Optimized genetic tools for modifying bacteria

Abstract

The present invention relates to the transformation and genetic modification of bacteria belonging to the phylum Firmicutes. The present invention therefore relates to methods, tools and kits allowing such genetic modification, in particular to nucleic acid sequences for facilitating the transformation of said bacteria, said sequences comprising i) all or part of the sequence SEQ ID NO: 126, and ii) sequences allowing the modification of the genetic material of the bacteria and / or the expression in said bacteria of DNA sequences partially or completely absent in the genetic material present in the wild-type version of said bacteria. The present invention also relates to the genetically modified bacteria obtained and to their use, in particular for the production of solvents, preferably on an industrial scale.

Description

[0001] The present invention relates to the transformation and genetic modification of bacteria, in particular bacteria belonging to the phylum Firmicutes, typically solvent-producing bacteria such as Clostridium, preferably bacteria that, in the wild state, have both a bacterial chromosome and at least one DNA molecule (or a natural plasmid) different from the chromosomal DNA. The present invention therefore relates to methods, tools and kits that allow for such genetic modification, in particular to nucleic acid sequences for promoting the transformation of said bacteria, said sequences comprising i) all or part of the sequence SEQ ID NO: 126, and ii) sequences that allow for modification of the genetic material of the bacteria and / or expression in said bacteria of DNA sequences that are partially or completely absent from the genetic material present in the wild-type version of said bacteria. The present invention also relates to the genetically modified bacteria obtained and their use, in particular for the production of solvents, preferably on an industrial scale. Background Art

[0002] The genus Clostridium, belonging to the phylum Firmicutes, contains Gram-positive, obligately anaerobic, spore-forming bacteria. Clostridia are an important group for the scientific community for several reasons. The first reason is that a number of serious diseases (e.g., tetanus, botulism) are caused by infection with pathogenic members of this family (John & Wood, 1986; Gonzales et al., 2014). The second reason is the possibility of using so-called acidogenic or solventogenic strains in biotechnology (Moon et al., 2016). These non-pathogenic Clostridia naturally have the ability to convert a wide range of sugars to produce chemicals of interest, especially acetone, butanol, and ethanol, in a process known as ABE fermentation (John & Wood, 1986). Likewise, IBE fermentation is possible in certain strains, during which acetone is reduced to isopropanol in variable proportions (Chen et al., 1986; George et al., 1983), due to the presence of genes encoding secondary alcohol dehydrogenase (s-ADH; Ismael et al., 1993; Hiu et al., 1987) in the genomes of these strains.

[0003] Solvent-producing species of Clostridium share significant phenotypic similarities, making their classification difficult before the advent of modern sequencing technology (Rogers et al., 2006). With the ability to sequence the complete genomes of these bacteria, the genus can now be divided into four major species: C. acetobutylicum, C. saccharoperbutylacetonicum, C. saccharobutylicum, and C. beijerinckii. A recent publication, based on a comparative analysis of the complete genomes of 30 strains, proposed the division of these solvent-producing Clostridia into four major clades ( Figure 1 ).

[0004] Specifically, these research groups separated the species C. acetobutylicum from C. beijerinckii using the corresponding references C. acetobutylicum ATCC 824 (also known as DSM 792 or LMG 5710) and C. beijerinckii NCIMB 8052. C. beijerinckii NCIMB 8052 is a model strain used to study ABE fermentations.

[0005] The number of Clostridium strains that are naturally capable of IBE fermentation is small and mainly belongs to the species Clostridium beijerinckii (see Zhang et al., 2018, Table 1). These strains are generally selected from Clostridium butyricum LMD 27.6, C. aurantibutyricum NCIB 10659, C. beijerinckii LMD 27.6, C. beijerinckii VPI2968, C. beijerinckii NRRLB-593, C. beijerinckii ATCC 6014, C. beijerinckii McClung 3081, C. isopropylicum IAM19239, C. beijerinckii DSM 6423, C. sp. A1424, C. beijerinckii optinoii and C. beijerinckii BGS1.

[0006] Despite being used in industry for more than a century, the understanding of bacteria, particularly those belonging to the genus Clostridium, has long been limited by the difficulties encountered in their genetic modification. In recent years, various genetic tools have been developed to optimize strains of this genus, the latest generation being based on the use of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) technology. This approach is based on the use of enzymes called nucleases (typically Cas-type nucleases in the case of CRISPR / Cas genetic tools, such as the Cas9 protein of Streptococcus pyogenes), which are guided by RNA molecules to make double-strand breaks within a DNA molecule (target sequence of interest). The sequence of the guide RNA (gRNA) determines the nuclease cleavage site, providing it with very high specificity ( Figure 1 ).

[0007] Since double-strand breaks in essential DNA molecules are lethal to an organism, the survival of the latter depends on its ability to repair said breaks (see, for example, Cui & Bikard, 2016). In bacteria of the genus Clostridium, the repair of double-strand breaks relies on the homologous recombination mechanism, which requires a complete copy of the cut sequence. By providing the bacterium with a DNA fragment that allows it to carry out this repair while modifying the original sequence, the microorganism can be forced to integrate the desired changes in its genome. By modifying the target sequence or the PAM site ( Figure 2 ), the modifications that have been made will no longer allow the genomic DNA to be targeted by the Cas9-gRNA ribonucleoprotein complex.

[0008] Various methods have been described to try to make this genetic tool work in Clostridium bacteria. In fact, these microorganisms are known to be difficult to genetically modify due to low transformation and homologous recombination frequencies. Several methods are based on the use of Cas9 constitutively expressed in Clostridium beijerinckii and Clostridium ljungdahlii (Wang et al., 2015; Huang et al., 2016) or Cas9 under the control of an inducible promoter in Clostridium beijerinckii, Clostridium saccharoaceticum and Clostridium autoethanogenum (C. authoethanogenum) (Wang et al., 2016; Nagaraju et al., 2016; Wang et al., 2017). Other authors have described the use of a modified version of the nuclease Cas9n, which makes single-strand breaks rather than double-strand breaks within the genome (Xu et al., 2015; Li et al., 2016). This choice was made because the toxicity of Cas9 was observed to be too high for its use in Clostridium bacteria under the experimental conditions tested. Most of the above tools are based on the use of a single plasmid. Finally, endogenous CRISPR / Cas systems can also be used when they are identified in the genome of a microorganism, such as in C. pasteurianum (Pyne et al., 2016).

[0009] Unless they use (as in the last case mentioned above) the endogenous machinery of the strain to be modified, tools based on CRISPR technology have the major disadvantage of significantly limiting the size of the nucleic acid of interest that can be inserted into the bacterial genome (according to Xu et al., 2015, about 1.8 kb is optimal) (and therefore the number of coding sequences or genes).

[0010] The present inventors have developed and described a more efficient genetic tool for modifying bacteria, suitable for Clostridium bacteria, based on the use of two different nucleic acids, typically two plasmids (WO2017064439, Wasels et al., 2017 and Figure 3), thereby specifically solving this problem. In a particular embodiment, the first nucleic acid of the tool allows the expression of cas9, and the second nucleic acid specific for the modification to be implemented contains one or more gRNA expression cassettes and a repair matrix, allowing a portion of the bacterial DNA targeted by Cas9 to be replaced with a sequence of interest. By placing the cas9 and / or gRNA expression cassette under the control of an inducible promoter, the toxicity of this system is limited. The inventors have recently improved this tool, providing a very significant increase in transformation efficiency, thereby obtaining genetically modified bacteria of interest in useful numbers and quantities (particularly in the case of selecting robust strains for industrial-scale production) (reference FR 18 / 548356). In this improved tool, at least one nucleic acid comprises a sequence encoding an anti-CRISPR protein ("acr") placed under the control of an inducible promoter. This anti-CRISPR protein makes it possible to suppress the activity of the DNA endonuclease / guide RNA complex. The expression of the protein is regulated to allow it to be expressed only during the transformation step of the bacteria.

[0011] Recently, the inventors have also succeeded in genetically modifying bacteria that, in their wild-type state, contain genes that confer resistance to one or more antibiotics, rendering them sensitive to said antibiotics, making their genetic tools, based on the use of at least two nucleic acids, more readily applicable. Thus, they succeeded in genetically modifying the Clostridium beijerinckii DSM 6423 strain, which naturally produces isopropanol. Specifically, they succeeded in removing from this strain a native plasmid identified in this specification as "pNF2" that is nonessential to the strain (see FR 18 / 73492).

[0012] Subsequently, the present inventors discovered and disclosed for the first time herein that removal of the plasmid pNF2 allows for an increase in the efficiency of introduction (i.e., transformation) of genetic material by approximately 10 1 Up to 5x 10 3 As explained below, the inventors have also succeeded in significantly improving genetic tools based on the use of at least two nucleic acids by using a portion of the plasmid pNF2 to design specific nucleic acids with sequences that allow them to modify the genetic material of bacteria and / or express DNA sequences in the bacteria that are absent from the genetic material present in the wild-type version of the bacteria. These nucleic acids and the new tools have dramatically increased the efficiency of bacterial transformation, particularly in bacteria that had previously been cleared of one or more native plasmids that they harbor in the wild.

