Linear nucleic acid templates for high-efficient cell-free protein expression

Abstract

96 New PCT-Patent Application based on EP 24 186 635.9 Insempra GmbH Vossius Ref.: AG4141 PCT S3 Abstract The present invention relates to a linear double-stranded deoxyribonucleic acid (dsDNA) molecule comprising one or more Tus protein (Tus) binding site(s) at the 5'-terminus, one or more Lac repressor protein (LacI) binding site(s) at the 3'-terminus, and a segment comprising a DNA sequence of interest (DOI) between said Tus binding site(s) and said LacI binding site(s). The present invention further relates to a non-naturally or naturally occurring RNA molecule as encoded by or transcribable / transcribed from the linear dsDNA molecule of the invention, and to an expression DNA cassette comprising a promoter, an RBS, a GOI encoding a POI, and one or more LacI binding site(s), wherein said expression cassette does not comprise a terminator sequence between the 3´ end of said GOI and said one or more LacI binding site(s). The present invention also relates to a non-naturally or naturally occurring RNA molecule as encoded by or transcribable / transcribed from said expression cassette. The present invention further relates to method of protecting a linear deoxyribonucleic acid (DNA) molecule from exonuclease degradation by adding one or more Tus binding site(s) at the first terminus of the DNA molecule and adding one or more LacI binding site(s) at the other terminus of the DNA molecule. The present invention further relates to a method of synthesizing a protein of interest (POI) in a cell- free protein synthesis (CFPS) reaction mixture by using the (ds)DNA and / or RNA molecules of the invention, and also to a cell-free biological system or CFPS reaction mixture comprising the (ds)DNA, expression cassette and / or RNA molecules of the invention.

