMT KGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIK FIEENHENIMACAKSPLENTWWAEQDSPFFCFLAFCFEYAG VQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVN LLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYG SKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKL IWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILR KRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPT INTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHE KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCD VLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDF AFA

Claims

1. 1. A system for recombinant cell-free expression comprising: - At least the following ingredients: - multiple initiation factors (IFs); - several elongation factors (EF); - multiple peptide release factors (RFs); - at least one ribosome recycling factor (RRF); - several aminoacyl-tRNA-synthetases (RS); and - have at least one methionyl-tRNA transformylase (MTF) , a recombinant core protein mixture; and at least one nucleic acid synthesis template, a reaction mixture having the cell-free reaction components necessary for in vitro macromolecule synthesis; Including, - the components are located in a bioreactor configured for cell-free expression of macromolecules It's a type of thing.

2. The components of the recombinant core protein mixture are selected from the group consisting of a bacterial-derived recombinant core protein mixture, The system of claim 1 comprising:

3. The recombinant core protein mixture derived from bacteria is a recombinant core protein mixture comprising a recombinant core protein mixture, at least one component of which is derived from a thermophilic bacterium; The system of claim 2.

4. The thermophilic bacterium may be a thermophilic Bacillaceae bacterium or a Geobacterium.

4. The method according to claim 2, comprising the step of: The system described above.

5. The Geobacillus thermophilic bacterium is Geobacillus subterraneus, and Geobacillus stearothermo 5. The system of claim 4, wherein the system is selected from the group consisting of:

6. The recombinant core protein mixture derived from bacteria is a recombinant core protein mixture At least one of the components is a non-thermophilic bacterium or a combination of a non-thermophilic bacterium and a thermophilic bacterium.

2. The system of claim 1, comprising a recombinant core protein mixture derived from a

7. 7. The method of claim 6, wherein the non-thermophilic bacterium comprises Escherichia coli. The system described.

8. The method according to claim 1, wherein the plurality of initiation factors (IFs) comprises a plurality of initiation factors derived from thermophilic bacteria. The system described above.

9. The multiple initiation factors derived from thermophilic bacteria include IF1, IF2, IF3, or any one thereof.

9. The system of claim 1, comprising a fragment or variant of:

10. The multiple initiation factors include amino acid sequences consisting of SEQ ID NOs: 2, 4, 6, 70, 72, and 74. or a sequence having at least 90% sequence identity. and 9. The system according to any one of claims 1 to 9.

11. 2. The method of claim 1, wherein the plurality of elongation factors (EFs) comprises a plurality of elongation factors derived from thermophilic bacteria. The system described above.

12. The multiple elongation factors derived from thermophilic bacteria include EF-G, EF-Tu, EF-Ts, EF- 4, EF-P, or any fragment or variant thereof.

10. The system according to any one of claims 1 to 9.

13. The plurality of elongation factors are SEQ ID NOs: 8, 10, 12, 14, 16, 76, 78, 80, 8 2, and 84, or a group of amino acid sequences having at least 90% sequence identity.

13. The system of any one of claims 1, 11 and 12, selected from the series:

14. The multiple peptide release factors (RFs) are derived from thermophilic bacteria or Bacillus s) a plurality of bacterially derived peptide release factors.

15. The multiple peptide release factors from thermophilic bacteria are RF1, RF2, and RF3, or 15. The method of claim 1, including any fragment or variant thereof. System of.

16. The multiple peptide terminators include amino acids consisting of SEQ ID NOs: 18, 20, 22, 86, and 88. or a sequence having at least 90% sequence identity.

16. A system according to any one of claims 1, 14 and 15.

17. The ribosome recycling factor (RRF) is a ribosome recycling factor derived from a thermophilic bacterium. The system of claim 1 , comprising a Kling factor.

18. The ribosome recycling factor is derived from Geobacillus 18. The system of any one of claims 1 and 17, wherein

19. The ribosome recycling factor has the amino acid sequence SEQ ID NO: 14 and 90 or at least and a ribosome recycling factor according to a sequence having 90% sequence identity with the ribosome recycling factor.

19. The system according to any one of paragraphs 1, 17 and 18.

20. The plurality of aminoacyl-tRNA-synthetases (RS) are derived from thermophilic bacteria or Escherichia coli.

1. The method of claim 1, comprising: The system described.

21. The plurality of aminoacyl-tRNA-synthetases include AlaRS; ArgRS; As nRS; AspRS; CysRS; GlnRS; GluRS; GlyRS; HisRS; IleRS; LeuRS; LysRS; MetRS; PheRS (a); PheRS (b ); ProRS; SerRS; ThrRS; TrpRS; TyrRS; and ValRS, or any fragment or variant thereof. The system described.