[0013] The present invention therefore very advantageously facilitates the transformation efficiency of said bacteria and thus their use, in particular on an industrial scale. Summary of the Invention

[0014] In the context of the present invention, the inventors have described for the first time a nucleic acid (also designated herein as nucleic acid "OPT") that facilitates the transformation of bacteria by improving the maintenance of all introduced genetic material within the bacteria. The nucleic acid OPT comprises: i) all or part of the sequence of SEQ ID NO: 126, and ii) sequences that allow modification of the genetic material of the bacterium and / or expression in the bacterium of DNA sequences that are partially or completely absent from the genetic material present in the wild-type version of the bacterium. The sequence of SEQ ID NO: 126 is also designated herein as nucleic acid "OREP."

[0015] The inventors have succeeded in increasing the frequency of transformation of nucleic acids in the bacterium Clostridium beijerinckii DSM 6423, in particular by suppressing the sequence OREP in said bacterium and advantageously using all or part of this sequence OREP to construct nucleic acids and / or genetic tools allowing the modification of the genetic material of the bacterium and / or the expression in said bacterium of DNA sequences that are partially or completely absent in the genetic material present in the wild-type version of said bacterium.

[0016] The sequence OREP comprises a nucleotide sequence (SEQ ID NO: 127) encoding a protein involved in the replication of the nucleic acid of interest OPT. This protein involved in replication is also designated herein as protein "REP" (SEQ ID NO: 128—"MNNNNTESEELKEQSQLLLDKCTKKKKKNPKFSSYIEPLVSKKLSERIKECGDFLQMLSDLNLENSKLHRASFCGNRFCPMCSWRIACKDSLEISILMEHLRKEESKEFIFLTLTTPNVKGADLDNSIKAYNKAFKKLMERKEVKSIVKGYIRKLEVTYNLDKSSKSYNTYHPHFHVVLAVNRSYFKKQNLYINHHRWLSLWQESTGDYSITQVDVRKAKINDYKEVYELAKYSAKDSDYLINREVFTVFYKSLKGKQVLVFSGLFKDAHKMYKNGELDLYKKLDTIEYAYMVSYNWLKKKYDTSNIRELTEEEKQKFNKNLIEDVDIE"). The protein REP has a domain called "COG 5655" of sequence SEQ ID NO: 129 (plasmid rolling circle replication initiator protein REP) which is conserved in Firmicutes.

[0017] The present invention also describes a genetic tool that allows the optimized transformation of the genetic material of bacteria and subsequent modification by homologous recombination and / or allows the expression in said bacteria of DNA sequences that are partially or completely absent in the genetic material of bacteria belonging to the phylum Firmicutes, such as bacteria of the genera Clostridium, Bacillus or Lactobacillus (Hidalgo-Cantabrana, C. et al.; Yadav, R. et al.).

[0018] In certain embodiments, the tools for modification by homologous recombination generally have the following characteristics:

[0019] i) It contains at least:

[0020] a "first" nucleic acid encoding at least one DNA endonuclease, such as a Cas9 enzyme, wherein the sequence encoding said DNA endonuclease is placed under the control of a promoter, and

[0021] - at least one "second" nucleic acid containing a repair matrix allowing the replacement of a portion of the bacterial DNA targeted by said endonuclease with a sequence of interest by the mechanism of homologous recombination,

[0022] wherein ii) at least one of the nucleic acids further encodes one or more guide RNAs (gRNAs) or wherein the genetic tool further comprises one or more guide RNAs, each guide RNA comprising an RNA structure for anchoring to a DNA endonuclease and a sequence complementary to the targeted part of the bacterial DNA, and preferably iii) wherein at least one of the nucleic acids further comprises a sequence encoding an anti-CRISPR protein placed under the control of an inducible promoter or wherein the genetic tool further comprises a "third" nucleic acid encoding an anti-CRISPR protein placed under the control of an inducible promoter.

[0023] In particular, a class of genetic tools is described comprising at least:

[0024] - a "first" nucleic acid encoding at least one DNA endonuclease, wherein the sequence encoding said DNA endonuclease is placed under the control of a promoter, and

[0025] - an "other" nucleic acid comprising or consisting of a sequence of the "nucleic acid OREP", i.e., said other nucleic acid comprising or consisting of: i) all or part of the sequence of SEQ ID NO: 126, and ii) a sequence allowing modification of the genetic material of the bacterium and / or expression in said bacterium of a DNA sequence that is partially or completely absent in the genetic material present in the wild-type version of said bacterium.

[0026] In certain embodiments, the "second nucleic acid comprising a repair matrix" as described above comprises such an "other" nucleic acid.

[0027] The inventors have also described a method for transforming and preferably for genetically modifying, for example by homologous recombination, bacteria belonging to the phylum Firmicutes, such as bacteria of the genera Clostridium, Bacillus or Lactobacillus, typically solvent-producing bacteria, as well as the (transformed and typically genetically modified) bacteria obtained using said method. This method advantageously comprises a step of transforming said bacteria by introducing into said bacteria all or part of the genetic tools described herein, in particular nucleic acids ("nucleic acids OREP") comprising or consisting of: i) all or part of the sequence of SEQ ID NO: 126, and ii) sequences allowing modification of the genetic material of the bacterium and / or expression in said bacterium of DNA sequences that are partially or completely absent from the genetic material present in the wild-type version of said bacterium.

[0028] In a particular embodiment, this method advantageously comprises the steps of:

[0029] a) introducing into said bacteria a genetic tool as described herein, preferably in the presence of an agent for inducing expression of said anti-CRISPR protein, and

[0030] b) culturing the transformed bacteria obtained at the end of step a) on a culture medium that does not contain the agent for inducing the expression of the anti-CRISPR protein and generally allows the expression of the DNA endonuclease / gRNA ribonucleoprotein complex, such as Cas9 / gRNA.

[0031] The inventors have also described a kit for transforming and preferably genetically modifying bacteria belonging to the phylum Firmicutes, such as bacteria of the genera Clostridium, Bacillus, or Lactobacillus, or for producing at least one solvent, such as a mixture of solvents, using such bacteria. Such a kit preferably comprises the nucleic acids described herein and an inducer of an inducible promoter for expression of a selected anti-CRISPR protein suitable for use in the genetic tools described herein. In a specific embodiment, the kit comprises all or a portion of the elements of the genetic tools described herein.

[0032] The present invention also describes the use of nucleic acids or genetic tools disclosed for the first time herein for transforming and optionally genetically modifying bacteria belonging to the phylum Firmicutes, such as bacteria of the genera Clostridium, Bacillus or Lactobacillus, preferably bacteria that in the wild have both a bacterial chromosome and at least one DNA molecule different from the chromosomal DNA (usually a natural plasmid).

[0033] The present invention also describes the use of nucleic acids, genetic tools, methods for transforming and preferably genetically modifying such bacteria, bacteria obtained by this method and / or kits disclosed for the first time herein, which allow the production of solvents or mixtures of solvents, preferably acetone, butanol, ethanol, isopropanol or mixtures thereof, typically isopropanol / butanol, butanol / ethanol or isopropanol / ethanol mixtures, preferably on an industrial scale. DETAILED DESCRIPTION

[0034] Despite being used in industry for over a century, our understanding of solventogenic bacteria, particularly those belonging to the genus Clostridium, remains limited by the difficulties encountered in their genetic modification. For example, naturally occurring isopropanol-producing Clostridium bacteria, which typically possess in their genomes the adh gene encoding a primary / tertiary alcohol dehydrogenase that reduces acetone to isopropanol, are genetically and functionally distinct from bacteria capable of naturally performing ABE fermentations.

[0035] Advantageously, in the present context, the inventors have succeeded in transforming and genetically modifying bacteria of the genus Clostridium that naturally produce isopropanol, namely the bacterium Clostridium beijerinckii DSM 6423 and the reference strain Clostridium acetobutylicum DSM 792.

[0036] Some of the work described in the experimental section was performed in a strain capable of IBE fermentation, Clostridium beijerinckii strain DSM 6423, whose genomic and transcriptomic analysis has been recently described by the present inventors (Máté de Gerando et al., 2018).

[0037] During the assembly of the genome of this strain, the inventors discovered in particular the presence of mobile genetic elements in addition to the chromosome (accession number PRJEB11626 - https: / / www.ebi.ac.uk / ena / data / view / PRJEB11626): two natural plasmids (pNF1 and pNF2) and a linear phage (Φ6423).

[0038] The Clostridium beijerinckii strain DSM 6423 is naturally sensitive to erythromycin but resistant to thiamphenicol. Patent application number FR18 / 73492 describes a specific strain, Clostridium beijerinckii strain DSM 6423ΔcatB (also designated herein as Clostridium beijerinckii IFP962ΔcatB), which was made sensitive to thiamphenicol. In a specific embodiment of the present invention, the inventors succeeded in removing its natural plasmid pNF2 from Clostridium beijerinckii strain DSM 6423 and obtained Clostridium beijerinckii strain DSM6423ΔcatBΔpNF2 (also designated herein as Clostridium beijerinckii IFP963ΔcatBΔpNF2). This strain was characterized for the first time in the text of this application. It was registered with the BCCM-LMG Collection on February 20, 2019 under the deposit number LMGP-31277. The strain lacks the catB gene of sequence SEQ ID NO: 18 and the plasmid pNF2 (wild type). The present description also relates to any derivative bacteria, clones, mutants or genetically modified versions of the latter, which also typically lack the catB gene of sequence SEQ ID NO: 18 and the plasmid pNF2 (wild type). The present invention also relates more generally to any bacterium that, in the wild state, has both a bacterial chromosome and at least one DNA molecule other than chromosomal DNA (designated herein as "non-chromosomal (bacterial) DNA" or "natural (bacterial) plasmid"), which has been genetically modified using the nucleic acids and / or genetic tools described in the context of the present invention so that it no longer contains at least one of its non-chromosomal DNA molecules, typically several of its non-chromosomal DNA molecules (e.g., 2, 3 or 4 non-chromosomal DNA molecules), preferably all of its non-chromosomal DNA molecules.