Description

[0001]New PCT-Patent Application based on EP 24186635.9 Insempra GmbH Vossius Ref.: AG4141 PCT S3 Linear nucleic acid templates for high-efficient cell-free protein expression The present invention relates to linear nucleic acid molecules, for example linear (double- stranded; ds) deoxyribonucleic acid molecules ((ds)DNA) or ribonucleic acid molecules (RNA), which are suitable as expression templates, in particular in cell-free protein synthesis (CFPS). Linear (ds)DNA molecules of the invention comprise one or more Tus protein (Tus) binding site(s) at the 5’-terminus, one or more Lac repressor protein (LacI) binding site(s) at the 3’- terminus, and a segment comprising a DNA sequence of interest (DOI) between said Tus binding site(s) and said LacI binding site(s). Linear nucleic acid dsDNA molecules of the invention may also lack a 5’ DNA protection group. The present invention further relates to a (non-naturally occurring) RNA molecule as encoded by or transcribable / transcribed from the linear (ds)DNA molecule of the invention (e.g. a non-naturally occurring, IVT-synthesized or chemically synthesized, RNA molecule having one or more lacO sequence(s) at the 3’-terminus). The present invention further relates to an expression DNA cassette comprising a promoter, a ribosomal binding site (RBS; or other 5´ untranslated region / other translation initiation / ribosome recruitment sequence), a gene of interest (GOI) encoding a protein of interest (POI), and one or more LacI binding site(s) at the 3’ terminus. A DNA molecule or expression cassette of the invention may not comprise a terminator between the 3´ end of said GOI and said one or more LacI binding site(s). The present invention also relates to a (non- naturally occurring) RNA molecule as encoded by or transcribable / transcribed from said expression cassette. The present invention further relates to a method of protecting a linear DNA molecule, or another nucleic acid molecule, from exonuclease degradation by adding at least one or more LacI binding site(s) (or respective nucleotide sequence(s)) at one terminus of the DNA molecule, or of the other nucleic acid molecule, e.g. at the 3’ terminus. One or more Tus binding site(s) (or respective nucleotide sequence(s)) at the other terminus of the DNA molecule, or of the other nucleic acid molecule, may also be added, e.g. also at the 5’ terminus. The present invention further relates to a method of synthesizing a POI in a CFPS reaction mixture by using the nucleic acid molecules ((ds)DNA and / or RNA molecules) of the invention (as expression templates for the POI). The present invention also relates to a cell-free biological system, CFPS reaction mixture or CFPS buffer comprising a nucleic acid molecule of the invention (for example a (ds)DNA molecule, an expression cassette and / or an RNA molecule of the invention. Cell-free production of enzymes and other proteins or (poly)peptides, also termed cell-free protein synthesis (CFPS), is a cutting-edge technique in biotechnology that allows for the synthesis of proteins and other biomolecules outside of living cells. This approach, in principle, offers several advantages over traditional cell-based systems, such as simplified control over the production process, faster reaction rates, easier purification, and increased speed and scalability (e.g. multiple testing in parallel). Cell-free systems are thus widely used in research, drug development, and also in some industrial applications, e.g. primarily for high-throughput screening of libraries of variant peptides and enzymes. Due to the costs and complexity, however, CFPS is currently mainly used for basic research purposes and for rapid screening of various enzyme sequences. In the mid-term, CFPS systems are desired to be used also for large-scale (industrial) production of biomolecules. Of critical importance for such (industrial) systems, however, are (i) sufficient scalability (e.g., the number of samples which could be handled), (ii) speed, and (iii) expression efficiency, i.e., the (final) level of protein expression. To date, three main approaches try to address these issues / challenges, namely, the use of plasmid-based systems, the use of linear DNA-based systems, and the use of linear DNA-based systems with terminal protection groups. In the context of plasmid-based systems (as, for example, disclosed in Jewett, Biotechnol Bioeng.86(1), 2004, 19-26; Kwon, Scientific Reports 5, 2005, 8663), plasmids enable researchers to use microbial strains for easy DNA amplification. Further, plasmids are relatively stable and can be stored for extended periods. They are less susceptible to degradation as compared to common (unmodified) linear DNA. However, producing plasmid DNA is a tedious and time-consuming process because it includes designing DNA, synthesizing, PCR, ligation to parent plasmid, transformation, cell growth, isolation, and analysis. Especially the cell-based steps are time consuming. It usually takes many days after receipt of the synthetic genes and respective expression cassettes before the final plasmid can be purified from a pure culture of a strain. Further, plasmids typically contain additional elements, like selection markers (e.g., antibiotic resistance genes) and origins of replication, which may add undesired complexity to cell-free systems which use plasmids as expression templates. Linear DNA-based systems (as, for example, disclosed in Hunt, Nat Commun 14(1), 2023, 3897 (doi: 10.1038 / s41467-023-38965-w)) offer the advantage that linear DNA can be produced fast and straight forward, because it only includes designing DNA, synthesizing and PCR. Linear DNA can be amplified by PCR and does not require the step of being brought and multiplied in a cell. Linear DNA usually contains only the desired essential components and lacks the additional elements as, for example, found in plasmids, thus simplifying the cell-free system and reducing potential complications arising from extra sequences. Thus, linear DNA requires less time for preparation, reduces complexity and has the potential for automation. However, linear DNA is more susceptible to degradation as compared to plasmids. This is a concern, especially for large scale industrial production approaches and in cases where the cell- free reaction needs to be carried out over an extended period. Most relevantly, the (final) protein expression level is often unsatisfactorily low when using linear DNA-based systems; especially in cases where linear DNA is degraded at an undesirable rate. Attempts for addressing the the drawbacks of linear DNA-based CFPS systems have been made to date, e.g. by adding terminal protection groups to the linear DNA. For example, US-A1 2005 / 0202480 describes stabilizing double-stranded (ds) linear DNA against exonucleolytic attack by adding a target sequence for one particular DNA-binding protein to each of both ends of the linear ds DNA. In particular, US-A12005 / 0202480 discloses the use of the E. coli Lac operator sequence (lacO) in combination with the E. coli Lac repressor protein (LacI) for this purpose. For achieving high relative differences between protected and unprotected DNA templates, however, LacI needs to be coexpressed in the CFPS system or added as exogenous protein to the reactions. Further, since the protein synthesis capacity in the CFPS system needs to be shared by both, the synthesis of the target protein and the synthesis of the coexpressed LacI protein, the absolute yield of the target protein is lowered. Furthermore, WO-A1 2022 / 160049 and Norouzi (ACS Synth Biol. 10(7), 2021, 1615-24; doi: 10.1021 / acssynbio.1c00110) disclose the protection of linear dsDNA in cell-free extracts from E. coli and Vibrio natriegens. In this context, the terminal incorporation of ter binding sites for the DNA-binding protein Tus were used to protect the linear expression templates. In particular, the system requires one or more ter sites at the 5’ terminus and one or more ter sites at the 3’ terminus of the linear construct. However, also the Tus protein needs to be added to the system exogenously / externally as purified Tus, needs to be provided from a Tus-expressing bacterial strain or needs to be overexpressed in the desired strain before lysate preparation. Furter, by using an in vitro plasmid replication system, Jameson (J. Biol. Chem.297(6), 2021, 1- 14; https: / / doi.org / 10.1016 / j.jbc.2021.101409) has performed basic research concerning the termination of DNA replication at Tus / ter barriers of the replication fork resulting in under- replication of template DNA. In this context, Jameson loc. cit. created a circular plasmid-based system with Tus / ter- and LacI / lacO-blocks to control the fusion of two replication forks directly at a Tus / ter complex. There is, however, still an unmet need in the field of protein peptide production by CFPS, namely the need for less complex / more simple but efficient and productive CFPS systems which would allow for (industrial) large-scale CFPS (e.g. CFPS systems with appropriate scalability, speed and expression efficiency). The problem underlying the present invention is therefore the provision of means and methods for economic CFPS, in particular for industrial purposes. The technical problem is solved by the provision of the embodiments as disclosed and described herein and as characterized in the claims. The present invention solves the technical problem because, as documented herein below and in the appended examples, linear (ds)DNA (namely linear (ds)DNA expression constructs) having at least one copy (e.g. two copies) of a Tus binding site (e.g. a ter site; also termed ter sequence or simply ter) at the 5’-terminus and at least one copy (e.g. two copies) of a LacI binding site (e.g. a lacO site; also termed Lac operator or simply lacO) at the 3’-terminus renders CFPS less complex / more simple but highly efficient. In particular, no external protecting protein needs to be added to the CFPS system during the reaction and no protecting protein needs to be coexpressed therein. For example, while earlier described CFPS systems require the overexpression and / or addition of the binding protection proteins, like Tus or LacI (e.g. Norouzi loc. cit.; US-A12005 / 0202480), no such proteins need to be (extra) expressed and / or (exogenously / externally) added to the CFPS reaction mixture in the context of the invention (i.e. when using the linear (ds)DNA or other nucleic acid molecules of the invention). The protection proteins which are endogenously present in the CFPS are sufficient to prevent degradation of linear DNA. Without being bound by theory, the overall amount of protection proteins which are endogenously present in the CFPS reaction mixture (e.g. deriving from the basis (bacterial) lysate), e.g. the sum of both, the amount of Tus and the amount of LacI, is sufficient to saturate all available protection protein binding sites of the linear nucleic acid molecules / (ds)DNA of the invention, e.g. all (5´) ter and all (3´) lacO sites (for example only one or two copies of each). In contrast, and also without being bound by theory, the amount of only one kind of protection protein which is endogenously present in the CFPS reaction mixture (e.g. deriving from the basis (bacterial) lysate), e.g. the amount of solely Tus or the amount of solely LacI, may not be sufficient to saturate all available, single-kind protection protein binding sites of a DNA template of the prior art, e.g. all (5´ and 3´) ter sites or all (5´ and 3´) lacO sites (for example two or more copies of each, like three or four copies in sum). As a consequence, nucleic acid molecule protection and stability, and thus expression efficiency, is expected to be lower in CFPS reaction mixtures, in particular in CFPS reaction mixtures without (extra) expressed and / or without (exogenously) added protection proteins, when using solely one kind of protection for the employed linear (ds)DNA template, e.g. solely (5´ and 3´) Tus / ter protection or solely (5´ and 3´) LacI / lacO protection. Respective experimental evidence is also provided herein and in the appended examples. Furthermore, in CFPS systems with coexpressed protecting proteins (like the LacI in US-A12005 / 0202480), the protein synthesis capacity needs to be shared by both, the synthesis of the target protein and the synthesis of the coexpressed protecting protein. As consequence, the the absolute yield of the target protein is lowered. This drawback can be avoided by relying on nucleic acid molecules of the present invention. More specifically, it has been shown in the context of this invention and the appended examples that, for all exemplified protein-encoding genes of interest (GOI), the linear (ds)DNA of the invention (also termed (5´)ter / (3´)lacO construct or (5´)ter / (3´)lacO design) performs better than the unprotected construct (e.g. in terms of protein yield), especially in an CFPS reaction mixture without (extra) expressed and without (exogenously) added protection proteins). Besides, the (5´)ter / (3´)lacO design performs also better as, or at least equivalent to, the (5´)ter / (3´)ter and the (5´)lacO / (3´)lacO protected constructs / designs (e.g. in terms of protein yield). Further, it has been demonstrated in the context of the invention and the appended examples that the (5´)ter / (3´)lacO design performs extraordinary well in CFPS wherein the construct encoding the protein to be synthesized (POI), in particular the resulting RNA encoding the POI, has a medium or low intrinsic stability (e.g. a stability of ≤ 510 AU (e.g. in an assay as described in Example 3 and Fig. 2, respectively; see also http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi)). Moreover, evidence has been provided in the context of the invention and the appended examples that a strong (lacO) hairpin at the 3’ end of the constructs (for example as resulting from the one or more LacL binding site(s)) can prevent also single-stranded sequences from further degradation, e.g. also RNA. This leads to higher (final) expression levels in CFPS. The experimental evidence provided in the context of the present invention thus also leads to further alternative constructs and CFPS approaches, respectively. In this context, the alternative linear DNA and RNA constructs of the invention have lacO protection sequence(s) at the 3’-end downstream of the GOI, with no terminator sequences (between the GOI and the LacI binding site(s) / lacO protection sequence(s); cf. also Example 7; Fig. 6A (lower panel) and Fig. 6B). Without being bound by theory, the single-stranded lacO sequence forms a stronger and more stable hairpin than a (T7) terminator sequence in the RNA transcript (cf. Fig. 6B). In one particular aspect, a 5´ (ter / Tus) protection on DNA level may even be omitted in the alternative linear constructs of the invention. It is also documented herein below and in the appended examples that the linear nucleic acid molecules / constructs as disclosed and described herein, especially when used in CFPS, work particularly well if the POI is expressed with an upstream N-terminal leader peptide, in particular with a highly expressed upstream N-terminal leader peptide, like an upstream nGFP (the first 20 amino acids of super-folded green fluorescence protein (sfGFP)). Further, upstream to the POI, a 2A self-cleavage peptide may be present (thereby, a poly-POI may advantageously be expressed / synthesized; i.e. two (or more) POIs may be expressed / synthesized from a single ORF in a multicistronic expression template). As appropriate, other upstream / N-terminal structures / peptides (e.g. an RBS) can be used; e.g. to calibrate POI expression to a desired level; for example, to a lower or higher level. Furthermore, it is documented herein below and in the appended examples that in CFPS, especially when using the linear nucleic acid molecule / (ds)DNA of the invention as expression templates, the level of expression can advantageously be assessed / monitored by linking to the POI a (C-terminal or N-terminal) tag, for example a peptide tag, like the Nanoluciferase binding peptide tag (e.g. a HiBit tag; Promega). When using the linear nucleic acid constructs of the invention (DNA and RNA constructs), for example the linear (ds)DNA of the invention, as an expression template, CFPS is also substantially faster than the established CFPS systems. For example, CFPS according to the invention is finished after about 3 hours (or even less); instead of, for example, after about 20 hours (as in conventional CFPS systems). The linear (ds)DNA (or RNA) of the invention requires also less time for preparation, for example as compared to the preparation of circular plasmid DNA (no expression constructs for the binding protection proteins need to be prepared / added; no exogenously / externally protection proteins need to be added to the respective CFPS system). The linear (ds)DNA of the invention is also less complex; as compared to expression templates which additionally encode protection compounds (like the Tus or LacI proteins). Moreover, the CFPS system in accordance with the invention is more simple (no (extra) expressed or (exogenously / externally) added protection compounds (like the Tus or LacI proteins) need to be provided). Moreover, it is documented herein below and in the appended examples that CFPS, especially when using the linear constructs of the invention ((ds)DNA and / or RNA), works particularly well with glucose (e.g. at a concentration of 10 to 30 mM (preferably 20 mM)) in combination with phosphoenolpyruvate (PEP) (e.g. at a concentration of 20 to 50 mM, preferably at about 28 to 38 mM, more preferably at about 33 mM) as (ATP-regenerating) energy sources in the CFPS reaction mixture. These advantages allow for efficient and high-yielding CFPS, as well as for scalable, fast and high-throughput CFPS and POI quantification and / or real-time CFPS monitoring. All in all, the linear constructs of the invention ((ds)DNA and RNA), especially when used in CFPSs (as disclosed herein), provide the potential for automation and high-efficiency / high yield (industrial) large-scale POI production. In general, the present invention relates to advantageously protected nucleic acid molecules. In particular, the present invention relates to advantageously protected linear nucleic acid molecules. These nucleic acid molecules are particularly suitable as expression templates in CFPS and respective CFPS systems; and may perform extraordinary well as such in CFPS and respective CFPS systems. For example, the use of the nucleic acid molecules of the invention as expression templates in CFPS and respective CFPS systems leads to high-efficient cell-free protein / peptide expression and / or an increased level of protein / peptide expression and increased level of (final) protein / peptide yield, respectively. This is mainly due to an increased stability of the nucleic acid molecules of the invention; and / or an increased translation efficiency and / or an increased transcription efficiency (resulting from (an increased stability of) the nucleic acid molecules when used as expression templates). In principle, a nucleic acid molecule of the invention may be an (advantageously protected) deoxyribonucleic acid (DNA) or an (advantageously protected) ribonucleic acid (RNA). Further, an (advantageously protected) nucleic acid molecule of the invention may, in principle, be a single-stranded DNA (ssDNA) or a double-stranded DNA (ds DNA). Although RNA, in particular mRNA, is typically ssRNA, also dsRNA is, in principle, within the invention; in particular (m)RNA, with (a) (partly) ds segment(s), like (a) (3’ terminal) hairpin structure(s). In a first aspect, the present invention relates to a linear nucleic acid molecule being linear DNA comprising (a) one or more Tus binding site(s) at the 5’-terminus; and (b) one or more LacI binding site(s) at the 3’-terminus; and (c) between (a) and (b) a segment comprising a DNA sequence of interest (DOI). The DOI of this linear DNA comprises a DNA sequence which encodes a protein / peptide of interest (POI). The DNA sequence (and also the corresponding RNA sequence) which encodes a POI is also termed herein gene of interest (GOI). This linear DNA of the invention may, in principle, be a ssDNA (optionally with (a) terminal hairpin structure). It is preferred, however, that this DNA of the invention is a dsDNA. This linear DNA of the invention may, or may not, comprise a terminator / terminator sequence between the GOI and the one or more LacI binding site(s) at the 3’-terminus. In a further aspect, the present invention relates to a linear nucleic acid molecule comprising (a) a particularly strong protection at the 3’-terminus, like one or more LacI binding site(s) (or the respective (lacO) sequence(s) on RNA level); and (b) at an upstream location of the 3’-terminal protection a segment comprising a nucleotide sequence of interest (DOI / GOI) (encoding a POI). This linear nucleic acid molecule of the invention may be a DNA or an RNA. In case of DNA, it may be a ssDNA or, preferably, a dsDNA. In case of RNA, it may be a ssRNA. In case of ssDNA or ssRNA, (a) (partly) ds segment(s) may be comprised; e.g. (a) ds segment(s) at the 3’-terminus, like (a) ds one or more LacI binding site(s) or the respective nucleic acid sequence(s) (for example one or more lacO hairpin(s)). This linear nucleic acid molecule of the invention may, or may not (preferred), comprise a terminator / terminator sequence, e.g. between the GOI and the particularly strong protection at the 3’-terminus (like one or more LacI binding site(s) or the respective nucleic acid sequence(s)). In one embodiment, this linear nucleic acid molecule of the invention may not comprise a 5’ nucleic acid protection group (e.g. like a 5’ Tus binding site and ter sequence(s), respectively). In the case of a linear DNA, it may not comprise a 5’ DNA protection group (e.g. like a 5’ Tus binding site). Especially in the context of this embodiment, the linear nucleic acid molecule may not comprise a terminator / terminator sequence. As mentioned, the nucleic acid molecules of the invention are particularly useful as expression templates in CFPS and respective CFPS systems (also sometimes termed just cell-free systems (CFSs)). In general, the technical meanings of CFPS, and of CFPS system, are known in the art; and are, for example, evident from Kim (Biotechnol Bioeng. 74(4), 2001, 309-16), Jewett (Biotechnol Bioeng. 86(1), 2004, 19-26), Calhoun (Biotechnol Bioeng. 90(5), 2005, 606-13), Rasor (Methods Mol Biol 2433, 2022, 199-215), WO-A12022 / 160049, Norouzi loc. cit. and US- A12005 / 0202480. If not explicitly described in a different manner or contradicted by context, the terms “CFPS”, and “CFPS system”, respectively, are used herein accordingly. Typically, CFPS is performed in a CFPS reaction mixture; a CFPS reaction mixture typically comprises a CFPS buffer and a cell free cellular extract / lysate (for translation and / or transcription). Instead of a cell free cellular extract / lysate, also respective reconstituted components may be comprised (see below for further details). In general, the meaning of the term “CFPS reaction mixture” is likewise well known in the art; and is, for example, also evident from Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., Rasor loc. cit., WO-A1 2022 / 160049, Norouzi loc. cit. and US-A1 2005 / 0202480 (if not explicitly described herein differently or contradicted by context). The technical meaning of expression template (also termed just template herein), particularly in the context of CFPS and CFPS systems, is also known in the art; and is, for example, evident from WO-A12022 / 160049, Norouzi loc. cit., US-A12005 / 0202480, Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., and Rasor loc. cit. If not explicitly described in a different manner or contradicted by context, the term “template” is used herein accordingly. Typically, a template is a nucleic acid molecule (DNA or RNA molecule) which encodes a POI; and on the basis of which a POI is expressed (by the translational and / or transcriptional l machinery; for example in the context of a cellular system, or, preferred herein, in the context of a CFPS system). Typically, a DNA template is transcribed into RNA (typically mRNA); by the transcriptional machinery (including transcription initiation, elongation, termination). An RNA template (typically mRNA) is translated into a (poly)peptide or protein (POI) by the translational machinery (including translation initiation, elongation, termination). In the context of the invention, template covers both, a DNA template (for transcription), and an (m)RNA template (for translation). A template in accordance with the invention may thus be a template for transcription or a template for translation. An (m)RNA resulting from transcription on the basis of a template for transcription may constitute / act as a template for translation (for example in transcription / translation cell-free systems). An (expression) template / nucleic acid molecule in accordance with the invention is envisaged to comprise all components necessary for (m)RNA and / or protein expression (particularly in CFPS and CFPS systems, respectively). A template / nucleic acid molecule in accordance with the invention is thus envisaged to comprise a nucleotide sequence coding for a POI. Typically, a template in accordance with the invention comprises a (DNA) expression cassette for a POI. A (DNA) expression cassette typically comprises (in the 5’- to 3’-direction) a promoter, an RBS, a GOI and a terminator. In some particular templates / nucleic acid molecules of the invention, a terminator may not be present; transcription would stop only at the 3´-end of the template in that particular case. The (expression) template / nucleic acid molecule of the invention may comprise a DNA sequence coding for an (m)RNA which encodes a POI; or it may comprise an (m)RNA which encodes a POI. If not explicitly indicated otherwise or contradicted by context, (expression) template refers to the entire nucleic acid molecule of the invention; including, for example, the 5’-terminal and / or 3’-terminal protection group(s), a promoter sequence, an RBS, a start codon, a GOI, (optionally) a tag-encoding sequence, a stop codon and / or (optionally) a (transcription) terminator / terminator sequence. In case the template / nucleic acid molecule of the invention is an (m)RNA, it includes, for example, an RBS, a start codon, a GOI, (optionally) a tag-encoding sequence, a stop codon, and / or (optionally) a terminator sequence; and (optionally) (a) strong 3’-terminal protection group(s). In any case, a template / nucleic acid molecule of the invention, is envisaged to be suitable for CFPS and in CFPS systems. Based on the common general knowledge and the teaching of the invention, the skilled person is readily able to establish such suitable templates / nucleic acid molecules. The skilled person also knows the relevant components to be comprised therein (e.g., as mentioned, a 5’-terminal and / or 3’-terminal protection group(s) (5´ Tus and 3´ LacO binding site(s)), a promoter sequence, an RBS, a start codon, a GOI, (optionally) a tag-encoding sequence, a stop codon, and / or (optionally) a (transcription) terminator / terminator sequence). Although the predominantly preferred nucleic acid molecules of the invention comprise the Tus binding site(s) at the 5’-terminus and the LacO binding site(s) at the 3’-terminus ((5´)ter / (3´)lacO design), nucleic acid molecules which comprise the LacO binding site(s) at the 5’-terminus and the Tus binding site(s) at the 3’- terminus (5´)lacO / (3´)ter design are, in principle, also within the scope of the invention (however, in the context of less preferred embodiments). If not explicitly indicated otherwise or contradicted by context, a DOI in the context of the invention refers to all components of a template / nucleic acid molecule, with the exception of the 5’-terminal and / or 3’-terminal protection group(s). For example (in case of a DNA), a DOI refers to a promoter sequence, an RBS, a start codon, a GOI, (optionally) a tag-encoding sequence, a stop codon, and / or (optionally) a (transcription) terminator / terminator sequence. In the context of the invention, the term DOI is also meant to cover the respective components of a transcribed (m)RNA (e.g. an RBS, a start codon, a GOI, (optionally) a tag-encoding sequence, a stop codon, and / or (optionally) a terminator sequence). The DOI is envisaged to encode a (poly)peptide / protein to be synthesized (by CFPS / in a CFPS system), i.e. a protein of interest (POI). The DOI may comprise (a) (further) functional sequence(s). In case the template / nucleic acid molecule is DNA, a functional sequence may be a functional DNA sequence. Functional in this respect means that a (biological) function is exhibited. In one aspect, functional (DNA) sequence is meant to include a (DNA) sequence that is transcribed and / or bound by particular proteins or RNA molecules (for example during transcription (and / or translation)). Non-limiting examples of functional (DNA) sequences include a gene (e.g GOI), a regulatory sequence (e.g. a promoter or a terminator), a splice site, a binding site, (a) primer sequence(s), (an) aptamer sequence(s), and the like. In the context of the present invention, expression is a predominant (biological) function of a functional sequence. The meaning of expression covers gene expression (transcription of a gene resulting in (m)RNA), transcription (resulting in (m)RNA), (m)RNA expression (resulting in (m)RNA upon gene expression / transcription of a gene), protein or (poly)peptide (POI) expression (resulting from translation; or from translation proceeded by gene expression / transcription). A predominant functional sequence in the context of the present invention is a coding nucleotide sequence, in particular a DNA sequence, which comprises an expression cassette (for example for a POI to be synthesized by CFPS / in a CFPS system). A (DNA) expression cassette may typically comprise (in the 5’- to 3’-direction) a promoter sequence, an RBS, a start codon, a GOI, (optionally) a tag-encoding sequence, a stop codon, and / or (optionally) a (transcription) terminator / terminator sequence. In general, the meanings of “expression cassette” and “expression cassette for a POI”, respectively, are well known in the art (cf., for example, Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., and Rasor loc. cit.). Typically, an expression cassette comprises (in this order) a promotor sequence (e.g., a bacterial promotor or a promoter functional in bacteria, like a T7 promotor), an RBS / RBS sequence, a start codon, the nucleic acid sequence which encodes the POI (also termed GOI), a stop codon, (optionally) a terminator sequence (e.g., a bacterial terminator or a terminator functional in bacteria, like a T7 terminator). Certain variations of this typical organization of an expression cassette are within the scope of the invention. Particular examples of such variations are described herein elsewhere. For example, in a specific (non- limiting) expression cassette of the invention, and in the respective nucleic acid construct, a terminator may not be present (transcription would stop only at the 3’-end of the nucleic acid construct in that particular case). The GOI as such is meant to be a coding sequence for a respective POI (including the start and stop codon; optionally including a tag-encoding sequence (in frame)). The term GOI thus also means the open reading frame encoding for the respective POI (optionally including a tag); the open reading frame is flanked by the start and stop codon, respectively). In the context of the invention, the term GOI is meant to cover both, POI-encoding DNA and POI-encoding (m)RNA; and the respective components (i.e. open reading frame / GOI (optionally including a tag- encoding sequence) flanked by / including the start and stop codons). The meaning of GOI as such in accordance with the invention does not include the other components of the template / nucleic acid molecule of the invention, or the other components of the DOI; e.g. (a) 5’-terminal and / or (a) 3’-terminal protection group(s), a promoter sequence, an RBS, and / or a (transcription) terminator / terminator sequence. The skilled person will acknowledge that, within the template / nucleic acid molecule of the invention, the relevant functional components (e.g. 5’-terminal and / or 3’-terminal protection group(s), promoter sequence, RBS, start codon, GOI, (optionally) tag-encoding sequence, stop codon, and / or (optionally) (transcription) terminator / terminator sequence) are operatively linked to each other. This means that they are linked to each other so that they exhibit their respective (biological) function. For example, a promoter is operatively linked (in an upstream position) to an open reading frame / GOI so that it actually promotes the expression / transcription of the open reading frame / GOI (e.g. (m)RNA synthesis by a DNA- dependent RNA polymerase). Further, in, for example, a linear DNA molecule of the invention, one or more Tus binding site(s) at the 5’-terminus, a segment comprising a DOI and one or more LacI binding site(s) at the 3’-terminus are operatively linked in the in the 5’- to 3’-direction. This means that the one or more Tus and LacI binding site(s) protect the DOI (from (exo)nuclease degradation). Based on the common general knowledge and the teaching provided herein, the skilled person is readily in the position to operatively link the (functional) components of the template / nucleic acid molecule of the invention. In this context, the skilled person can, for example, rely on the herein-provided teaching, including the respective linker sequences (e.g. DNA or RNA linker sequences), like the buffer regions disclosed and described herein elsewhere. Illustrative, non-limiting examples of templates and nucleic acid molecules of the invention, and of the respective (functional) components, are depicted in Fig.6. In general, the technical meanings of the terms Tus and Tus binding site, and LacI and LacI binding site, are well known in the art (see, for example Berghuis, Crit. Rev. Biochem. Mol. Biol. 53, 2018, 49-63; WO-A12022 / 160049; Norouzi, ACS Synth Biol. 10(7), 2021, 1615-24; US-A1 2005 / 0202480; Jameson, J. Biol. Chem.297(6), 2021, 1-14); and these terms are used herein accordingly (unless explicitly indicated otherwise or contradicted by context). The “Tus-ter” E. coli DNA replication termination system is, for example, described in Berghuis loc.cit., WO-A1 2022 / 160049, Norouzi loc.cit. The “Tus-ter” system has homologues across many y- proteobacterial strains (Galli, Sci. Rep. 9, 2019, 1-11). The native “Tus-ter” system in E. coli involves a protein module, the Tus protein (or simply Tus), and a 23 base pair cognate DNA sequence module (Tus binding site), the ter sequence (or simply ter). Tus and ter have a remarkable equilibrium binding constant (KD) of 3.4 x 10-13M (Galli loc.cit.). In vivo, the high- affinity binding of Tus to the ter sequence strongly inhibits the progress of helicase-containing complexes towards any DNA sequence preceding the ter site (Gottlieb, Crit. Rev. Biochem. Mol. Biol.53, 2018, 49-63; Lee, PNAS USA 86, 1989, 9104-8), even in eukaryotic systems. As such, the Tus-ter system has been proposed as a system to regulate replication fork arrest and can be utilized for disrupting DNA replication. The E. coli “LacI-lacO” system is another example for binding of a repressor (LacI) to an operator (lacO). The E. coli “LacI-lacO” system is, for example, described in US-A12005 / 0202480 and Jameson loc.cit. In vivo, in a situation where no lactose, or no lactose analog, is present, transcription of the Lac operon is blocked by the Lac repressor (LacI), a 37-kDa protein with high affinity for a specific DNA sequence (lacO), that is to say the Lac operator. The Lac repressor is synthesized by the LacI gene present in the E. coli genome. The nucleotide sequence of the operator site (lacO) shows a nearly perfect inverted repeat, indicating that the DNA in this region has an approximate twofold axis of symmetry. Symmetry in the operator site (lacO) usually corresponds to symmetry in the repressor protein (LacI) that binds the operator site. In the E. coli cell, the LacI (in the absence of lactose, or a lactose analog (like IPTG)) binds to lacO and blocks transcription. LacI can exist as a dimer of 37 kDa subunits, and two dimers often come together to form a tetramer. In the absence of lactose, or a lactose analog, LacI binds very tightly and rapidly to lacO. When LacI is bound to DNA in vivo, it prevents bound RNA polymerase from locally unwinding the DNA to expose the bases that will act as the template for the synthesis of the RNA strand. LacI binds 4x106times as strongly to operator sequence (lacO) as it does to random sites in the genome. This high degree of selectivity allows the LacI to find the lacO efficiently, even with a large excess (4.6x106) of other sites within the E. coli genome. The dissociation constant for the LacI / lacO complex is approximately 0.1 pM (10-13M). The rate constant for association (1010M-1s-1) is also strikingly high. Without being bound by theory, this indicates that the repressor finds the operator by diffusing along a DNA molecule corresponding to a one-dimensional search rather than encountering it from the aqueous medium corresponding to a three-dimensional search. In principle, a nucleic acid molecule of the invention may be in a form where one or more Tus is / are bound to the 5’ Tus binding site(s) and / or one or more LacI is / are bound to the 3’ LacI binding site(s); or a nucleic acid molecule of the invention may be in a form where no Tus is bound to a 5’ Tus binding site and / or no LacI is bound to a 3’ LacI binding site. Thus, a nucleic acid molecule of the invention may comprise one or more Tus bound to the 5’ Tus binding site(s) and / or one or more LacI bound to the 3’ LacI binding site(s); or a nucleic acid molecule of the invention may not comprise Tus bound to a 5’ Tus binding site and / or no LacI bound to a 3’ LacI binding site. During CFPS and in CFPS systems, it is preferred that one or more Tus is / are bound to the 5’ Tus binding site(s) and / or one or more LacI is / are bound to the 3’ LacI binding site(s). Most preferably, during CFPS and in CFPS systems, all 5’ Tus binding sites and 3’ LacI binding sites of a nucleic acid molecule of the invention are envisaged to be saturated with Tus and LacI, respectively. Without being bound by theory, the more protecting proteins are bound to a nucleic acid molecule of the invention, the higher is the protecting efficacy. Thus, a nucleic acid molecule of the invention may comprise one, preferably two, or even more, Tus protein(s); or, preferably and, one, preferably two, or even more, LacI protein(s ); bound to the respective 5’- and 3´-binding site(s). It is one predominant advantage of the present invention that the Tus and LacI binding sites of the nucleic acid molecule of the invention can be protected with Tus and LacI, respectively, even in case no (exogeneous) Tus / LacI are added (to the respective CFPS system), and no Tus / LacI are additionally expressed (in the respective CFPS system). Without being bound by theory, the