22. The aminoacyl-tRNA-synthetases are selected from the group consisting of SEQ ID NOs: 26, 28, 32, 3 ４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５４、５６、５８、６０ 、６２、６４、６６、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１ 10, 112, 114, 116, 118, 120, 122, 124, 126, 128, and and 130 amino acid sequences, or sequences having at least 90% sequence identity.

22. The system according to any one of claims 1, 20 and 21, wherein the system is selected from:

23. The methionyl-tRNA transformylase (MTF) is a methionyl-tRNA transformylase derived from a thermophilic bacterium. The system of claim 1, comprising an onyl-tRNA transformylase.

24. The methionyl-tRNA transformylase was obtained from Geobacillus 24. The system according to claims 1 and 23, which is derived from Bacillus subtilis.

25. The methionyl-tRNA transformylase has the amino acid sequence SEQ ID NO:68 and 1 32, or a methionyl-tRNA tRNA tRNA according to a sequence having at least 90% sequence identity.

25. The system of any one of claims 1, 23 and 24, comprising a phosphotransformylase.

26. The system of claim 1 , wherein the nucleic acid synthesis template comprises a DNA template.

27. The DNA template is - at least one target sequence operably linked to a promoter, optionally the target sequence, optionally optimized; - at least one ribosome binding site (RBS); - at least one expression product cleavage site; and - at least one tag, 27. The system of claim 26, comprising a linear DNA template having:

28. The system of claim 1 , wherein the nucleic acid synthesis template comprises an RNA template.

29. The reaction mixture comprises the following components: - a quantity of ribosomes, and optionally a quantity of ribosomes from thermophilic bacteria; - an amount of a ribonuclease inhibitor; - a certain amount of RNA polymerase; - an amount of tRNA, and optionally an amount of tRNA from a thermophilic bacterium; buffer solutions; and - a certain amount of amino acids, 10. The system of claim 1, comprising one or more of:

30. The reaction mixture comprises the following components: - Tris-acetic acid; -Mg(OAc)2; -K ＋ - glutamate; amino-acetate; -NaCl; -KCl; -MgCk; - DTT; -octyl-b-glycoside; -NAD; -NADP; - sorbitol; -FADH; -CoA; -PLP; and -SAM, 30. The system of claim 29, further comprising one or more of:

31. 30. The system of any one of claims 1 and 29, further comprising an energy source.

32. 33. The method of claim 32, wherein the energy source comprises a quantity of nucleotide triphosphates (NTPs). The system described above.

33. The nucleotide triphosphates include adenine triphosphate (ATP); guanosine triphosphate (G TP), uridine triphosphate UTP, and cytidine triphosphate (CTP).

33. The system of claim 32, comprising one or more of the nucleotide triphosphates selected from the group consisting of nucleotide triphosphates.

34. 32. The method of claim 31 , wherein the energy source comprises an inorganic polyphosphate-derived energy regeneration system.

34. The system according to any one of claims 32 and 33.

35. The inorganic polyphosphate-derived energy regeneration system comprises: - a cellular adenosine triphosphate (ATP) energy regeneration system, - a certain amount of adenosyl kinase (Gst AdK) enzyme; - a certain amount of the enzyme polyphosphate kinase (TaqPPK); an amount of inorganic polyphosphate (PPi); and - Cells containing a certain amount of adenosine monophosphate (AMP), adenosine triphosphate (ATP) ) including energy regeneration systems, The AdK and PPK enzymes act synergistically to convert PPi and AMP to cellular ATP 35. The system of claim 34, which regenerates energy.

36. 10. The system of claim 1, wherein the bioreactor comprises a continuous flow bioreactor.

37. 1. A recombinant cell-free expression reaction mixture comprising: - multiple initiation factors (IFs); - several elongation factors (EF); - multiple release factors (RFs); - at least one ribosome recycling factor (RRF); - several aminoacyl-tRNA-synthetases (RS); and Recombinant methionyl-tRNA transformylase (MTF) Cell-free expression reaction mixture.

38. 38. The method of claim 37, wherein the plurality of initiation factors (IFs) comprises a plurality of initiation factors from a thermophilic bacterium. The system described.

39. The multiple initiation factors derived from thermophilic bacteria include IF1, IF2, IF3, or any one thereof.

39. The system of any one of claims 37 and 38, comprising a fragment or variant of:

40. The initiation factors are selected from the group consisting of amino acid sequences of SEQ ID NOs: 2, 4, 6, 70, 72 and 74. 37, 38, or a sequence having at least 90% sequence identity. and 39. The system according to any one of claims 39 to 40.

41. 38. The method of claim 37, wherein the plurality of elongation factors (EFs) comprises a plurality of elongation factors from thermophilic bacteria. The system described.

42. The multiple elongation factors derived from thermophilic bacteria include EF-G, EF-Tu, EF-Ts, EF- 4, EF-P, or any fragment or variant thereof.