[0039] Thus, the inventors describe in the present application a solvent-producing bacterium belonging to the Firmicutes phylum, such as a bacterium of the genus Clostridium, Bacillus or Lactobacillus, more particularly a bacterium of the genus Clostridium, which is naturally capable (i.e. capable in the wild state) of producing isopropanol, in particular naturally capable of performing IBE fermentation, which has been genetically modified and, as a result of this genetic modification, has in particular lost at least one natural plasmid (i.e. a plasmid naturally present in the wild-type version of said bacterium), preferably has lost all of its natural plasmids, and also describe the tools, in particular genetic tools, for obtaining said bacterium.

[0040] These tools offer the advantage of greatly facilitating the transformation and genetic modification of bacteria. Experiments carried out by the inventors have demonstrated the possible use of the tools and more generally the technology described herein for genetically modifying bacteria capable of producing isopropanol in the wild, in particular performing IBE fermentation, in particular bacteria belonging to the phylum Firmicutes, such as Clostridium, Bacillus or Lactobacillus, in particular Clostridium, in particular those carrying genes encoding resistance to antibiotics, in particular genes encoding amide alcohol-O-acetyltransferases, such as chloramphenicol-O-acetyltransferase or thiamphenicol-O-acetyltransferase.

[0041] Thus, in a particular embodiment, the inventors have succeeded in sensitizing bacteria that naturally carry (in the wild) the genes encoding the enzymes responsible for resistance to these antibiotics to antibiotics belonging to the class of amides.

[0042] Other preferred bacteria contain in the wild both a bacterial chromosome and at least one DNA molecule other than chromosomal DNA.

[0043] In addition, preferred bacteria contain both a bacterial chromosome and at least one DNA molecule different from the chromosomal DNA in the wild state, as well as a gene that confers resistance to antibiotics. In a specific embodiment, this gene encodes an amide alcohol-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase or thiamphenicol-O-acetyltransferase.

[0044] The first object described by the inventors relates to a nucleic acid (designated herein as nucleic acid "OPT") which can be advantageously used to promote the transformation of bacteria by improving the maintenance of all introduced genetic material in said bacteria. This nucleic acid OPT comprises i) all or part of the sequence of SEQ ID NO: 126 (sequence "OREP") or a functional variant thereof, and ii) a sequence (also designated herein as "sequence of interest") which allows modification of the genetic material of the bacterium and / or expression in said bacterium of a DNA sequence which is partially or completely absent from the genetic material present in the wild-type version of said bacterium.

[0045] The sequence OREP (SEQ ID NO: 126) comprises the nucleotide sequence of SEQ ID NO: 127. The sequence SEQ ID NO: 127 preferably comprises a sequence encoding a protein involved in the replication of the nucleic acid OPT. The protein believed to be involved in the replication is also designated herein as protein "REP" (SEQ ID NO: 128). The protein REP has a domain designated "COG 5655" of sequence SEQ ID NO: 129 that is conserved in Firmicutes.

[0046] In a specific embodiment, the nucleic acid OPT comprises a portion of the sequence OREP (SEQ ID NO: 126), typically one or more fragments of the sequence OREP, preferably comprising at least a sequence encoding the protein REP (SEQ ID NO: 128) or a variant or functional fragment thereof (i.e., a fragment involved in replication), typically the sequence SEQ ID NO: 127 or a variant thereof, or a fragment encoding a fragment within the protein REP that participates in the replication of the OPT nucleic acid. Functional fragments of the sequence OREP that encode a fragment present within the protein REP that participates in the replication of the nucleic acid OPT comprise the domain of the sequence SEQ ID NO: 129. These examples of nucleic acid fragments encoding functional fragments of the protein REP and its variants can be readily prepared by a skilled artisan. Typical examples of variants have between 70% and 100%, preferably between 85 and 99%, more preferably between 95 and 99%, for example 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence homology to the sequence of SEQ ID NO: 127.

[0047] In a preferred embodiment, a fragment or functional variant of the sequence OREP encodes a protein involved in the replication of the nucleic acid OPT.

[0048] In a preferred embodiment of the invention, the fragment or functional variant of the sequence OREP comprises, in addition to a sequence encoding a protein involved in the replication of the nucleic acid OPT (for example the protein REP) (for example a genetic construct of the plasmid type) or a variant or functional fragment thereof, a site of 1 to 150 bases, preferably 1 to 15 bases, such as a sequence rich in A and T bases (Rajewska et al.), preferably the site of the sequence SEQ ID NO: 118 present in the plasmid pNF2, which allows the fixation of a protein that permits the replication of the nucleic acid OPT.

[0049] The sequence of interest allowing the modification of the genetic material of the bacterium is generally a modification matrix that allows, for example, the replacement of a portion of the genetic material of the bacterium with the sequence of interest by a homologous recombination mechanism, for example according to one of the methods described herein. The sequence of interest allowing the modification of the genetic material of the bacterium may also be a sequence that recognizes (at least partially binds to) and preferably targets, i.e. recognizes and allows the cleavage of at least one strand of the following sequences in the genome of the bacterium of interest: i) a target sequence, ii) a sequence that controls the transcription of the target sequence, or iii) a sequence flanking the target sequence.

[0050] Said sequence of interest which allows expression in said bacterium of a DNA sequence which is partially or completely absent in the genetic material present in the wild-type version of said bacterium generally allows said bacterium to express one or more proteins which are not expressed or not expressed in sufficient amounts in the wild state.

[0051] Depending on the specific situation, the "nucleic acid OPT" also includes iii) a sequence encoding a DNA nuclease such as Cas9, and / or iv) one or more guide RNAs (gRNAs), each gRNA comprising an RNA structure for fixing the DNA nuclease and a sequence complementary to the targeted part of the genetic material of the bacterium.

[0052] According to another particular aspect, said "nucleic acid OPT" does not exhibit methylation at the level of the units recognized by methyltransferases of the Dam and Dcm type.

[0053] Preferably, the "nucleic acid OPT" is selected from an expression cassette and a vector, preferably a plasmid, such as a plasmid having a sequence selected from SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 124 and SEQ ID NO: 125.

[0054] Another object described by the inventors relates to a genetic tool that can be used to transform and / or genetically modify a bacterium of interest, typically a bacterium belonging to the phylum Firmicutes as described herein, such as a bacterium of the genus Clostridium, Bacillus, or Lactobacillus, preferably a bacterium of the genus Clostridium, which is naturally capable (i.e., capable in the wild) of producing isopropanol, in particular naturally capable of IBE fermentation, preferably a bacterium that is naturally resistant to one or more antibiotics, such as Clostridium beijerinckii. Preferred bacteria have both a bacterial chromosome and at least one DNA molecule different from chromosomal DNA in the wild.

[0055] In the context of the present specification, "bacteria belonging to the phylum Firmicutes" means bacteria belonging to the classes Clostridia, Mollicutes, Bacilli, or Togobacteria, preferably bacteria belonging to the classes Clostridia or Bacilli.

[0056] Specific bacteria belonging to the phylum Firmicutes include, for example, bacteria of the genus Clostridium, bacteria of the genus Bacillus, or bacteria of the genus Lactobacillus.

[0057] "Clostridium bacteria" refers specifically to so-called Clostridium species of industrial interest, typically solventogenic or acetogenic bacteria of the genus Clostridium. The expression "Clostridium bacteria" encompasses wild-type bacteria as well as strains derived therefrom that have been genetically modified to improve their properties (e.g., overexpression of ctfA, ctfB, and adc genes) but have not been exposed to the CRISPR system.

[0058] By "industrially interesting Clostridium species" is meant a Clostridium species capable of producing solvents and acids such as butyric acid or acetic acid by fermentation from sugars or monosaccharides, typically sugars containing 5 carbon atoms such as xylose, arabinose or fructose, sugars containing 6 carbon atoms such as glucose and mannose, polysaccharides such as cellulose or hemicellulose, and / or any other carbon source that can be assimilated and utilized by bacteria of the genus Clostridium (e.g., CO, CO2 and methanol). Examples of interesting solventogenic bacteria are acetone-, butanol-, ethanol- and / or isopropanol-producing bacteria of the genus Clostridium, such as strains identified in the literature as "ABE strains" [strains that perform fermentations allowing the production of acetone, butanol and ethanol] and "IBE" strains [strains that perform fermentations allowing the production of isopropanol (by reducing acetone), butanol and ethanol]. The solvent-producing bacteria of the genus Clostridium can, for example, be selected from Clostridium acetobutylicum, C. cellulolyticum, C. phytofermentans, C. beijerinckii, C. saccharobutylicum, C. saccharoaceticum, C. sporogenes, C. butyricum, C. aureobutyricum and C. tyrobutyricum, preferably from Clostridium acetobutylicum, C. beijerinckii, C. butyricum, C. tyrobutyricum and C. cellulolyticum, even more preferably from Clostridium acetobutylicum and C. beijerinckii.