Tus and LacI binding sites of the nucleic acid molecule of the invention may be saturated in this respect with Tus and LacI as endogenously present in the respective CFPS system. The number of the Tus binding site(s) and / or the LacI binding site(s) to be comprised in the nucleic acid molecule of the invention is in principle, not particularly limited. At least one Tus binding site(s) and / or at least one LacI binding site(s), however, is to be comprised in the nucleic acid molecule of the invention. Two Tus binding sites and / or two LacI binding sites are preferred. Three, four or even more Tus binding sites and / or three, four or even more LacI binding site(s) may also be comprised. As mentioned, Tus proteins and LacI proteins are well known in the art; and likewise are proteins which bind to a Tus binding site and a LacI binding site, respectively. A non-limiting example of a Tus protein in accordance with the invention is an E. coli Tus protein, like a Tus protein which is (or comprises) the E. coli Tus as depicted in SEQ ID NO. 16. A non-limiting example of a LacI protein in accordance with the invention is an E. coli LacI protein, like a LacI protein which is (or comprises) the E. coli LacI as depicted in SEQ ID NO.17. A Tus protein in accordance with the invention may thus be or comprise an amino acid sequence as depicted in SEQ ID NO. 16. A LacI protein in accordance with the invention may thus be or comprise an amino acid sequence as depicted in SEQ ID NO.17. Other examples of Tus proteins / Tus-like proteins which may be employed in accordance with the invention may be selected from homologs of the Tus protein as depicted in SEQ ID NO.16 (also these homologs are envisaged to be capable of binding to a Tus binding site as defined herein). Other examples of LacI proteins / LacI-like proteins which may be employed in accordance with the invention may be selected from homologs of the LacI protein as depicted in SEQ ID NO.17 (also these homologs are envisaged to be capable of binding to a LacI binding site as defined herein). The origin, nature and amino acid sequence of a Tus protein / Tus-like protein and a LacI protein / LacI-like protein to be employed in accordance with the invention is not particularly limited. The Tus protein / Tus-like protein and a LacI protein / LacI-like protein must be capable of binding to a Tus binding site and LacI binding site, respectively; for example to a Tus binding site and LacI binding site, respectively, as defined herein. Once bound to the respective Tus and LacI binding site, a Tus and LacI in accordance with the invention must be capable of stabilizing and / or protecting the respective nucleic acid molecule of the invention, for example from degradation (by (exo)nucleases). Assays, means and methods for testing whether a given protein fulfils these properties are well known in the art (Norouzi loc. cit.; US-A12005 / 0202480; WO-A1 2022 / 160049; Galli loc.cit. etc.) and are also disclosed in the appended examples. Besides the particular Tus and LacI as depicted in SEQ ID NO.16 and 17, respectively, variant Tus proteins / Tus-like proteins and a LacI proteins / LacI-like proteins are also within the scope of the invention. These variants may, for example, comprise or consist of an amino acid sequence which shares ≥80%, ≥85%, ≥90%, ≥95%, ≥98% or ≥99% sequence identity with SEQ ID NO.16 and 17, respectively (in principle, the higher sequence identity values are preferred). Further variants may be provided which may even have a different amino acid sequence, but a similar (three-dimensional) structure so as to be capable of binding to a Tus binding site and LacI binding site, respectively; for example to a Tus binding site and LacI binding site, respectively, as defined herein (like the ter and lacO sequence(s)). Modelling tools as known in the art may be used in this respect. These may be based on artificial intelligence (AI) applications and the further variants may be AI-generated Tus binding site-binding- and LacI binding site-binding- proteins, respectively. Generative AI tools for protein design based on, for example, large language models may be used in this 15 aspect; e.g. those trained on protein sequence and functional data. One such tool is the one that is developed by the company https: / / www.cradle.bio / . Further respective guidance is also provided in the art (e.g. Model RFdiffusion (Watson, Nature 620, 2023, 1089–100); Model ProteinMPNN (Dauparas, Science 378(6615), 2022, 49-56). A Tus binding site in accordance with the invention may be, or may comprise, a ter sequence. The one, two, three (or more) Tus binding sites to be comprised in the templates / nucleic acid molecules of the invention may consist of, or may comprise, one, two, three (or more) ter sequences, respectively. ter sequences are the typical Tus binding sites, and are well known in the art (e.g. Norouzi loc. cit.; WO-A12022 / 160049; Jameson loc. cit.). Several particular ter sequences are known. Examples are terA (as, for example, depicted in SEQ ID NO.1), terB (as, for example, depicted in SEQ ID NO.3), terC (as, for example, depicted in SEQ ID NO.4), terD (as, for example, depicted in SEQ ID NO.5), terE (as, for example, depicted in SEQ ID NO.6). terA is a preferred ter sequence to be used in accordance with the invention. The origin, nature and particular nucleotide sequence of a ter sequence to be employed in accordance with the invention is not particularly limited. The ter sequence must be capable of exhibiting the respective (biological) function(s). In particular the ter sequence must be capable of serving in the protection and / or stabilization of the templates / nucleic acid molecules of the invention (for example protection from degradation (by (exo)nucleases)). In particular, it is envisaged that ter sequence acts as a Tus binding site. In other words, the nature (nucleotide sequence) of a ter sequence in accordance with the invention is envisaged to be so that the Tus protein / Tus-like protein to be employed in accordance with the invention (and as defined herein) is capable of binding to it (for example with a KD of 10-12to 10-14M or 1 – 5 x10-13M; e.g. (about) 3.9 x 10-13M; for example in an assay like the one as disclosed in Galli loc. cit. Once bound the ter sequence, it is envisaged that a Tus in accordance with the invention is capable of stabilizing and / or protecting the respective nucleic acid molecule of the invention; for example protecting from degradation (by (exo)nucleases). Assays, means and methods for testing whether a given ter sequence fulfils these properties are well known in the art (Norouzi loc. cit.; WO-A1 2022 / 160049; Galli loc.cit. etc.) and are also disclosed in the appended examples. Besides the particular ter sequences as exemplified herein, like terA (as, for example, depicted in SEQ ID NO.1), terB (as, for example, depicted in SEQ ID NO.3), terC (as, for example, depicted in SEQ ID NO.4), terD (as, for example, depicted in SEQ ID NO.5), terE (as, for example, depicted in SEQ ID NO.6), variant ter sequences are also within the scope of the invention. These variants may, for example, comprise or consist of a nucleotide sequence which shares ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥98% or ≥99% sequence identity with any of SEQ ID NOs.1, 3, 4, 5 and 6 (in principle, the higher sequence identity values are preferred). Nucleotide sequences which comprise a nucleotide sequence as depicted in SEQ ID NO. 7 (the “ter consensus sequence”) are also envisaged to be ter sequences in accordance with the invention (provided that they exhibit the respective (biological) function(s), like Tus binding). Variants of terA are preferred ter sequence variants to be used in accordance with the invention. These variants are envisaged to share a higher sequence identity with terA as compared to any other ter sequence, like terB, terC, terD, terE. All the herein disclosed and described ter sequences, as well as the herein defined ter sequence variants, are meant to be encompassed in the meaning of “ter sequence”. A LacI binding site in accordance with the invention may be, or may comprise, a lacO sequence. The one, two, three (or more) LacI binding sites to be comprised in the templates / nucleic acid molecules of the invention may consist of, or may comprise, one, two, three (or more) lacO sequences, respectively. lacO sequences are the typical LacI binding sites, and are well known in the art (e.g. US-A12005 / 0202480; Jameson loc. cit.). Several particular lacO sequences are known. Examples of lacO sequences are lacO1 (as, for example, depicted in SEQ ID NO. 10), lacO2 (as, for example, depicted in SEQ ID NO.14), lacO3 (as, for example, depicted in SEQ ID NO. 15), Gilbert (as, for example, depicted in SEQ ID NO. 13), Symmetric (as, for example, depicted in SEQ ID NO.12), pET (as, for example, depicted in SEQ ID NO.11). A further example of a particular lacO sequence to be used in accordance with the invention is or comprises a nucleotide sequence as depicted in SEQ ID NO.2 (termed “Insempra”). Another example of a particular lacO sequence to be used in accordance with the invention is or comprises a nucleotide sequence as depicted in SEQ ID NO.9 (termed “Core”). Insempra is a preferred lacO sequence to be used in accordance with the invention. The origin, nature and particular nucleotide sequence of a lacO sequence to be employed in accordance with the invention is not particularly limited. The lacO sequence must be capable of exhibiting the respective (biological) function(s). In particular the lacO sequence must be capable of serving in the protection and / or stabilizationof the templates / nucleic acid molecules of the invention (for example protection from degradation (by (exo)nucleases)). In particular, it is envisaged that lacO sequence acts as a LacI binding site. In other words, the nature (nucleotide sequence) of a lacO sequence in accordance with the invention is envisaged to be so that the LacI protein / LacI- like protein to be employed in accordance with the invention (and as defined herein) is capable of binding to it (for example with a dissociation construct in the range of 10-12to 10-14M; e.g. (about) 10-13M; for example in an assay like the one as disclosed in Galli loc. cit.). Once bound to the lacO sequence, it is envisaged that a LacI in accordance with the invention is capable of stabilizing and / or protecting the respective nucleic acid molecule of the invention; for example protecting from degradation (by (exo)nucleases). Assays, means and methods for testing whether a given lacO sequence fulfils these properties are well known in the art (US-A1 2005 / 0202480; Jameson loc. cit. etc.) and are also disclosed herein and in the appended examples. Besides the particular lacO sequences as exemplified herein, like lacO1 (as, for example, depicted in SEQ ID NO.10), lacO2 (as, for example, depicted in SEQ ID NO.14), lacO3 (as, for example, depicted in SEQ ID NO.15), Gilbert (as, for example, depicted in SEQ ID NO. 13), Symmetric (as, for example, depicted in SEQ ID NO.12), pET (as, for example, depicted in SEQ ID NO. 11), Insempra (as, for example, depicted in SEQ ID NO.2), Core (as, for example, depicted in SEQ ID NO.9), variant lacO sequences are also within the scope of the invention. These variants may, for example, comprise or consist of a nucleotide sequence which shares ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥98% or ≥99% sequence identity with any of SEQ ID NOs. 2, 9, 10, 11, 12, 13, 14 and 15 (in principle, the higher sequence identity values are preferred). Nucleotide sequences which comprise a nucleotide sequence as depicted in SEQ ID NO.8 (the “lacO consensus sequence”), or which consist or comprise a nucleotide sequence as depicted in SEQ ID NO.8 (“Core”) are also envisaged to be lacO sequences in accordance with the invention (provided that they exhibit the respective (biological) function(s), like LacI binding). Variants of Insempra are preferred lacO sequence variants to be used in accordance with the invention. These variants are envisaged to share a higher sequence identity with Insempra as compared to other lacO sequence, like lacO1, lacO2, lacO3, Gilbert, Symmetric, pET. All the herein disclosed and described lacO sequences, as well as the herein defined lacO sequence variants, are meant to be encompassed in the meaning of “lacO sequence”. A template / nucleic acid molecule of the invention (in particular in the case of a DNA molecule) may comprise a promoter / promoter sequence. Typically, a promotor / promoter sequence indicates the start of the transcription; it defines the start of the transcriptional unit of a gene. Further, a promotor / promoter sequence is typically a region of DNA upstream of a gene where the relevant proteins of the transcriptional machinery (such as RNA polymerase and transcription factors) bind to initiate transcription of the gene. The resulting transcription produces an RNA molecule (such as mRNA). Promoters in general, and as used in the context of CFPS / CFPS systems, are well known in the art. Promoters which are particularly suitable in accordance with the invention are those promoters which initiate transcription in the respective CFPS system, i.e. to which the relevant proteins of the transcriptional machinery (such as RNA polymerase and transcription factors) present in the respective CFPS system bind and initiate transcription. For example, in case the CFPS system is of bacterial origin / derived from bacterial cells, the promoter may be a bacterial promoter / active in bacteria, in case the CFPS system is of archaebacterial origin / derived from archaebacterial cells, the promoter may be an archaebacterial promoter / active in archaebacteria, in case the CFPS system is of eukaryotic (e.g. plant or animal) origin / derived from eukaryotic (e.g. plant or animal) cells, the promoter may be an eukaryotic (e.g. plant or animal) promoter / active in eukaryotes, and so on. Non-limiting examples of typical bacterial promoters are / can be sigma factors (e.g. σ70 (RpoD), σ19 (FecI), σ24 (RpoE), σ28 (RpoF / FliA), σ32 (RpoH), σ38 (RpoS), σ54 (RpoN)). Non-limiting examples of typical eukaryotic promoters are / comprise TATA boc, GC box, CAAT box. A prominent promoter used in CFPS and CFPS systems, respectively, is the T7 promoter of the T7 bacteriophage expression system (used in combination with the (exogenously added or endogeneously co-expressed) T7 RNA polymerase). This promoter is a preferred promoter to be used in the context of the invention. However, alternatives to this promoter are also available; e.g. the Trc, T3, K11, and SP6 promoters. Based on the common general knowledge and the teaching provided herein, the skilled person is readily in the position to choose suitable promoters to be comprised in the templates / nucleic acid molecules of the invention. Typically, in the templates / nucleic acid molecules of the invention, a promoter is the first (functional) component of the DOI (in the 5’ to 3’ direction). As such, the promoter is located upstream of the RBS (or other 5´UTR / other translation initiation / ribosome recruitment sequence), and GOI (and downstream of the one or more Tus binding sites). A template / nucleic acid molecule of the invention (in particular in the case of a DNA molecule) may comprise a terminator / terminator sequence. Typically, a terminator / terminator sequence indicates the end of the transcription; it defines the end of the transcriptional unit of a gene. Further, a terminator / terminator sequence typically initiates the process of releasing the synthesized (m)RNA from the transcription machinery (RNA polymerase etc.). Terminators in general, and as used in the context of CFPS / CFPS systems, are well known in the art. Terminators which are particularly suitable in accordance with the invention are those terminators which terminate transcription in the respective CFPS system, i.e. which release the synthesized (m)RNA from the transcription machinery (RNA polymerase etc.) present in the respective CFPS system. For example, in case the CFPS system is of bacterial origin / derived from bacterial cells, the terminator may be a bacterial terminator / active in bacteria, in case the CFPS system is of archaebacterial origin / derived from archaebacterial cells, the terminator may be an archaebacterial terminator / active in archaebacteria, in case the CFPS system is of eukaryotic (e.g. plant or animal) origin / derived from eukaryotic (e.g. plant or animal) cells, the terminator may be an eukaryotic (e.g. plant or animal) terminator / active in eukaryotes, and so on. Examples of typical bacterial terminators are Rho-dependent terminators (sequence in DNA which transcribes into signal sequence in the mRNA to which the rho-protein binds and signals for cleavage) and Rho-independent terminators (sequence in DNA which transcribes into a stable RNA secondary structure; like hairpin loop (GC-rich hairpin), also known as stem-loop; this loop is typically followed by multiple uracil nucleotides (U-rich tract)). Typical eukaryotic terminators include (a) polyadenylation signal(s). A prominent terminator used in CFPS and CFPS systems, respectively, is the T7 terminator of the T7 bacteriophage expression system. This terminator is a preferred terminator to be used in the context of the invention. Alternatives to this terminator are also available. Based on the common general knowledge and the teaching provided herein, the skilled person is readily in the position to choose suitable terminators to be comprised in the templates / nucleic acid molecules of the invention. Typically, in the templates / nucleic acid molecules of the invention, a terminator is the last functional component of the DOI (in the 5’ to 3’ direction). As such, the terminator is located downstream of the GOI (optionally including a tag-encoding sequence) (and upstream of the one or more LacI binding sites). A template, nucleic acid molecule, DOI, expression cassette in accordance with the invention may, or may not, comprise a terminator / terminator sequence. In case a terminator / terminator sequence is comprised, also the (m)RNA resulting from transcription of the template, nucleic acid molecule, DOI, expression cassette, respectively, may contain a terminator sequence (downstream of the GOI (optionally including a tag-encoding sequence)). Such a terminator sequence may be (a part of) a rho-protein-binding signal sequence or a stem- loop sequence (see above). In case a terminator / terminator sequence is not comprised, transcription may end only at the end of the template / nucleic acid molecule (i.e. after the one or more LacI binding sites). The resulting (m)RNA may thus comprise also a 3´-terminal RNA sequence which corresponds to the one or more 3´-terminal LacI binding sites (of the respective DNA template). This 3´-terminal RNA sequence may be, or may comprise, one or more lacO sequence(s). A template / nucleic acid molecule of the invention may comprise a 5′ untranslated region (5′UTR; sometimes also termed transcript leader or leader RNA). A 5′UTR typically is a region of an mRNA that is located from transcription start site till translation start site, for example (directly) upstream of the start codon (and downstream of a promoter; translation may start from an AUG start codon or from a non-AUG start codon). A 5′UTR may be involved in the regulation of translation of a transcript (different mechanisms underly this regulation in viruses, prokaryotes and eukaryotes). In many organisms the 5′UTR forms a secondary structure to regulate translation. In principle, unless explicitly indicated otherwise or contradicted by context, the term “5′UTR” herein refers to all kind of 5′UTRs.5′UTRs, in general, and which may be employed in the context of CFPS / CFPS systems, are well known in the art; and may also be employed accordingly in the context of the invention. The kind of 5′UTR to be employed in accordance with the invention is not particularly limited. Suitable 5′UTRs can readily be provided by the skilled person based on the common general knowledge and the teaching provided herein. The kind of 5′UTR which is particularly suitable in accordance with the invention is so that sufficient / high expression / synthesis of the POI occurs, for example, in the respective CFPS system which may be used. A 5′UTR to be employed in accordance with the invention may consist of / constitute or comprise an RBS / RBS sequence, preferably an RBS / RBS sequence as disclosed and defined herein (see below for details). Non-limiting particular examples of 5′UTR sequences are provided herein elsewhere; and are depicted in SEQ ID NOs. 55 to 66. The skilled person is readily in the position to choose suitable / respective 5′UTRs. Based on the common general knowledge and the teaching provided herein, the skilled person is readily in the position to choose suitable 5′UTRs to be comprised in the templates / nucleic acid molecules of the invention. Typically, in the templates / nucleic acid molecules of the invention, a 5′UTR is located (directly) upstream of the start codon for the POI (and downstream of a promoter). A template / nucleic acid molecule of the invention may comprise an RBS / RBS sequence. An RBS / RBS sequence indicates the initiation of translation, and the recruitment of the ribosome, respectively. Typically, “RBS / RBS sequence” as such refers to bacterial sequences / expression systems. However, analogous internal ribosome entry sites (IRES) have also been described in eukaryotic mRNAs or in mRNAs of viruses that infect eukaryotes. Further, ribosome recruitment in eukaryotes is usually mediated by the 5'-cap present on eukaryotic mRNAs. In principle, unless explicitly indicated otherwise or contradicted by context, the term “RBS” or “RBS sequence” herein refers to all kind of translation initiation / ribosome recruitment; although this may not be based on a particular sequence present in the (m)RNA, and on DNA level in the respective coding DNA; like in typical eukaryotic systems. When used in the context of procaryotic / bacterial systems, the terms “RBS” or “RBS sequence” are used according to the respective particular meaning in the procaryotic / bacterial context. RBS / RBS sequences, and translation initiation / ribosome recruitment, respectively, in general, and to be employed in the context of CFPS / CFPS systems, are / is well known in the art; and may also be employed accordingly in the context of the invention. The kind of RBS / RBS sequences, and of translation initiation / ribosome recruitment, respectively, to be employed in accordance with the invention is not particularly limited. Suitable RBS / RBS sequences, and translation initiation / ribosome recruitment sequences, respectively, can readily be provided by the skilled person based on common general knowledge and the teaching provided herein. In this context, respective online calculators may be used, like, for example, the Salis calculator (Salis, Nature Biotechnology 27, 2009, 946–50; doi:https: / / doi.org / 10.1038 / nbt.1568). The kind of RBS / RBS sequences, and of translation initiation / ribosome recruitment, respectively, which is particularly suitable in accordance with the invention, is so that translation initiation / ribosome recruitment can occur in the respective CFPS system which may be used. For example, in case the CFPS system is of bacterial origin / derived from bacterial cells, the RBS / RBS sequences, and of translation initiation / ribosome recruitment, respectively, may be of bacterial origin / active in bacteria; in case the CFPS system is of archaebacterial origin / derived from archaebacterial cells, the RBS / RBS sequences, and of translation initiation / ribosome recruitment, respectively, may be of archaebacterial origin / active in archaebacteria; in case the CFPS system is of eukaryotic (e.g. plant or animal) origin / derived from eukaryotic (e.g. plant or animal) cells, the translation initiation / ribosome recruitment, and respective sequences, may be of eukaryotic (e.g. plant or animal) origin / active in eukaryotes, and so on. The RBS in prokaryotes usually is a region upstream of the start codon. This region of the mRNA typically has the consensus 5'-AGGAGG- 3', also called the Shine-Dalgarno (SD) sequence. The complementary sequence (CCUCCU), called the anti-Shine-Dalgarno (ASD), is typically contained in the 3’ end of the 16S region of the smaller (30S) ribosomal subunit. Upon encountering the Shine-Dalgarno sequence, the ASD of the ribosome base pairs with it, after which translation is initiated. Variations of the 5'- AGGAGG-3' sequence have been found in archaebacteria as highly conserved 5′-GGTG-3′ regions; 3 to 12 basepairs (e.g.5 basepairs) upstream of the start site. Ribosome recruitment in eukaryotes usually happens when eukaryote initiation factors, like elF4F and poly(A)-binding protein (PABP), recognize the 5'-capped mRNA; and recruit the 43S ribosome initiation complex at the 5’ cap. Translation initiation happens following recruitment of the ribosome and scanning until it reaches the start codon (underlined) found within the Kozak consensus sequence, A / GxxAUGG (-3 position may be A / G and +4 position may be a G (+1 is designated to A in AUG)). Since the Kozak sequence itself is not involved in the direct binding of the ribosome to the mRNA, it is not considered an RBS in the stricter sense. Further, as mentioned eukaryotic ribosomes may also bind to transcripts in a 5' cap-independent mechanism, i.e. unlike the one involving the 5' cap, namely at an IRES sequence. This process is not dependent on the full set of translation initiation factors (although this depends on the specific IRES). This process is commonly found in the translation of viral mRNA. Typically, an RBS / RBS sequence (or a translation initiation / ribosome recruitment sequence) is comprised in or constitutes a 5´UTR in accordance with the invention (see also above). Non-limiting particular examples of (sequences comprising) RBS / RBS sequences are provided herein elsewhere; and are depicted in SEQ ID NOs. 55 to 66. These may be used together with the respective GOIs. Based on the common general knowledge and the teaching provided herein, the skilled person is readily in the position to choose suitable RBS / RBS sequences (or translation initiation / ribosome recruitment sequences) to be comprised in the templates / nucleic acid molecules of the invention. Typically, in the templates / nucleic acid molecules of the invention, an RBS / RBS sequence (or a translation initiation / ribosome recruitment sequence) is located upstream of (the start codon in) the GOI (and downstream of the promoter). A template / nucleic acid molecule of the invention may comprise a nucleic acid sequence encoding a leader peptide. A leader peptide may be located directly downstream of the start codon. A leader peptide may be involved in the regulation of the expression of the POI In principle, unless explicitly indicated otherwise or contradicted by context, the term “leader peptide” herein refers to all kind of leader peptides. Leader peptides in general, and to be employed in the context of CFPS / CFPS systems, are well known in the art; and may also be employed accordingly in the context of the invention. The kind of leader peptides to be employed in accordance with the invention is not particularly limited. Suitable leader peptides can readily be provided by the skilled person based on the common general knowledge and the teaching provided herein. The kind of leader peptides which is particularly suitable in accordance with the invention is so that sufficient / high expression / synthesis of the POI occurs, for example, in the respective CFPS system which may be used. It is preferred that a leader peptide is sufficiently / highly expressed, and leads to sufficient / high expression / synthesis of the POI, respectively. The skilled person is readily in the position to choose suitable / respective leader peptides. A preferred nucleotide sequence to be used in in accordance with the present invention encodes a leader peptide comprising (or consisting of) nGFP, like the nGFP leader peptide with its RBS (e.g. from pJL1). The nGFP leader peptide may be (or comprise) the first 20 amino acids of sfGFP, the amino acid sequence as depicted in SEQ ID NO.28, or the amino acid sequence as encoded by SEQ ID NO.18. The coding nucleotide sequence may accordingly be (or comprise) the first 60 bases of the nucleotide sequence encoding sfGFP, or of the nucleotide sequence as depicted in SEQ ID NO. 43. Based on the common general knowledge and the teaching provided herein, the skilled person is readily in the position to choose suitable coding nucleotide sequences to be comprised in the templates / nucleic acid molecules of the invention. Typically, in the templates / nucleic acid molecules of the invention, a nucleic acid sequence encoding a leader peptide would be located (directly) downstream of the start codon and upstream of the POI-encoding sequence. A template / nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a self-cleavage peptide. In general, the technical meaning of self-cleavage peptide is known in the art. Self-cleavage peptides are, for example, described in Szymczak-Workman (Cold Spring Harb Protoc 2, 2012, 199-204), US 10738325, Kim (PLoS One 6(4), 2011, e18556), Szymczak (Nature Biotech.22, 2004, 589-94), Tan (Biologicals 38, 2010, 586-93), Liu (Scientific Reports 7(1), 2017, 2193), Karuna and Sudipto (World Scientific, 2010, 51–52; ISBN 978-981- 4464-89-5), Luke (The Journal of General Virology 89(4), 2008, 1036-42), Yang (Frontiers in Microbiology 8, 2017, 1373), Donnelly (The Journal of General Virology 82(5), 2001, 1013–25), https: / / en.wikipedia.org / wiki / 2A_self-cleaving_peptides. If not explicitly described in a different manner or contradicted by context, the term “self-cleavage peptide” is used herein accordingly. Typically, self-cleavage peptides may be of viral origin. They are typically used in the design and construction of multicistronic vectors to be used to express multiple proteins / peptides from a single ORF. Within a peptide / protein chain, a self-cleavage peptide leads to the separation of the peptide / protein chain during translation at the site of the self- cleavage peptide (typically at the end of the self-cleavage peptide; and upstream of a proline (P) residue which may be comprised at the end of / in the self-cleavage peptide; the resulting first / upstream peptide / protein typically comprises the self-cleavage peptide sequence (without the proline residue) and the resulting second / downstream peptide / protein typically comprises the proline residue as the N-terminal amino acid residue). A preferred self-cleavage peptide in accordance with the invention may thus contain a proline residue (P) at its C-terminus. In principle, unless explicitly indicated otherwise or contradicted by context, the term “self- cleavage peptide” herein refers to all kind of self-cleavage peptide. The kind of self-cleavage peptide to be employed in accordance with the invention is not particularly limited. Suitable self-cleavage peptides can readily be provided by the skilled person based on the common general knowledge and the teaching provided herein. A self-cleavage peptide to be used in accordance with the present invention may be a 2A peptide. A 2A peptide is a type of self- cleaving peptide as encoded by some RNA viruses, such as picornaviruses.2A peptides typically comprise 18 to 22 amino acid residues.2A peptides function by promoting the ribosome to skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the downstream peptide (see, for example, Kim, PLoS One 6(4), 2011, e18556). The “cleavage” occurs between the glycine and proline residues found on the C-terminus of the 2A peptide. Exemplary 2A peptides include, but are not limited to, the 2A peptides encoded by thosea asigna virus (TaV), equine rhinitis A virus (ERAV), porcine teschovirus-1 (PTV1) and foot and mouth disease virus (FMDV)). A self-cleavage peptide to be used in accordance with the present invention may comprise the amino acid sequence as depicted in SEQ ID NO. 19 (the “2A consensus sequence”; including the C-terminal P) or as depicted in SEQ ID NO. 20 (the “2A core sequence”; including the C-terminal P). A preferred self-cleavage peptide to be used in accordance with the present invention may be (or comprise) the P2A self-cleavage peptide (for example as depicted in SEQ ID NO.21 (more preferred) or in SEQ ID NO.22 (most preferred); or as encoded by SEQ ID NOs: 29 (more preferred) or by SEQ ID NO.30 (most preferred)). Other examples of self-cleavage peptides are the F2A, E2A or T2A self-cleavage peptides (for example as depicted in SEQ ID NOs: 23, 24 or 25, respectively, or as encoded by SEQ ID NOs: 31, 32 or 33, respectively). A self-cleavage peptide to be used in accordance with the invention may comprise a Gly-Ser-Gly (GSG) motif at the N-terminus (cf. SEQ ID NO. 22). This motif may improve cleavage efficiency. Based on the common general knowledge and the teaching provided herein, the skilled person is readily in the position to choose suitable self-cleavage peptides to be encoded by respective nucleotide sequences which may be comprised in the templates / nucleic acid molecules of the invention. A (nucleotide sequence encoding a) self-cleavage peptide may advantageously be used in accordance with the invention, for example for multicistronic expression / synthesis of two or more POIs from a single ORF. Typically, in the templates / nucleic acid molecules of the invention, a nucleotide sequence encoding a self-cleavage peptide is located downstream of the promoter, and downstream of the (optionally comprised) leader peptide-encoding sequence, and / or downstream of a first POI-encoding sequence; and upstream of a second POI-encoding sequence / GOI, respectively. A template / nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a tag. In general, the technical meaning of a tag in protein / peptide synthesis is known in the art. If not explicitly described in a different manner or contradicted by context, the term “tag” is used herein accordingly. Typically, a tag is a peptide tag, i.e. a (short) amino acid stretch (e.g. about 5 to 100, 10 to 80, 10 to 50, 30 to 50 or 30 to 60 amino acid residues). The tag may act as a marker and / or as a binding tag of a POI to be synthesized. The tag may be an affinity tag, a signal tag or an epitope tag. The tag may be suitable for / useful in quantification of a POI (e.g. during / after CFPS), monitoring the synthesis of a POI (e.g. during CFPS), assessing the level of expression of a POI (e.g. during / after CFPS) and / or determining the concentration of the POI (e.g. during / after CFPS). The quantification, monitoring, assessing, and determining may be in real-time (during CFPS). Respective (peptide) tags may be suitable for / useful in, for example, fast and / or high-throughput CFPS. In principle, unless explicitly indicated otherwise or contradicted by context, the term “tag” or “peptide tag” herein refers to all kind of tags or peptide tags that can be used in the field of protein synthesis. The particular kind of tag to be employed in accordance with the invention is not particularly limited in this respect. Suitable tags can readily be provided by the skilled person based on the common general knowledge and the teaching provided herein. Non-limiting examples of tags which may be used in accordance with the present invention are Myc-tags, MBP-tags, HA-tags, His-tags and FLAG- tags and Strep-tags. In the context of a particular aspect of the invention, the tag may be a (Nano-)luciferase binding tag, in particular a (Nano-)luciferase binding peptide tag (like, for example, described in US10107800 and US10288605). An examplary Nanoluciferase binding peptide tag may be a HiBiT tag (as provided by Promega; see SEQ ID NO.26; and SEQ ID NO.34 for the respective coding sequence). A preferred example of a Nanoluciferase binding peptide tag may be the peptide tag as depicted in SEQ ID NO.27: (see SEQ ID NO.35 for the respective coding sequence (RNA; including the stop codon); see SEQ ID NO.97 for the respective coding sequence (DNA; including the stop codon). In the templates / nucleic acid molecules of the invention, a nucleotide sequence encoding a (peptide) tag may be located upstream or downstream of the GOI; typically, it is located downstream of the GOI. In the resulting peptide / protein, the tag may likewise be located upstream or downstream of the POI; typically downstream of the POI. A template / nucleic acid molecule of the invention may comprise a 3′ untranslated region (3′UTR). A 3′UTR typically is a region of an mRNA that is located (directly) downstream of the stop codon (and upstream of a terminator). In principle, unless explicitly indicated otherwise or contradicted by context, the term “3′UTR” herein refers to all kind of 3′UTRs.3′UTRs, in general, and which may be employed in the context of CFPS / CFPS systems, are well known in the art; and may also be employed accordingly in the context of the invention. The kind of 3′UTR to be employed in accordance with the invention is not particularly limited. Suitable 3′UTRs can readily be provided by the skilled person based on the common general knowledge and the teaching provided herein. The kind of 3′UTR which is particularly suitable in accordance with the invention is so that sufficient / high expression / synthesis of the POI occurs, for example, in the respective CFPS system which may be used. Non-limiting particular examples of 3′UTR sequences are provided herein elsewhere; and are depicted in sequences disclosed herein. The skilled person is readily in the position to choose suitable / respective 3′UTRs. Based on the common general knowledge and the teaching provided herein, the skilled person is readily in the position to choose suitable 3′UTRs to be comprised in the templates / nucleic acid molecules of the invention. Typically, in the templates / nucleic acid molecules of the invention, a 3′UTR is located (directly) downstream of the stop codon for the POI (and upstream of a terminator). In case no terminator / terminator sequence is comprised in the templates / nucleic acid molecules of the invention, a 3′UTR is typically located between the stop codon for the POI and the 3’ LacI binding site(s). Templates / nucleic acid molecules as defined herein, but which do not comprise a 3′UTR are also within the scope of the invention (less preferred). A template / nucleic acid molecule of the invention may comprise one or more buffer region(s). (A) buffer region(s) may be comprised between (sequences encoding) functional components of the template / nucleic acid molecule, or POI, of the invention; like the 5’ Tus binding site(s), the DOI or GOI, and / or the 3’ LacI binding site(s). Typically, (a) buffer region(s) may be between the 5’ Tus binding site(s) and the promoter and / or between the terminator and the 3’ LacI binding site(s). In case no terminator / terminator sequence is comprised, a 3’ UTR may be comprised between the GOI (optionally including the peptide tag) and the 3’ LacI binding site(s). A buffer region in the context of the invention is meant to be a nucleotide sequence stretch (especially DNA stretch). A buffer region, as such, may not be functional (e.g. not functional in expression regulation or not encoding a POI (or a (functional) part thereof)). However, a buffer region may facilitate / contribute to the function of (a) (neighbouring) functional component(s) (e.g. by avoiding spatial hindering between (two or more) (neighbouring) functional components). (A) buffer region(s) may also prevent direct exo-degradation of the functional parts (e.g. the promoter). In general, (a) suitable buffer region(s) can readily be provided by the skilled person and is / are known in the art. If not explicitly described in a different manner or contradicted by context, the term “buffer region” is used herein accordingly. In principle, unless explicitly indicated otherwise or contradicted by context, the term “buffer region” herein refers to all kind of buffer regions that can be used in the field of expression templates for protein synthesis (e.g. in CFPS). The particular kind of buffer region to be employed in accordance with the invention is not particularly limited in this respect. Suitable buffer regions can readily be provided by the skilled person based on the common general knowledge and the teaching provided herein. A template / nucleic acid molecule of the invention may comprise a 5’ buffer region (buffer region 1); i.e. a 5’ buffer region downstream of the 3’ end of the Tus binding site(s) (and upstream of (a promoter at) the 5’ end of the DOI). A template / nucleic acid molecule of the invention may comprise a 3’ buffer region (buffer region 2); i.e. a 3’ buffer region downstream of the 3’ end of the DOI. In case the template / nucleic acid molecule of the invention comprises a terminator, a 3’ buffer region (buffer region 2) may be upstream of the 5’ end of the LacI binding site(s) and downstream of the 3’ end of a terminator. In case the template / nucleic acid molecule of the invention does not comprise a terminator, a 3´UTR may be downstream of the 3’ end of the GOI (optionally including a tag) and upstream of the 5’ end of the lacL binding site(s). In case of such template / nucleic acid molecule, the 3’ buffer region (buffer region 2) would be transcribed to the RNA and become an extension (part) of the 3’ UTR. The buffer region 1 may consist of 0 to 200 base pairs (bp), preferably 70 to 150 bp; e.g. about 46 bp and / or about 55 bp, respectively. The buffer region 2 may consist of 0 to 500 bp, preferably 70 to 300 bp; e.g. about 193 bp. Non-limiting examples of buffer region sequences are given in the appended examples and sequences exemplified herein. A buffer region as comprised in the template / nucleic acid molecule in accordance with the invention may, for example, be a buffer region as comprised in SEQ ID NO.98 or 99, or a buffer region which is ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥98% or ≥99% identical to a buffer region as comprised in SEQ ID NO.98 or 99. The POI to be encoded / expressed by the template / nucleic acid molecule of the invention may be encoded / expressed as a single protein or single (poly)peptide, as a fusion protein or fusion (poly)peptide, or as a poly-protein or poly-(poly)peptide. A fusion protein / (poly)peptide, or a poly-protein / (poly)peptide may comprise, for example, two or more (e.g. three or four) proteins and / or (poly)peptides, respectively. The template / nucleic acid molecule of the invention may thus also be multicistronic; i.e. encoding two (or more; e.g. three or four) POIs. As such, the template / nucleic acid molecule of the invention may be suitable for multigene delivery; in particular for CFPS and in CFPS systems, respectively. In general, the kind of POI to be encoded / expressed / synthesized in accordance with the invention is not particularly limited. Any POI desired to be produced may be encoded / expressed / synthesized in accordance with the invention. The particular POIs as described herein (e.g. the exemplified POIs as encoded by SEQ ID NOs 43, 44, 45, 46, 47, 48, 49, 50, 54, 52, 53 and 54) are non-binding respective examples. The DOI as comprised in the nucleic acid molecule of the invention (for example when being (ds)DNA) may comprise in the 5’ to 3’ direction (a) a promotor sequence (active in bacteria; or in CFPS systems based on other cells or being artificial; e.g. the T7 promoter; see herein elsewhere for details); (b) a (DNA) sequence encoding an RBS / RBS sequence (or other 5´UTR / other translation initiation / ribosome recruitment sequence); and (c) a (DNA) sequence (GOI) encoding a POI; and (d) optionally, a (transcription) terminator / terminator sequence (active in bacteria; or in CFPS systems based on other cells or being artificial; e.g. the T7 terminator; see herein elsewhere for details). In one aspect of the invention, the DOI as comprised in the nucleic acid molecule of the invention (for example when being (ds)DNA) may comprise in the 5’ to 3’ direction (a) a promotor sequence (active in bacteria; or in CFPS systems based on other cells or being artificial; e.g. the T7 promoter; see herein elsewhere for details); (b) a (DNA) sequence encoding an RBS / RBS sequence (or other 5´UTR / other translation initiation / ribosome recruitment sequence); and (c) a (DNA) sequence encoding a (highly expressed) leader peptide; and (d) optionally, a (DNA) sequence encoding a self-cleavage peptide (one additional proline (P) may be encoded in downstream direction of said self-cleavage peptide); and (e) a GOI encoding a POI; and (f) optionally, a (transcription) terminator / terminator sequence (active in bacteria; or in CFPS systems based on other cells or being artificial; e.g. the T7 terminator; see herein elsewhere for details). In one aspect of the invention, the DOI as comprised in the nucleic acid molecule of the invention (for example when being (ds)DNA) may comprise in the 5’ to 3’ direction (a) a promotor sequence (active in bacteria; or in CFPS systems based on other cells or being artificial; e.g. the T7 promoter; see herein elsewhere for details); (b) a (DNA) sequence encoding an RBS / RBS sequence (or other 5´UTR / other translation initiation / ribosome recruitment sequence); and (c) optionally, a (DNA) sequence encoding a (highly expressed) leader peptide; and (d) a (DNA) sequence encoding a self-cleavage peptide (one additional proline (P) may be encoded in downstream direction of said self-cleavage peptide); and (e) a GOI encoding a POI; and (f) optionally, a (transcription) terminator / terminator sequence (active in bacteria; or in CFPS systems based on other cells or being artificial; e.g. the T7 terminator; see herein elsewhere for details). A 3´UTR may also be comprised in any of the above three DOIs ((directly) downstream of the GOI encoding a POI (as mentioned in item (c) and items (e), respectively)). What has been said above (or herein elsewhere), for example with respect to the DOI, nucleic acid molecule of the invention, promotor / promotor sequence, 5´UTR, RBS / RBS sequence, translation initiation / ribosome recruitment sequence, buffer region(s), leader peptide, self-cleavage peptide, 3´UTR and / or (transcription) terminator / terminator sequence, also applies here and to these aspects of the invention, mutatis mutandis. In a further aspect of the invention, the DOI as comprised in the nucleic acid molecule of the invention (for example when being (ds)DNA or RNA), may comprise, for example in addition to (one, more or all of) the components of (a), (b), (c), (d), above, or of (a), (b), (c), (d), (e), (f), above, a ((ds)DNA or RNA) sequence encoding a tag; or the POI may be expressed together with a tag. What has been said above (or herein elsewhere), in particular with respect to the tag (and the DOI, nucleic acid molecule of the invention, promotor / promotor sequence, RBS (or other 5´UTR / other translation initiation / ribosome recruitment sequence), buffer region(s), leader peptide, self-cleavage peptide, and / or (transcription) terminator / terminator sequence), also applies here and to this further aspect of the invention, mutatis mutandis. In particular, in the context of this further aspect of the invention, the tag may be suitable for / useful in quantification of a / the POI (e.g. during / after CFPS), monitoring the synthesis of a / the POI (e.g. during CFPS), assessing the level of expression of a / the POI (e.g. during / after CFPS) and / or determining the concentration of a / the POI (e.g. during / after CFPS). The quantification, monitoring, assessing, and determining may be in real-time (during CFPS; cf., for example, Fig. 5). A respective (peptide) tag may be suitable for / useful in, for example, fast and / or high- throughput CFPS. In the context of this aspect of the invention, the tag may be suitable for (bio)luminescence detection. The tag may be a (Nano-)luciferase binding tag, in particular a (Nano-)luciferase binding peptide tag. An example of a respective Nanoluciferase binding peptide tag may be a HiBiT tag (as provided by Promega; see SEQ ID NO.26; see SEQ ID NO.34 for the respective coding sequence). A preferred example of a Nanoluciferase binding peptide tag may be the peptide tag as depicted in SEQ ID NO.27: (see SEQ ID NO.35 for the respective coding sequence (RNA; including the stop codon); see SEQ ID NO.97 for the respective coding sequence (DNA; including the stop codon). The nucleic acid molecule according to this further aspect of the invention (for example linear (ds)DNA / or linear RNA) may be used in CFPS methods and in CFPS systems, respectively; and in respective methods of quantification, monitoring, assessing, and determining. Respective non-limiting examples are (real-time) CFPS monitoring and / or detection methods (cf., for example, Fig.5). These methods are also within the scope of the present invention. In accordance with this aspect of the invention, the CFPS system, CFPS reaction mixture and / or CFPS puffer, for example as used in the respective methods, may (further) comprise components / reagents which are suitable / necessary for detecting the tag, for example in the context of these methods. For example, the CFPS system, CFPS reaction mixture and / or CFPS puffer may (further) comprise a complement binding partner of the tag and / or any other / further component(s) / reagent(s) which are required for detecting the tag; e.g. respective substrates, and the like. When a (Nano-)luciferase binding (peptide) tag is used (like the ones described above), the CFPS system, CFPS reaction mixture and / or CFPS puffer may (further) comprise the component(s) / reagent(s) of the respective buffer (for example a Nano-Glo® buffer (for cellular / liquid detection; Promega)), the complement binding partner (for example the HiBiT complementation partner, like the LgBiT protein (18 kDa; Promega)) and / or the respective substrate (for example a luciferase assay substrate (e.g. Furimazine), like the Nano-Glo® Luciferase Assay Substrate (Promega)). For example, (components of) the Nano-Glo® HiBiT Lytic Detection System (Promega) may be used in this respect. Respective CFPS systems, CFPS reaction mixtures and CFPS puffers are also within the scope of the present invention. In another aspect of the invention, the DOI as comprised in the nucleic acid molecule of the invention (for example when being (ds)DNA or RNA), or the expression cassette as comprised therein, may not comprise a (transcription) terminator / terminator sequence (e.g. neither on DNA-level nor on RNA level). In the context of this other aspect, the DOI, or the expression cassette, may comprise (one, more or all of) the other components of (a), (b), (c), (d), (e), above. It is envisaged that the nucleic acid molecule of this other aspect of the invention comprises at least the one or more 3´-terminal LacI binding site(s) (or respective nucleic acid sequence(s)). In one embodiment of this other aspect of the invention, the nucleic acid molecule may comprise both, the one or more 5´-terminal Tus binding site(s) and the one or more 3´-terminal LacI binding site(s). In one embodiment of this other aspect of the invention, the nucleic acid molecule comprises (as the only terminal protection group(s)) only a particularly strong 3’ terminal DNA / RNA protection site / protection group, like the one or more 3´-terminal LacI binding site(s) (or respective nucleic acid sequence(s)), (but no 5´-terminal DNA / RNA protection site / protection group (e.g. like a 5´-terminal Tus binding site or respective nucleic acid sequence). What has been said above (or herein elsewhere), in particular with respect to the DOI, nucleic acid molecule of the invention, promotor / promotor sequence, RBS / RBS sequence, buffer region(s), leader peptide, self-cleavage peptide, tag, and / or one or more 3´-terminal LacI binding site(s), also applies here and to this embodiment of the other aspect of the invention, mutatis mutandis. In the context of this other aspect of the invention, due to the lack of the transcription terminator / terminator sequence, transcription (e.g. during CFPS, and in CFPS systems, respectively) terminates only at the ultimate 3´ end (i.e. after the strong 3’ terminal DNA / RNA protection site / protection group, like the one or more 3´-terminal LacI binding site(s)). Thus, on RNA level, the nucleic acid molecule of this particular other aspect of the invention comprises the RNA sequence(s) of this site / group, e.g. the RNA sequence(s) of the one or more 3´-terminal lacO sequence(s)); but no terminator sequence. The present invention also relates to this particular kind of RNA (see, for example, also Fig.6, 4thscheme (with or without HiBit); for illustration purposes only). The present invention further relates to an RNA / RNA molecule transcribed from or transcribable from the nucleic acid molecule according to this other aspect of the invention (e.g. from a respective linear (ds)DNA molecule; see, for example, also Fig.6, 4thscheme (with or without HiBit); for illustration purposes only). The RNA / RNA molecule of the invention may result from transcription of a respective (linear) (ds)DNA template of the invention (for example during CFPS, and in CFPS systems, respectively); or the RNA / RNA molecule of the invention may be produced separately (for example synthetically or recombinantly). One specific embodiment this other aspect of the invention relates to a DNA expression cassette (or to a DNA molecule comprising the same), wherein said expression DNA cassette comprises in the 5’ to 3’ direction (i) a promoter (active in bacteria; or in CFPS systems based on other cells or being artificial; see herein elsewhere for details); (ii) an RBS / RBS sequence (or other 5´UTR / other translation initiation / ribosome recruitment sequence); (iii) a GOI encoding a POI; and (iv) one or more LacI binding site(s), wherein the expression cassette (or DNA molecule comprising it) does not comprise a (transcription) terminator / terminator sequence; especially not between the 3´ end of said GOI of (iii) and said one or more LacI binding site(s) of (iv). A 3´UTR may also be comprised in the DNA expression cassette (or in the DNA molecule comprising the same) ((directly) downstream of the GOI encoding a POI; as mentioned in item (iii)). Further, a buffer region (e.g buffer region 1 as defined herein elsewhere) may also be comprised in the DNA expression cassette (or in the DNA molecule comprising the same) ((directly) upstream of the promoter; as mentioned in item (i)). What has been said above (or herein elsewhere), for example with respect to the DOI, nucleic acid molecule of the invention, buffer region(s), promotor / promotor sequence, 5´UTR, RBS / RBS sequence, translation initiation / ribosome recruitment sequence, leader peptide, self- cleavage peptide, 3´UTR and / or (transcription) terminator / terminator sequence, also applies here and to these aspects of the invention, mutatis mutandis. One further specific embodiment this other aspect of the invention relates to a (non-naturally occurring) RNA molecule comprising (i) an RBS / RBS sequence (or other 5´UTR / other translation initiation / ribosome recruitment sequence); (ii) a sequence encoded by a GOI encoding a POI; and (iii) a sequence of the one or more LacI binding site(s); like the one or more LacO sequences), wherein said RNA molecule does not comprise a terminator sequence (and no other protecting sequence(s)) between the end of said sequence of (ii) and said one or more LacI binding site(s) of (iii). Said sequence of the one or more LacI binding site(s) may be as defined herein elsewhere (respective RNA sequences). The sequence(s) of the one or more LacI binding site(s) (like the LacO sequence(s)) are envisaged to be capable of forming a hair-pin (especially on RNA level). A 3´UTR may also be comprised in the RNA molecule ((directly) downstream of the sequence encoded by a GOI encoding a POI; as mentioned in item (ii)). What has been said above (or herein elsewhere), for example with respect to the DOI, nucleic acid molecule of the invention, promotor / promotor sequence, 5´UTR, RBS / RBS sequence, translation initiation / ribosome recruitment sequence, leader peptide, self-cleavage peptide, 3´UTR and / or (transcription) terminator / terminator sequence, also applies here and to these aspects of the invention, mutatis mutandis. “Non-naturally occurring” in accordance with the invention means that respective molecules (e.g. RNA molecules) do not normally occur in nature (like, for example, (IVT-synthesized or chemically synthesized) RNA molecules according to the invention having one or more lacO sequence(s) at the 3’-terminus). Examples of typical naturally-occurring RNA molecules are mRNA molecules which comprise the components RBS (or other 5´ UTR / other translation initiation / ribosome recruitment sequence), POI-encoding sequence, 3´UTR and terminator sequence (but no further components which would normally not be comprised in naturally- occurring RNA molecules; like (recombinantly / chemically introduced) leader peptides, tags, self-cleavage peptide; 3´ lacO sequence(s) etc.). RNA molecules may be considered as naturally- occurring if they occur as such in nature / natively in vivo; but also if they were recombinantly produced, IVT-synthesized or chemically synthesized but comprise only the typical components of RNAs which as such occur in nature / natively in vivo and no components which would normally not be comprised in naturally-occurring RNA molecules, respectively. A non-limiting, exemplary and illustrative, model template / nucleic acid molecule of the invention and that may be used in accordance with the invention is depicted in SEQ ID NO.98 (model DNA template / nucleic acid molecule). This template / nucleic acid molecule is an example of a template / nucleic acid molecule as depicted at the bottom of Figure 6A. A further non-limiting, exemplary and illustrative, model template / nucleic acid molecule of the invention and that may be used in accordance with the invention is depicted in SEQ ID NO.99 (model DNA template / nucleic acid molecule). This template / nucleic acid molecule is an example of a template / nucleic acid molecule as depicted at the beginning of Figure 6A. Other templates / nucleic acid molecules can be generated by relying on these model templates / nucleic acid molecules. In this context, one, more or all of the respective components of these model templates / nucleic acid molecules (e.g. the Tus binding site(s), promoter, RBS, leader sequence, self-cleavage peptide, GOI, tag, terminator, 5´and / or 3´ UTR(s), terminator, buffer region(s), LacI binding site(s)) may be replaced by (an)other respective component(s) as described herein (e.g. (an)other Tus binding site(s), promoter, RBS, leader sequence, self-cleavage peptide, GOI, tag, terminator, 5´and / or 3´ UTR(s), terminator, buffer region(s), LacI binding site(s), respectively, as described herein). Certain components may also be omitted in this respect (e.g. the leader peptide, self-cleavage peptide and / or terminator; see also the third template / nucleic acid molecule as depicted in Figure 6A for respective illustration). In general, the skilled person will appreciate that not only the herein disclosed and exemplified specific nucleic acid molecules / sequences, nucleic acid constructs and amino acid sequences, and the respective specific components as disclosed herein (like, e.g. the Tus binding sites, promoters, RBS´, leader sequences, GOIs / POIs, tags, terminators, buffer regions, UTRs etc.) may be provided / used in accordance with the invention, but also respective variants thereof. It is preferred that such variants exhibit the same (biological) function than the respective specific basis molecules / sequences, constructs, components. Further, such variants preferably share high sequence identities with the respective specific basis molecules / sequences, constructs, components; for example, sequence identities of ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥98% or ≥99%. It is particularly preferred that, within the sequence identity ranges / sequence variances, those particular nucleotide or amino acid residues which contribute to the respective (biological) function of the respective basis molecules / sequences, constructs, components are not altered / modified. Those having skill in the art will know how to determine percent variance / percent identity between / among amino acid sequences or between / among nucleotide sequences. In this context, algorithms such as those based on CLUSTALW computer program (Thompson (1994) Nucl. Acids Res. 2:4673-4680), CLUSTAL Omega (Sievers (2014) Curr. Protoc. Bioinformatics 48:3.13.1-3.13.16) or FASTDB (Brutlag (1990) Comp App Biosci 6: 237-245) may, for example be used. Also available to those having skill in the art are the BLAST, which stands for Basic Local Alignment Search Tool, and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389- 3402; Altschul (1990) J. Mol. Biol.215:403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLOSUM62 scoring matrix (Henikoff (1992) Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. In order to determine whether a nucleotide residue or an amino acid residue in a given nucleotide sequence or amino acid sequence, respectively, corresponds to a certain position compared to another nucleotide sequence or amino acid sequence, respectively, the skilled person can also use means and methods well known in the art, e.g., alignments, either manually or by using computer programs such as those mentioned herein. For example, BLAST 2.0 can be used to search for local sequence alignments. BLAST or BLAST 2.0, as discussed above, produces alignments of (nucleotide ort amino acid) sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST or BLAST 2.0 is especially useful in determining exact matches or in identifying similar or identical sequences. The present further relates to a method of protecting a (linear) nucleic acid molecule (e.g. a (linear) (ds)DNA molecule or a (linear) RNA molecule)), in particular a linear nucleic acid molecule having a free 5’ terminus and a free 3’ terminus, more particular a linear nucleic acid molecule as described and as defined herein (e.g. a respective (linear) (ds)DNA molecule or a respective (linear) RNA molecule). Most relevantly, protection in the context of the method of protecting of the invention is protection from nuclease degradation, more particular from exonuclease degradation; as, for example typically occurring during CFPS, and in CFPS systems, respectively. The method of protecting a nucleic acid molecule according to the invention comprises (the step of) adding at least one or more LacI binding site(s) (or the respective DNA or RNA sequences) at the 3´ terminus of a / the nucleic acid molecule (e.g. one or more 3´- terminal lacO sequence(s)). The method may further comprise (the step of) allowing a LacI protein to bind to (each of) the LacI binding site(s). The method may further comprise (the step of) adding one or more Tus binding site(s) at the 5´ terminus of a / the nucleic acid molecule (e.g. one or more 5´-terminal ter sequence(s)). The method may further comprise (the step of) allowing a Tus protein to bind to (each of) the Tus binding site(s); especially on DNA level. In one embodiment, the method of protecting a (linear) nucleic acid molecule of the invention comprises (the steps of) (a) adding one or more Tus binding site(s) at the first terminus of a DNA molecule (preferably at the 5´ end) and adding one or more LacI binding site(s) at the other terminus of the DNA molecule (preferably at the 3´ end); and (b) allowing a Tus protein to bind to (each of) the Tus binding site(s) and a LacI protein to bind to (each of) the LacI binding site(s). What has been said above (or herein elsewhere), in particular with respect to the one or more Tus binding site(s) and / or the one or more LacI binding site(s), and the Tus and / or the LacI, and the DOI, nucleic acid molecule of the invention, promotor / promotor sequence, RBS / RBS sequence (or other 5´UTR / other translation initiation / ribosome recruitment sequence), buffer region(s), leader peptide, self-cleavage peptide, tag etc., also applies here and to the method of protecting of the invention, mutatis mutandis. The means and methods for nucleic acid molecule protection (e.g.5´ Tus / 3´ LacO binding site(s); 5´ ter / 3´ lacO design) as disclosed and described herein enhance the stability of linear nucleic acid molecules (on DNA and / or on RNA level); especially in CFPS systems. The means and methods for nucleic acid molecule protection as disclosed and described herein may thus be particularly useful for linear nucleic acid molecules with a low stability (intrinsic stability; especially on RNA level; for example due to no or only a few intrinsic hairpin structures). The skilled person is readily in the position to determine whether a nucleic acid molecule has, or is expected to have, a low (intrinsic) stability (or a high (intrinsic) stability). In this context, the skilled person can rely on the common general knowledge (see also http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) and the herein-provided teaching (see, for example, Example 3 and Fig.2 for respective illustration and guidance). The skilled person is readily able to provide / establish respective assays. In the context of such assays, and / or when determining whether a nucleic acid molecule has, or is expected to have, a low (intrinsic) stability (or a high (intrinsic) stability), (RNA) stability may be converted from the free energy of the thermodynamic ensemble (with arbitrary unit (AU)). The thermodynamic ensemble may be predicted by an RNAfold calculator (for example, available at http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi). For example, a nucleic acid molecule may have a low (intrinsic) stability in case it shows a stability of ≤510 AU (for example as determined by an assay like the one in Example 3; cf. also Fig.2). A nucleic acid molecule may have a high (intrinsic) stability in case it shows a stability of >510 AU or, preferably, ≥600AU (for example as determined by an assay like the one in Example 3; cf. also Fig. 2). A nucleic acid molecule may have a low (intrinsic) stability in case only ≤30% of its sequence forms intrinsic hairpin structures. The present invention further relates to a method of producing / synthesizing a protein or (poly)peptide of interest (POI), in particular by (applying) CFPS, wherein a nucleic acid construct as disclosed and described herein is used a / the template for expressing the POI. The present invention also relates to a method of producing / synthesizing a POI by using a CFPS system with a CFPS reaction mixture and a CFPS buffer / buffer composition, respectively, wherein a nucleic acid construct as disclosed and described herein is used as a / the template for expressing the POI. The present invention also relates to a method of producing / synthesizing a POI in a CFPS reaction mixture, comprising a CFPS buffer / buffer composition and CFPS extract / lysate, wherein the CFPS reaction mixture and the CFPS buffer / buffer composition, respectively, comprises a template / nucleic acid construct as disclosed and described herein. The present invention further relates to a method of producing / synthesizing a POI in a CFPS reaction mixture, said method comprising (the steps of): (a) providing a template / nucleic acid construct of the invention (e.g. as defined herein above (or herein elsewhere); e.g. comprising an expression cassette for a / the POI); (b) adding said template / nucleic acid construct to a / the CFPS reaction mixture; and (c) synthesizing said POI in said CFPS reaction mixture. The method of producing / synthesizing a POI of the invention may further comprise (one or, preferably, all two of) the following steps: (d) providing a CFPS reaction mixture; and (e) adding to said CFPS reaction mixture glucose and PEP. The added glucose may be in un- purified or, preferably, in purified form and / or the added PEP may be in un-purified or, preferably, purified form. Preferably, this / these step(s) are performed before step (c), above, more preferably before step (b), above. It is particularly preferred that the step of adding glucose and PEP to said CFPS reaction mixture is performed prior to the synthesizing of said POI in said CFPS reaction mixture, or at least at the beginning of said synthesizing (for example during the first quarter or first eighth of said synthesizing / CFPS reaction). However, adding glucose and PEP to said CFPS reaction mixture at a later stage during said synthesizing is, in principle, also within the scope of the invention. In the context of the method of producing / synthesizing a POI according to the invention, the CFPS reaction mixture, and CFPS extract / lysate and CFPS buffer, respectively, to be employed is / are envisaged to contain all components needed for transcription or translation, preferably all components needed for transcription and translation. All or any of said components, in particular of the cell extract / lysate, may be of endogenous origin (i.e. deriving from the cells from which the CFPS extract / lysate originates / is derived from). All or any of said components may also be of heterologous origin (i.e. not taken / deriving from the cells from which the extract / lysate originates / is derived from, but from other cells and / or from an artificial / recombinant origin). All or any of said components may also be recombinantly produced. The heterologously originating / recombinantly produced components may be added exogenously / derived from an exogenous source. In principle, however, it is preferred that a component to be comprised in the CFPS reaction mixture and cell extract / lysate, respectively, is of endogenous origin (the (T7) RNA polymerase may be one exception). Relevantly, the CFPS reaction mixture and cell extract / lysate comprises an RNA polymerase (for transcription; e.g. a T7 RNA polymerase) and / or ribosomes, translation factors, tRNAs, aminoacyl-transferases (for translation). As mentioned, the RNA polymerase may be the endogenous RNA polymerase (e.g. the bacterial RNA polymerase; or, for example, a phage RNA expressed in bacteria, like the T7 RNA polymerase); or an exogenous RNA polymerase, like a recombinant RNA polymerase or a phage RNA polymerase (like a T7 RNA polymerase). For ATP regeneration, the CFPS reaction mixture, and cell CFPS extract / lysate, may comprise (cytosolic) glycolysis enzymes, TCA enzymes, oxidative phosphorylation enzymes (in small vesicles) etc. The method of producing / synthesizing a POI according to the invention, in particular the step of synthesizing the POI in the CFPS reaction mixture according to the invention (step (c)), can be performed at any suitable temperature; or at any suitable range of temperatures. The skilled person is readily able to choose or determine (a) respective suitable temperature(s) and temperature range(s). The skilled person is also readily able to determine or choose (an) optimal temperature(s) or respective range(s); for example where a maximum final protein yield and / or a maximal protein expression (e.g. amount of POI per time) can be achieved. If necessary, the skilled person is also readily able to change / adapt the temperature during the course of the method of producing / synthesizing a POI according to the invention, for example during the step of synthesizing the POI in the CFPS reaction mixture according to the invention (step (c)). Ranges of temperatures at which the method of producing / synthesizing a POI according to the invention, in particular the step of synthesizing the POI in the CFPS reaction mixture according to the invention (step (c)), may be performed are 10 °C to 40 °C, 15 °C to 40 °C, 20 °C to 40 °C, 30 °C to 40 °C, 35 °C to 40 °C, 36 °C to 38 °C, 25 °C to 35 °C, and 20 °C to 25 °C. As regards the choice, determination or testing of suitable / optimal temperatures / temperature ranges, the skilled person may rely on the means and methods for CFPS as described herein and in the appended examples. In the context of the invention, the temperature / temperature range may also be adapted to / chosen for a certain kind of cellular extract / lysate to be comprised in the CFPS reaction mixture according to the invention, for example to / for a certain kind of origin of the cellular extract / lysate; i.e. to / for an organism from which the cellular extract / lysate originates. For example, when the cellular extract / lysate originates from a plant (e.g. a wheat germ extract / lysate), the temperature may be in the range of 20 °C to 40 °C, preferably in the range of 20 °C to 30 °C, more preferably in the range of 20 °C to 25 °C. For example, when the cellular extract / lysate originates from an animal, in particular from a mammal (e.g. from rabbit reticulocytes), the temperature may be in the range of 20 °C to 39 °C, preferably in the range of 25 °C to 37, preferably in the range of 35 °C to 37, more preferably in the range of 29 °C to 34 °C. For example, when the cellular extract / lysate originates from yeast cells (e.g. from Saccharomyces cerevisiae cells), the temperature may be in the range of 25 °C to 35 °C, preferably in the range of 27 °C to 33 °C, more preferably in the range of 28 °C to 32 °C. For example, when the cellular extract / lysate originates from bacteria (e.g. from Escherichia coli), the temperature may be in the range of 25 °C to 37 °C, preferably in the range of 27 °C to 33 °C, more preferably in the range of 28 °C to 32 °C (e.g.30 °C). Non-limiting examples of cellular extracts / lysates, their origin, and respective preferred temperatures / temperature ranges for the CFPS, which may be used / applied in accordance with the invention, e.g. as a basis for the POI synthesis method, CFPS reaction mixture or CFPS lysate of the invention, are given in Table 2, below. Besides the indicated preferred temperatures / temperature ranges, also other temperatures / temperature ranges may be applied, depending on the particular CFPS settings. The method of producing / synthesizing a POI according to the invention, in particular the step of synthesizing the POI in the CFPS reaction mixture according to the invention (step (c)), can be performed at any suitable pH value; or within any suitable range of pH value. The skilled person is readily able to choose or determine (a) respective suitable pH value(s) and pH value range(s). Moreover, the skilled person is readily able to determine or choose (an) optimal pH value(s) or respective range(s); for example where a maximum final protein yield and / or a maximal protein expression (e.g. amount of POI per time) can be achieved. If necessary, the skilled person is also readily able to change / adapt the pH value during the course of the method of producing / synthesizing a POI according to the invention, for example during the step of synthesizing the POI in the CFPS reaction mixture according to the invention (step (c)). The skilled person is also readily able to adapt the pH value / pH value range to the need of the components, or to the need of (a) certain component(s), which are comprised in the CFPS reaction mixture and / or cell extract / lysate, respectively (for example the RNA polymerase, e.g. T7 RNA polymerase; and / or the aminoacyl-transferases etc.). In accordance with the invention, the pH value in the method of producing / synthesizing a POI according to the invention, in particular during the step of synthesizing the POI in the CFPS reaction mixture according to the invention (step (c)), may typically be in the range of pH6 to pH8, more preferably in the range of pH6.5 to pH8, more preferably in the range of pH6.8 to pH7.8, more preferably in the range of pH7.2 to pH7.5. As regards the choice, determination or testing of suitable / optimal pH values / pH ranges, the skilled person may rely on the means and methods for CFPS as described herein and in the appended examples. It is envisaged that the CFPS reaction mixture and cell extract / lysate, respectively, comprises components, for example proteins and / or hairpin structures, which protect the template / nucleic acid molecule (DNA and / or RNA) from degradation (by, for example, (exo)nucleases; upon, for example, binding to the template / nucleic acid molecule (in case of protecting proteins). Respective protecting components are known in the art and are described herein. A particularly preferred example of a respective protecting component is the herein described Tus (of the “Tus-Ter” E. coli DNA replication termination system; cf., e.g., Berghuis loc.cit.; WO-A1 2022 / 160049; Norouzi loc.cit.). Another particularly preferred example of a respective protecting component is the herein described LacI (of the “LacI-lacO” E. coli nucleic acid molecule protection system; cf., e.g., US-A1 2005 / 0202480; Jameson loc.cit.). These protecting components / proteins may bind to (the 5’-terminus and / or the 3’-terminus of) the nucleic acid molecules (e.g. RNA or DNA template) which may be comprised in the CFPS reaction mixture and CFPS buffer, respectively, in accordance with the invention, and which comprise (a) respective binding site(s) (e.g. one or more Tus binding site(s) (e.g. one or more ter sequence(s)) and / or one or more LacI binding site(s) (e.g. one or more lacO sequence(s))). The nucleic acid molecules are thereby stabilized / protected (from (exo)nuclease degradation). The protecting components / proteins may likewise be endogenous proteins, i.e. they may originate from the cells from which the cell CFPS extract / lysate is derived; or they may be recombinant and / or exogenously added (e.g. from an exogenous, non-endogenous (heterologous) source). The may also be recombinantly produced within the the CFPS reaction mixture and cell extract / lysate, respectively (e.g. during the CFPS / synthesis of the POI). In one embodiment, the CFPS reaction mixture may comprise an endogenous (e.g. procaryotic or bacterial) RNA polymerase or an exogenous RNA polymerase (exogenously added; e.g. a (recombinant) phage RNA polymerase), the endogenous Tus (and / or (an)other endogenous protecting component(s); preferred) or a recombinant and / or exogenously added Tus (and / or (an)other recombinant and / or exogenously added protecting component(s)); and / or the endogenous LacI (and / or (an)other endogenous protecting component(s); preferred) or a recombinant and / or exogenously