10. The system according to any one of claims 1 to 9.

43. The plurality of elongation factors are SEQ ID NOs: 8, 10, 12, 14, 16, 76, 78, 80, 8 2, and 84, or a group of amino acid sequences having at least 90% sequence identity.

43. The system of any one of claims 37, 41 and 42, selected from the series:

44. The multiple peptide release factors (RFs) are derived from thermophilic bacteria or Bacillus sp.

38. The system of claim 37, comprising multiple release factors derived from bacteria (Lactobacillus sp.).

45. The multiple peptide release factors may be RF1, RF2, and RF3, or any fragment thereof.

45. The system of any one of claims 37 and 44, comprising a fragment or variant thereof.

46. The multiple peptide terminators include amino acids consisting of SEQ ID NOs: 18, 20, 22, 86, and 88. or a sequence having at least 90% sequence identity. 37, 44 and 45. A system according to any one of claims 37, 44 and 45.

47. The ribosome recycling factor (RRF) is a ribosome recycling factor derived from a thermophilic bacterium.

38. The system of claim 37, comprising a Kling factor.

48. The ribosome recycling factor is derived from Geobacillus 48. The system of any one of claims 37 and 47, wherein

49. The ribosome recycling factor has the amino acid sequence SEQ ID NO: 14 and 90 or at least and a ribosome recycling factor according to a sequence having 90% sequence identity with the ribosome recycling factor.

49. A system according to any one of paragraphs 37, 47 and 48.

50. At least one of the plurality of aminoacyl-tRNA-synthetases (RSs) 38. The method of claim 37, comprising multiple aminoacyl-tRNA-synthetases derived from thermophilic bacteria. The system described.

51. The plurality of aminoacyl-tRNA-synthetases include AlaRS; ArgRS; As nRS; AspRS; CysRS; GlnRS; GluRS; GlyRS; HisRS; IleRS; LeuRS; LysRS; MetRS; PheRS (a); PheRS (b ); ProRS; SerRS; ThrRS; TrpRS; TyrRS; and ValRS, or any fragment or variant thereof. The system described in

52. The aminoacyl-tRNA-synthetases are selected from the group consisting of SEQ ID NOs: 26, 28, 32, 3 ４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５４、５６、５８、６０ 、６２、６４、６６、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１ 10, 112, 114, 116, 118, 120, 122, 124, 126, 128, and and 130 amino acid sequences, or sequences having at least 90% sequence identity.

52. The system of any one of claims 37, 50 and 51, wherein the system is selected from:

53. The methionyl-tRNA transformylase (MTF) is a methionyl-tRNA transformylase derived from a thermophilic bacterium.

38. The system of claim 37, comprising an onyl-tRNA transformylase.

54. The methionyl-tRNA transformylase was obtained from Geobacillus 54. The system of any one of claims 37 and 53, which is derived from Bacillus gracilis.

55. The methionyl-tRNA transformylase has the amino acid sequence SEQ ID NO:68 and 1 32, or a methionyl-tRNA tRNA tRNA according to a sequence having at least 90% sequence identity.

55. The system of any one of claims 37, 53 and 54, comprising a phosphotransformylase.

56. 1. An isolated nucleotide comprising: SEQ ID NOs: 1, 3, 5, 69, 71, and 73; SEQ ID NOs: 7, 9, 11, 13, 15, 75, 77, 79, 81, and 83; SEQ ID NOs: 17, 19, 21, 85, and 87; SEQ ID NOs: 23 and 89; and SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 4 ７、４９、５１、５３、５５、５７、５９、６１、６３、６５、６７、９１、９３、９５ 、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１ 17, 119, 121, 123, 125, 127, 129, and 131. An isolated nucleotide comprising a selected nucleotide.

57. At least one of the nucleotide sequences of claim 56 operably linked to a promoter. an expression vector comprising at least one

58. 58. A bacterium transformed with one of the expression vectors of claim 57.

59. 59. The transformed bacterium of claim 58, wherein the bacterium comprises E. coli.

60. A peptide, SEQ ID NOs: 2, 4, 6, 70, 72, and 74; SEQ ID NOs: 8, 10, 12, 14, 16, 76, 78, 80, 82, and 84; SEQ ID NOs: 18, 20, 22, 86, 88; - SEQ ID NOs: 14 and 90; SEQ ID NOs: 26, 28, 32, 34, 36, 38, 40, 42, 44, 46, 48, 5 0, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, SEQ ID NO: 98, 1 ００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１１８、１ 20, 122, 124, 126, 128, and 130; and - consisting of SEQ ID NOs: 68 and 132, or any fragment or variant thereof A peptide comprising an amino acid sequence selected from the group:

61. 61. A cell-free expression system using at least one of the peptides of claim 60.

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