[0059] Bacteria capable of producing isopropanol in the wild, in particular capable of performing IBE fermentation in the wild, can be, for example, selected from Clostridium beijerinckii, Clostridium diolis, Clostridium puniceum, Clostridium butyricum, Clostridium saccharoaceticum, Clostridium botulinum, Clostridium drakei, Clostridium scatologenes, Clostridium perfringens, and Clostridium tunisiense, preferably selected from Clostridium beijerinckii, Clostridium diolis, Clostridium puniceum, and Clostridium saccharoaceticum. A particularly preferred bacterium that is naturally capable of producing isopropanol in the wild, in particular capable of performing IBE fermentation, is Clostridium beijerinckii.

[0060] Acetogenic bacteria of interest are bacteria that produce acids and / or solvents starting from CO and H. Acetogenic bacteria of the genus Clostridium can, for example, be selected from the group consisting of C. aceticum, C. thermoaceticum, C. ljungdahlii, C. autoethanogenum, C. difficile, C. coprolificum, and C. carboxydivorans.

[0061] In a particular embodiment, the Clostridium bacterium in question is an "ABE strain", preferably Clostridium acetobutylicum strain DSM 792 (also known as ATCC 824 or LMG 5710) or Clostridium beijerinckii strain NCIMB 8052.

[0062] In another specific embodiment, the Clostridium bacterium under consideration is an "IBE strain," preferably a bacterium selected from the group consisting of a clade of Clostridium beijerinckii selected from DSM 6423, LMG 7814, LMG 7815, NRRL B-593, NCCB 27006, or Clostridium aureobutyricum DSZM 793 (Georges et al., 1983), and a clade of Clostridium beijerinckii or Clostridium aureobutyricum bacteria that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to strain DSM 6423. Particularly preferred Clostridium beijerinckii bacteria or particularly preferred clades of Clostridium beijerinckii bacteria do not contain the plasmid pNF2.

[0063] The corresponding genomes of the clades LMG 7814, LMG 7815, NRRL B-593 and NCCB27006 on the one hand and DSZM793 on the other hand have a sequence identity percentage of at least 97% with the genome of the clade DSM 6423.

[0064] The present inventors conducted fermentation experiments and confirmed that Clostridium beijerinckii bacteria of the evolutionary branches DSM 6423, LMG 7815 and NCCB 27006 are capable of producing isopropanol in the wild (see Table 1).

[0065] [Table 1]

[0066]

[0067] Balance of glucose fermentation tests using the naturally isopropanol producing strains Clostridium beijerinckii DSM 6423, LMG 7815 and NCCB 27006. In a particularly preferred embodiment of the present invention, the Clostridium beijerinckii bacterium is a bacterium of the clade DSM6423.

[0068] In another preferred embodiment of the present invention, the Clostridium beijerinckii bacterium is Clostridium beijerinckii strain IFP963ΔcatBΔpNF2 (deposited with the BCCM-LMG Collection under accession number LMG P-31277 on February 20, 2019, and also designated herein as Clostridium beijerinckii DSM 6423ΔcatBΔpNF2) or a genetically modified version thereof. The Clostridium beijerinckii IFP963ΔcatBΔpNF2 or the genetically modified version thereof lacks the catB gene of SEQ ID NO: 18 and the pNF2 plasmid.

[0069] The “bacteria of the genus Bacillus” specifically means B. amyloliquefaciens, B. thurigiensis, B. coagulans, B. cereus, B. anthracis or B. subtilis.

[0070] During recent experiments, the inventors observed that the removal of the natural plasmid pNF2 has significant advantages for the introduction and maintenance of other natural or synthetic genetic elements (e.g., expression cassettes or plasmid expression vectors). Thus, strain IFP963ΔcatBΔpNF2 can be expressed at a rate 10 to 5 x 10 higher than its wild-type homolog or strain DSM 6423ΔcatB (also designated herein as IFP962ΔcatB). 3 Times the efficiency conversion.

[0071] The bacteria to be transformed and preferably genetically modified are preferably bacteria which have been exposed to a first step of transformation using the nucleic acids or genetic tools according to the invention and to a first step of genetic modification, said exposure making it possible to remove at least one extrachromosomal DNA molecule (generally at least one plasmid) naturally present in said bacteria in the wild.

[0072] One particular genetic tool described by the inventors is characterized by:

[0073] i) It contains:

[0074] a "first" nucleic acid encoding at least one DNA endonuclease, such as a Cas9 enzyme, wherein the sequence encoding said DNA endonuclease is placed under the control of a promoter, and

[0075] - at least one "second" nucleic acid containing a repair matrix allowing the replacement of a portion of the bacterial DNA targeted by said endonuclease with a sequence of interest by the mechanism of homologous recombination,

[0076] wherein ii) at least one of the nucleic acids further encodes one or more guide RNAs (gRNAs), or wherein the genetic tool further comprises one or more guide RNAs, each guide RNA comprising an RNA structure for anchoring to a DNA endonuclease and a sequence complementary to the targeted portion of the bacterial DNA.

[0077] The example of the genetic tool described by the inventors is just like the CRISPR / Cas system, containing two different essential components, i.e., i) endonuclease, in this case, the nuclease (Cas or "CRISPR-associated protein") Cas associated with the CRISPR system, and ii) guide RNA. The guide RNA takes the form of a chimeric RNA consisting of a combination of bacterial CRISPR RNA (crRNA) and tracrRNA (trans-activating CRISPR RNA) (Jinek et al., Science 2012). The gRNA combines the targeting specificity of the crRNA corresponding to the "spacer sequence" of the guidance serving as the Cas protein and the conformational properties of the tracrRNA in a single transcript. When the gRNA and Cas protein are expressed simultaneously in a cell, the target genomic sequence is typically advantageously permanently modified due to the repair matrix provided.

[0078] The genetic tool according to the present invention preferably further has the following feature: iii) at least one of the ("first" and "second") nucleic acids further comprises a sequence encoding an anti-CRISPR protein under the control of an inducible promoter, or wherein the genetic tool further comprises a third nucleic acid encoding an anti-CRISPR protein under the control of an inducible promoter.

[0079] In particular, a genetic tool is described, comprising at least:

[0080] - a first nucleic acid encoding at least one DNA endonuclease, wherein the sequence encoding said DNA endonuclease is placed under the control of a promoter, and

[0081] - another nucleic acid (or "nth nucleic acid") comprising or consisting of the nucleic acid sequence "OPT", i.e. a sequence comprising: i) all or part of the sequence SEQ ID NO: 126 ("OREP"); and ii) a sequence allowing modification of the genetic material of a bacterium and / or expression in said bacterium of a DNA sequence that is partially or completely absent from the genetic material present in the wild-type version of said bacterium, at least one of said nucleic acids of this particular genetic tool further comprising a sequence encoding an anti-CRISPR protein under the control of an inducible promoter, or said particular genetic tool preferably further comprises a third nucleic acid encoding an anti-CRISPR protein under the control of an inducible promoter.

[0082] In a specific embodiment, the "second" or "nth" nucleic acid containing the repair matrix as described above comprises or consists of the "other nucleic acid".

[0083] In another specific embodiment, the "first nucleic acid" further encodes one or more guide RNAs (gRNAs).

[0084] In the sense of the present invention, "nucleic acid" means any natural, synthetic, semisynthetic or recombinant DNA or RNA molecule that is optionally chemically modified (i.e., comprises unnatural bases, modified nucleotides comprising, for example, modified bonds, modified bases and / or modified sugars) or optimized so that the codons of the transcripts synthesized from the coding sequence are the codons most commonly found in the bacterium of the genus Clostridium in which it is intended to be used. In the case of Clostridium, the optimized codons are typically codons rich in adenine bases ("A") and thymine bases ("T").

[0085] In the peptide sequences described in this document, amino acids are represented by their single-letter codes according to the following nomenclature: C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan and Y: tyrosine.

[0086] The genetic tools described in the context of the present invention comprise a first nucleic acid encoding at least one DNA endonuclease (also indicated herein as "nuclease"), typically a nuclease of the Cas type such as Cas9 or MAD7.

[0087] "Cas9" means the Cas9 protein (also known as CRISPR-associated protein 9, Csn1, or Csx12), or a functional protein, peptide, or polypeptide fragment thereof, that is, an enzyme (nuclease) activity that is capable of interacting with one or more guide RNAs and performing double-strand breaks in the DNA of a target genome. Thus, "Cas9" may refer to a protein that has been modified, for example, truncated to remove protein domains that are not essential for the intended function of the protein, particularly domains that are not essential for interaction with one or more gRNAs.

[0088] In addition, the nuclease MAD7 (whose amino acid sequence corresponds to the sequence SEQ ID NO: 72), also designated "Cas12" or "Cpf1", can be advantageously used in the context of the present invention by combining it with one or more gRNAs known to those skilled in the art that are capable of binding to this nuclease (see Garcia-Doval et al., 2017 and Stella S. et al., 2017).

[0089] According to a specific embodiment, the sequence encoding the nuclease MAD7 is a sequence optimized for easy expression in a Clostridium strain, preferably the sequence SEQ ID NO: 71.

[0090] According to another specific aspect, the sequence encoding the nuclease MAD7 is a sequence optimized to be easily expressed in a Bacillus strain, preferably the sequence SEQ ID NO: 132.

[0091] A sequence encoding Cas9 (the complete protein or a fragment thereof), such as that which can be used in examples of possible embodiments of the present invention, can be obtained starting from any known Cas9 protein (Makarova et al., 2011). Examples of Cas9 proteins that can be used in the present invention include, but are not limited to, Cas9 proteins from Streptococcus pyogenes (reference application WO 2017 / 064439, SEQ ID NO: 1 and NCBI accession number: WP_010922251.1), Streptococcus thermophilus, Streptococcus mutans, Campylobacter jejuni, Pasteurella multocida, Francisella novicida, Neisseria meningitidis, Neisseria lactamica, and Legionella pneumophila (see Fonfara et al., 2013; Makarova et al., 2015).