added LacI (and / or (an)other recombinant and / or exogenously added protecting component(s)). In the context of a preferred embodiment of the present invention, the CFPS reaction mixture is envisaged to comprise only the endogenous Tus (or only endogenous Tus and (an)other endogenous protecting component(s)) and only the endogenous LacI (or only the endogenous LacI and (an)other endogenous protecting component(s)). In the context of this embodiment, no (purified / surplus) Tus and LacI (or other protecting component(s)) is / are to be added to the CFPS reaction mixture (for example in form of recombinantly produced and / or exogenously added LacI and / or Tus; or in in form of LacI and / or Tus which is recombinantly expressed within the CFPS reaction mixture). In the context of one particular embodiment of the method of producing / synthesizing a POI according to the invention, the CFPS reaction mixture to be employed contains both, glucose and PEP (as (ATP-regenerating) energy sources). The method of producing / synthesizing a POI according to the invention may further comprise (the step of) adding to the CFPS reaction mixture glucose and PEP. The added glucose may be in un-purified or, preferably, purified form and / or the added PEP may be in un-purified or, preferably, purified form. The (step of) adding to the CFPS reaction mixture glucose and PEP may be performed before the step of synthesizing said POI in said CFPS reaction mixture, more preferably before the step of adding the template / nucleic acid construct to a / the CFPS reaction mixture. Typically, a CFPS reaction mixture contains a cell extract; typically a cell lysate (also termed herein “CFPS cell extract / lysate”, or just “CFPS extract” or “CFPS lysate”). Respective reference is made herein also to just “(cell) extract” or just “(cell) lysate”. The terms “(cell) extract” and “(cell) lysate”, and “(cellular) extract” and “(cellular) lysate”, respectively, are used interchangeably herein. A cell-free extract and cell-free lysate, respectively, as typically used in CFPS systems is meant in this respect (see, for example, Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., Rasor loc. cit.). “CFPS (cell) extract / lysate” means that the respective (cell) extract / lysate is suitable for CFPS (when contained, together with the respective CFPS buffer, in the resulting respective CFPS reaction mixture). The cell extract / lysate may be a crude cell extract / lysate or a (partially-)purified cell extract / lysate (e.g. essentially (partially) free of (unsoluble) cell debris and / or (unsoluble) membrane components). In principle, the cell extract / lysate may be of any (eukaryotic, archaebacterial or bacterial) cellular origin; however prokaryotic extracts / lysates are preferred in the context of the present invention, bacterial cell extracts / lysates are particularly preferred. Non-limiting examples of respective bacteria (i.e., from which the bacterial extract / lysate, and respective CFPS reaction mixture, is derived) may be selected from bacteria of the genus selected from the group consisting of Escherichia, Vibrio, Bacillus, Corynebacterium, Klebsiella, and Pseudomonas. Non- limiting examples of respective particular bacteria are bacteria selected from the group consisting of E. coli, V. natriegens, B. subtilis, C. glutamate, K. pneumoniae, P. fluorescens and B. megaterium. Respective cells may endogenously express for example recombinantly a suitable RNA polymerase; like the E. coli BL21 (DE3) cells (expressing T7 RNA polymerase). A typical cellular extract / lysate for use in CFPS contains all components which are necessary for the processes of translation and / or transcription; starting from a nucleic acid template which contains the nucleic acid sequence (also termed “gene of interest” (GOI)) which encodes the respective (m)RNA and POI, respectively). The respective components are well known in the art and are, for example, described in Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., and Rasor loc. cit. Non-limiting examples of cellular extracts / lysates which are commercially available for use in CFPS are the E. coli S30 extract (e.g. the E. coli S30 Extract System for Linear Templates; Promega), myTXTL (Arborbioscience) and PUREfrex® 2.0 (PUREfrex). The skilled person is also readily able to produce a CFPS cell extract / lysate to be used in accordance with the invention. Respective guidance is provided herein and in the appended examples (e.g. Example 1). Further respective guidance is provided in the prior art (e.g Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., Rasor loc. cit.). Besides the extract / lysate, the CFPS reaction mixture typically further contains the respective buffer / buffer composition for CFPS (also termed herein “CFPS buffer / CFPS buffer composition”, or just “buffer”; the CFPS buffer is sometimes also termed “(CFPS) reaction buffer”). The composition of a CFPS buffer is, in principle, also known in the art (cf., for example, Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., and Rasor loc. cit.). In principle, the composition of a CFPS buffer / buffer composition in accordance with the invention (and of a CFPS reaction mixture, respectively) is not particularly limited. However, the CFPS buffer / buffer composition (and respective CFPS reaction mixture) of the invention has (one, more or all of) the herein described modifications, supplements and / or optimizations (like, for example, the addition of glucose (in addition to PEP); and / or the addition to GMP, DTT, an increased HEPES concentration, and / or an increased magnesium ion concentration). In general, a CFPS reaction mixture and CFPS buffer / buffer composition, respectively, are so that CFPS is promoted. This means that a CFPS reaction mixture and respective buffer promotes at least the translation from a template (m)RNA into a protein or (poly)peptide. Transcription from a template DNA into the template (m)RNA may also be part of the CFPS in accordance with the invention, i.e., the respective CFPS reaction mixture and buffer may also promote transcription from a template DNA into a (m)RNA template for translation. Promoting of both, transcription and translation is preferred in accordance with the invention. Highly promoting transcription and translation is, for example, achieved by increasing the ATP regeneration by a combination of glucose and PEP (both, transcription and translation, require ATP as energy source) and / or by improving transcription priming with GMP. The basis / initial CFPS reaction mixture (i.e. before (an) ATP-regenerating energy source(s) is / are added; for example together with the buffer) may comprise some marginal amounts of, for example, glucose and / or PEP (e.g., natively deriving from the cellular extract / lysate as comprised in the CFPS reaction mixture). For example, (about) 3 mM intracellular PEP may be comprised in the cells from which the extract / lysate is derived (e.g., bacterial cells; see, for example, doi: 10.1128 / MMBR.00024-06. and 10.3390 / microorganisms11061588). Thus, (about) ≤500 µM, ≤450 µM or ≤400 µM PEP may be comprised in the basis / initial CFPS reaction mixture (the lower values are preferred). Likewise, (about) 6 mM intracellular glucose may be contained in the cells from which the extract / lysate derives (e.g., CHO cells; cf., for example, doi: 10.1371 / journal.pone.0034512). Thus, (about) ≤1000 µM, ≤900 µM ≤700 µM glucose may be comprised in the basis / initial CFPS reaction mixture (the lower values are preferred). Such marginal amounts of PEP and / or glucose which may derive just from cell extracts / lysates to be comprised in typical basis / initial CFPS reaction mixtures, however, are not sufficient for promoting CFPS to acceptable yields. Thus, in appropriate CFPS systems, i.e. in CFPS reaction mixtures, including respective buffers, compounds which act as (ATP-regenerating) energy sources are to be added (cf., for example, Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., and Rasor loc. cit.). The particular (ATP-regenerating) energy sources and respective amount(s) / concentration(s) thereof which may be comprised in the CFPS reaction mixture, and respective buffer, in accordance with the invention, and which may accordingly be added in accordance with the method of producing / synthesizing according to the invention, are described herein below. Standard CFPS reaction mixtures and respective CFPS buffers are, for example, described in Kim loc. cit. (“Materials and Methods”), Jewett loc. cit. (“Cell-Free Protein Synthesis”; “Table I” (the PANOx, PANOx-SP, and Cytomim systems)), Calhoun loc. cit. (“Cell-Free Protein Synthesis”), Rasor loc. cit. (“Materials”; 2.1 and 2.2). The person skilled in the art is readily able to determine which particular reagents are to be comprised in a suitable CFPS reaction mixture and suitable CFPS buffer, and at which particular amounts / concentrations. Respective guidance is, for example, provided in Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., Rasor loc. cit., in Table 1 herein below, and in the appended examples. A CFPS reaction mixture and respective buffer in accordance with the invention may, for example, comprise (e.g. in addition to glucose and / or PEP as described herein elsewhere) (one, more or all of the) reagents as exemplified in Table 1A and / or B; or in Table 1A, B, and C. The respective amounts / concentrations of the comprised reagents may be like those amounts / concentrations as specified in Table 1, preferably as in the last column thereof (some deviation from the particular numeric values of Table 1 may be allowed in the context of the invention; e.g., deviations from 0-10%). In general, at least the components as indexed with “3” in Table 1A and B are envisioned to be comprised in the CFPS reaction mixture and respective buffer of the invention (the components essential for CFPS (for basic production)). In principle, the amount / concentration of glucose and / or PEP to be comprised in accordance with the invention in the CFPS reaction mixture, and CFPS buffer, respectively, is not particularly limited. In accordance with the invention, both, glucose and PEP, may be comprised in the CFPS reaction mixture, and CFPS buffer, respectively, in accordance with one aspect of the invention. (An) only marginal respective content(s) is / are not sufficient (for example, as deriving only from the used cellular extract / lysate). Glucose may, for example, be comprised in a similar amount as in CFPS systems of the prior art (e.g., Calhoun loc. cit.; e.g., 30 mM). PEP may, for example, be comprised in a similar amount as in CFPS systems of the prior art (e.g., Kim loc. cit. and Jewett loc. cit., Jewett loc. cit. and Rasor loc. cit.; e.g., 30 or 33 mM). In principle, the skilled person is able to determine suitable amounts of glucose and / or PEP to be comprised in the CFPS reaction mixture and buffer in accordance with the invention. This determination can, for example, be based on the protein expression and / or final protein yield as achieved with the respective CFPS. For example, the skilled person is readily able to titrate the optimal amount of glucose to be comprised (in addition to a given amount of comprised PEP; e.g., 30 or 33 mM), on the basis of protein expression efficiency and / or final protein yield. Respective guidance is provided herein and in the appended examples. Vice versa, the skilled person is readily able to titrate the optimal amount of PEP to be comprised (in addition to a given amount of comprised glucose; e.g. 10-30 mM) on the basis of the protein expression and / or final protein yield. Respective guidance is also provided herein and in the appended examples. In particular, in accordance with the invention, the CFPS reaction mixture and CFPS buffer, respectively, may comprise (i) between (about) 10 and (about) 30 mM of glucose, between (about) 15 and (about) 30 mM of glucose or between (about) 15 and (about) 25 mM of glucose; and / or (ii) between (about) 20 and (about) 50 mM PEP, (about) 25 to 40 mM PEP or (about) 28 to 38 mM PEP. The ranges may include or exclude the respective particular limit values. A non-limiting example for glucose is (about) 10 mM, (about) 20 mM or (about) 30 mM. In one particular embodiment, the glucose content is below (<) 33 mM or below (<) 30 mM. Non- limiting examples for PEP are (about) 28 mM, (about) 33 mM or (about) 38 mM. In one particular embodiment, the PEP content is below (<) 28 mM, below (<) 33 mM or below (<) 38 mM. In one embodiment, the CFPS reaction mixture and buffer may comprise (about) 20 mM of glucose (± 1-3 mM) and <33 mM PEP or <30 mM PEP; or the CFPS reaction mixture and buffer may comprise (about) 20 mM of glucose (±1-3 mM) and (about) 33 mM or (about) 30 mM PEP (± 1-5 mM). In principle, it is desired that a CFPS reaction mixture and buffer may comprise as much as glucose and / or PEP as suitable / possible (to achieve a maximum CFPS capacity and final yield of the POI). However, the maximal respective values have some limits. For example, the addition of more than 30-40 mM glucose may not further enhance the protein expression efficiency and final yield, respectively. As mentioned, however, the skilled person is readily able to titrate the respective maximal amounts of PEP and glucose on the basis of the common general knowledge and the teaching provided herein. What has been said above with respect to the optimal amounts / concentrations of glucose and / or PEP also applies, in principle, to the optimal amounts / concentrations of the other reagents that may be comprised in accordance with the invention in the CFPS reaction mixture and buffer. Also for these reagents, the skilled person is readily in the position to determine / titrate the optimal respective amounts / concentrations based on the common general knowledge and the teaching provided herein. In general, an amount or concentration given herein for a component of the CFPS reaction mixture or CFPS buffer in accordance with the invention, unless explicitly indicated otherwise or contradicted by context, refers to the respective amount or concentration in the final CFPS reaction mixture; i.e., the CFPS reaction mixture in which the CFPS, and POI synthesis, respectively, actually took place. For example, if an amount or concentration of a given component is given for a CFPS buffer, the amount or concentration, respectively, in the (final) CFPS reaction mixture (which comprises both, the CFPS extract / lysate and the CFPS buffer) is actually meant. In accordance with the invention, the CFPS reaction mixture and buffer may comprise between (about) 30 and 300 mM HEPES, preferably between (about) 50 and 200 mM HEPES, more preferably (about) 70 to 150 mM HEPES e.g. (about) 100 mM HEPES. Preferably, HEPES is HEPES(KOH). The respective pH is preferably ph 7.5 (a variation of ± a ph of 0.1-0.3 may be allowed). In accordance with the invention, the CFPS reaction mixture and buffer may comprise phosphate, preferably between (about) 5 and 20 mM phosphate, preferably between (about) 7.5 and 15 mM phosphate, e.g. (about) 10 mM phosphate. The respective ph may be 7.2 (preferred). The phosphate may be provided in form of and / or by adding potassium phosphate or respective buffer (preferred) or sodium phosphate or respective buffer (to reach / in the respective concentration and / or the respective pH). Typically, a mixture of mono, di- and / or tri- potassium phosphate at equilibrium is used. Practically, di- and mono-potassium phosphate solutions may be mixed in a certain ratio to reach the desired concentration and target pH (cf. Table 1 in Ganesh, Biochem Biophys Rep 9, 2017, 121-7; illustratively exemplified for sodium phosphate). In accordance with the invention, the CFPS reaction mixture and buffer may comprise 1.5 - 4.5 mM ATP (e.g. (about) 3 mM ATP), 1.5 - 4.5 mM GTP (e.g. (about) 3 mM GTP), 1.0 - 3.0 mM CTP (e.g. (about) 2 mM CTP) and / or 1.0 - 3.0 mM UTP (e.g. (about) 2 mM UTP). In accordance with the invention, the CFPS reaction mixture and buffer may comprise between (about) 10 and 30 mM Mg2+ions, preferably between (about) 15 and 25 mM Mg2+ions, more preferably between (about) 18 and 24 mM Mg2+ions, e.g. (about) 23 mM Mg2+ions (± 1-3 mM; most preferred) or (about) 19 mM Mg2+ions (± 1-3 mM). The Mg2+ions may be provided in form of and / or by adding magnesium diglutamate (Mg(Glu)2) (to reach / in the respective concentration). In accordance with the invention, the CFPS reaction mixture and buffer may comprise GMP, preferably between (about) 1 and 10 mM GMP, more preferably between (about) 3 and 7 mM GMP, e.g. (about) 5 mM GMP (± 1 mM; most preferred). In accordance with the invention, the CFPS reaction mixture and buffer may comprise DTT, preferably between (about) 1 and 15 mM DTT, more preferably between (about) 3 and 12 mM DTT, e.g. (about) 5 mM DTT (± 1 mM; most preferred). In accordance with the invention, the CFPS reaction mixture and buffer may comprise an RNA polymerase (e.g. a T7 RNA polymerase); for example, 20 to 150 µg / mL RNA polymerase or 30 to 100 µg / mL RNA polymerase (e.g. (about) 30 µg / mL RNA polymerase or, preferably, (about) 100 µg / mL RNA polymerase). In accordance with the invention, the CFPS reaction mixture may comprise between (about) 0.1 to 0.6 volume of cell extract / lysate (CFPS lysate), more preferably between (about) 0.2 to 0.5 volume of cell extract / lysate, e.g. (about) 0.24 volume of cell extract / lysate (± 0.1-0.2 volume) or (about) 0.4 volume of (± 0.1-0.2 volume; most preferred). Volume part of the entire (final) CFPS reaction mixture is meant in this respect (for example, 0.24 volume of cell extract / lysate means that 24% of the volume of the entire (final) CFPS reaction mixture is cell extract / lysate). This may correspond to a final concentration of total proteins as derived from the cell extract / lysate (e.g. E. coli cell extract / lysate), and to be comprised as such in the CFPS reaction mixture, of between (about) 4.2 to 33 mg / mL, more preferably between (about) 8.3 to 21 mg / mL, e.g. (about) 10 mg / mL (±1-2 mg / mL) or (about) 17 mg / mL (± 1-3 mg / mL; most preferred). For example, a 0.24 volume of an E.coli S30 extract corresponds to about 9.9 mg / mL final total E.coli protein in the CFPS reaction mixture (cf., for example, Jewett loc. cit.). The skilled person is readily in the position to determine suitable volumes / amounts of the cell extract / lysate to be comprised in the CFPS reaction / CFPS reaction mixture. Optimal respective volumes / amounts can also be determined (e.g. volumes / amounts which result in the desired (maximal) yield of the POI to be synthesized / produced). Suitable / optimal volumes / amounts of the cell extract / lysate may be determined by titration. In this context, the skilled person can rely on the guidance provided herein, in the appended examples and in the art (e.g. Jewett loc. cit.; Calhoun loc. cit.). In principle, the origin of the cellular extract / lysate to be comprised in the CFPS reaction mixture in accordance with the invention is not particularly limited. The cellular extract / lysate may be of prokaryotic, plant or animal origin. Examples of cellular extracts / lysates of plant origin may be derived from wheat germ, rice, tobacco (e.g., BY-2) and Arabidopsis. Examples of cellular extracts / lysates of animal origin may be derived from K-562 cells, CHO cells, insect cells, rabbit reticulocytes, human blood cells, HeLa cells, HEK cells and Leishmania tarentolae cells. Examples of non-prokaryotic microbial cellular extracts / lysates are derived from yeast cells. However, a preferred origin of the cellular extract / lysate to be comprised in the CFPS reagent mixture in accordance with the invention is a prokaryotic origin; the most preferred origin of the cellular extract / lysate to be comprised in the CFPS reagent mixture in accordance with the invention is a bacterial origin. Thus, preferably, the CFPS reaction mixture and cellular extract / lysate, respectively, is of procaryotic origin / is derived from procaryotic cells; most preferably, the CFPS reaction mixture and cellular extract / lysate, respectively, is of bacterial origin / is derived from bacterial cells. A CFPS reaction mixture which is of prokaryotic / bacterial origin / is derived from prokaryotic / bacterial cells, respectively means that it comprises a cellular extract / lysate which is of prokaryotic / bacterial origin / is derived from prokaryotic / bacterial cells, respectively (in addition to the CFPS buffer / buffer composition as disclosed herein). It is preferred in the context of the invention that the origin of the CFPS reaction mixture and cellular extract / lysate, respectively, is of cells which are capable of exhibiting endogenous glycolysis. Bacteria that possess endogenous glycolysis are preferred. The CFPS reaction mixture and cellular extract / lysate, respectively, in accordance with the invention is thus envisaged to contain glycolytic enzymes, preferably the endogenous glycolytic enzymes of the respective basis cellular extract / lysate (for example as deriving from bacterial cells). CFPS in accordance with the invention is preferably achieved by lysate-based protein expression; CFPS may also be achieved be on the basis of (partially) artificial and / or (partially) recombinant CFPS systems. As mentioned, in the context of a preferred embodiment of the invention, the lysate originates / is derived from procaryotic cells. In principle, procaryotic cells encompass both, bacterial cells and archaebacterial cells; however, bacterial cells are the more preferred origin / basis of the extract / lysate to be used in accordance with the invention. The amount / concentration of the nucleic acid construct to be added (as an expression template to the CFPS reaction mixture) in the context of the method of producing / synthesizing a POI of the invention, or which may be comprised in the CFPS reaction mixture or CFPS buffer, respectively, in accordance with the invention, is not particularly limited. Amounts / concentrations of the nucleic acid construct which are suitable in CFPS are known in the art (see, for example, Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., Rasor loc. cit.). Such amounts / concentrations may also be used in the context of the invention (unless explicitly indicated otherwise or contradicted by context). For example, the amount / concentration of the nucleic acid construct to be added in the context of the method of synthesizing of the invention, or which may be comprised in the CFPS reaction mixture or CFPS buffer, respectively, in accordance with the invention, may be 5 to 20 µg / mL, 7 to 18 µg / mL, or 9 to 15 µg / mL, or 10 to 16 µg / mL; e.g.13 µg / mL (± 1-2 µg / mL). Also in this respect, the amount / concentration as in the final CFPS reaction mixture is indicated (i.e. µg nucleic acid construct per mL CFPS reaction mixture). One specific embodiment of the invention relates to a method of producing / synthesizing a POI in a CFPS reaction mixture, said method comprising (the steps of): (i) providing a linear dsDNA comprising an expression cassette for a POI, (ii) adding said linear dsDNA to the CFPS reaction mixture; and (iii) synthesizing said POI, said POI may be expressed as a fusion protein precursor or poly-protein comprising in the N- terminus to C-terminus direction (a) a (highly-expressed) leader peptide or a (highly expressed) peptide (preferably nGFP) (e.g. as defined herein elsewhere); (b) a (2A) self-cleavage peptide (e.g. as defined herein elsewhere); and (c) the sequence of said POI; and wherein said fusion protein precursor / poly-protein optionally further comprises (preferably in the C-terminal direction after (c)), (d) a tag (e.g. for quantification of the POI and the like (e.g., a HiBit tag; e.g. as defined herein elsewhere). Also in this respect, said CFPS reaction mixture, said linear dsDNA, and / or said expression cassette and / or the other components / features may be as defined herein elsewhere. The present invention further relates to a CFPS system as disclosed and described herein; and to a CFPS system to be employed in the context of the method of synthesizing of the invention; wherein said CFPS system comprises a template / nucleic acid construct of the invention. The CFPS system of the invention may be a biological CFPS system (i.e. based on a biological cells-derived cell extract / lysate). The CFPS system of the invention may also be a (partly) reconstituted CFPS system (i.e. the relevant components for translation and / or transcription may (partly) be of reconstituted and / or recombinant origin and / or exogenously added). Biological CFPS systems are known in the art (e.g. Kim loc. cit., Jewett loc. cit., Calhoun loc. cit., and Rasor loc. cit.); and reconstituted CFPS systems are known in the art (e.g. Shimizu, Nature Biotechnology 19, 2001,751–5; Shimizu, Methods 36(3) 2005, 299-304). Reconstituted CFPS systems may be reconstituted cell-free protein synthesis systems, like reconstituted in vitro coupled transcription / translation Systems. The components of a reconstituted CFPS system are provided / purified separately (e.g. by recombinant and / or chemical synthesis) and then mixed in the cell-free reactions. Most relevantly, a (reconstituted or biological) CFPS system comprises the transcription- and( / or) translation-related factors. In other words, a (reconstituted or biological) CFPS system comprises those proteins and factors which are necessary for transcription, translation, aminoacylation, energy regeneration etc. The components of a (reconstituted or biological) CFPS system thus may comprise RNA polymerase (e.g. the T7 RNA polymerase), NTPs, ribosomes, the translation(-related) factors, the tRNAs, the amino acyltransferases, enzymes for ATP regenerations, etc. In reconstituted CFPS systems, the proteinous factors and ribosome may be provided individually (e.g. by recombinant and / or purification techniques); and may be assembled with substrates, such as amino acids, NTPs, and tRNAs, in the CFPS buffer. Translation factors may include (one, more or all of) the following: IF1 / IF2 / IF3 (as initiation factors), EF-Tu / EF-Ts / EF-G (as elongation factors), RF1 / RF2 / RF3 (as release factors), ribosome recycling factor (RRF), 20 kinds of aminoacyl-tRNA synthetase (ARS), methionyl-tRNA transformylase. In general, the skilled person is readily in the position to choose all essential components of a CFPS system of the invention (and the respective amounts / concentrations) based on the common general knowledge, the citations made herein and the teaching provided herein. The CFPS system of the invention may be a CFPS system like a biological CFPS system or like a reconstituted CFPS system as known in the art (and may have (one, more or all of) the herein described modifications, supplements and / or optimizations (like, for example, the addition of glucose (in addition to PEP; and / or in addition to GMP, DTT, an increased HEPES concentration, and / or an increased magnesium ion concentration)). A particular example of a reconstituted CFPS system known in the art is the PURE (Protein synthesis Using Recombinant Elements) system (PUREfrex®; GeneFrontier Corp.; https: / / purefrex.genefrontier.com / pure_system / puresystem.html). A particular example of a reconstituted CFPS system of the invention is based on the PURE system (e.g. PUREfrex®); but may have (one, more or all of) the herein described modifications, supplements and / or optimizations (like, for example, the addition of glucose (in addition to PEP; and / or in addition to GMP, DTT, an increased HEPES concentration, and / or an increased magnesium ion concentration)). In principle, further (purified and / or recombinantly produced) components may be added to a CFPS system of the invention; for example to enhance certain functions. For example, more (T7) RNA polymerase may be added (for higher transcription rates); more tRNA an / or more amino acids may be added (for higher translation rates). (More) creatine phosphate and (more) creatine kinase may be added (for (better) ATP regeneration). In the context of one specific, non-limiting, embodiment, no protecting proteins like Tus and LacI may intrinsically be comprised in the CFPS system (or in the respective CFPS reaction mixture); for example in reconstituted CFPS systems (see also above) or in eukaryotic CFPS systems (see also above). When such particular CFPS systems are used, protecting proteins like Tus and LacI may also be (exogenously) added to or recombinantly expressed (endogenously) in such particular CFPS systems in accordance with the invention. Even if no protecting proteins like Tus and LacI would be added to such particular CFPS systems or expressed therein, the structure of the respective binding sites as defined herein (e.g. the intrinsic (5´- and 3´) hairpin structure(s), like the intrinsic 5´-ter and 3´-lacO hairpin structure(s), or like the at least strong 3´-lacO hairpin structure) would provide for a stabilizing effect in accordance with the invention. The CFPS system of the invention may comprise a CFPS reaction mixture as disclosed and described herein or a CFPS reaction mixture to be employed in the context of the method of synthesizing of the invention; and / or the CFPS system of the invention may comprise a CFPS buffer as disclosed and described herein or a CFPS buffer to be employed in the context of the method of synthesizing of the invention. The CFPS system of the invention is envisaged to comprise a nucleic acid construct as disclosed and described herein or a nucleic acid construct to be employed in the context of the method of synthesizing of the invention; and / or the CFPS system of the invention may, or may not, comprise a CFPS lysate as disclosed and described herein or a CFPS lysate to be employed in the context of the method of synthesizing of the invention. In any case, however, a suitable nucleic acid construct and a suitable CFPS lysate is to be added to the CFPS reaction mixture in the context of the method of synthesizing of the invention. The present invention further relates to a CFPS reaction mixture as disclosed and described herein; and to a CFPS reaction mixture to be employed in the context of the method of synthesizing of the invention; wherein said CFPS reaction mixture comprises the template / nucleic acid construct of the invention. The CFPS reaction mixture of the invention is envisaged to comprise a lysate as disclosed and described herein or a lysate to be employed in the context of the method of synthesizing of the invention; and a CFPS buffer as disclosed and described herein or a CFPS buffer to be employed in the context of the method of synthesizing of the invention. The CFPS reaction mixture of the invention is envisaged to comprise a nucleic acid construct as disclosed and described herein or a nucleic acid construct to be employed in the context of the method of synthesizing of the invention. A suitable nucleic acid construct is to be added to the CFPS reaction mixture in the context of the method of synthesizing of the invention. The present invention further relates to a CFPS buffer as disclosed and described herein; to a CFPS buffer to be employed in the context of the method of synthesizing of the invention; and to a CFPS buffer to be comprised in the CFPS reaction mixture of the invention; wherein said CFPS buffer comprises the template / nucleic acid construct of the invention. The CFPS buffer of the invention is envisaged to comprise a nucleic acid construct as disclosed and described herein or a nucleic acid construct to be employed in the context of the method of synthesizing of the invention. A suitable nucleic acid construct is to be added to the CFPS buffer and CFPS reaction mixture, respectively, in the context of the method of synthesizing of the invention. Any of the CFPS system, CFPS reaction mixture and CFPS buffer of the invention comprises a nucleic acid molecule / construct of the invention. Any of the CFPS system, CFPS reaction mixture and CFPS buffer may (further) comprise any other of the herein described components (which may be employed in the context of the method of producing / synthesizing of the invention). What has been said for the nucleic acid molecule / construct, and for any other of the herein described components (e.g. the components / proteins which protect nucleic acid molecules; e.g. Tus and LacI and respective binding sites and sequences), also applies here, mutatis mutandis. The present invention further relates to a kit or to a kit of contents. Said kit / kit of contents may contain a nucleic acid molecule or nucleic acid template, e.g. a linear dsDNA molecule, an RNA molecule and / or an expression cassette, as described and disclosed herein. Said kit / kit of contents may, in addition, contain a CFPS system, a CFPS reaction mixture, a CFPS lysate and / or a CFPS reaction buffer as described and disclosed herein. Within said kit, said nucleic acid molecule or nucleic acid template, e.g. linear dsDNA molecule, RNA molecule or expression cassette, may be contained in a separate vial. Likewise, said CFPS system, a CFPS reaction mixture, a CFPS lysate and / or a CFPS reaction buffer may be contained in a separate vial. The kit / kit of contents of the invention may be used for / in the context of any of the herein described and disclosed methods. What has been said herein elsewhere for the nucleic acid molecule or nucleic acid template, e.g. linear dsDNA molecule, RNA molecule and expression cassette, and for the respective components, also applies here, mutatis mutandis. Likewise, what has been said herein elsewhere for the CFPS system, CFPS reaction mixture, CFPS lysate and CFPS reaction buffer, and for the respective components and contents, also applies here, mutatis mutandis. The present invention also relates to the following items. 1. A linear double-stranded deoxyribonucleic acid (dsDNA) molecule comprising (a) one or more Tus protein (Tus) binding site(s) at the 5’-terminus; and (b) one or more Lac repressor protein (LacI) binding site(s) at the 3’-terminus; and (c) between (a) and (b) a segment comprising a DNA sequence of interest (DOI). 2. The linear dsDNA molecule of item 1, further comprising one or more Tus protein(s) bound to the 5’ Tus binding site(s) and / or one or more LacI protein(s) bound to the 3’ LacI binding site(s). 3. The linear dsDNA molecule of item 1 or 2 comprising two Tus binding sites at the 5’- terminus and / or two LacI binding sites at the 3’-terminus. The linear dsDNA molecule of any one of items 1 to 3, wherein at least one of said one or more Tus binding site(s) is or comprises a ter sequence and / or wherein at least one of said one or more LacI binding site(s) is or comprises a Lac operator (lacO) sequence. The linear dsDNA molecule of item 3 or 4, wherein said two Tus binding sites are or comprise two ter sequences, respectively, and / or wherein said two LacI binding sites are or comprise two lacO sequences, respectively. The linear dsDNA molecule of item 4 or 5, wherein said ter is terA (preferred), terB, terC, terD or terE and / or wherein said lacO is lacO1, lacO2, or lacO3. The linear dsDNA molecule of any one of item 4 - 6, wherein said ter sequence comprises a nucleotide sequence as depicted in SEQ ID NO.7 (“ter consensus sequence”) and / or wherein said lacO sequence comprises a nucleotide sequence as depicted in SEQ ID NO. 8 (“lacO consensus sequence”). The linear dsDNA molecule of any one of item 4 - 7, wherein said ter sequence is or comprises a nucleotide sequence as depicted in SEQ ID NO.1 (terA; preferred), SEQ ID NO. 3 (terB), SEQ ID NO. 4 (terC), SEQ ID NO. 5 (terD) or SEQ ID NO. 6 (terE) and / or wherein said lacO sequence is or comprises SEQ ID NO.2 (“Insempra”; preferred), SEQ ID NO. 9 (“Core”), SEQ ID NO. 10 (“lacO1”), SEQ ID NO. 11 (“pET”), SEQ ID NO. 12 (“Symmetric”), SEQ ID NO. 13 (“Gilbert”), SEQ ID NO. 14 (“lacO2”) or SEQ ID NO. 15 (“lacO3”). The linear dsDNA molecule of any one of items 1 to 8, wherein said Tus is or comprises an amino acid sequence as depicted in SEQ ID NO. 16 and / or wherein said LacI is or comprises an amino acid sequence as depicted in SEQ ID NO.17. The linear dsDNA molecule of any of items 1 to 9, wherein said one or more 3’ LacI binding site(s) is / are located downstream of a buffer region (which may be preceded by a (transcription) terminator sequence which is located 3’ in / of the DOI (e.g. of the stop- codon for a protein of interest (POI) which may be encoded by said DOI)). The linear dsDNA molecule of any of items 1 to 10, wherein said DOI encodes a protein of interest (POI). The linear dsDNA molecule of any of items 1 to 11, wherein said DOI is or comprises a functional DNA sequence. The linear dsDNA molecule of item 12, wherein said functional DNA sequence is an expression cassette for a POI. The linear dsDNA molecule of any one of items 1 - 13, wherein said DOI comprises in the 5’ to 3’ direction (a) a promotor sequence (active in bacteria); (b) a DNA sequence encoding an RBS (or another 5´UTR / other translation initiation / ribosome recruitment sequence); and / or (c) a DNA sequence (GOI) encoding a POI (optionally followed by a 3´UTR); and / or (d) optionally, a (transcription) terminator sequence (active in bacteria). The linear dsDNA molecule of any one of items 1 to 14 further comprising a 5’ DNA buffer region (buffer region 1; 5´ overhang; 5´ UTR) downstream of the 3’ end of said Tus binding site(s) (upstream of the 5’ end of said DOI) and / or a 3´UTR downstream of the 3’ end of said DOI (upstream of the 5’ end of a / said terminator) and / or a 3’ DNA buffer region (buffer region 2; 3´ overhang) upstream of the 5’ end of said LacI binding site(s) (downstream of the 3’ end of a / said terminator). The linear dsDNA molecule of item 15, wherein said buffer region 1 and / or said 3´UTR consists of between 0 to 200 base pairs (bp) (preferably 70 – 150 bp; e.g. about 46 bp and / or about 55 bp, respectively) and / or wherein said buffer region 2 consists of between 0 to 500 bp (preferably 70 – 300 bp; e.g. about 193 bp). The linear dsDNA molecule of any one of items 11 - 16, wherein said POI is expressed as a fusion protein or a poly-protein. The linear dsDNA molecule of any one of items 1 - 17, wherein said DOI comprises in the 5’ to 3’ direction (a) a promotor sequence (active in bacteria; e.g. T7 promoter); (b) a DNA sequence encoding an RBS (or another 5´UTR / other translation initiation / ribosome recruitment sequence); (c) a DNA sequence encoding a (highly expressed) leader peptide; (d) optionally, a DNA sequence encoding a self-cleavage peptide (one additional proline (P) may be encoded in downstream direction of said self-cleavage peptide); and / or (e) a GOI encoding a POI (optionally followed by a 3´UTR); and / or (f) optionally, a (transcription) terminator sequence (active in bacteria; e.g. the T7 terminator). The linear dsDNA molecule of item 18, wherein said leader peptide is or comprises nGFP (the first 20 amino acids of sfGFP); e.g. as encoded by SEQ ID NO.18 or as depicted in SEQ ID NO.28. The linear dsDNA molecule of item 18 or 19, wherein said self-cleavage peptide is or comprises a 2A self-cleavage peptide, e.g. a P2A (preferred), F2A, E2A or T2A self- cleavage peptide; e.g. as depicted in SEQ ID NOs: 21 (more preferred) / 22 (most preferred), 23, 24 or 25, respectively, or as encoded by SEQ ID NOs: 29 (more preferred) / 30 (most preferred), 31, 32 or 33. The linear dsDNA molecule of any one of items 18 to 20, wherein said self-cleavage peptide comprises the amino acid sequence as depicted in SEQ ID NO. 19 (the “2A consensus sequence”) or as depicted in SEQ ID NO.20 (the “2A core sequence”). The linear dsDNA molecule of any one of items 11 to 21, wherein said POI comprises, or is expressed together with, a tag (e.g. for quantification of said / a POI). The linear dsDNA molecule of item 22, wherein said tag is or comprises a Nanoluciferase binding peptide tag (e.g. as depicted in SEQ ID NO.26 or SEQ ID NO.27 (preferred), or as encoded by SEQ ID NO.34 or SEQ ID NO.35 / 97 (preferred)). The linear dsDNA molecule of any one of items 17 - 21, wherein said poly-protein comprises in the N-terminus to C-terminus direction (a) a sequence coding for a highly expressed peptide (preferably nGFP (e.g. as defined in item 19), or a fragment thereof); and (b) a (2A) self-cleavage peptide (e.g. as defined in item 20 or 21); and (c) the sequence of a POI; and, wherein said poly-protein optionally further comprises (preferably in the C-terminal direction after (c)) (d) a tag (for quantification of the POI (e.g., a HiBit tag; e.g. as defined in item 23). The linear dsDNA molecule of any of items 1 to 9 and 11 to 24, wherein said DOI, in particular said expression cassette as defined in any one of items 13 to 24, does not comprise a (transcription) terminator sequence. A linear dsDNA molecule as defined in item 25, wherein said linear dsDNA molecule does not comprise a 5’ DNA protection group (e.g. like a 5’ Tus binding site). A (non-naturally occurring) RNA molecule transcribable from the linear dsDNA molecule of item 25 or 26. A method of protecting a linear deoxyribonucleic acid (DNA) molecule having a free 5’ terminus and a free 3’ terminus from exonuclease degradation, said method comprising (the steps of): (a) adding one or more Tus binding site(s) at the first terminus of the DNA molecule and adding one or more LacI binding site(s) at the other terminus of the DNA molecule; and (b) allowing a Tus protein to bind to (each of) the Tus binding site(s) and a LacI protein to bind to (each of) the LacI binding site(s). The method of item 28, wherein said one or more Tus binding site(s) is / are at the 5’ terminus (“5’ Tus binding site(s)”) of the DNA molecule and the one or more LacI binding site(s) is / are at the 3’ terminus (“3’ LacI binding site(s)”) of the DNA molecule. The method of item 28 or 29, wherein said one or more Tus binding site(s) and / or said one or more LacI binding site(s) are defined as in any one of items 3 - 8. The method of any one of items 28 - 30, wherein said Tus and / or said LacI is defined as in item 9. The method of any one of items 28 - 31, wherein said linear DNA molecule is defined as the linear dsDNA molecule of any one of items 1 - 26. A method of synthesizing a POI in a cell-free protein synthesis (CFPS) reaction mixture, said method comprising (the steps of): (a) providing a linear dsDNA molecule as defined in any of items 1 - 26, wherein said DOI is or comprises a GOI encoding said POI (broader: a nucleic acid construct as defined herein, e.g. comprising an expression cassette for a POI); (b) adding said linear dsDNA to the CFPS reaction mixture; and (c) synthesizing said POI in said CFPS reaction mixture. The method of any one of item 33, wherein said CFPS reaction mixture comprises a CFPS lysate which is of procaryotic origin / is derived from procaryotic cells; preferably, said CFPS reaction mixture comprises a CFPS lysate which is of bacterial origin / is derived from bacterial cells. The method of any one of items 33 to 35, wherein said CFPS reaction mixture or said CFPS lysate is derived from bacteria of the genus selected from the group consisting of Escherichia, Vibrio, Bacillus, Corynebacterium, Klebsiella, and Pseudomonas. The method of any one of items 33 to 36, wherein said CFPS reaction mixture or said CFPS lysate is derived from bacteria selected from the group consisting of E. coli, V. natriegens, B. subtilis, C. glutamate, K. pneumoniae, P. fluorescens and B. megaterium. The method of any one of items 33 to 37, wherein the concentration of (endogenous) Tus in said CFPS reaction mixture is ≤15µM, preferably ≤13µM, and / or the concentration of (endogenous) LacI in said CFPS reaction mixture is ≤15µM, preferably ≤13µM. The method of any one of items 33 to 38, wherein said CFPS reaction mixture or said CFPS lysate comprises an endogenous (e.g. procaryotic or bacterial) RNA polymerase or an exogenous RNA polymerase (exogenously added; e.g. a (recombinant) phage RNA polymerase), the endogenous Tus (and / or (an)other endogenous protecting component(s)) or a recombinant and / or exogenously added Tus (and / or (an)other recombinant and / or exogenously added protecting component(s)); and / or the endogenous LacI (and / or (an)other endogenous protecting component(s)) or a recombinant and / or exogenously added LacI (and / or (an)other recombinant and / or exogenously added protecting component(s)). The method of any one of items 33 to 39, wherein said CFPS reaction mixture or said CFPS lysate comprises the endogenous Tus (and / or (an)other endogenous protecting component(s)) and the endogenous LacI (and / or (an)other endogenous protecting component(s)); and / or wherein no (purified / surplus) Tus or lacO is added to said CFPS reaction mixture (for example in form of recombinant and / or exogenously added LacI and / or Tus). The method of any one of items 33 to 40, wherein said CFPS reaction mixture comprises both, glucose and phosphoenolpyruvate (PEP). The method of any one of items 33 to 41, further comprising, before (c), preferably before (b), (the steps of) (a) providing a CFPS reaction mixture; and (b) adding to said CFPS reaction mixture (purified) glucose and (purified) PEP. The method of any one of items 33 to 421, wherein said CFPS reaction mixture comprises (i) 10 to 30 mM of glucose, preferably 15 to 25 mM of glucose; and / or (ii) 20 to 50 mM PEP, preferably 28 to 38 mM PEP. The method of any one of items 33 to 43, wherein said CFPS reaction mixture comprises 20 mM glucose (± 1-3 mM) and / or 33 mM PEP (± 1-5 mM). The method of any one of items 33 to 44, wherein said CFPS reaction mixture comprises 30 to 300 mM HEPES (HEPES(KOH); ph 7.5), preferably 50 to 200 mM HEPES (HEPES(KOH); ph 7.5), more preferably 70 to 150 mM HEPES (HEPES(KOH); ph 7.5); e.g. (about) 100 mM HEPES (HEPES(KOH); ph 7.5). The method of any one of items 33 to 45, wherein said CFPS reaction mixture comprises phosphate (ph 7.2; e.g. in form of / by adding potassium phosphate buffer (e.g. K2HPO4 and KH2PO4)), preferably 5 to 20 mM phosphate (ph 7.2; e.g. in form of / by adding potassium phosphate buffer (e.g. K2HPO4 and KH2PO4)), preferably 7.5 to 15 mM phosphate (ph 7.2; e.g. in form of / by adding potassium phosphate buffer (e.g. K2HPO4and KH2PO4)); e.g. (about) 10 mM phosphate (ph 7.2; e.g. in form of / by adding potassium phosphate buffer (e.g. K2HPO4and KH2PO4)). The method of any one of items 33 to 46, wherein said CFPS reaction mixture comprises 1.5 to 4.5 mM ATP (e.g. (about) 3 mM ATP), 1.5 to 4.5 mM GTP (e.g. (about) 3 mM GTP), 1.0 to 3.0 mM CTP (e.g. (about) 2 mM CTP) and / or 1.0 to 3.0 mM UTP (e.g. (about) 2 mM UTP). The method of any one of items 33 to 47, wherein said CFPS reaction mixture comprises (e.g. in form of / by adding magnesium diglutamate (Mg(Glu)2)) 10 to 30 mM Mg2+ions, preferably 15 to 25 mM Mg2+ions, more preferably 18 to 24 mM Mg2+ions; e.g.23 mM Mg2+ions (± 1-3 mM; most preferred) or 19 mM Mg2+ions (± 1-3 mM). The method of any one of items 33 to 48, wherein said CFPS reaction mixture comprises guanosine monophosphate (GMP), preferably 1 to 10 mM GMP, more preferably 3 to 7 mM GMP; e.g.5 mM GMP (± 1 mM; most preferred). The method of any one of items 33 to 49, wherein said CFPS reaction mixture comprises DTT, preferably 1 to 15 mM DTT, more preferably 3 to 12 mM DTT; e.g.5 mM DTT (± 1 mM; most preferred). The method of any one of items 33 to 50, wherein said CFPS reaction mixture comprises 0.1 to 0.8 volume of a CFPS lysate, more preferably 0.2 to 0.5 volume of a CFPS lysate; e.g.0.24 volume of a CFPS lysate (± 0.05-0.1 volume) or 0.4 volume of a CFPS lysate (± 0.05-0.1 volume). A cell-free biological system (e.g. CFPS system, like, for example, a bacterial lysate CFPS system) comprising the linear dsDNA molecule of any one of items 1 to 26, the RNA molecule of item 27 and / or the CFPS reaction mixture as defined in any one of items 33 to 51. A method of synthesizing a POI in a CFPS reaction mixture, said method comprising (the steps of): (a) providing a linear dsDNA comprising an expression cassette for a POI, (b) adding said linear dsDNA to the CFPS reaction mixture; and (c) synthesizing said POI, wherein said POI is expressed as a fusion protein precursor comprising in the N-terminus to C-terminus direction (a) a sequence coding for a highly expressed peptide (preferably nGFP (e.g. as defined in item 19); (b) a (2A) self-cleavage peptide (e.g. as defined in item 20 or 21); and (c) the sequence of said POI; and, wherein said poly-protein optionally further comprises (preferably in the C-terminal direction after (c)) optionally, (d) a tag (for quantification of the POI (e.g., a HiBit tag; e.g. as defined in item 23). The method of item 53, wherein said CFPS reaction mixture is defined as in any one of items 33 - 51; wherein said linear dsDNA is defined as in any one of items 1 - 25; and / or wherein said expression cassette is defined as in any one of items 18 – 23. An expression DNA cassette comprising in the 5’ to 3’ direction (i) a promoter (active in bacteria); (ii) an RBS (or other 5´UTR / other translation initiation / ribosome recruitment sequence); (iii) a GOI encoding a POI (optionally followed by a 3´UTR); and (iv) one or more LacI binding site(s), wherein the expression cassette does not comprise a (transcription) terminator sequence (active in bacteria) between the 3´ end of said GOI of (iii) and said one or more LacI binding site(s) of (iv). A non-naturally occurring RNA molecule comprising (i) an RBS (or other 5´UTR / other translation initiation / ribosome recruitment sequence); (ii) a sequence encoded by a GOI encoding a POI (optionally followed by a 3´UTR); and (iii) one or more LacI binding site(s), wherein said RNA molecule does not comprise a terminator sequence (or (an)other protecting sequence(s)) between the end of said sequence of (ii) and said one or more LacI binding site(s) of (iii). 57. The expression DNA cassette of item 55 or the RNA molecule of item 56, wherein said one or more LacI binding site(s) is / are defined as in any one of items 2 – 9 (respective DNA or RNA sequences). 58. The (non-naturally occurring) RNA molecule of item 27, 56 and 57, wherein said lacO sequence(s) or said one or more LacI binding site(s) is / are capable of forming a hair-pin. If not indicated differently herein elsewhere, and unless explicitly indicated otherwise or contradicted by context, “inhibiting”, “decreasing”, “blocking”, “suppressing” or “reducing”, and the like, in the context of the present invention is envisaged to mean that the reference / initial status (for example status of POI expression or yield) is lowered by, for example, at least 10%, by at least 20%, by at least 30%, by at least 50%, by at least 80%, by at least 90%, at least 95%, by at least 99% or even by 100% (in principle, the higher values of percentage are preferred). The skilled person is readily in the position to test the respective degree of “inhibiting”, “decreasing”, “blocking”, “suppressing” or “reducing”. Likewise, if not indicated differently herein elsewhere, and unless explicitly indicated otherwise or contradicted by context, “increasing”, “inducing” or “improving”, and the like, in the context of the present invention particularly means that the reference / initial status (for example status of POI expression or yield) is increased by, for example, at least 10%, by at least 20%, by at least 30%, by at least 50%, by at least 80%, by at least 90%, at least 95%, by at least 99% or by at least 100%; or by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold (in principle, the higher values of percentage are preferred). In particular, “increasing” means that there is already an initial degree of activity / status (baseline) which is further “increased”. In particular, “inducing” means that there is (substantially) no initial degree of activity / status which is then “induced”. The skilled person is readily in the position to test the respective degree of “increasing”, “inducing” or “improving”. As used herein, in particular with respect to the disclosed components and methods of synthesizing, and unless explicitly indicated otherwise or contradicted by context, the terms "optional", "optionally" and "may" denote that the indicated feature may be present but can also be absent, or may be replaced by another (corresponding) feature. Whenever the term "optional", "optionally" or "may" is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression "X is optionally substituted with Y" (or "X may be substituted with Y") means that X is either substituted with Y or is unsubstituted. Likewise, if a component (of a reaction mixture, buffer or construct) is indicated to be "optional", the invention specifically relates to both possibilities, i.e., that the corresponding component is present (e.g contained in the reaction mixture, buffer or construct) or that the corresponding component is absent (e.g from the reaction mixture, buffer or construct). It is to be understood that where a list of groups is preceded by the expression "optionally substituted", the expression "optionally substituted" applies to each one of the respective groups in that list, not just to the first item in the list. As used herein, unless explicitly indicated otherwise or contradicted by context, the terms "a", "an" and "the" are used interchangeably with "one or more" and "at least one". Thus, for example, a reaction mixture, buffer or construct comprising "a" component in accordance with the present invention can be interpreted as referring to a reaction mixture, buffer or construct comprising "one or more" respective component(s). As used herein, the term "comprising" (or "comprise", "comprises", "contain", "contains", or "containing"), unless explicitly indicated otherwise or contradicted by context, has the meaning of "containing, inter alia", i.e., "containing, among further optional elements, …". In addition thereto, this term may also include the narrower meanings of "consisting essentially of". For example, the term "A comprising B and C" has the meaning of "A containing, inter alia, B and C", wherein A may contain further optional elements (e.g., "A containing B, C and D" would also be encompassed). This term may also include the meaning of "A consisting essentially of B and C" (i.e., no other components than B and C are comprised in A). As used herein, the terms "about", “similar”, or “slight(ly)” different, “essential(ly)” the same etc., unless explicitly indicated otherwise or contradicted by context, has the meaning of ± ≤20%, ± ≤10%, ± ≤5%, ± ≤3%, ± ≤2% or ± ≤1%. In principle, the lower values are preferred in this respect. The present invention is further described by reference to the following non-limiting figures and examples. The Figures show: Figure 1. Protein expression levels of linear DNAs with different end-protection in CPFS. A: Linear DNA templates comprising genes of interest (GOI) encoding sfGFP, EcHpaC, RrBLVRB, RsTYR, ScKDC and EcHpaB (as respective exemplary POI) were prepared with different end- protections, i.e., no protection, ter / lacO, lacO / ter, ter / ter and lacO / lacO (1stposition is 5’ end and 2ndposition refers to 3’ end, e.g., 5’ter / 3’lacO; in all cases with end-protection, two binding sequences were used per each kind of binding site, e.g., 2x5’ter / 2x3’lacO; i.e., for example, “ter / lacO” means 2x5’ter / 2x3’lacO, “lacO / ter” means 2x5’lacO / 2x3’ter etc.). CFPS was carried out in the PANOx-SP buffer system (Jewett loc.cit.; Rasor loc.cit.; see also material and method section) at 30°C for 3hr. If not explicitly described otherwise, the experiments (e.g. (template) preparation and testing conditions) underling the following figures were as in those underlying Fig. 1 and Example 2, respectively. B: Statistical significance between templates with no protection and ter / lacO protection was shown for templates encoding sfGFP, EcHpaC, RrBLVRB, RsTYR, ScKDC and EcHpaB by applying a two-tailed paired Student’s t-test. Significance is indicated by a single asterisk (*). Figure 2. Linear DNA with ter / lacO protection strengthened protein expression from RNAs with less stability in a more pronounced manner. Fold of protein expression from templates encoding RsTYR, ScKDC, RrBLVRB, sfGFP, PcAAS, SvVlmR, EcYahK or EcTyrB as the respective POI was calculated by dividing the final protein concentration of CFPS from ter / lacO-protected DNA by the one from non-protected DNA and shown in grey bars. ≤ 1-fold protein expression means no effect of ter / lacO protection on the linear DNA. The RNA stability is converted from the free energy of the thermodynamic ensemble predicted by RNAfold calculator (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) with arbitrary unit and shown in the white bars. Preparation and testing conditions were as in Fig.1. Figure 3. Protein expression levels of plasmid and linear DNAs in CFPS. RBS and coding sequences of ScKDC and RsTYR genes were constructed either on a plasmid or on a linear DNA and driven by a T7 promoter. The sequences upstream of the T7 promoter and downstream of the GOIs were identical on both, plasmid and linear DNA. The end-protections were added to the linear DNAs at 46 bases upstream of the T7 promoter and 223 bases downstream of the T7 terminator. Preparation and testing conditions were as in Fig.1. Figure 4. Protein expression levels of linear DNAs with P2A leader peptide or RBS. Four GOI (encoding EcHpaC, EcHpaB, SvVlmR, PcAAS) were either expressed by an individual bespoke RBS, which was predicted by Salis calculator for the highest expression level for each GOI; or expressed downstream of a N-terminal leader peptide with nGFP and a self-cleavage P2A peptide in the CFPS reactions with PANOx-SP buffer (see herein elsewhere for respective sequences used). Linear DNAs with ter / lacO protection were used in this experiment. The P value between the RBS and P2A group is < 0.05 from a two-tailed paired Student’s t-test. Statistical significance is indicated by a single asterisk (*). Preparation and testing conditions were as in Fig.1. Figure 5. Illustrative scheme showing real-time protein quantification in CFPS. Protein quantification shown in the previous figures was an end-point measurement at 3 hr of the CFPS reaction. The present figure shows functionality of the CFPS system of the invention also during the reaction time (“real-time”). For real-time protein quantification in CFPS, the Nanoluciferase binding peptide tag is linked to the C-terminal of the proteins to be synthesized, while its complementary partner, LgBiT (18 kDa), and luminescent substrate, furimazine, are added to the CFPS reaction at the desired time points; starting from the beginning (time 0). The LgBiT may be enriched endogenously in the lysate prior to the lysate preparation or added exogenously as a recombinant protein to the lysate. Figure 6. Illustration of (exemplary) DNA constructs and resulting RNA molecules of the invention; as well as of illustrative examples of respective protection groups (e.g. the 2xTer and 2xLacO end-protection groups and the T7 protection group). A: Exemplary illustration of the linear DNA constructs of the invention; and the resulting RNA molecules. Illustrative example of the linear DNA construct of the invention (upper pannel): lacO protection sequence(s) was / were constructed at the 3’-end downstream of a (T7) terminator. The resulting transcripts contain RNA sequences until the (T7) terminator sequence (transcribed (T7) terminator sequence; no transcribed lacO protection sequence(s)). It is known that RNA transcripts with (T7) terminator sequence perform better in CFPS, because the secondary structure of the (T7) terminator sequence (see, for example, Fig. 6B) protects RNA from exonuclease degradation (cf. Jin-Ho Ahn, Biochem Biophys Res Commun 338(3), 2005, 1346- 52). Illustrative example of alternative linear DNA construct (middle pannel): lacO protection sequence(s) is / are constructed at the 3’-end downstream of the GOI, with no (T7) terminator. (T7) RNA polymerase will transcribe whole sequences downstream of the GOI and run off from the linear DNA at the 3’-end. The resulting RNA transcripts contain transcribed lacO protection sequence(s) (no ter sequence(s) at the 5’end of the RNA because transcription start is downstream of ter sequence(s) in DNA template). lacO sequence(s) / hairpin(s) (cf., for example, Fig. 6B) is thus used as terminator and as a protection group also on RNA level. As single- stranded lacO sequence forms a stronger and more stable hairpin than a (T7) terminator in RNA transcript (cf., B, below). Further illustrative example of a linear DNA construct of the invention (lower pannel): As in the upper panel, however, including, between the promoter and the GOI, an nGFP leader peptide with its RBS (from pJL1) and a self-cleavage peptide. In one particular aspect, the depicted 5´ (ter / Tus) protection on DNA level may also be omitted in the alternative linear DNA construct. B: Illustration of protection groups used in the context of the invention (exemplarily depicted on RNA level). Left panel: 2xter sequences (SEQ ID NO. 100; illustrative example of 5´protection group; cf. A, above). Middle panel: 2xlacO sequences (SEQ ID NO.101; illustrative example of 3´protection group (also on RNA level in the alternative approach of the invention); cf. A, above). Right panel: T7 terminator sequence (SEQ ID NO. 102; illustrative example of 3´protection group on RNA level; cf. A, above; cf. Jin-Ho Ahn loc. cit.). Both, (2x)ter and (2x)lacO sequences, form hairpin structure (when they are single-stranded). Single- stranded (2x)lacO hairpin structure is more stable than the T7-sequence structure, which is considered a strong (i.e. stable) secondary structure. As used herein, the term “comprising” is intended to mean that the compounds, compositions methods, etc. include the respective recited elements, but do not exclude others. “Consisting of” or “consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein may not exclude, for example, trace contaminants from an isolation and purification method, pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. Embodiments defined by each of these transition terms are within the scope of this disclosure. In the detailed description of the invention, a number of individual elements, characterizing features, techniques and / or steps are disclosed. It is readily recognized that each of these has benefit not only individually when considered or used alone, but also when considered and used in combination with one another. Accordingly, to avoid exceedingly repetitious and redundant passages, this description has refrained from reiterating every possible combination and permutation. Nevertheless, whether expressly recited or not, it is understood that such combinations are entirely within the scope of the presently disclosed subject matter. All technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Reference is made herein also to the following table(s): Table 1: Exemplary reagents as comprised in a CFPS reaction mixture (including extract / lysate and buffer) and in the respective buffer (w / o extract / lysate); e.g. in addition to PEP and / or Glucose (as ATP-regenerating energy sources; see herein elsewhere for details). Amounts / concentrations are given with respect to the final CFPS reaction mixture. A: Reagents which may be unmodified in CFPS reaction mixture and buffer of the invention. Reagent exemplary exemplary content exemplary content unit (prior art) (invention) NH4(OAc)3mM 10 10 Oxalic Acid mM 4 4 CoA mM 0.27 0.27 Folinic acid mg / mL 0.034 0.034 tRNA mixture (E. coli) mg / mL 0.171 0.171 Putrescine mM 1 1 Spermidine mM 1.5 1.5 AAs mix (each of 20 AAs) mM 2 2 (DNA) template (plasmid)1; 3µg / mL 13.3 13.3 B: Reagents the content of which may be modified in CFPS reaction mixture and buffer of the invention. NAD mM 0.33 or 0.4 0.4 K(Glu)3mM 130 140 ATP3mM 1.2 3 GTP3mM 0.85 3 CTP3mM 0.85 2 UTP3mM 0.85 2 Mg(Glu)23mM 8 19 extract / lysate2; 3v / v % 0.24 0.4 Hepes(KOH), pH7.5 mM 57 100 C: Reagents which may additionally present in CFPS reaction mixture and buffer of the invention. KHPO4, pH7.2 mM 0 10 GMP mM 0 5 DTT mM 0 5 nuclease inhibitor mg / mL 0 0.03 RNase inhibitor (murine) Unit / µL 0 1.01may not be present in the buffer as such or in the CFPS reaction mixture as such2may not be present in the buffer as such3components essential for CFPS (for basic synthesis) Table 2: Non-limiting examples of cellular extracts / lysates, and respective temperatures / temperature ranges, which may be used / applied in accordance with the invention, e.g. as a basis of the CFPS reaction mixture, CFPS lysate or method as disclosed herein. Origin of cell free extract Optimal temperature for performance of CFPS Wheat germ 25^C Rabbit reticulocytes30^C (see, for example, https: / / www.promega.de / en / products / protein- expression / cell-free-protein-expression / rabbit-reticulocyte-lysate- system-nuclease-treated / ?catNum=L4960#protocols) Nicotiana tabacum 25^C (see, for example, https: / / www.leniobio.com / product / alice-for- research-cell-free-protein-expression-midi-kit-24-reactions / ) Yeast species 30^C Escherichia coli30^CV. natriegens 28-32^C B. subtilis28-32^CC. glutamicum25-30^CK. pneumoniae 28-32^C P. fluorescens25-30^CB. megaterium 25-30^C Herein exemplified GOI and respective POI sfGFP: super-folded green fluorescence protein RsTYR: Ralstonia solanacearum Tyrosinase ScKDC: Saccharomyces cerevisiae Phenylpyruvate decarboxylase EcHpaB: Escherichia coli 4-hydroxyphenylacetate 3-monooxygenase oxygenase EcHpaC: Escherichia coli 4-hydroxyphenylacetate 3-monooxygenase reductase SvVlmR: Streptomyces viridifaciens NADPH-flavin oxidoreductase PcAAS: Petroselinum crispum 4-hydroxyphenylacetaldehyde synthase EcYahK: Escherichia coli Aldehyde reductase RrBLVRB: Rattus rattus Flavin reductase EcTyrC: Escherichia coli Cyclohexadienyl dehydrogenase EcTyrB: Escherichia coli Aromatic-amino-acid aminotransferase EcGluD: Escherichia coli Glutamate dehydrogenase Reference is made herein also to the following nucleotide sequences (and also to the respective encoded amino acid sequences, as the case may be), as well as to the following amino acid sequences (and also to the respective coding nucleotide sequences, as the case may be). SEQ ID NO.1: terA nucleotide sequence; AATTAGTATGTTGTAACTAAAGT; 23 nucleotides SEQ ID NO. 2: “Insempra” lacO nucleotide sequence (LacI-binding site); cccgggaattgtgagcggataacaattcccggg; 33 nucleotides SEQ ID NO.3: terB nucleotide sequence; AATaAGTATGTTGTAACTAAAGT; 23 nucleotides SEQ ID NO.4: terC nucleotide sequence; AtaTAGgATGTTGTAACTAAtaT; 23 nucleotides SEQ ID NO.5: terD nucleotide sequence;cATTAGTATGTTGTAACTAAAtg; 23 nucleotides SEQ ID NO.6: terE nucleotide sequence; ttaaAGTATGTTGTAACTAAg--; 21 nucleotides SEQ ID NO.7: ter consensus sequence; AGxATGTTGTAACTAA SEQ ID NO.8: lacO consensus sequence;GTGAGCGxxTxxCASEQ ID NO.9: LacI-binding site, Core; --------TTGTGAGCGGATAACAA--------; 17 nucleotides SEQ ID NO. 10: LacI-binding site, lacO1; ------AATTGTGAGCGGATAACAATT------; 21 nucleotides SEQ ID NO.11: LacI-binding site, pET; ----GGAATTGTGAGCGGATAACAATTCC----; 25 nucleotides SEQ ID NO. 12: LacI-binding site, Symmetric; ------AATTGTGAGCGCTCACAATT-------; 20 nucleotides SEQ ID NO. 13: LacI-binding site, Gilbert; ---TGGAATTGTGAGCGGATAACAATT------; 24 nucleotides SEQ ID NO. 14: LacI-binding site, lacO2; ------AAaTGTGAGCGagTAACAAcc------; 21 nucleotidesSEQ ID NO. 15: LacI-binding site, lacO3;------ggcaGTGAGCGcAacgCAATT------; 21nucleotides SEQ ID NO.16: amino acid sequence of E. coli Tus protein (UniProt: A0A140NBS0, E. coli BL21- DE3 strain) MARYDLVDRLNTTFRQMEQELAAFAAHLEQHKLLVARVFSLPEVKKEDEHNPLNRIEVKQHLGNDAQSLALRHFRH LFIQQQSENRSSKAAVRLPGVLCYQVDNLSQAALVSHIQHINKLKTTFEHIVTVESELPTAARFEWVHRHLPGLIT LNAYRTLTVLHDPATLRFGWANKHIIKNLHRDEVLAQLEKSLKSPRSVAPWTREEWQRKLEREYQDIAALPQNAKL KIKRPVKVQPIARVWYKGDQKQVQHACPTPLIALINRDNGAGVPDVGELLNYDADNVQHRYKPQAQPLRLIIPRLH LYVAD SEQ ID NO.17: amino acid sequence of E. coli LacI protein (UniProt: A0A140NB96, E. coli BL21- DE3 strain) MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLAL HAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLD VSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSG FQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSV DRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ SEQ ID NO.18: nucleotide sequence encoding nGFP atgagcaaaggtgaagaactgtttaccggcgttgtgccgattctggtggaactggatggc SEQ ID NO.19: Consensus sequence of 2A self-cleavage peptide: LxxxGDVExNPGP SEQ ID NO.20: Core sequence motif of 2A self-cleavage peptide:DVExNPGPSEQ ID NO.21: P2A self-cleavage peptide; ATNFSLLKQAGDVEENPGP; 19 amino acid residues SEQ ID NO. 22: P2A self-cleavage peptide (herein exemplified version);GSGATNFSLLKQAGDVEENPGP; 22 amino acid residuesSEQ ID NO.23: F2A self-cleavage peptide; VKQTLNFDLLKLAGDVESNPGP; 22 amino acid residues SEQ ID NO.24: E2A self-cleavage peptide; --QCTNYALLKLAGDVESNPGP; 20 amino acid residues SEQ ID NO.25: T2A self-cleavage peptide; ----EGRGSLLTCGDVEENPGP; 18 amino acid residues SEQ ID NO.26: Nanoluciferase binding peptide tag (HiBiT; Promega): VSGWRLFKKIS SEQ ID NO. 27: Nanoluciferase binding peptide tag (herein exemplified version): SSG VSGWRLFKKIS SEQ ID NO.28: amino acid sequence of nGFP MSKGEELFTGVVPILVELDG SEQ ID NO. 29: nucleotide sequence encoding P2A self-cleavage peptide: gctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacct SEQ ID NO. 30: nucleotide sequence encoding P2A self-cleavage peptide (herein exemplified version): ggaagcggagctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacct SEQ ID NO. 31: nucleotide sequence encoding F2A self-cleavage peptide: GTGAAACAGACCCTGAACTTTGATCTGTTGAAGTTAGCGGGCGACGTTGAAAGCAATCCGGGTCCT SEQ ID NO. 32: nucleotide sequence encoding E2A self-cleavage peptide: CAGTGCACCAACTATGCGTTACTGAAACTGGCCGGCGATGTGGAAAGCAATCCGGGTCCT SEQ ID NO. 33: nucleotide sequence encoding T2A self-cleavage peptide: GAAGGTCGCGGCAGCTTACTGACCTGCGGAGATGTGGAAGAGAACCCTGGCCCG SEQ ID NO.34: coding sequence for nanoluciferase binding peptide tag (HiBiT; Promega): GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC SEQ ID NO. 35: coding sequence for nanoluciferase binding peptide tag (herein exemplified version; RNA; including STOP codon): AGCUCCGGUGUGAGCGGCUGGCGGCUGUUCAAGAAGAUUAGCUAA SEQ ID NO.36: Example of 5’ overhang (“buffer region 1”): atgcgtccggcgtagaggatcgagatcgatctcgatcccgcgaaat SEQ ID NO.37: Example of 3’ UTR: gtcgaccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataa SEQ ID NO.38: Example of 3’ overhang (“buffer region 2”): ctgaaagccaattctgagtcatagctgtttcctggcggccgctattagacgcgatcgcccgggagctgcatgtgtc agaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgt catgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtt SEQ ID NO.39: T7 Promoter: TAATACGACTCACTATAGGG SEQ ID NO.40: T7 terminator: CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG SEQ ID NO.41: Example of 5’ Overhang + T7 Promoter atgcgtccggcgtagaggatcgagatcgatctcgatcccgcgaaatTAATACGACTCACTATAGGG SEQ ID NO.42: Example of 3’ UTR + T7 terminator + 3’ Overhang gtcgaccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataaCTAGCATAACC CCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGctgaaagccaattctgagtcatagctgtttcctggcggc cgctattagacgcgatcgcccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaa agggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttt tcggggaaatgtgcgcggaacccctatttgtt SEQ ID NO.43: sfGFP: super-folded green fluorescence protein atgagcaaaggtgaagaactgtttaccggcgttgtgccgattctggtggaactggatggcgatgtgaacggtcaca aattcagcgtgcgtggtgaaggtgaaggcgatgccacgattggcaaactgacgctgaaatttatctgcaccaccgg caaactgccggtgccgtggccgacgctggtgaccaccctgacctatggcgttcagtgttttagtcgctatccggat cacatgaaacgtcacgatttctttaaatctgcaatgccggaaggctatgtgcaggaacgtacgattagctttaaag atgatggcaaatataaaacgcgcgccgttgtgaaatttgaaggcgataccctggtgaaccgcattgaactgaaagg cacggattttaaagaagatggcaatatcctgggccataaactggaatacaactttaatagccataatgtttatatt acggcggataaacagaaaaatggcatcaaagcgaattttaccgttcgccataacgttgaagatggcagtgtgcagc tggcagatcattatcagcagaataccccgattggtgatggtccggtgctgctgccggataatcattatctgagcac gcagaccgttctgtctaaagatccgaacgaaaaaggcacgcgggaccacatggttctgcacgaatatgtgaatgcg gcaggtattacg SEQ ID NO.44: RsTYR: Ralstonia solanacearum Tyrosinase ATGGTTGTTAGACGCACAGTTCTCAAAGCCATTGCTGGCACCTCTGTAGCGACCGTTTTCGCCGGAAAACTTACGG GACTGAGTGCGGTGGCTGCAGACGCAGCACCCTTGAGAGTTCGTCGTAATTTACACGGGATGAAAATGGATGATCC AGATTTGTCCGCGTACCGTGAATTTGTGGGTATTATGAAGGGAAAAGATCAAACCCAAGCCTTATCTTGGCTTGGT TTCGCAAATCAACATGGCACTCTGAATGGTGGGTATAAATATTGTCCACATGGAGATTGGTATTTCTTACCGTGGC ATCGAGGATTTGTCCTTATGTATGAACGTGCGGTAGCGGCCTTAACGGGTTATAAAACTTTTGCAATGCCTTATTG GAATTGGACTGAGGATCGGTTGTTACCAGAGGCGTTTACTGCGAAAACTTATAATGGTAAAACTAATCCCCTGTAT GTCCCGAATAGAAACGAATTGACTGGTCCGTATGCTTTAACTGATGCAATAGTGGGACAGAAAGAAGTAATGGATA AAATATACGCGGAGACGAATTTTGAGGTGTTTGGTACTTCACGCAGTGTTGATCGGTCCGTTCGTCCACCATTAGT GCAAAATTCGCTTGATCCGAAGTGGGTACCAATGGGCGGTGGAAATCAAGGTATATTAGAACGTACTCCTCATAAT ACTGTGCATAATAATATTGGAGCATTTATGCCAACTGCAGCTTCTCCACGGGACCCAGTATTTATGATGCATCATG GAAACATTGATCGTGTCTGGGCAACGTGGAATGCACTTGGTCGTAAGAATAGTACCGATCCTTTATGGCTTGGTAT GAAATTTCCGAATAATTATATTGACCCCCAAGGTCGATATTATACCCAGGGTGTTTCTGACTTGTTGTCCACAGAA GCCCTTGGTTATCGTTACGATGTGATGCCTAGAGCAGATAATAAAGTAGTAAATAATGCGCGCGCTGAACACTTAT TGGCGTTGTTTAAAACTGGGGATTCGGTTAAATTAGCAGACCACATACGTTTGCGTTCAGTTTTAAAAGGAGAGCA CCCAGTGGCAACTGCAGTGGAGCCCCTTAATTCAGCAGTGCAATTTGAAGCAGGTACGGTTACAGGCGCATTAGGA GCGGACGTGGGAACGGGGAGCACAACTGAGGTAGTTGCACTGATTAAGAATATTCGAATACCGTACAATGTGATAT CAATAAGAGTTTTCGTAAATTTACCTAATGCTAATCTGGACGTCCCCGAGACAGATCCTCATTTTGTGACTTCACT GAGTTTTCTGACACACGCAGCAGGTCACGATCATCACGCTCTCCCGTCCACTATGGTCAATCTCACTGATACCTTA AAAGCACTGAATATTCGTGATGATAATTTCAGCATAAATCTTGTAGCTGTTCCACAGCCAGGTGTAGCGGTCGAAT CGTCAGGCGGGGTTACTCCAGAGAGCATTGAAGTTGCAGTTATA SEQ ID NO.45: ScKDC: Saccharomyces cerevisiae Phenylpyruvate decarboxylase ATGGCCCCGGTGACTATAGAGAAATTTGTTAACCAAGAGGAGAGACATCTCGTGAGTAATCGGAGCGCGACCATCC CATTCGGCGAGTATATTTTCAAGCGTCTGTTATCAATTGACACAAAGTCCGTGTTTGGAGTGCCAGGGGATTTTAA TCTCAGCCTGCTGGAGTACCTTTACTCGCCGTCAGTAGAGAGTGCCGGACTGCGGTGGGTTGGAACATGCAACGAG TTAAATGCTGCCTACGCTGCAGATGGGTACAGTCGGTATTCCAACAAAATAGGTTGCCTTATCACAACATACGGTG TCGGAGAGCTGTCTGCGTTAAATGGCATTGCTGGCAGTTTTGCGGAGAACGTGAAGGTGCTTCATATCGTAGGAGT AGCCAAAAGTATTGACAGCCGGTCCTCAAATTTCTCAGACCGTAATCTGCACCACCTGGTTCCGCAACTGCACGAC TCCAACTTCAAGGGCCCTAACCACAAGGTTTACCACGACATGGTTAAGGACCGCGTGGCATGTAGTGTTGCTTATC TGGAAGACATAGAGACGGCGTGCGATCAGGTAGACAACGTGATTAGAGACATCTATAAATACAGTAAGCCGGGCTA CATCTTCGTCCCGGCTGACTTCGCAGATATGTCGGTGACCTGCGACAACCTGGTGAACGTACCTCGGATTTCACAG CAGGACTGCATCGTTTATCCGAGCGAGAATCAGTTAAGTGATATCATTAATAAAATAACATCGTGGATTTACTCGT CGAAGACCCCAGCCATATTGGGCGATGTGCTTACGGACCGCTACGGGGTTTCCAATTTCTTAAATAAATTGATTTG TAAGACCGGAATATGGAACTTCAGCACCGTGATGGGTAAGTCCGTGATAGACGAAAGTAATCCGACCTACATGGGC CAGTACAACGGGAAGGAAGGCCTTAAACAGGTATACGAGCACTTCGAGTTGTGTGATCTGGTACTGCACTTCGGTG TTGATATAAACGAGATCAACAACGGTCACTACACCTTCACCTACAAGCCGAACGCAAAGATAATACAGTTCCACCC TAACTACATACGTTTAGTAGATACCCGTCAAGGTAACGAACAGATGTTTAAGGGGATTAACTTCGCGCCGATCTTA AAGGAGTTGTATAAAAGAATAGATGTCAGTAAGTTGTCATTACAGTACGACAGTAACGTCACCCAGTACACAAATG AGACGATGCGTCTTGAGGACCCCACGAACGGCCAGAGTTCAATAATAACTCAGGTGCATCTGCAGAAAACCATGCC GAAGTTCCTGAATCCCGGAGACGTAGTAGTGTGCGAGACGGGATCTTTCCAGTTTTCAGTGCGCGACTTTGCATTC CCCTCACAGCTGAAGTACATTTCTCAGGGGTTCTTTCTGAGTATAGGGATGGCTTTACCGGCGGCTCTGGGCGTAG GGATCGCGATGCAGGATCATTCCAATGCCCATATTAACGGCGGAAATGTGAAGGAAGATTACAAACCGCGACTTAT ACTGTTCGAGGGCGATGGCGCTGCTCAAATGACGATTCAGGAGTTGAGTACAATACTCAAATGTAACATCCCGTTA GAGGTAATAATCTGGAATAACAATGGATATACAATCGAGCGTGCTATTATGGGTCCCACAAGATCCTACAATGATG TAATGAGCTGGAAGTGGACAAAGTTGTTCGAGGCCTTTGGTGATTTTGATGGTAAATACACAAACTCTACGCTGAT CCAGTGCCCGAGCAAGCTCGCGCTGAAGCTCGAAGAATTGAAGAACAGCAATAAGCGTTCGGGCATTGAGTTACTT GAGGTTAAGCTCGGGGAGCTTGACTTTCCTGAGCAACTGAAATGTATGGTAGAGGCGGCAGCTCTGAAGCGCAACA AGAAG SEQ ID NO.46: EcHpaB: Escherichia coli 4-hydroxyphenylacetate 3-monooxygenase oxygenase atgaaacctgaagatttccgcgccagtacgcaacgtccttttacaggggaagagtatcttaaaagcctgcaagatg gtcgtgagatctatatctatggcgagcgagtaaaagatgtcaccactcatccggcatttcgtaatgcggcagcgtc tgttgcccagctgtatgacgcattgcacaaaccggaaatgcaggactctctgtgttggaataccgataccggcagc ggcggctatacgcataaattctttagagtggcgaaaagtgccgacgacttacgccagcaacgcgatgccatcgctg aatggtcacgcctgagctatggatggatgggccgtacccccgactacaaagccgcttttggatgcgcactgggcgc gaatccgggcttttacggtcagttcgagcagaacgcccgtaactggtacacccgtattcaggaaactggactctac tttaaccacgcgattgttaacccacccattgatcgtcatttgccgaccgataaagtgaaagacgtttacatcaagc tggaaaaagaaacggacgccgggattatagtcagcggtgcgaaagtggttgccacaaattcggcgctgactcatta caacatgattggcttcggcACCCTGGAAgtgatgggcgaaaacccggactttgcgttaatgttcgttgcgcctatg gatgccgatggcgtaaaattaatctcgcgagcctcttatgagatggtcgcgggtgctaccggctcaccatacgact acccgctctccagccgcttcgatgagaacgatgcgattctggtgatggataacgtgctgattccctgggaaaatgt gctgatttaccgcgattttgatcggtgccgtcgctggacgatggaaggcggctttgcccgtatgtatccgcttcaa gcctgtgtgcgcctggcagtgaaattagacttcattacggcactgctgaaaaaatcactcgaatgtaccggcaccc tggagttccgtggtgttcaggccgatctaggtgaagtggtagcgtggcgcaacacattttgggcattgagtgactc gatgtgctcagaagcaacgccgtgggtcaacggggcttatttaccggatcatgcggcacttcaaacctatagggta ctggcaccaatggcgtacgcgaagatcaaaaacattatcgaacgcaacgttaccagtggcctcatttatctacctt ccagcgcccgtgacctgaataatccgcagatcgatcagtatctggcgaagtatgtacggggttcgaacgggatgga tcatgtccagcgcataaagattctcaaactgatgtgggatgctattggaagcgaatttggtggtcgtcacgaatta tatgaaatcaattacagcggtagccaggatgagattcgcttgcagtgcttgcgccaggcgcaaaactccggcaata tggataagatgatggcgatggttgatcgctgcctgtcggaatacgaccaggatggatggactgtgccgcaccttca caataatgacgatattaatatgttagataagctgcttaaa SEQ ID NO.47: EcHpaC: Escherichia coli 4-hydroxyphenylacetate 3-monooxygenase reductase atgcaattagatgaacaacggctgcgctttcgtgacgcgatggccagcttatcggcggcggtaaatattatcacca ccgaaggcgacgccggacagtgtgggattacggcaaccgccgtatgttctgtcacggatacaccaccctccctgat ggtgtgcattaacgccaattcagctatgaacccggtttttcagggcaacggcaagttgtgcgttaatgtcctcaac cacgagcaggaactgatggcacgacacttcgcgggcatgaccggcatggcgatggaagagcgtttcagcctctcat gctggcagaaaggtccgctggcgcagcctgtgctaaaaggtagcttggccagtcttgaaggtgagatccgcgatgt gcaggcaattggcactcatctggtgtatctggtggagattaaaaatatcatactgtcggcagaaggtcatggactt atctactttaaacgccgtttccatccggttatgctggaaatggaagctgcgatt SEQ ID NO.48: SvVlmR: Streptomyces viridifaciens NADPH-flavin oxidoreductase ATGACCCCTAGTGCGGCTGCGACCGGCCATGAAGCAGCCGACGAACAGCGTCTGCGCGAATTAAGGGGCCTCACCC GCCAGTTGCCGACGGGTGTGGCGGTGGTGACGGCCCAGGACGGCGAAGTGGCACACGGGGCGACTGTCAGTACCGT CAGCGTACTTTCGCAACAGCCGCTGCGCATTGGTGTTAGCCTGCGACGCGGTTCGTATTTGACAGGTCTGATCCGC CAACGTCGCGTGTTCGCATTGAATGTTCTGTCCTCCCGCCAGTCAGCGGTGGCGGATTGGTTTGCAAACCCGGAAC GACCGCGTGGTTGGCGCCAGTTTGATTACGTCCGTTGGACTGCGCATCCGAAAGCCGGGATGCCGGTTCTGGAGGA TGCGTTAGCCCAGCTTCACTGCCGGTTAACGGACCTGATTCCCCTCGGAGCCAGCGATGATCTGCTGGTCGCCGAA GTTCTGGATGGCCGTGGACGGAACGGCCGTCCACTGGTGAATTTTAACGGCCGTCTACATGATGTAGAGTTCCGCG GCGTTGTGCGCGTAAGCCGTGACCAACCTTCTGCTGTAACCTCACTTGAG SEQ ID NO.49: PcAAS: Petroselinum crispum 