[0092] In a specific embodiment, the Cas9 protein or a functional protein, peptide or polypeptide fragment thereof encoded by one of the nucleic acids of the genetic tools according to the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 75, or any other amino acid sequence that is at least 50%, preferably at least 60%, identical thereto and contains at least two aspartic acids ("D") occupying positions 10 ("D10") and 840 ("D840") of the amino acid sequence of SEQ ID NO: 75.

[0093] In a preferred embodiment, Cas9 comprises or consists of the following sequence: a Cas9 protein (NCBI accession number: WP_010922251.1, SEQ ID NO: 75) encoded by the cas9 gene from Streptococcus pyogenes strain M1 GAS (NCBI accession number: NC_002737.2SPy_1046, SEQ ID NO: 76), or a Cas9 protein encoded by a version of the cas9 gene that has undergone optimization at the origin of its transcript ("optimized version"), the optimized version containing codons preferentially used by bacteria of the genus Clostridium, typically codons rich in adenine ("A") and thymine ("T") bases, allowing for the promotion of expression of the Cas9 protein in this bacterial genus. These optimized codons adhere to the codon usage pattern specific to each bacterial strain, which is well known to those skilled in the art.

[0094] According to a specific embodiment, the Cas9 domain is composed of a complete Cas9 protein, preferably a Cas9 protein of Streptococcus pyogenes or an optimized version thereof.

[0095] Each nucleic acid of the genetic tools described herein, typically the "first" nucleic acid and the "second" or "nth" nucleic acid of the genetic tool, is composed of different entities and is typically in the form of an expression cassette (or "construct"), for example a nucleic acid comprising at least one transcription promoter operably linked (in the sense understood by those skilled in the art) to one or more (coding) sequences of interest, for example operably linked to an operon comprising several coding sequences of interest whose expression products contribute to the performance of a function of interest in the bacterium, or a nucleic acid further comprising transcription activation and / or transcription termination sequences; or in the form of a circular or linear single-stranded or double-stranded vector, such as a plasmid, phage, cosmid, artificial or synthetic chromosome, comprising one or more expression cassettes as defined above. Preferably, the vector is a plasmid.

[0096] The nucleic acid of interest, typically an expression cassette or expression vector, can be constructed by conventional techniques familiar to those skilled in the art and can include one or more promoters, bacterial replication origins (ORI sequences), terminator sequences, selection genes such as antibiotic resistance genes, and sequences ("flanking regions") that allow the targeted insertion of the expression cassette or vector. Additionally, these expression cassettes and vectors can be integrated into the bacterial genome by techniques familiar to those skilled in the art.

[0097] The ORI sequence of interest may be selected from pIP404, pAMβ1, repH (origin of replication in C. acetobutylicum), ColE1 or rep (origin of replication in E. coli) or any other origin of replication allowing the vector, typically a plasmid, to be maintained in bacterial cells, such as Clostridium or Bacillus cells.

[0098] In the context of the present invention, a preferred ORI sequence is the ORI sequence present within the sequence OREP (SEQ ID NO: 126) of the plasmid pNF2 (SEQ ID NO: 118).

[0099] The termination sequence of interest may be selected from the termination sequences of the adc, th1 genes, the bcs operon, or any other terminator familiar to those skilled in the art that allows the cessation of transcription in bacterial cells, such as Clostridium or Bacillus cells.

[0100] The selection gene (resistance gene) of interest may be selected from ermB, catP, bla, tetA, tetM and / or any other gene that confers resistance to ampicillin, erythromycin, chloramphenicol, thiamphenicol, spectinomycin, tetracycline or any other antibiotic familiar to those skilled in the art that can be used for the selection of bacteria, e.g., bacteria of the genus Clostridium or Bacillus.

[0101] Optionally, a sequence encoding a DNA endonuclease, such as Cas9, present in one of the nucleic acids of the genetic tool according to the present invention can be placed under the control of a promoter. Such a promoter can be a constitutive promoter or an inducible promoter. In a preferred embodiment, the promoter controlling the expression of the nuclease is an inducible promoter.

[0102] Examples of constitutive promoters that can be used in the context of the present invention can be selected from the promoters of the th1 gene, the ptb gene, the adc gene, the BCS operon or derivatives thereof, preferably functional but shorter (truncated) derivatives such as the "miniPth1" derivative of the th1 gene promoter of Clostridium acetobutylicum (Dong et al., 2012), or any other promoter familiar to those skilled in the art that allows the expression of proteins in bacteria of interest, such as Clostridium bacteria.

[0103] Examples of inducible promoters that can be used in the context of the present invention can be selected, for example, from promoters whose expression is controlled by the transcriptional repressor TetR, such as the promoter of the tetA gene (a tetracycline resistance gene originally present on the Escherichia coli transposon Tn10); promoters whose expression is controlled by L-arabinose, such as the promoter of the ptk gene (Zhang et al., 2015), which is preferably combined with the araR regulatory expression cassette in Clostridium acetobutylicum to construct the ARAi system (Zhang et al., 2015); promoters whose expression is controlled by laminaribiose (a dimer of β-1,3 glucose); promoters whose expression is controlled by lactose, such as the promoter of the bgaL gene (Banerjee et al., 2014); promoters whose expression is controlled by xylose, such as the promoter of the xylB gene (Nariya et al., 2011); and promoters whose expression is controlled by UV exposure, such as the promoter of the bcn gene (Dupuy et al., 2005).

[0104] Promoters derived from one of the above-mentioned promoters, preferably shorter (truncated) functional derivatives, can also be used in the context of the present invention.

[0105] Other inducible promoters that can be used in the context of the present invention are also described in, for example, Ransom et al. (2015), Currie et al. (2013), and Hartman et al. (2011).

[0106] A preferred inducible promoter is a promoter derived from tetA that can be induced by anhydrotetracycline (aTc; which is less toxic than tetracycline and can relieve the repression of the transcription repressor TetR at lower concentrations), which is selected from Pcm-2tetO1 and Pcm-2tetO2 / 1 (Dong et al., 2012).

[0107] Another preferred inducible promoter is a promoter that can be induced by lactose, such as the promoter of the bgaL gene (Banerjee et al., 2014).

[0108] The nucleic acid of particular interest, typically an expression cassette or vector, comprises one or more expression cassettes, each encoding a gRNA.

[0109] Within the meaning of the present invention, the term "guide RNA" or "gRNA" refers to an RNA molecule capable of interacting with a DNA endonuclease in order to guide it to a target region of the bacterial chromosome. The specificity of the cleavage is determined by the gRNA. As explained above, each gRNA contains two regions:

[0110] - a first region at the 5' end of the gRNA (often referred to as the "SDS" region) that is complementary to the target chromosomal region and mimics the crRNA of the endogenous CRISPR system, and

[0111] - A second region at the 3' end of the gRNA (often referred to as the "handle" region), which mimics the base pairing interaction between tracrRNA ("trans-activating crRNA") and the crRNA of the endogenous CRISPR system and has a double-stranded stem-loop structure ending in a substantially single-stranded sequence at the 3' end. This second region is essential for the binding of the gRNA to the DNA endonuclease.

[0112] The first region of the gRNA (the "SDS" region) varies depending on the chromosomal sequence being targeted.

[0113] The "SDS" region of the gRNA that is complementary to the target chromosomal region comprises at least 1 nucleotide, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides, typically between 1 and 40 nucleotides. Preferably, this region has a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0114] The second region ("handle" region) of the gRNA has a stem-loop structure (or hairpin structure). The "handle" region of each gRNA is independent of the selected chromosomal target.

[0115] According to a particular embodiment, the "handle" region comprises or consists of a sequence of at least 1 nucleotide, preferably at least 1, 50, 100, 200, 500 and 1000 nucleotides, typically between 1 and 1000 nucleotides. Preferably, this region has a length of 40 to 120 nucleotides.

[0116] The total length of the gRNA is generally 50 to 1000 nucleotides, preferably 80 to 200 nucleotides, more particularly preferably 90 to 120 nucleotides. According to a specific embodiment, the gRNA used in the present invention has a length between 95 and 110 nucleotides, for example, a length of about 100 or about 110 nucleotides.

[0117] Those skilled in the art can easily determine the sequence and structure of the gRNA according to the chromosomal region to be targeted using well-known techniques (see, for example, DiCarlo et al., 2013).

[0118] The targeted DNA region / part / sequence within the bacterial genome, e.g. the bacterial chromosome, may correspond to the non-coding part of the DNA or the coding part of the DNA.

[0119] In a specific embodiment consisting of modifying a given sequence, the targeted portion of the bacterial DNA is essential for bacterial survival. For example, it corresponds to any region of the bacterial chromosome or any region located on non-chromosomal DNA, such as a mobile genetic element that is essential for the survival of the microorganism under specific growth conditions, such as any region on a plasmid containing a resistance marker for an antibiotic when the desired growth conditions require that the bacteria be cultured in the presence of the antibiotic.

[0120] In another specific embodiment aimed at removing genetic elements that are nonessential under the specific growth conditions associated with cultivation of the microorganism, the targeted portion of the bacterial DNA may correspond to any region of the non-chromosomal bacterial DNA.