4-hydroxyphenylacetaldehyde synthase ATGGGGTCAATTGACAACCTGACGGAGAAACTGGCATCACAATTCCCTATGAACACCTTAGAACCAGAGGAATTTA GACGACAGGGACACATGATGATCGACTTCCTGGCAGACTATTACCGCAAGGTCGAGAACTACCCGGTGCGTAGCCA AGTAAGCCCAGGGTACCTGCGGGAGATCCTGCCCGAGAGTGCGCCGTATAATCCTGAGAGCTTGGAGACGATATTA CAGGACGTGCAGACTAAGATCATTCCGGGAATAACCCACTGGCAGTCTCCGAATTTCTTCGCGTACTTCCCCAGCA GCGGCTCCACCGCCGGGTTCCTGGGCGAGATGCTGTCAACAGGCTTTAACGTGGTGGGTTTCAATTGGATGGTGTC TCCAGCCGCAACTGAACTTGAGAATGTCGTTACTGACTGGTTTGGTAAAATGTTACAGTTACCGAAGAGCTTCCTG TTTTCCGGTGGCGGTGGTGGAGTTTTACAGGGTACCACGTGTGAAGCGATTCTCTGCACTTTGGTAGCGGCTCGTG ATAAGAATTTACGTCAGCACGGTATGGACAACATCGGTAAACTGGTGGTCTACTGCAGCGATCAGACACACAGTGC ACTCCAGAAAGCGGCTAAGATAGCCGGTATAGACCCTAAGAATTTTCGCGCTATTGAGACGACCAAGAGTTCTAAC TTTCAACTTTGCCCAAAACGGTTAGAGTCAGCAATTCTCCACGATCTCCAGAACGGACTCATACCTCTGTATCTGT GCGCCACCGTCGGTACGACTTCTTCCACGACAGTAGACCCGCTGCCTGCCCTCACCGAAGTTGCTAAGAAATATGA CTTGTGGGTACACGTTGACGCCGCTTACGCCGGCTCGGCATGTATTTGTCCTGAGTTCAGACAATACTTAGATGGG GTCGAGAACGCTGACAGCTTCTCTCTGAACGCTCATAAATGGTTCCTGACCACTCTGGACTGCTGCTGCCTGTGGG TTCGTAACCCCTCAGCGTTGATTAAATCACTCAGCACTTACCCAGAATTTCTGAAGAACAACGCCAGCGAGACTAA TAAAGTAGTTGACTATAAGGATTGGCAGATCATGCTGTCCAGACGTTTCCGTGCCCTGAAGCTGTGGTTCGTGCTC CGTTCTTATGGCGTCGGCCAATTGCGCGAATTTATACGAGGACACGTGGGTATGGCAAAATACTTTGAGGGCCTTG TAAATATGGATAAACGATTTGAGGTGGTGGCCCCGCGGCTCTTCTCAATGGTTTGCTTCCGAATAAAACCCAGTGC CATGATAGGAAAGAACGACGAGGACGAGGTCAATGAAATTAATCGCAAACTGCTGGAAAGTGTTAACGACTCAGGG CGCATTTACGTCTCGCATACCGTCCTTGGCGGCATATATGTTATACGTTTCGCAATTGGCGGTACGCTGACGGACA TCAATCACGTTTCGGCTGCGTGGAAAGTCCTGCAAGATCACGCGGGTGCACTGCTGGACGACACATTTACATCTAA CAAATTAGTAGAGGTTCTCAGT SEQ ID NO.50: EcYahK: Escherichia coli Aldehyde reductase atgaagatcaaagctgttggtgcatattccgctaaacaaccacttgaaccgatggatatcacccggcgtgaaccgg gaccgaatgatgtcaaaatcgaaatcgcttactgtggcgtttgccattccgatctccaccaggtccgttccgagtg ggcggggacggtttacccctgcgtgccgggtcatgaaattgtggggcgtgtggtagccgttggtgatcaggtagaa aaatatgcgccgggcgatctggtcggtgtcggctgcattgtcgacagttgtaaacattgcgaagagtgtgaagacg ggttggaaaactactgtgatcacatgaccggcacctataactcgccgacgccggacgaaccgggccatactctggg cggctactcacaacagatcgtcgttcatgagcgatatgttctgcgtattcgtcacccgcaagagcagctggcggcg gtggctcctttgttgtgtgcagggatcaccacgtattcgccgctacgtcactggcaggccgggccgggtaaaaaag tgggcgtggtcggcatcggcggtctgggacatatggggattaagctggcccacgcgatgggggcacatgtggtggc atttaccacttctgaggcaaaacgcgaagcggcaaaagccctgggggccgatgaagttgttaactcacgcaatgcc gatgagatggcggctcatctgaagagtttcgatttcattttgaatacagtagctgcgccacataatctcgacgatt ttaccaccttgctgaagcgtgatggcaccatgacgctggttggtgcgcctgcgacaccgcataaatcgccggaagt tttcaacctgatcatgaaacgccgtgcgatagccggttctatgattggcggcattccagaaactcaggagatgctc gatttttgcgccgaacatggcatcgtggctgatatagagatgattcgggccgatcaaattaatgaagcctatgagc gaatgctgcgcggtgatgtgaaatatcgttttgttatcgataatcgcacactaacagac SEQ ID NO.51 RrBLVRB: Rattus rattus Flavin reductase atgacagtaaagaaaaacgcgaatggcgcaacgggccgcaccggcactacggcagccgtggcgggttatgtcacgg tggtgcgcgatagctcacgctccggtgcgcatgtcgtggtgggtgatgttcgcgccgcagacgtagacaaaacggt ggcgggcgatgccgtaaatgtgggcactgggaacgatagtaccaccgttatgagtggtacccgtaacaatgttgcc gcgatgaaggcgcacggcgtagacaaagtggtcgcttgcacctcggcttgggatagcaaagtgagcaacgttactg atgatcacaaccgaatgcataaaaattcaggtaaatacgtggccgttatgcacaacggagataccggagcatacac cgttaccgacggacgtggctcgcgtgtcaattctaaacatgatgggcacatgcggtgtacgacacattacgatggc aagacctatagccattatgac SEQ ID NO.52: EcTyrC: Escherichia coli Cyclohexadienyl dehydrogenase aTGACCGTCTTTAAGCATATTGCCATTATCGGATTAGGACTGATCGGTTCCTCTGCGGCACGGGCAACAAAGGCCT ATTGTCCTGATGTAACGGTCAGTCTCTATGACAAAAGCGAATTTGTCCGCGACAGAGCTAGAGCGCTCAATCTCGG CGACAATGTCACCGATGATATTCAAGATGCGGTTCGTGAGGCTGATCTGGTGCTGCTATGCGTGCCAGTCAGGGCA ATGGGTATCGTCGCGGCAGCGATGGCACCGGCACTGAAAAAAGACGTTATTATCTGCGATACAGGTTCGGTAAAAG TCAGCGTTATAAAAACGCTGCAAGACAATTTACCCAATCACATTATTGTTCCCAGCCATCCTTTGGCTGGGACTGA AAATAACGGACCCGATGCCGGTTTTGCTGAATTATTCCAAGATCATCCTGTTATTTTGACCCCCGATGCCCATACA CCAGCACAGGCTATCGCCTATATCGCCGATTATTGGGAAGAAATTGGTGGGCGTATCAATCTGATGTCGGCGGAAC ATCACGATCACGTTTTGGCGCTTACCAGCCATTTGCCTCATGTCATTGCATACCAACTTATCGGGATGGTATCGGG TTATGAGAAAAAAAGCCGGACACCCATTATGCGTTATTCGGCAGGCAGCTTTCGGGATGCGACGCGGGTAGCGGCT TCGGAACCGCGTCTCTGGCAAGATATTATGCTGGAAAATGCGCCCGCCCTTTTACCAGTGCTGGATCATTTTATCG CAGATCTCAAAAAATTGCGGACAGCCATTGCTTCGCAAGATGGGGATTATCTTCTTGAGCATTTCAAAGAATCGCA GAAAGCGCGTTTAGCCTTAAAAACAGACCACGATATTCGCCCT SEQ ID NO.53: EcTyrB: Escherichia coli Aromatic-amino-acid aminotransferase ATGtttcaaaaagttgacgcctacgctggcgacccgattcttacgcttatggagcgttttaaagaagaccctcgca gcgacaaagtgaatttaagtatcggtctgtactacaacgaagacggaattattccacaactgcaagccgtggcgga ggcggaagcgcgcctgaatgcgcagcctcatggcgcttcgctttatttaccgatggaagggcttaacagctatcgc catgccattgcgccgctgctgtttggtgcggaccatccggtactgaaacaacagcgcgtagcaaccattcaaaccc ttggcggctccggggcattgaaagtgggcgcggatttcctgaaacgctacttcccggaatcaggcgtctgggtcag cgatcctacctgggaaaaccacgtagcaatattcgccggggctggattcgaagtgagtacttacccctggtatgac gaagcgactaacggcgtgcgctttaatgacctgttggcgacgctgaaaacattacctgcccgcagtattgtgttgc tgcatccatgttgccacaacccaacgggtgccgatctcactaatgatcagtgggatgcggtgattgaaattctcaa agcccgcgagcttattccattcctcgatattgcctatcaaggatttggtgccggtatggaagaggatgcctacgct attcgcgccattgccagcgctggattacccgctctggtgagcaattcgttctcgaaaattttctccctttacggcg agcgcgtcggcggactttctgttatgtgtgaagatgccgaagccgctggccgcgtactggggcaattgaaagcaac agttcgccgcaactactccagcccgccgaattttggtgcgcaggtggtggctgcagtgctgaatgacgaggcattg aaagccagctggctggcggaagtagaagagatgcgtactcgcattctggcaatgcgtcaggaattggtgaaggtat taagcacagagatgccagaacgcaatttcgattatctgcttaatcagcgcggcatgttcagttataccggtttaag tgccgctcaggttgaccgactacgtgaagaatttggtgtctatctcatcgccagcggtcgcatgtgtgtcgccggg ttaaatacggcaaatgtacaacgtgtggcaaaggcgtttgctgcggtgatgt SEQ ID NO.54: EcGluD: Escherichia coli Glutamate dehydrogenase atgagtggcaaagatgtaaatgtcttcgagatggcgcaaagccaagtcaagaatgcatgtgacaaattgggcatgg aaccggcagtttatgaattgttgaaagaacctatgcgtgtgattgaggtgtcaattccagtaaaaatggatgacgg ttcgatcaagacctttaaaggatttcgctcccagcataatgatgcagtagggccgacaaaaggtggcattcgattc caccagaacgttagccgtgacgaagtcaaagccctgtctatctggatgaccttcaagtgcagcgtgacgggcattc cctatggtggcggtaaaggcggcatcattgtcgatccgagcacgttaagccagggtgaactggaacgtctgagtcg cggatacatcgatggaatctataaattaatcggtgagaaagttgacgttcctgcaccggacgtaaatacgaacggc cagattatgtcgtggatggttgatgagtacaataaactgactggacagagttctattggtgtcattaccggcaagc cggtggaatttggtggttcgctgggcaggaccgcggcgactggctttggggtggcggttacagcacgggaagccgc ggcgaaactaggcattgatatgaaaaaagccaaaattgcggtgcaaggtatcggcaacgtgggctcctataccgtg ctgaactgtgagaaacttggtgggacggtggtagctatggctgaatggtgcaagagtgaaggtagctatgccatct acaatgaaaatggccttgatgggcaggccatgttagattatatgaaagaacatgggaacctgctcaacttccccgg cgccaagcgcatctctttagaagagttctgggcctcagatgttgatatagtcataccagcggcactcgaaaacagc attaccaaagaagtggccgaaagcattaaggctaaactggtgtgcgaggccgcgaatggaccgactacgccggagg ccgatgaagtatttgcggaacgtggaattgtgcttaccccggacattctgaccaacgcgggcggcgttaccgtgtc ctattttgagtgggtgcagaacctgtatggctactactggtcagaagaagaagtcgagcagaaagaagaaattgcg atggttaaagcatttgaatcgatttggaaaatcaaagaagagtacaacgttacaatgcgcgaagctgcgtacatgc actcaattaagaaggtggccgaagcgatgaaactgcgcggctggtac SEQ ID NO.55: RBS for, for example, sfGFP: super-folded green fluorescence protein (from pJL1) agaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatatacat SEQ ID NO.56: RBS for, for example, RsTYR: Ralstonia solanacearum Tyrosinase ATTCCCCAAGGACAAATACATTTAAGGAGGTTTTTT SEQ ID NO. 57: RBS for, for example, ScKDC: Saccharomyces cerevisiae Phenylpyruvate decarboxylase AGGAGAATATCCACAAACCACAACTAAGGAGGTATTTTT SEQ ID NO. 58: RBS for, for example, EcHpaB: Escherichia coli 4-hydroxyphenylacetate 3- monooxygenase oxygenase TCCGAATCTCAGCACCAATTTTAAGGAGGTTATTT SEQ ID NO. 59: RBS for, for example, EcHpaC: Escherichia coli 4-hydroxyphenylacetate 3- monooxygenase reductase CACTCGACTCTTTCCAGATAAATAAGGAGGTATTAT SEQ ID NO. 60: RBS for, for example, SvVlmR: Streptomyces viridifaciens NADPH-flavin oxidoreductase AGCAGGAATTAAAACAGATAAGTAAGGAGGATTTTTT SEQ ID NO. 61: RBS for, for example, PcAAS: Petroselinum crispum 4- hydroxyphenylacetaldehyde synthase CGAGCACCAACGGGCTCGAGACATACAAGTATACATAAGGAGGTAATTTT SEQ ID NO.62: RBS for, for example, EcYahK: Escherichia coli Aldehyde reductase CCTCGGTTCGAACGAGTAAATTTAGGTATTAAGGAGGTTATTTTAA SEQ ID NO.63: RBS for RrBLVRB: Rattus rattus Flavin reductase CGACAAAGAAGCTCAAAAAGATAAATAAGGAGGTATTTT SEQ ID NO.64: RBS for, for example, EcTyrC: Escherichia coli Cyclohexadienyl dehydrogenase CGCTTCCGCAGGAGAGTAAAGCTCTCCCTTAATATAAGAGAGAAATATA SEQ ID NO. 65: RBS for, for example, EcTyrB: Escherichia coli Aromatic-amino-acid aminotransferase TGCAATTAGATACAGTTCAGCTCCCGCTGAGTTAAGGAGGTTTTTT SEQ ID NO.66: RBS for, for example, EcGluD: Escherichia coli Glutamate dehydrogenase CTAGATACGCGAAAGTAGGATAAGGAGGTTTTTTT SEQ ID NO. 97: coding sequence for nanoluciferase binding peptide tag (herein exemplified version; DNA; including STOP codon): AGCTCCGGTGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTAA SEQ ID NO. 98: illustrative example of template / nucleic acid molecule of the invention (including EcHpaC coding sequence; including a P2A cleavage tag) AATTAGTATGTTGTAACTAAAGTAATTAGTATGTTGTAACTAAAGTatgcgtccggcgtagaggatcgagatcgat ctcgatcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaac tttaagaaggagatatacatatgagcaaaggtgaagaactgtttaccggcgttgtgccgattctggtggaactgga tggcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctatgcaattagatgaa caacggctgcgctttcgtgacgcgatggccagcttatcggcggcggtaaatattatcaccaccgaaggcgacgccg gacagtgtgggattacggcaaccgccgtatgttctgtcacggatacaccaccctccctgatggtgtgcattaacgc caattcagctatgaacccggtttttcagggcaacggcaagttgtgcgttaatgtcctcaaccacgagcaggaactg atggcacgacacttcgcgggcatgaccggcatggcgatggaagagcgtttcagcctctcatgctggcagaaaggtc cgctggcgcagcctgtgctaaaaggtagcttggccagtcttgaaggtgagatccgcgatgtgcaggcaattggcac tcatctggtgtatctggtggagattaaaaatatcatactgtcggcagaaggtcatggacttatctactttaaacgc cgtttccatccggttatgctggaaatggaagctgcgattAGCTCCGGTGTGAGCGGCTGGCGGCTGTTCAAGAAGA TTAGCtaagtcgaccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataacta gcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaagccaattctgagtcatagctgtttc ctggcggccgctattagacgcgatcgcccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgc gagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggt ggcacttttcggggaaatgtgcgcggaacccctatttgttcccgggaattgtgagcggataacaattcccgggccc gggaattgttatccgctcacaattcccggg SEQ ID NO. 99: illustrative example of template / nucleic acid molecule of the invention (including sfGFP coding sequence; not including a P2A cleavage tag) AATTAGTATGTTGTAACTAAAGTAATTAGTATGTTGTAACTAAAGTatgcgtccggcgtagaggatcgagatcgat ctcgatcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaac tttaagaaggagatatacatatgagcaaaggtgaagaactgtttaccggcgttgtgccgattctggtggaactgga tggcgatgtgaacggtcacaaattcagcgtgcgtggtgaaggtgaaggcgatgccacgattggcaaactgacgctg aaatttatctgcaccaccggcaaactgccggtgccgtggccgacgctggtgaccaccctgacctatggcgttcagt gttttagtcgctatccggatcacatgaaacgtcacgatttctttaaatctgcaatgccggaaggctatgtgcagga acgtacgattagctttaaagatgatggcaaatataaaacgcgcgccgttgtgaaatttgaaggcgataccctggtg aaccgcattgaactgaaaggcacggattttaaagaagatggcaatatcctgggccataaactggaatacaacttta atagccataatgtttatattacggcggataaacagaaaaatggcatcaaagcgaattttaccgttcgccataacgt tgaagatggcagtgtgcagctggcagatcattatcagcagaataccccgattggtgatggtccggtgctgctgccg gataatcattatctgagcacgcagaccgttctgtctaaagatccgaacgaaaaaggcacgcgggaccacatggttc tgcacgaatatgtgaatgcggcaggtattacgAGCTCCGGTGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCta agtcgaccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataac cccttggggcctctaaacgggtcttgaggggttttttgctgaaagccaattctgagtcatagctgtttcctggcgg ccgctattagacgcgatcgcccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacga aagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactt ttcggggaaatgtgcgcggaacccctatttgttcccgggaattgtgagcggataacaattcccgggcccgggaatt gttatccgctcacaattcccggg SEQ ID NO.100: 2xter sequences (RNA) AAUUAGUAUGUUGUAACUAAAGUAAUUAGUAUGUUGUAACUAAAGU SEQ ID NO.101: 2xlacO sequences (RNA) CccgggaauugugagcggauaacaauucccgggCccgggaauugugagcggauaacaauucccggg SEQ ID NO.102: T7 terminator sequence (RNA) cuagcauaaccccuuggggccucuaaacgggucuugagggguuuuuug Selection of abbreviations used herein GOI: gene of interest POI: protein of interest pUC: in-house high copy E. coli plasmid pUC-GOI: pUC plasmid constructed with respective GOI as described ter / lacO: 2 repeats of ter sequence at 5’ end and 2 repeats of lacO sequences at 3’ end of a linear DNA lacO / ter: 2 repeats of lacO sequence at 5’ end and 2 repeats of ter sequences at 3’ end of a linear DNA ter / ter: 2 repeats of ter sequence at both 5’ and 3’ end of a linear DNA lacO / lacO: 2 repeats of ter sequence at both 5’ and 3’ end of a linear DNA Nanoluciferase binding peptide: A small 11-amino acid peptide tag from Promega CFPS: cell-free protein synthesis PANOx-SP: PEP, amino acids, NAD, oxalate, spermidine, putrescine PEP: phosphoenolpyruvate NAD: nicotinamide adenine dinucleotide ATP: adenosine triphosphate GMP: guanosine monophosphate DTT: dithiothreitol The invention will now be described by reference to the following examples. These are merely illustrative and are not to be construed as a limitation of the scope of the present invention. Example 1: Materials and Methods Cell extract preparation E. coli BL21 (ED3) cells (purchased from Thermo Scientific) were inoculated in 2X YTPG medium (10 g / L yeast extract, 16 g / L tryptone, 5 g / L sodium chloride (NaCl), 7 g / L dipotassium hydrogen phosphate (K2HPO4), 3 g / L potassium dihydrogen phosphate (KH2PO4) and 18 g / L glucose) and grown overnight at 37 °C with shaking at 180 rpm. The overnight culture was diluted with fresh 1 L 2X YTPG medium in a 2-L baffled flask at OD600 of 0.1 and grown at 37 °C with shaking at 180 rpm. The cells were induced with 0.5 mM IPTG at OD600 of 0.6–0.8 for T7 RNA polymerase expression and harvested at OD600 of 2.8-3.2 by centrifuging at 4000 rpm and 4 °C for 10 mins. The cells were resuspended in the wash buffer (10 mM Tris-acetate (pH8.2), 14 mM magnesium acetate, 60 mM potassium acetate and 2 mM dithiothreitol (DTT)) and pelleted again by centrifuging at 4000 rpm and 4 °C for 10 mins, this was repeated three times. The final pellets were frozen in liquid nitrogen and stored at -80 °C. The frozen cell pellets were resuspended on ice in the lysis buffer (20 mM Tris-acetate (pH8.2), 14 mM magnesium acetate, 60 mM potassium acetate, 10 mM DTT, 1 tablet / 100ml buffer cOmplete™ EDTA-free Protease Inhibitor Cocktail (Merck)) at 1 ml buffer per gram of cells and aliquoted 1 mL into microcentrifuge tubes. Each aliquot of cell resuspension was sonicated in an ice water bath using QSonica Q125 Sonicator with 3.175 mm diameter probe at 50% amplitude with alternating 15 s on and 15 s off mode until the energy input reached 400-450 Joule.3 mM DTT were added to the lysate immediately after each sonication. The lysate was centrifuged twice at 10,000 g and 4 °C for 10 mins and the supernatants were collected without disturbing the cell debris. The lysates were frozen in liquid nitrogen and stored at -80 °C. Plasmid preparation A high copy number of E. coli plasmid, pUC, was constructed with an ampicillin resistance gene, a pUC ori, a T7 promoter, an in-frame C-terminal Nanoluciferase binding peptide tag and a T7 terminator. The gene of interest and the respective ribosome binding site (RBS) are conjugated downstream of the T7 promoter, in-frame with the C-terminal nanoluciferase binding peptide tag, and upstream of the T7 terminator by Gibson assembly (NEBuilder® HiFi DNA Assembly kit). For example, the sequence of a super-folded green fluorescence protein (sfGFP) and its bespoke RBS was constructed on the pUC plasmid under the T7 promoter with a C-terminal nanoluciferase binding peptide tag and upstream of a T7 terminator. For highly expressed P2A plasmid, first 60 bases of sfGFP (first 20 amino acid residues of sfGFP) and its pJL1-RBS are used as leader peptide and a P2A sequence is inserted at the position +61 (position of A in AUG is designated as +1), where the gene of interests is conjugated in-frame and downstream of the P2A sequence. All plasmids were replicated and isolated from NEB 10-beta strain following standard plasmid preparation protocol (NucleoSpin Plasmid Mini kit, MACHEREY-NAGEL) without the addition of RNase A in any of the solutions. Linear DNA preparation The coding sequence of the genes of interests (GOIs) were codon-optimized for E. coli and the double-stranded DNA fragments of the GOIs were synthesized by commercial producers (Integrated DNA Technologies and / or Twist Bioscience). RBSs with different predicted expression levels of a GOI were calculated from an online calculator (De Novo DNA; Salis, Nature Biotechnology 27, 2009, 946–50; doi:https: / / doi.org / 10.1038 / nbt.1568) (see RBS list herein elsewhere), except for sfGFP where its RBS is from pJL1 (Stark, Sci Adv 4(8), 2018, eaat5107, 1- 10; doi:10.1126 / sciadv.aat5107; Addgene plasmid #102634). T7 promoter and RBS were introduced to the GOI fragment in the forward primer that binds to the 5’ end of the GOI and Nanoluciferase binding peptide tag was introduced in-frame in the reverse primer that binds to the 3’ end of the GOI excluding the stop-codon by standard PCR reactions with synthesized GOI fragment as PCR template (see the Frw and Rev primers below). The elongated DNA fragments with T7 promoter and RBS at the 5’ end and Nanoluciferase binding peptide tag at the 3’ end were conjugated to the pUC plasmid backbone by Gibson assembly with respective overlapping regions in vitro (in vitro pUC-GOI). To introduce the end protections to each linear DNA with ter sequence at the 5’ end and lacO sequence at the 3’ end (ter / lacO protection), a forward primer with an overhang of 2 repeats of ter sequence and a reverse primer with an overhang of 2 repeats of lacO sequence were designed to bind at 46 and 223 bases upstream of the T7 promoter and downstream of the T7 terminator, respectively (see the F2 and R2 primers below). For other end-protection combinations (lacO / ter, ter / ter or lacO / lacO), 2 repeats of ter or lacO sequences were introduced accordingly as overhangs to the forward or reverse primers that bind to the same position on the plasmid. Forward and reverse primers without ter or lacO overhangs were used for the synthesis of the linear DNA fragment without end protection (termed F2 w / o protection group and R2 w / o protection group in the table below). The linear DNA fragments were synthesized by standard PCR reactions with respective primer sets and in vitro pUC-GOIs as template. The PCR products were purified with FastGene Gel / PCR Extraction Kit (NIPPON Genetics) following the manufacturer manual. SEQ ID NOs. 98 and 99 depict exemplary nucleic acid templates which have been generated as described above (including end-protection; with an EcHpaC- and sfGFP-encoding GOI, respectively). Nucleic acid templates with other GOIs, and / or with other respective components as described herein, and / or without end-protection (e.g. without the 2xTer and / or without the 2xLacO end-protection groups), can be generated analogously. The following primer were used in the context of the linear DNA fragment preparation: GOI Frw primer Rev primer Gtagaggatcgagatcgatctcgatcccgcgaaattaatac FP gactcactatagggagaccacaacggttt cacgaatatgtgaatgcggcaggtattacgAGCTCCGG sfG ccctctagaaata attttgtttaactttaagaaggagatatacatatgag (SEQ TGTGAGCGGCTGGCGGCTGTTCAAGAAGATT ID NO.67) AGCtaa (SEQ ID NO.68) cgtagaggatcgagatcgatctcgatcccgcgaaattaatac gcagccggtcgacttaGCTAATCTTCTTGAACAGC RsTYR gactcactatagggATTCCCCAAGGACAAATACATT CGCCAGCCGCTCACACCGGAGCTTATAACTG TAAGGAGGTTTTTTATGGTTG (SEQ ID NO.69) CAACTTCAATGCTCTCTGGAGTAACCCCGCCT GACGATTCGACCGC (SEQ ID NO.70) gtagaggatcgagatcgatctcgatcccgcgaaattaatacg gccggtcgacttaGCTAATCTTCTTGAACAGCCGC ScKDC actcactatagggagaccacaacggtttccctctagaaataa CAGCCGCTCACACCGGAGCTCTTCTTGTTGCG ttttgtttaactttaagaaggagatatacatatgag (SEQ ID CTTCAGAGCTGCCGCCTCTACCATACATTTCA NO.71) GTTGCTC (SEQ ID NO.72) cgtagaggatcgagatcgatctcgatcccgcgaaattaatac gtcgacttaGCTAATCTTCTTGAACAGCCGCCAG EcHpaB gactcactatagggTCCGAATCTCAGCACCAATTTT CCGCTCACACCGGAGCTtttaagcagcttatctaac AAGGAGGTTATTTatgaaacc (SEQ ID NO.73) atattaatatcgtcattattgtgaaggtgcggcacagtccat ccatcctg (SEQ ID NO.74) cggcgtagaggatcgagatcgatctcgatcccgcgaaatta EcHpaC atacgactcactatagggCACTCGACTCTTTCCAGAT ttaGCTAATCTTCTTGAACAGCCGCCAGCCGCT AAATAAGGAGGTATTATatgcaattagatgaacaac CACACCGGAGCTaatcgcagcttccatttccagcata gcctgc (SEQ ID NO.75) accggatggaaacgg (SEQ ID NO.76) gtccggcgtagaggatcgagatcgatctcgatcccgcgaaat gcagccggtcgacttaGCTAATCTTCTTGAACAGC SvVlmR taatacgactcactatagggAGCAGGAATTAAAACA CGCCAGCCGCTCACACCGGAGCTCTCAAGTG GATAAGTAAGGAGGATTTTTTATGACCCCTAG AGGTTACAGCAGAAGGTTGGTCACGGCTTAC TGCGGCTGCG (SEQ ID NO.77) (SEQ ID NO.78) gcgtccggcgtagaggatcgagatcgatctcgatcccgcga CTAATCTTCTTGAACAGCCGCCAGCCGCTCAC PcAAS aattaatacgactcactatagggCGAGCACCAACGGG ACCGGAGCTACTGAGAACCTCTACTAATTTGT CTCGAGACATACAAGTATACATAAGGAGGTAA TAGATGTAAATGTGTCGTCCAGCAG (SEQ ID TTTTATGGGG (SEQ ID NO.79) NO.80) atgcgtccggcgtagaggatcgagatcgatctcgatcccgcg gccggtcgacttaGCTAATCTTCTTGAACAGCCGC EcYahK aaattaatacgactcactatagggCCTCGGTTCGAACG CAGCCGCTCACACCGGAGCTgtctgttagtgtgcg AGTAAATTTAGGTATTAAGGAGGTTATTTTAAa attatcgataacaaaacgatatttcacatcaccgcgcagca tgaag (SEQ ID NO.81) ttcgctc (SEQ ID NO.82) agaggatcgagatcgatctcgatcccgcgaaattaatacga gcagccggtcgacttaGCTAATCTTCTTGAACAGC RrBLVRB ctcactatagggCGACAAAGAAGCTCAAAAAGAT CGCCAGCCGCTCACACCGGAGCTgtcataatggc AAATAAGGAGGTATTTTatgacagtaaagaaaaacg tataggtcttgccatcgtaatgtgtcgtacaccgc (SEQ cgaatggcg (SEQ ID NO.83) ID NO.84) gatcgagatcgatctcgatcccgcgaaattaatacgactcac ttaGCTAATCTTCTTGAACAGCCGCCAGCCGCT EcTyrC tatagggCGCTTCCGCAGGAGAGTAAAGCTCTC CACACCGGAGCTCGGATGGATGTCGTGATCG (SEQ ID NO.85) GTCTTCAG (SEQ ID NO.86) atgcgtccggcgtagaggatcgagatcgatctcgatcccgcg gccggtcgacttaGCTAATCTTCTTGAACAGCCGC EcTyrB aaattaatacgactcactatagggTGCAATTAGATACA CAGCCGCTCACACCGGAGCTcatcaccgcagcaa GTTCAGCTCCCGCTGAGTTAAGGAGGTTTTTTA acgcctttgccacacgttgtacatttgccgtatttaaccc TGtttc (SEQ ID NO.87) (SEQ ID NO.88) cgtagaggatcgagatcgatctcgatcccgcgaaattaatac gccggtcgacttaGCTAATCTTCTTGAACAGCCGC EcGluD gactcactatagggCTAGATACGCGAAAGTAGGAT CAGCCGCTCACACCGGAGCTgtaccagccgcgca AAGGAGGTTTTTTTatgagtgg (SEQ ID NO.89) gtttcatcgcttcggccaccttc (SEQ ID NO.90) F2 w / o cgtacggccacctaggTTGACAATTAATCATCCGGC protection TCGTATAATGtgtaaaacgacggccagtgaattcgagct catgcgtccggcgtagaggatcgagatcgatctcgatcccgc group g (SEQ ID NO.91) R2 w / o protection aacaaataggggttccgcgcacatttccccgaaaag (SEQ ID NO.92) group AATTAGTATGTTGTAACTAAAGTAATTAGTATG F22Xter TTGTAACTAAAGTatgcgtccggcgtagaggatcgaga tcgatctcgatcccgcgaaattaatacgactcactataggg (SEQ ID NO.93) cccgggaattgtgagcggataacaattcccgggcccgggaa F22XlacO ttgttatccgctcacaattcccgggtagaggatcgagatcgat ctcgatcccgcgaaattaatacgactcactataggg (SEQ ID NO.95) AATTAGTATGTTGTAACTAAAGTAATTAGTAT R22XterGTTGTAACTAAAGTaacaaataggggttccgcgcac atttccccgaaaagtgccacctgacgtctaagaaaccatta ttatcatgac (SEQ ID NO.94) cccgggaattgtgagcggataacaattcccgggcccggga R22XlacOattgttatccgctcacaattcccgggaacaaataggggttc cgcgcacatttccccgaaaagtgccacctgacgtctaag (SEQ ID NO.96) Cell-Free protein synthesis (CFPS) buffer and reaction The PANOx-SP buffer was first described by Jewett (loc. cit.) and has been further adapted by the same group (Rasor loc. cit.), which has become one of the most popular buffer systems for CFPS nowadays. The PANOx-SP buffer consists of 57 mM HEPES (pH7.5) (in Rasor loc. cit.), 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate (decrease from 175 mM to 130 mM in Rasor loc. cit.), 33 mM phosphoenolpyruvate (PEP), 1.5 mM spermidine, 1 mM putrescine, 4 mM oxalate (increase from 2.7 mM to 4 mM in Rasor loc. cit.), 1.2 mM adenosine triphosphate (ATP), 0.85 mM of each GTP, UTP and CTP, 34 µg / mL folinic acid, 170.6 µg / mL of E. coli tRNA mixture, 2 mM each of 20 amino acids, 0.4 mM nicotinamide adenine dinucleotide (NAD) (increase from 0.33 mM to 0.4 mM in Rasor loc. cit.), 0.27 mM coenzyme-A (CoA). The PANOx-SP buffer is usually supplemented with 13.3 µg / mL (linear) DNA template; and with 0.24 volume of lysate. In the context of the present examples, the optimized PANOx-SP buffer system from Rasor loc. cit. was used as the basis / control buffer; and referred to as “PANOx-SP” buffer. One modified / supplemented buffer in the context of the invention consisted of 100 mM HEPES (pH7.5) 10mM potassium phosphate, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 33 mM phosphoenolpyruvate (PEP), 1.5 mM spermidine, 1 mM putrescine, 4 mM oxalate, 1.2 mM adenosine triphosphate (ATP), 0.85 mM of each GTP, UTP and CTP, 34 µg / mL folinic acid, 170.6 µg / mL of E. coli tRNA mixture, 2 mM each of 20 amino acids, 0.4 mM nicotinamide adenine dinucleotide (NAD), 0.27 mM coenzyme- A (CoA). The modified / supplemented buffer was supplemented with 13.3 µg / mL (linear) DNA template; and with 0.4 volume of lysate. One modified / supplemented buffer in the context of the invention consisted of 57 mM HEPES (pH7.5), 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 33 mM phosphoenolpyruvate (PEP), 1.5 mM spermidine, 1 mM putrescine, 4 mM oxalate, 1.2 mM adenosine triphosphate (ATP), 0.85 mM of each GTP, UTP and CTP, 34 µg / mL folinic acid, 170.6 µg / mL of E. coli tRNA mixture, 2 mM each of 20 amino acids, 0.4 mM nicotinamide adenine dinucleotide (NAD), 0.27 mM coenzyme-A (CoA). The modified / supplemented buffer was supplemented with 13.3 µg / mL (linear) DNA template; and with 0.4 volume of lysate.5mM GMP and / or 5mM DTT may optionally be added. An optimized buffer in the context of the invention consisted of 100 mM HEPES (pH7.5), 23 mM magnesium glutamate, 10 mM ammonium acetate, 140 mM potassium glutamate, 33 mM PEP, 1.5 mM spermidine, 1 mM putrescine, 4 mM oxalate, 3 mM ATP and GTP, 2 mM UTP and CTP, 5 mM guanosine monophosphate (GMP), 34 µg / mL folinic acid, 170.6 µg / mL of E. coli tRNA mixture, 2 mM each of 20 amino acids, 0.4 mM NAD, 0.27 mM CoA, 20 mM glucose, 10 mM potassium phosphate (pH7.2), 5 mM dithiothreitol (DTT), 0.03 mg / mL GamS nuclease inhibitor (NEW ENGLAND Biolabs), 1 units / µL murine RNase inhibitor (NEW ENGLAND Biolabs). The optimized buffer in the context of the present examples was supplemented with 13.3 µg / mL (linear) DNA template; and with 0.4 volume of lysate. The higher concentration of HEPES in the modified / optimized buffer functions to balance the acidic byproducts from glucose metabolism; and the addition of phosphate buffer functions to replenish the phosphate consumption during ATP regeneration from glucose and glutamate. DTT ensured the reducing environment for the full activity of T7 RNA polymerase and GMP improved the transcription priming step of T7 RNA polymerase, while higher magnesium is included to compensate the chelating of free magnesium ion by the elevated concentration of total nucleotides in the buffer. GamS and murine RNase inhibitor were added to further protect linear DNA and newly transcribed RNA in the cell-free reaction. All cell-free protein synthesis reactions were performed in 15 µl volume in a 2-mL microcentrifuge tube or a 2-mL 96 deep-well plate with a seal at 30 °C for 3 hr. After the CFPS reactions, newly synthesized proteins were quantified directly by the luminescence assay. Notably, no exogenous / recombinant Tus or LacI proteins were added to the present CFPS reactions. These are based on merely the endogenous Tus and LacI proteins which are present in the lysate; and are apparently active in a sufficient manner in linear DNA protection (by binding to the LacO (and Ter) sequence(s) as comprised in respective templates). Quantification of protein by luminescence assay The fresh CFPS samples were diluted 100- to 10000-fold in the dilution buffer (50 mM HEPES (pH7.5), 10 mM ammonium acetate, 150 mM potassium glutamate, 8 mM magnesium glutamate and 10 mM DTT). Meanwhile, Nano-Glo® Nanoluciferase binding peptide lytic detection reagent (Promega) was prepared following manufacture manual. The equal volume of the diluted CFPS samples and Nanoluciferase binding peptide lytic detection reagent was mixed in a 96-well plate and the luminescence signal was measured in a plate-reader (Synergy). 5, 2.5, 1.25, 0.625, 0 nM of the Nanoluciferase binding peptide control protein (Promega N3010) were used to generate a calibration curve and measured in the same 96-well plate as other CFPS samples, which correlates the luminescence signal and protein concentration. All samples were measured in triplicates and the mean value of the triplicates was used in analyses. Example 2: Protein expression levels of linear DNAs with different end-protection in CPFS. Linear DNA templates comprising GOI encoding sfGFP, EcHpaC, RrBLVRB, RsTYR, ScKDC and EcHpaB (as the respective exemplified POI) were prepared with different end-protections, i.e., no protection, ter / lacO, lacO / ter, ter / ter and lacO / lacO (1stposition is 5’ end and 2ndposition refers to 3’ end, e.g., 5’ter / 3’lacO; in all cases with end-protection, two binding sequences were uses per each kind of binding site, e.g., 2x5’ter / 2x3’lacO; i.e., for example, “ter / lacO” means 2x5’ter / 2x3’lacO, “lacO / ter” means 2x5’lacO / 2x3’ter etc.; see SEQ ID NOs.99 as an illustrative example). CFPS was carried out in the PANOx-SP buffer system (Jewett loc.cit.; Rasor loc.cit.; see also material and method section) at 30°C for 3hr. The protein concentrations were determined by comparing the luminescence signals of C-terminal Nanoluciferase binding peptide-tagged RsTYR and ScKDC to the calibration curve of a known-concentration Nanoluciferase binding peptide control protein (HaloTaq, Promega). In both cases, i.e. for RsTYR and ScKDC, the ter / lacO construct performs better than the no- protection construct, the ter / ter construct and the lacO / lacO construct (Fig.1A). Surprisingly, when positions of ter / lacO are swapped to the opposite lacO / ter construct, lacO / ter design does not show the same protection as ter / lacO (Fig.1A). Without being bound by theory, the reason for this could be that a stronger protection at the 3’-end of the DNA is critical, as there are more 3’ exonuclease in the bacterial lysate. As there are more endogenous housekeeping LacI proteins than conditionally expressed Tus proteins (only during replication of chromosomes) in the bacteria lysate, 3' lacO protects linear DNAs better than 3’ ter. Besides, and again without being bound by theory, when one strand of lacO sequence is degraded by the exonuclease, single-stranded lacO sequence itself could form a very stable hairpin structure, which prevents further degradation (see also Example 7; the alternative constructs / approach). Additionally, high GC content of lacO sequence at the 5’ end might potentially hinder the binding of T7 RNA polymerase to the T7 promoter and decrease the transcription efficiency. In any case, however, the 5´ter / 3´lacO protection of the present invention constitutes the most advantageous kind of protection. Statistical significance between templates with no protection and ter / lacO protection was demonstrated for templates encoding sfGFP, EcHpaC, RrBLVRB, RsTYR, ScKDC and EcHpaB by applying a two-tailed paired Student’s t-test (Fig.1B). If not explicitely described otherwise, the experiments (e.g. (template) preparation and testing conditions) of the following examples were as in Example 1 and Example 2, respectively. Example 3: Linear DNA with ter / lacO protection strengthened protein expression from RNAs with less stability in a more pronounced manner. Fold of protein expression from DNA templates encoding RsTYR, ScKDC, RrBLVRB, sfGFP, PcAAS, SvVlmR, EcYahK or EcTyrB as the respective POI was calculated by dividing the final protein concentration of CFPS from ter / lacO-protected DNA by the one from non-protected DNA (cf. SEQ ID NOs.99 as an illustrative DNA template for the construct format). The stability of the RNA resulting from the respective templates was converted from the free energy of the thermodynamic ensemble predicted by RNAfold calculator (http: / / rna.tbi.univie.ac.at / cgi- bin / RNAWebSuite / RNAfold.cgi) with arbitrary unit. Preparation and testing conditions were as in Example 2. The results are shown in Fig.2. Without being bound by theory, RNA with higher stability has higher secondary structure and, therefore, is less prone to nuclease degradation; but may also reduce accessibility by ribosomes. On the other hand, RNA with less stability is considered to be prone to degradation but may be more efficient for ribosome recruitment due to its less-structured nature. In the results demonstrated herein, the constructs with lower RNA stability (less than 510 AU) show higher protein expression with ter / lacO protection. Without being bound by theory, ter / lacO protected linear DNA enables longer transcription and higher amounts of RNA for translation. Templates resulting in RNA with high stability (over 600 AU) are, without being bound by theory, considered to be limited at the translation step due to the inhibition of RNA structure to the ribosome recruitment. Therefore, protection of linear DNA might not significantly increase protein production once the intrinsic stability of the resulting RNA would anyway lead to higher amounts of RNA. Example 4: Protein expression levels of plasmid and linear DNAs in CFPS. RBS and coding sequences of ScKDC and RsTYR genes were constructed either on a plasmid or on a linear DNA and driven by a T7 promoter. The sequences upstream of the T7 promoter and downstream of the GOIs were identical on both, plasmid and linear DNA. The end-protections were added to the linear DNAs at 46 bases upstream of the T7 promoter and 223 bases downstream of the T7 terminator (cf. SEQ ID NOs. 99 as an illustrative DNA template for the construct format). Preparation and testing conditions were as in Example 2. The results are shown in Fig.3. For both genes, ter / lacO-protected linear DNA apparently rescued the DNA instability and outperformed the plasmid constructs in CFPS reactions. Surprisingly, ter / lacO linear DNAs even resulted in higher protein production than circular plasmids. Without being bound by theory, this is considered to result from the better transcription efficiency on a linear DNA than on a circular plasmid. Furter, and again without being bound by theory, as the plasmid also contains an antibiotic resistance gene and ori site and carryover of E. coli endogenous mRNA during plasmid preparation in the absence of RNase, a portion of the translation machinery could be bound to the non-desired transcripts, which might further reduce the target protein expression level. Example 5. Protein expression levels of linear DNAs with P2A leader peptide or RBS. Four GOI (encoding EcHpaC, EcHpaB, SvVlmR, PcAAS; cf. SEQ ID NOs.98 as an illustrative DNA template for the construct format) were either expressed by an individual bespoke RBS, which was predicted by Salis calculator for the highest expression level for each GOI; or expressed downstream of a N-terminal leader peptide with nGFP and a self-cleavage P2A peptide in the CFPS reactions with PANOx-SP buffer. Linear DNAs with ter / lacO protection were used in this experiment. Preparation and testing conditions were as in Example 2. The P value between the RBS and P2A group was established by a two-tailed paired Student’s t-test and is < 0.05. Preparation and testing conditions were as in Fig.1. The results are shown in Fig.4. N-terminal leader peptide with nGFP and P2A peptide is known to have efficient translation initiation, which is the rate-limiting step during translation reactions. The present data indicate that, as soon as the barrier of inefficient translation initiation is eliminated by using the leader peptides, all GOI are expressed at high levels, around 1.5-10 fold higher than the ones driven by their respective own RBS. Even though the Salis calculator may have predicted a highly expressed RBS for each GOI, an in-silica prediction may not always reflect a good expression level in vitro or in vivo. The CFPS system of the present invention, however, allows researchers to test RBSs for target GOI in an efficient and time-saving way. Furthermore, by changing the N-terminal peptides, the expression of target proteins can also be adjusted to a desired level. Example 6: Fast, high throughput and / or real-time protein quantification in CFPS. Protein quantification performed in the previous examples was an end-point measurement at 3 hr of the CFPS reaction. The present example shows functionality of the CFPS system of the invention in fast, high throughput CFPS and / or during CFPS reaction time (“real-time”; Fig.5). For these purposes, e.g. for real-time protein quantification in CFPS, the Nanoluciferase binding peptide tag (e.g. HiBit tag; Promega) is linked to the C-terminus of the proteins to be synthesized, while its complementary partner, LgBiT, and luminescent substrate, e.g. furimazine, are added to the CFPS reaction at the desired time points. To monitor the protein synthesis in real-time, excess amount of LgBiT and furimazine is added to the lysate from the beginning of the CFPS reaction and the luminescence signals reflect the formation of the C-terminal Nanoluciferase binding peptide-tagged target proteins. The LgBiT may be enriched endogenously in the lysate prior to the lysate preparation or added exogenously as a recombinant protein to the lysate. Experimental particulars can be performed as described in the manufacturer´s manual (e.g. system for live imaging in mammalian cell as provided by Promega; LgBit Expression Vector and Stable Cell Line). Example 7: Alternative linear DNA construct: lacO protection sequence(s) also at the RNA level. It was surprisingly found in the context of the invention that, when the positions of ter / lacO are swapped to the opposite lacO / ter positions, the lacO / ter design does not show the same protection as ter / lacO (cf. Fig.1A; Example 2). It is thus expected that, when one strand of lacO sequence is degraded by the exonuclease, single-stranded lacO sequence itself could still form stable hairpin structure, which prevents from further degradation. As 3’ endonuclease activity is stronger in bacteria than 5’ nuclease activity, a strong protection at the 3’ end is expected to be critical. A strong lacO hairpin at the 3’ end can thus prevent also single-stranded sequences from further degradation, i.e. also RNA. This leads to higher final expression levels in CFPS. The experimental evidence provided in the context of the present invention thus also leads to further alternative constructs and CFPS approach, respectively. In this context, the alternative linear DNA and RNA constructs have lacO protection sequence(s) constructed at the 3’-end downstream of the GOI, with no (T7) terminator sequences (cf. also Fig.6A (lower panel) and Fig.6B). (T7) polymerase will transcribe whole sequences downstream of the GOI and run off from the linear DNA at the 3’-end. The resulting RNA transcripts thus contain transcribed lacO protection sequence(s) (no ter sequence(s) at the 5’end of the RNA because transcription start is downstream of ter sequence(s) in DNA template; cf. Fig. 6A (lower panel)). lacO sequence / hairpin (see, for example, Fig. 6B) is thus used as terminator and as a protection group also on RNA level. The single-stranded lacO sequence forms a stronger and more stable hairpin than a (T7) terminator in RNA transcript (cf., Fig.6B). The RNA structure of lacO can thus also protect RNA from RNase degradation and, therefore, can prolong the lifetime of the target transcripts and can promotes extended translation reactions. As a result, alternative linear DNA constructs of the invention lead to higher expression level of RNA and higher final protein expression level in CFPS. In one particular aspect, the depicted 5´ (ter / Tus) protection on DNA level may also be omitted in the alternative linear DNA construct.