[0121] Specific examples of targeted DNA regions within Clostridium bacteria are the sequences used in Example 1 of the Experimental Section. These are, for example, sequences encoding the bdhA (SEQ ID NO: 77) and bdhB (SEQ ID NO: 78) genes. The targeted DNA region / region / sequence is followed by a "PAM" ("protospacer adjacent motif") sequence involved in the binding of DNA endonucleases.

[0122] The "SDS" region of a given gRNA is identical (100%) or at least 80%, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the targeted DNA region / portion / sequence within the bacterial genome, e.g., bacterial chromosome, and is capable of hybridizing to all or part of the complementary sequence of said region / portion / sequence, typically to a sequence comprising at least 1 nucleotide, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides, typically between 1 and 40 nucleotides, preferably a sequence comprising 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0123] In the context of the present invention, the nucleic acid of interest can comprise one or more gRNAs targeting a sequence ("target sequence," "targeted sequence," or "recognized sequence"). These different gRNAs can target the same or different chromosomal regions or regions belonging to non-chromosomal bacterial DNA (e.g., mobile genetic elements) that are optionally present within the microorganism.

[0124] The gRNA can be introduced into the bacterial cell in the form of a gRNA molecule (mature or precursor), a precursor or one or more nucleic acids encoding the gRNA. The gRNA is preferably introduced into the bacterial cell in the form of one or more nucleic acids encoding the gRNA.

[0125] When the one or more gRNAs are directly introduced into the cell in the form of RNA molecules, these gRNAs (mature or precursors) can contain modified nucleotides or chemical modifications that allow them, for example, to improve their resistance to nucleases and therefore improve their lifespan in the cell. Specifically, they can include at least one modified or non-natural nucleotide, for example, a modified base such as hypoxanthine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base that allows hybridization. The gRNA used according to the present invention can also be modified at the level of internucleotide linkages, such as phosphorothioate, H-phosphonate or alkyl-phosphonate, or at the level of the backbone, such as α-oligonucleotides, 2′-O-alkyl ribose or PNA (peptide nucleic acid) (Egholm et al., 1992).

[0126] The gRNA can be natural RNA, synthetic RNA or the RNA produced by recombinant technology. These gRNAs can be prepared by all methods known to those skilled in the art such as chemical synthesis, in vivo transcription or amplification technology.

[0127] When the gRNA is introduced into the bacterial cell in the form of one or more nucleic acids, the sequence encoding the one or more gRNAs is placed under the control of an expression promoter. This promoter can be constitutive or inducible.

[0128] When using several gRNAs, the expression of each gRNA can be controlled by a different promoter. Preferably, the promoter used for all gRNAs is the same. In a specific embodiment, the same promoter can be used to allow the expression of several, for example only some, or in other words part or all of the gRNAs to be expressed.

[0129] In a preferred embodiment, the promoter controlling the expression of the one or more gRNAs is an inducible promoter.

[0130] Examples of constitutive promoters that can be used in the context of the present invention can be chosen from the promoters of the th1 gene, the ptb gene or the BCS operon or their derivatives, preferably miniPth1, or any other promoter familiar to a person skilled in the art that allows the synthesis of (coding or non-coding) RNA in the bacterium of interest.

[0131] Examples of inducible promoters that can be used in the context of the present invention can be selected from the promoters of the tetA gene, xylA gene, lacI gene or bgaL gene or derivatives thereof, preferably 2tetO1 or tetO2 / 1. A preferred inducible promoter is 2tetO1.

[0132] The promoters controlling the expression of the DNA endonuclease and the one or more gRNAs can be identical or different and can be constitutive or inducible. In a particularly preferred embodiment, the promoters controlling the expression of the DNA endonuclease or the one or more gRNAs, respectively, are different promoters but can be induced by the same inducer.

[0133] Inducible promoters as described above allow advantageous control of the action of the DNA endonuclease / gRNA ribonucleoprotein complex, such as Cas9 / gRNA, and facilitate the selection of transformants that have undergone the desired genetic modification.

[0134] Advantageously, the genetic tool according to the present invention may also comprise a sequence encoding at least one anti-CRISPR protein, i.e. a protein capable of inhibiting or preventing / neutralizing the action of Cas and / or a protein capable of inhibiting or preventing / neutralizing the action of a CRISPR / Cas system, for example a type II CRISPR / Cas system when the nuclease is a Cas9 type nuclease. This sequence is typically placed under the control of an inducible promoter that is different from the promoter controlling the expression of the DNA endonuclease and / or one or more gRNAs, and can be induced by another inducer. In a preferred embodiment, the sequence encoding the anti-CRISPR protein is typically also located on one of the at least two nucleic acids present in the genetic tool. In a specific embodiment, the sequence encoding the anti-CRISPR protein is located on a nucleic acid different from the first two nucleic acids (typically a "third nucleic acid"). In another specific embodiment, both the sequence encoding the anti-CRISPR protein and the sequence encoding the transcriptional repressor of the anti-CRISPR protein are integrated into the bacterial chromosome.

[0135] In a preferred embodiment, the sequence encoding the anti-CRISPR protein is placed on a nucleic acid encoding the DNA endonuclease within the genetic tool (also designated herein as the "first nucleic acid"). In another embodiment, the sequence encoding the anti-CRISPR protein is placed on a nucleic acid within the genetic tool that is different from the nucleic acid encoding the DNA endonuclease, for example, on a nucleic acid designated herein as the "second nucleic acid" or on an "nth" (typically "third") nucleic acid optionally included in the genetic tool.

[0136] The anti-CRISPR protein is typically an "anti-Cas9" protein or an "anti-MAD7" protein, i.e., a protein that can inhibit or prevent / neutralize the action of Cas9 or CAS7.

[0137] Advantageously, the anti-CRISPR protein is an "anti-Cas9" protein, for example selected from AcrIIA1, AcrIIA2, AcrIIA3, AcrIIA4, AcrIIA5, AcrIIC1, AcrIIC2, and AcrIIC3 (Pawluk et al., 2018). Preferably, the "anti-Cas9" protein is AcrIIA2 or AcrIIA4. Even more preferably, the "anti-Cas9" protein is AcrIIA4. Such proteins are generally capable of very significantly limiting, and ideally preventing, the action of Cas9, for example by binding to the Cas9 enzyme (Dong et al., 2017; Rauch et al., 2017).

[0138] Another anti-CRISPR protein that can be used advantageously is an “anti-MAD7” protein, such as the AcrVA1 protein (Marino et al., 2018).

[0139] In a preferred embodiment, said anti-CRISPR protein is capable of inhibiting, preferably neutralizing, the action of said DNA endonuclease, preferably during the step of introducing the nucleic acid sequence of said genetic tool into the bacterial strain of interest.

[0140] The promoter that controls the expression of the sequence encoding the anti-CRISPR protein is preferably an inducible promoter. The inducible promoter is accompanied by a constitutively expressed gene, typically a gene responsible for allowing the expression of a protein that is repressed by transcription from the inducible promoter. Such a promoter can, for example, be selected from the promoters of the tetA gene, the xylA gene, the lacI gene, or the bgaL gene, or derivatives thereof.

[0141] An example of an inducible promoter that can be used in the context of the present invention is the Pbgal promoter (lactose inducible), which is present in the genetic tool and is present on the same nucleic acid as the bgaR gene, which is constitutively expressed and whose expression product allows transcriptional repression from Pbgal. In the presence of the inducer lactose, the transcriptional repression of the Pbgal promoter is released, allowing transcription of the gene placed downstream thereof. Preferably, the gene placed downstream corresponds to a gene encoding an anti-CRISPR protein, such as acrIIA4, in the context of the present invention.

[0142] Said promoter controlling the expression of the anti-CRISPR protein makes it possible to advantageously control the action of DNA endonucleases such as the Cas9 enzyme and thus facilitate the transformation of bacteria such as Clostridium, Bacillus or Lactobacillus and the production of transformants that have undergone the desired genetic modification.

[0143] In a specific embodiment, the present invention relates to a genetic tool comprising a plasmid vector having the sequence of SEQ ID NO: 23 as a "first" nucleic acid.

[0144] In another specific embodiment, the present invention relates to a genetic tool comprising a plasmid vector having a sequence selected from one of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 124 and SEQ ID NO: 125 as the "second" or "nth" nucleic acid.

[0145] In yet another specific embodiment, the present invention relates to a genetic tool comprising as "nucleic acid OPT" a plasmid vector having a sequence selected from the group consisting of SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 124, and SEQ ID NO: 125. In another specific embodiment, the genetic tool comprises several (e.g., at least 2 or 3) sequences selected from SEQ ID NO: 23, 79, 80, 119, 123, 124, and 125, said sequences being different from one another.

[0146] The inventors describe examples of nucleic acids of interest, generally DNA sequences of interest, which allow the expression in bacteria of DNA sequences that are partially or completely absent in the genetic material present in the wild-type version of said bacteria.

[0147] In a particular embodiment, expression of the DNA sequence of interest allows the bacteria, e.g., bacteria of the genus Clostridium, to ferment (usually simultaneously) several different sugars, e.g., at least two different sugars, typically at least two different sugars among sugars containing 5 carbon atoms (e.g., glucose or mannose) and / or sugars containing 6 carbon atoms (e.g., xylose, arabinose or fructose), preferably at least three different sugars, selected from, for example, glucose, xylose and mannose, glucose, arabinose and mannose, and glucose, xylose and arabinose.

[0148] In another specific embodiment, the DNA sequence of interest encodes at least one product of interest, preferably a product that promotes solvent production in bacteria such as bacteria of the genus Clostridium, Bacillus or Lactobacillus, typically at least one protein of interest, such as an enzyme, a membrane protein such as a transporter, a protein for the maturation of other proteins (chaperone protein), a transcription factor or a combination thereof.

[0149] In a preferred embodiment, the DNA sequence of interest promotes solvent production and is generally selected from i) sequences encoding enzymes, such as enzymes involved in the conversion of aldehydes to alcohols, such as sequences encoding alcohol dehydrogenases (e.g., sequences selected from adh, adhE, adhE1, adhE2, bdhA, bdhB and bdhC), sequences encoding transferases (e.g., sequences selected from ctfA, ctfB, atoA and atoB), sequences encoding decarboxylases (e.g., adc), sequences encoding hydrogenases (e.g., sequences selected from etfA, etfB and hydA), and combinations thereof, ii) sequences encoding membrane proteins, such as sequences encoding phosphotransferases (e.g., sequences selected from glcG, bglC, cbe4532, cbe4533, cbe4982, cbe4983, cbe0751), iii) sequences encoding transcription factors (e.g., sequences selected from sigL, sigE, sigF, sigG, sigH, sigK), and iv) combinations thereof.

[0150] The inventors have also described examples of nucleic acids of interest that recognize (at least partially bind) and preferably target, i.e. recognize and allow cleavage of at least one strand of the following sequences in the genome of the bacteria of interest: i) the target sequence, ii) a sequence that controls the transcription of the target sequence, or iii) a sequence flanking the target sequence.

[0151] The identified sequence is also designated herein as a "target sequence" or "targeted sequence."

[0152] Also described are genetic tools comprising or consisting of said nucleic acid of interest. In this case, said nucleic acid of interest is typically present in the "second" or "nth" nucleic acid of the genetic tools described herein.

[0153] In the context of the present specification, the nucleic acid of interest is typically used to inhibit a recognized sequence in the genome of the bacterium or to modify its expression, such as to regulate / modulate its expression, in particular to inhibit it, preferably to modify it so that the bacterium cannot express a protein, in particular a functional protein, from the sequence.

[0154] When the target sequence is a sequence encoding an enzyme that allows the bacterium of interest to grow in a culture medium containing the antibiotic to which it confers resistance, a sequence that controls the transcription of such a sequence, or a sequence flanking such a sequence, the antibiotic is typically an antibiotic belonging to the class of amide alcohols. In the context of the present specification, examples of amide alcohols of interest are chloramphenicol, thiamphenicol, chloramphenicol azido, and florfenicol (Schwarz S. et al., 2004), in particular chloramphenicol and thiamphenicol.

[0155] In a specific embodiment, the nucleic acid of interest comprises at least one region complementary to a target sequence, which has 100% identity or at least 80% identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the targeted DNA region / portion / sequence in the bacterial genome, and is capable of hybridizing to all or part of the complementary sequence of said region / portion / sequence, typically comprising at least 1 nucleotide, preferably at least 1, 2, 3, 4, 5, 10, 14, 15, 20, 25, 30, 35 or 40 nucleotides, typically between 1, 10 or 20 and 1000 nucleotides, for example 1, 10 or 20 to 900, 800 or more nucleotides. 29 or 30 nucleotides. The complementary region of the target sequence present in the nucleic acid of interest can correspond to the "SDS" region of the guide RNA (gRNA) used in the CRISPR tools described herein.

[0156] In another specific embodiment described, the nucleic acid of interest comprises at least two regions each complementary to a target sequence, which have 100% identity or at least 80% identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the targeted DNA region / portion / sequence within the bacterial genome. These regions are capable of hybridizing to a fully or partially complementary sequence to the region / portion / sequence, typically a sequence comprising at least 1 nucleotide, preferably at least 100 nucleotides, typically between 100 and 1000 nucleotides as described above. The complementary regions to the target sequence present in the nucleic acid of interest can recognize, preferably target, the genetic modification tools described herein, such as the genetic tools Genetic tools or The 5′ and 3′ flanking regions of the targeted sequence are described in the type of allelic exchange tool.

[0157] According to a specific aspect, the target sequence is a sequence encoding an amide alcohol-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase or thiamphenicol-O-acetyltransferase, a sequence that controls the transcription of such a sequence, or a sequence flanking such a sequence in the genome of a bacterium of interest, such as a Clostridium bacterium that is capable of growing in a culture medium containing one or more antibiotics belonging to the amide alcohol class, such as chloramphenicol and / or thiamphenicol.

[0158] The recognized sequence is, for example, the sequence corresponding to the catB gene (CIBE_3859) encoding chloramphenicol-O-acetyltransferase from Clostridium beijerinckii DSM 6423, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to the chloramphenicol-O-acetyltransferase, or a sequence that comprises all or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 18. In other words, the recognized sequence can be a sequence comprising at least 1 nucleotide of the sequence SEQ ID NO: 18, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides, usually between 1 and 40 nucleotides, preferably a sequence comprising 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides of the sequence SEQ ID NO: 18.

[0159] Examples of amino acid sequences that are at least 70% identical to the chloramphenicol-O-acetyltransferase encoded by the sequence SEQ ID NO: 18 correspond to the sequences indicated in the NCBI database under the following references: WP_077843937.1, SEQ ID NO: 44 (WP_063843219.1), SEQ ID NO: 45 (WP_078116092.1), SEQ ID NO: 46 (WP_077840383.1), SEQ ID NO: 47 (WP_077307770.1), SEQ ID NO: 48 (WP_103699368.1), SEQ ID NO: 49 (WP_087701812.1), SEQ ID NO: 50 (WP_017210112.1), SEQ ID NO: 51 (WP_077831818.1), SEQ ID NO: 52 (WP_077831818.1), SEQ ID NO: 53 (WP_077831820.1), SEQ ID NO: 54 (WP_077831831.1), SEQ ID NO: 55 (WP_077831832.1). NO: 52 (WP_012059398.1), SEQ ID NO: 53 (WP_077363893.1), SEQ ID NO: 54 (WP_015393553.1), SEQ ID NO: 55 (WP_023973814.1), SEQ ID NO: 56 (WP_026887895.1), SEQ ID NO 57 (AWK51568.1), SEQ ID NO: 58 (WP_003359882.1), SEQ ID NO: 59 (WP_091687918.1), SEQ ID NO: 60 (WP_055668544.1), SEQ ID NO: 61 (KGK90159.1), SEQ ID NO: 62(WP_032079033.1), SEQ ID NO: 63 (WP_029163167.1), SEQ ID NO: 64 (WP_017414356.1), SEQ ID NO: 65 (WP_073285202.1), SEQ ID NO: 66 (WP_063843220.1) and SEQ ID NO: 67 (WP_021281995.1).

[0160] Examples of amino acid sequences having at least 75% identity to the chloramphenicol-O-acetyltransferase encoded by the sequence SEQ ID NO: 18 correspond to the sequences WP_077843937.1, WP_063843219.1, WP_078116092.1, WP_077840383.1, WP_077307770.1, WP_103699368.1, WP_087701812.1, WP_01721011 2.1, WP_077831818.1, WP_012059398.1, WP_077363893.1, WP_015393553.1, WP_023973814.1, W P_026887895.1, AWK51568.1, WP_003359882.1, WP_091687918.1, WP_055668544.1 and KGK90159.1.

[0161] Examples of amino acid sequences that are at least 90% identical to the chloramphenicol-O-acetyltransferase encoded by the sequence SEQ ID NO: 18 are the sequences WP_077843937.1, WP_063843219.1, WP_078116092.1, WP_077840383.1, WP_077307770.1, WP_103699368.1, WP_087701812.1, WP_017210112.1, WP_077831818.1, WP_012059398.1, WP_077363893.1, WP_015393553.1, WP_023973814.1, WP_026887895.1 and AWK51568.1.

[0162] Examples of amino acid sequences that are at least 95% identical to the chloramphenicol-O-acetyltransferase encoded by the sequence SEQ ID NO: 18 correspond to the sequences WP_077843937.1, WP_063843219.1, WP_078116092.1, WP_077840383.1, WP_077307770.1, WP_103699368.1, WP_087701812.1, WP_017210112.1, WP_077831818.1, WP_012059398.1, WP_077363893.1, WP_015393553.1, WP_023973814.1, and WP_026887895.1.

[0163] Preferred amino acid sequences having at least 99% identity to the chloramphenicol-O-acetyltransferase encoded by the sequence SEQ ID NO: 18 are the sequences WP_077843937.1, SEQ ID NO: 44 (WP_063843219.1) and SEQ ID NO: 45 (WP_078116092.1).

[0164] A specific sequence identical to SEQ ID NO: 18 is the sequence identified as WP_077843937.1 in the NCBI database.

[0165] According to a specific example, the target sequence is the sequence of SEQ ID NO: 68, which corresponds to the catQ gene encoding a chloramphenicol-O-acetyltransferase of Clostridium perfringens, whose amino acid sequence corresponds to SEQ ID NO: 66 (WP_063843220.1), or a sequence that is at least 70%, 75%, 80%, 80%, 85%, 90% or 95% identical to the chloramphenicol-O-acetyltransferase, or a sequence that comprises all or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 68.

[0166] In other words, the recognized sequence can be a sequence comprising at least 1 nucleotide of the sequence SEQ ID NO: 68, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides, generally between 1 and 40 nucleotides, preferably a sequence comprising 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides of the sequence SEQ ID NO: 68.