Claims

New PCT-Patent Application based on EP 24186635.9 Insempra GmbH Vossius Ref.: AG4141 PCT S3 82 CLAIMS 1. A linear double-stranded deoxyribonucleic acid (dsDNA) molecule comprising (a) one or more Tus protein (Tus) binding site(s) at the 5’-terminus; and (b) one or more Lac repressor protein (LacI) binding site(s) at the 3’-terminus; and (c) between (a) and (b) a segment comprising a DNA sequence of interest (DOI).

2. The linear dsDNA molecule of claim 1, further comprising one or more Tus protein(s) bound to the 5’ Tus binding site(s) and / or one or more LacI protein(s) bound to the 3’ LacI binding site(s).

3. The linear dsDNA molecule of claim 1 or 2 comprising two Tus binding sites at the 5’- terminus and / or two LacI binding sites at the 3’-terminus.

4. The linear dsDNA molecule of any one of claims 1 to 3, wherein at least one of said one or more Tus binding site(s) is or comprises a ter sequence and / or wherein at least one of said one or more LacI binding site(s) is or comprises a Lac operator (lacO) sequence.

5. The linear dsDNA molecule of claim 3 or 4, wherein said two Tus binding sites are or comprise two ter sequences, respectively, and / or wherein said two LacI binding sites are or comprise two lacO sequences, respectively.

6. The linear dsDNA molecule of claim 4 or 5, wherein said ter is terA, terB, terC, terD or terE and / or wherein said lacO is lacO1, lacO2, or lacO3.

7. The linear dsDNA molecule of any one of claim 4 - 6, wherein said ter sequence comprises a nucleotide sequence as depicted in SEQ ID NO.7 (“ter consensus sequence”) and / or wherein said lacO sequence comprises a nucleotide sequence as depicted in SEQ ID NO.8 (“lacO consensus sequence”).

8. The linear dsDNA molecule of any one of claim 4 - 7, wherein said ter sequence is or comprises a nucleotide sequence as depicted in SEQ ID NO.1 (terA), SEQ ID NO.3 (terB), SEQ ID NO.4 (terC), SEQ ID NO.5 (terD) or SEQ ID NO.6 (terE) and / or wherein said lacO sequence is or comprises SEQ ID NO.2 (“Insempra”), SEQ ID NO.9 (“Core”), SEQ ID NO. 10 (“lacO1”), SEQ ID NO. 11 (“pET”), SEQ ID NO. 12 (“Symmetric”), SEQ ID NO. 13 (“Gilbert”), SEQ ID NO.14 (“lacO2”) or SEQ ID NO.15 (“lacO3”).

9. The linear dsDNA molecule of any one of claims 1 to 8, wherein said Tus is or comprises an amino acid sequence as depicted in SEQ ID NO. 16 and / or wherein said LacI is or comprises an amino acid sequence as depicted in SEQ ID NO.

17.

10. The linear dsDNA molecule of any of claims 1 to 9, wherein said one or more 3’ LacI binding site(s) is / are located downstream of a buffer region.

11. The linear dsDNA molecule of claim 10, wherein said buffer region is preceded by a transcription terminator sequence which is located 3’ in / of the DOI.

12. The linear dsDNA molecule of claim 10 or 11, wherein said transcription terminator sequence which is located downstream of a stop-codon for a protein, polypeptide or peptide of interest (POI) encoded by said DOI.

13. The linear dsDNA molecule of any of claims 1 to 12, wherein said DOI encodes a protein, polypeptide or peptide of interest (POI).

14. The linear dsDNA molecule of any of claims 1 to 13, wherein said DOI is or comprises a functional DNA sequence.

15. The linear dsDNA molecule of claim 14, wherein said functional DNA sequence is an expression cassette for a POI.

16. The linear dsDNA molecule of any one of claims 1 - 15, wherein said DOI comprises in the 5’ to 3’ direction (a) a promotor sequence; (b) a DNA sequence encoding a ribosomal binding site (RBS); or another 5´UTR / other translation initiation / ribosome recruitment sequence; and / or (c) a DNA sequence (GOI) encoding a POI; and / or (d) optionally, a transcription terminator sequence.

17. The linear dsDNA molecule of claim 16, wherein said promotor or terminator sequence is active in bacteria.

18. The linear dsDNA molecule of claim 16 or 17, wherein said GOI is followed by a 3´UTR.

19. The linear dsDNA molecule of any one of claims 1 to 18 further comprising a 5’ DNA buffer region (buffer region 1; 5´ overhang; 5´ UTR) downstream of the 3’ end of said Tus binding site(s), and / or a 3´ UTR downstream of the 3’ end of said GOI, and / or a 3’ DNA buffer region (buffer region 2; 3´ overhang) upstream of the 5’ end of said LacI binding site(s).

20. The linear dsDNA molecule of claim 19, wherein said 5’ DNA buffer region is located upstream of the 5’ end of said DOI, and / or wherein said 3´ UTR is located upstream of the 5’ end of a / said terminator, and / or wherein said 3’ DNA buffer region is located downstream of the 3’ end of a / said terminator.

21. The linear dsDNA molecule of claim 19 or 20, wherein said buffer region 1 and / or said 3´UTR consists of between 0 to 200 base pairs (bp), and / or wherein said buffer region 2 consists of between 0 to 500 bp.

22. The linear dsDNA molecule of any one of claims 19 to 21, wherein said buffer region 1 and / or said 3´UTR consists of between 70 – 150 bp, and / or wherein said buffer region 2 consists of between 70 – 300 bp.

23. The linear dsDNA molecule of any one of claims 19 to 22, wherein said buffer region 1 and / or said 3´UTR consists of about 46 bp and / or about 55 bp, respectively, and / or wherein said buffer region 2 consists of about 193 bp.

24. The linear dsDNA molecule of any one of claims 13 - 23, wherein said POI is expressed as a fusion protein or as a poly-protein.

25. The linear dsDNA molecule of any one of claims 1 - 24, wherein said DOI comprises in the 5’ to 3’ direction (a) a promotor sequence; (b) a DNA sequence encoding an RBS, or another 5´UTR / other translation initiation / ribosome recruitment sequence; (c) a DNA sequence encoding a leader peptide; (d) optionally, a DNA sequence encoding a self-cleavage peptide; and / or (e) a GOI encoding a POI; and / or (f) optionally, a transcription terminator sequence.

26. The linear dsDNA molecule of claim 25, wherein said promotor and terminator sequences are active in bacteria.

27. The linear dsDNA molecule of claim 25 or 26, wherein said leader peptide is a highly expressed leader peptide.

28. The linear dsDNA molecule of any one of claims 25 to 27, wherein one additional proline (P) is encoded in downstream direction of said self-cleavage peptide.

29. The linear dsDNA molecule of any one of claims 25 to 28, wherein said GOI is followed by a 3´UTR.

30. The linear dsDNA molecule of any one of claims 25 to 29, wherein said leader peptide is or comprises nGFP.

31. The linear dsDNA molecule of any one of claims 25 to 30, wherein said leader peptide is or comprises the first 20 amino acids of sfGFP.

32. The linear dsDNA molecule of any one of claims 25 to 31, wherein said leader peptide is or comprises the amino acid sequence as encoded by SEQ ID NO.18 or as depicted in SEQ ID NO.

28.

33. The linear dsDNA molecule of any one of claims 25 to 32, wherein said self-cleavage peptide is or comprises a 2A self-cleavage peptide.

34. The linear dsDNA molecule of any one of claims 25 to 32, wherein said self-cleavage peptide is or comprises a P2A, F2A, E2A or T2A self-cleavage peptide.

35. The linear dsDNA molecule of any one of claims 25 to 32, wherein said self-cleavage peptide is or comprises a 2A self-cleavage peptide as depicted in SEQ ID NOs: 21 / 22, 23, 24 or 25, or as encoded by SEQ ID NOs: 29 / 30, 31, 32 or 33.

36. The linear dsDNA molecule of any one of claims 25 to 35, wherein said self-cleavage peptide comprises the amino acid sequence as depicted in SEQ ID NO. 19 (the “2A consensus sequence”) or as depicted in SEQ ID NO.20 (the “2A core sequence”).

37. The linear dsDNA molecule of any one of claims 13 to 36, wherein said POI comprises, or is expressed together with, a tag.

38. The linear dsDNA molecule of claim 37, wherein said tag is for quantification of said / a POI.

39. The linear dsDNA molecule of claim 37 or 38, wherein said tag is or comprises a Nanoluciferase binding peptide tag.

40. The linear dsDNA molecule of any one of claims 37 to 39, wherein said tag is or comprises a Nanoluciferase binding peptide tag as depicted in SEQ ID NO.26 or in SEQ ID NO.27, or as encoded by SEQ ID NO.34 or by SEQ ID NO.35 / 97.

41. The linear dsDNA molecule of any one of claims 24 - 36, wherein said poly-protein comprises in the N-terminus to C-terminus direction (a) a sequence coding for a highly expressed peptide; and (b) a self-cleavage peptide; and (c) the sequence of a POI; and, wherein said poly-protein optionally further comprises (d) a tag.

42. The linear dsDNA molecule of claims 41, wherein said sequence coding for a highly expressed peptide is nGFP or a fragment thereof; wherein said self-cleavage peptide is defined as in any one of claims 33 to 36; and / or wherein said tag is for quantification of the POI.

43. The linear dsDNA molecule of claim 41 or 43, wherein said nGFP is defined as in any one of claims 30 to 32; and / or wherein said tag is a tag as defined in claim 39 or 40.

44. The linear dsDNA molecule of any of claims 1 to 10 and 13 to 43, wherein said DOI, in particular said expression cassette as defined in any one of claims 15 to 43, does not comprise a transcription terminator sequence.

45. A linear dsDNA molecule as defined in claim 44, wherein said linear dsDNA molecule does not comprise a 5’ Tus binding site or another 5’ DNA protection group.

46. An RNA molecule transcribable from the linear dsDNA molecule of claim 44 or 45.

47. The RNA molecule of claim 46, which is a non-naturally occurring RNA molecule.

48. A method of protecting a linear deoxyribonucleic acid (DNA) molecule having a free 5’ terminus and a free 3’ terminus from exonuclease degradation, wherein said method comprises: (a) adding one or more Tus binding site(s) at the first terminus of said DNA molecule and adding one or more LacI binding site(s) at the other terminus of said DNA molecule; and (b) allowing a Tus protein to bind to one, more of all of the Tus binding site(s) and a LacI protein to bind to one, more of all of the LacI binding site(s).

49. The method of claim 48, wherein said one or more Tus binding site(s) is / are at the 5’ terminus (“5’ Tus binding site(s)”) of the DNA molecule, and the one or more LacI binding site(s) is / are at the 3’ terminus (“3’ LacI binding site(s)”) of the DNA molecule.

50. The method of claim 48 or 49, wherein said one or more Tus binding site(s) and / or said one or more LacI binding site(s) are defined as in any one of claims 3 - 8.

51. The method of any one of claims 48 - 50, wherein said Tus and / or said LacI is defined as in claim 9.

52. The method of any one of claims 48 - 51, wherein said linear DNA molecule is defined as the linear dsDNA molecule of any one of claims 1 - 45.

53. A method of synthesizing a POI in a cell-free protein synthesis (CFPS) reaction mixture, wherein said method comprises: (a) providing a linear dsDNA molecule as defined in any one of claims 1 – 45 or an RNA molecule as defined in claim 46 or 47, wherein said DOI is or comprises a GOI encoding said POI; (b) adding said linear dsDNA or said RNA to the CFPS reaction mixture; and (c) synthesizing said POI in said CFPS reaction mixture.

54. The method of claim 53, wherein said dsDNA molecule or said RNA molecule comprises an expression cassette for said POI.

55. The method of claim 53 or 54, wherein said CFPS reaction mixture comprises a CFPS lysate which is a procaryotic cell lysate.

56. The method of any one of claims 53 to 55, wherein said CFPS reaction mixture comprises a CFPS lysate which is a bacterial cell lysate.

57. The method of claim 55 or 56, wherein said said CFPS lysate is a lysate of cells of bacteria of the genus selected from the group consisting of Escherichia, Vibrio, Bacillus, Corynebacterium, Klebsiella, and Pseudomonas.

58. The method of any one of claims 53 to 57, wherein said said CFPS lysate is a lysate of cells of bacteria selected from the group consisting of E. coli, V. natriegens, B. subtilis, C. glutamate, K. pneumoniae, P. fluorescens and B. megaterium.

59. The method of any one of claims 53 to 58, wherein the concentration of (endogenous) Tus in said CFPS reaction mixture is ≤15µM, and / or the concentration of (endogenous) LacI in said CFPS reaction mixture is ≤15µM.

60. The method of any one of claims 53 to 59, wherein the concentration of (endogenous) Tus in said CFPS reaction mixture is ≤13µM, and / or the concentration of (endogenous) LacI in said CFPS reaction mixture is ≤13µM.

61. The method of any one of claims 53 to 60, wherein said CFPS reaction mixture or said CFPS lysate comprises an endogenous RNA polymerase or an exogenous RNA polymerase; the endogenous Tus and / or (an)other endogenous protecting component(s)) or a recombinant and / or exogenously added Tus and / or (an)other recombinant and / or exogenously added protecting component(s); and / or the endogenous LacI and / or (an)other endogenous protecting component(s), or a recombinant and / or exogenously added LacI (and / or (an)other recombinant and / or exogenously added protecting component(s)).

62. The method of claim 61, wherein said endogenous RNA polymerase is a procaryotic or bacterial RNA polymerase.

63. The method of claims 61 or 62, wherein said exogenous RNA polymerase is a phage RNA polymerase.

64. The method of any one of claims 53 to 63, wherein said CFPS reaction mixture or said CFPS lysate comprises the endogenous Tus and / or (an)other endogenous protectingcomponent(s), and the endogenous LacI and / or (an)other endogenous protecting component(s); and / or wherein no Tus or LacI is added to said CFPS reaction mixture.

65. The method of any one of claims 53 to 64, wherein said CFPS reaction mixture comprises both, glucose and phosphoenolpyruvate (PEP).

66. The method of any one of claims 53 to 65, further comprising, before (b) or before (c), (a) providing a CFPS reaction mixture; and (b) adding to said CFPS reaction mixture glucose and PEP.

67. The method of any one of claims 53 to 66, wherein said CFPS reaction mixture comprises (i) 10 to 30 mM of glucose; and / or (ii) 20 to 50 mM PEP.

68. The method of any one of claims 53 to 67, wherein said CFPS reaction mixture comprises (i) 15 to 25 mM of glucose; and / or (ii) 28 to 38 mM PEP.

69. The method of any one of claims 53 to 68, wherein said CFPS reaction mixture comprises (i) 17 to 23 mM of glucose; and / or (ii) 30 to 36 mM PEP.

70. The method of any one of claims 53 to 69, wherein said CFPS reaction mixture comprises 20 mM glucose and / or 33 mM PEP.

71. The method of any one of claims 53 to 70, wherein said synthesizing is performed at a temperature in the range of 20 °C to 40 °C.

72. The method of any one of claims 53 to 71, wherein said synthesizing is performed at a temperature in the range of 25 °C to 37 °C.

73. The method of any one of claims 53 to 72, wherein said synthesizing is performed at a temperature in the range of 27 °C to 33 °C.

74. The method of any one of claims 53 to 73, wherein said synthesizing is performed at a temperature in the range of 28 °C to 32 °C.

75. The method of any one of claims 53 to 74, wherein said synthesizing is performed at 30 °C.

76. The method of any one of claims 53 to 75, wherein said synthesizing is performed at a pH in the range of pH6 to pH8 and / or wherein said CFPS reaction mixture has a pH in the range of pH6 to pH8.

77. The method of any one of claims 53 to 76, wherein said synthesizing is performed at a pH in the range of pH6.8 to pH7.8 and / or wherein said CFPS reaction mixture has a pH in the range of pH6.8 to pH7.

8.

78. The method of any one of claims 53 to 77, wherein said synthesizing is performed at a pH in the range of pH7.2 to pH7.5 and / or wherein said CFPS reaction mixture has a pH in the range of pH7.2 to pH7.

5.

79. The method of any one of claims 53 to 78, wherein said CFPS reaction mixture comprises 30 to 300 mM HEPES.

80. The method of any one of claims 53 to 79, wherein said CFPS reaction mixture comprises 50 to 200 mM HEPES.

81. The method of any one of claims 53 to 80, wherein said CFPS reaction mixture comprises 70 to 150 mM HEPES.

82. The method of any one of claims 53 to 81, wherein said CFPS reaction mixture comprises 100 mM HEPES.

83. The method of any one of claims 79 to 82, wherein said HEPES is comprised in form of a HEPES-KOH buffer.

84. The method of any one of claims 79 to 83, wherein said HEPES is comprised in form of a HEPES buffer with a ph of 7.

5.

85. The method of any one of claims 53 to 84, wherein said CFPS reaction mixture comprises phosphate.

86. The method of any one of claims 53 to 85, wherein said CFPS reaction mixture comprises 5 to 20 mM phosphate.

87. The method of any one of claims 53 to 86, wherein said CFPS reaction mixture comprises 7.5 to 15 mM phosphate.

88. The method of any one of claims 53 to 87, wherein said CFPS reaction mixture comprises 10 mM phosphate.

89. The method of any one of claims 85 to 88, wherein said phosphate is comprised in form of a potassium phosphate buffer.

90. The method of any one of claims 53 to 89, wherein potassium phosphate buffer is added to said CFPS reaction mixture.

91. The method of claim 89 or 90, wherein said phosphate buffer has a pH of 7.

2.

92. The method of any one of claims 85 to 91, wherein said phosphate is comprised in form of K2HPO4 and / or KH2PO4.

93. The method of any one of claims 53 to 92, wherein said CFPS reaction mixture comprises 1.5 to 4.5 mM ATP, 1.5 to 4.5 mM GTP, 1.0 to 3.0 mM CTP and / or 1.0 to 3.0 mM UTP.

94. The method of any one of claims 53 to 93 wherein said CFPS reaction mixture comprises 3 mM ATP, 3 mM GTP, 2 mM CTP and / or 2 mM UTP.

95. The method of any one of claims 53 to 94, wherein said CFPS reaction mixture comprises 10 to 30 mM Mg2+ions.

96. The method of any one of claims 53 to 95, wherein said CFPS reaction mixture comprises 15 to 25 mM Mg2+ions.

97. The method of any one of claims 53 to 96, wherein said CFPS reaction mixture comprises 18 to 24 mM Mg2+ions.

98. The method of any one of claims 53 to 97, wherein said CFPS reaction mixture comprises 23 mM Mg2+ions (± 1-3 mM) or 19 mM Mg2+ions (± 1-3 mM).

99. The method of any one of claims 95 to 98, wherein said Mg2+ions are comprised in form of magnesium diglutamate (Mg(Glu)2).

100. The method of any one of claims 53 to 99, wherein Mg(Glu)2is added to said CFPS reaction mixture.

101. The method of any one of claims 53 to 100, wherein said CFPS reaction mixture comprises guanosine monophosphate (GMP).

102. The method of any one of claims 53 to 101, wherein said CFPS reaction mixture comprises 1 to 10 mM GMP.

103. The method of any one of claims 53 to 102, wherein said CFPS reaction mixture comprises 3 to 7 mM GMP.

104. The method of any one of claims 53 to 103, wherein said CFPS reaction mixture comprises 5 mM GMP (± 1 mM).

105. The method of any one of claims 53 to 104, wherein said CFPS reaction mixture comprises DTT.

106. The method of any one of claims 53 to 105, wherein said CFPS reaction mixture comprises 1 to 15 mM DTT.

107. The method of any one of claims 53 to 106 wherein said CFPS reaction mixture comprises 3 to 12 mM DTT.

108. The method of any one of claims 53 to 107 wherein said CFPS reaction mixture comprises 5 mM DTT (± 1 mM).

109. The method of any one of claims 53 to 108, wherein said CFPS reaction mixture comprises 0.1 to 0.8 volume of a CFPS lysate.

110. The method of any one of claims 53 to 109, wherein said CFPS reaction mixture comprises 0.2 to 0.5 volume of a CFPS lysate.

111. The method of any one of claims 53 to 110, wherein said CFPS reaction mixture comprises 0.24 volume of a CFPS lysate (± 0.05-0.1 volume) or 0.4 volume of a CFPS lysate (± 0.05-0.1 volume).

112. A cell-free biological system comprising the linear dsDNA molecule of any one of claims 1 to 26, the RNA molecule of claim 27 and / or the CFPS reaction mixture as defined in any one of claims 53 to 111.

113. The cell-free biological system of claim 112, which is a CFPS system.

114. The cell-free biological system of claim 112 or 113, which is a bacterial lysate CFPS system.

115. A method of synthesizing a POI in a CFPS reaction mixture, wherein said method comprises: (a) providing a linear dsDNA comprising an expression cassette for a POI, (b) adding said linear dsDNA to the CFPS reaction mixture; and (c) synthesizing said POI, wherein said POI is expressed as a poly-protein or fusion protein precursor comprising in the N-terminus to C-terminus direction (i) a sequence coding for a highly expressed peptide; (ii) a self-cleavage peptide; and (iii) the sequence of said POI; and, wherein said poly-protein / fusion protein precursor optionally further comprises (iv) a tag.

116. The method of claim 115, wherein said sequence coding for a highly expressed peptide is defined as in any one of claims 30 to 32, said self-cleavage peptide is defined as in any one of claims 33 to 36, and / or said tag is defined as in any one of claims 37 to 40.

117. The method of claim 115 or 116, wherein said poly-protein / fusion protein precursor comprises said tag in the C-terminal direction after (iii).

118. The method of any one of claims 115 to 117, wherein said CFPS reaction mixture is defined as in any one of claims 53, 55 to 65, 67 to 70 and 76 to 111; wherein said lineardsDNA is defined as in any one of claims 1 - 45; and / or wherein said expression cassette is defined as in any one of claims 25– 43.

119. An expression DNA cassette comprising in the 5’ to 3’ direction (i) a promoter (active in bacteria); (ii) an RBS, or other 5´UTR / other translation initiation / ribosome recruitment sequence; (iii) a GOI encoding a POI; and (iv) one or more LacI binding site(s), wherein the expression cassette does not comprise a transcription terminator sequence between the 3´ end of said GOI of (iii) and said one or more LacI binding site(s) of (iv).

120. The expression DNA cassette of claim 119, wherein said promoter is active in bacteria and / or wherein said GOI is followed downstream by a 3´UTR.

121. A non-naturally occurring RNA molecule comprising (i) an RBS, or other 5´UTR / other translation initiation / ribosome recruitment sequence; (ii) a sequence encoded by a GOI encoding a POI; and (iii) one or more LacI binding site(s), wherein said RNA molecule does not comprise a terminator sequence or (an)other protecting sequence(s) between the end of said sequence of (ii) and said one or more LacI binding site(s) of (iii).

122. The non-naturally occurring RNA molecule of claim 1121, wherein said sequence encoded by a GOI is followed downstream by a 3´UTR.

123. The expression DNA cassette of claim 119 or 120 or the RNA molecule of claim 121 or 122, wherein said one or more LacI binding site(s) is / are defined as in any one of claims 2 – 9.

124. The RNA molecule of any one of claim 46, 47, 121, 122 and 123, wherein said lacO sequence(s) or said one or more LacI binding site(s) is / are capable of forming a hair-pin.

125. A kit / kit of contents comprising a linear dsDNA molecule, an RNA molecule or an expression cassette as defined in any of the preceding claims.

126. The kit / kit of contents of claim 125, further comprising a CFPS reaction mixture, a CFPS lysate and / or a CFPS reaction buffer as defined in any of the preceding claims.

127. The kit / kit of contents of claim 126, wherein said linear dsDNA molecule, RNA molecule or expression cassette and said CFPS reaction mixture, CFPS lysate and / or CFPS reaction buffer are contained in separate vials, respectively.

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