[0167] In another specific example, the recognized sequence is selected from the nucleic acid sequences catB (SEQ ID NO: 18), catQ (SEQ ID NO: 68), catD (SEQ ID NO: 69, Schwarz S. et al., 2004) or catP (SEQ ID NO: 70, Schwarz S. et al., 2004) known to those skilled in the art, which are naturally present in bacteria or artificially introduced into the bacteria.

[0168] As stated above, according to another specific example, the target sequence may also be a sequence that controls the transcription of a coding sequence as described above (encoding an enzyme that allows the bacterium of interest to grow in a culture medium containing an antibiotic to which it confers resistance), typically a promoter sequence, such as the promoter sequence of the catB gene (SEQ ID NO: 73) or the promoter sequence of the catQ gene (SEQ ID NO: 74).

[0169] The nucleic acid of interest then recognizes and is therefore typically capable of binding to sequences that control the transcription of the coding sequence as described above.

[0170] According to another specific example, the target sequence may be a sequence flanking a coding sequence as described above, for example a sequence flanking the catB gene of SEQ ID NO: 18, or a sequence having at least 70% identity thereto. The flanking sequence typically comprises 1, 10 or 20 to 1000 nucleotides, such as 1, 10 or 20 to 900, 800, 700, 600, 500, 400, 300 or 200 nucleotides, 1, 10 or 20 to 100 nucleotides, 1, 10 or 20 to 50 nucleotides, or 1, 10 or 20 to 40 nucleotides, such as 10 to 40 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 20 to 30 nucleotides, 15 to 40 nucleotides, 15 to 30 nucleotides or 15 to 20 nucleotides.

[0171] Depending on the particular case, the target sequence corresponds to a pair of sequences flanking the coding sequence, each flanking sequence usually comprising at least 20 nucleotides, usually between 100 and 1000 nucleotides, preferably between 200 and 800 nucleotides.

[0172] In the context of the present specification, specific examples of nucleic acids of interest used for transformation and / or genetic modification of bacteria of interest are the following DNA fragments: i) a DNA fragment that recognizes a coding sequence, ii) a DNA fragment that controls the transcription of a coding sequence, or iii) a DNA fragment flanking a coding sequence; the coding sequence encodes an enzyme of interest in the genome of a bacterium, such as the above-mentioned Clostridium bacteria, preferably an amide alcohol-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase or thiamphenicol-O-acetyltransferase.

[0173] As stated above, examples of nucleic acids of interest according to the present invention are capable of inhibiting a recognized sequence ("target sequence") in the genome of a bacterium or modifying its expression, e.g. regulating it, in particular inhibiting it, preferably modifying it such that the bacterium is unable to express a protein, e.g. an amidoalcohol-O-acetyltransferase, in particular a functional protein, starting from said sequence.

[0174] In a specific embodiment in which the enzyme encoding the recognized sequence is a sequence that confers resistance to chloramphenicol and / or thiamphenicol to the bacterium, the selection gene used is not a chloramphenicol and / or thiamphenicol resistance gene, and preferably is not one of the catB, catQ, catD or catP genes.

[0175] In certain embodiments, the nucleic acid of interest comprises one or more guide RNAs (gRNAs) that target, control the transcription of, or flank a coding sequence encoding an enzyme of interest, particularly an amide alcohol-O-acetyltransferase, and / or comprises a modification matrix (also referred to herein as an "editing matrix"), such as a matrix that can remove or modify all or part of the target sequence, preferably for the purpose of inhibiting or repressing the expression of the target sequence, typically comprising sequences located upstream and downstream of the target sequence. A matrix of homologous (corresponding) sequences, typically the sequences (homologous to the sequences located upstream and downstream of the target sequence) each comprise 10 or 20 base pairs to 1000, 1500 or 2000 base pairs, for example 100, 200, 300, 400 or 500 base pairs to 1000, 1200, 1300, 1400 or 1500 base pairs, preferably 100 to 1500 or 100 to 1000 base pairs, even more preferably 500 to 1000 base pairs or 200 to 800 base pairs.

[0176] In a particular embodiment, the nucleic acid of interest used for transformation and / or genetic modification of the bacterium of interest is a nucleic acid that is not methylated at the level of motifs recognized by methyltransferases of the Dam and Dcm types (prepared from Escherichia coli bacteria having the dam-dcm- genotype).

[0177] When the bacterium of interest to be transformed and / or genetically modified is a Clostridium beijerinckii bacterium, in particular belonging to one of the clades DSM 6423, LMG 7814, LMG 7815, NRRL B-593 and NCCB 27006, the nucleic acid of interest used as a genetic tool, such as a plasmid, is a nucleic acid that does not have methylation at the level of the motifs recognized by methyltransferases of the Dam and Dcm types, typically a nucleic acid in which the adenosine ("A") of the GATC motif and / or the second cytosine ("C") of the CCWGG motif (W can correspond to adenosine ("A") or thymine ("T")) is demethylated.

[0178] Nucleic acids that are not methylated at the motif level recognized by Dam and Dcm type methyltransferases can often be isolated from nucleic acids that have Dam - dcm -The same nucleic acid may contain other methylations, for example, by an ecoKI-type methyltransferase that targets adenine ("A") at the motifs AAC(N6)GTGC and GCAC(N6)GTT (N can correspond to any base).

[0179] In certain embodiments, the targeted sequence corresponds to a gene encoding an amidool-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase, such as the catB gene, a sequence that controls the transcription of the gene, or a sequence flanking the gene.

[0180] The nucleic acids of particular interest described by the inventors are, for example, vectors, preferably plasmids, such as the plasmid pCas9ind-ΔcatB of sequence SEQ ID NO: 21 or the plasmid pCas9ind-gRNA_catB of sequence SEQ ID NO: 38 described in the experimental part of the present specification (see Example 2), in particular versions of said sequences that do not have methylation at the level recognized by Dam and Dcm type methyltransferases.

[0181] The present description also relates to the use of nucleic acids of interest for transforming and / or genetically modifying the bacteria of interest described herein.

[0182] Another aspect described by the inventors relates to a method for transforming and preferably also genetically modifying bacteria belonging to the phylum Firmicutes, such as bacteria of the genera Clostridium, Bacillus, or Lactobacillus, typically solventogenic bacteria, particularly solventogenic bacteria of the genus Clostridium, using the genetic tools described herein, typically using the nucleic acids of interest described herein as described above. Advantageously, this method comprises a step of transforming the bacteria by introducing into the bacteria all or part of the genetic tools described herein, particularly a nucleic acid of interest described herein, preferably a "nucleic acid OPT" comprising or consisting of: i) all or part of the sequence SEQ ID NO: 126 (OREP), and ii) a sequence that allows modification of the genetic material of the bacteria and / or expression in the bacteria of DNA sequences that are partially or completely absent from the genetic material present in wild-type novel versions of the bacteria. The method may also comprise a step of obtaining, recovering, selecting, or isolating the transformed bacteria, i.e., the bacteria having the desired recombinant / modified / optimized bacterium.

[0183] In a particular embodiment, the method for transforming and preferably genetically modifying the bacteria described herein involves tools for genetic modification, for example selected from CRISPR tools, tools based on the use of group II introns (e.g. Tools or tools) and allelic exchange tools (e.g. The invention relates to a genetic modification tool for bacteria comprising a step of transforming the bacteria by introducing the nucleic acid of interest according to the present invention as described above into the bacteria.

[0184] Advantageously, the present invention is generally implemented in the case where the genetic modification tool chosen for transforming and preferably genetically modifying bacteria belonging to the phylum Firmicutes, such as bacteria of the genus Clostridium, is intended for use in bacteria, such as Clostridium beijerinckii, which, in the wild state, carries a gene encoding an enzyme responsible for resistance to one or more antibiotics and / or carries, in the wild state, at least one extrachromosomal DNA sequence, and the use of said genetic tool comprises a step of transforming said bacteria with a nucleic acid allowing the expression of a resistance marker towards the antibiotic to which said bacteria are resistant in the wild state, and / or a step of selecting the transformed and / or genetically modified bacteria using said antibiotic to which said bacteria are resistant in the wild state, preferably selecting among said bacteria that have lost said extrachromosomal DNA sequence.

[0185] A modification that can be advantageously performed using the present invention, for example using genetic modification tools selected from the group consisting of CRISPR tools, tools based on the use of group II introns and allelic exchange tools, consists in suppressing unwanted sequences, such as sequences encoding enzymes that provide the bacterium with resistance to one or more antibiotics, or rendering such unwanted sequences non-functional. Another modification that can be advantageously performed using the present invention consists in genetically modifying a bacterium that has previously been modified using the present invention to make it sensitive to an antibiotic to which it is resistant in the wild and / or to remove extrachromosomal DNA sequences present in the wild form of the bacterium in order to improve its performance, for example its performance in producing a solvent or solvent mixture of interest.

[0186] In a preferred embodiment, the method according to the present invention is based on the use (utilization) of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology / genetic tools, in particular CRISPR / Cas (CRISPR associated protein) genetic tools.

[0187] The present invention can be implemented using conventional CRISPR / Cas genetic tools, using a single plasmid containing the nuclease, gRNA and repair matrix, for example as described by Wang et al. (2015).

[0188] The sequence and structure of the gRNA can be easily determined by those skilled in the art using well-known techniques according to the chromosomal region or mobile genetic element to be targeted (see, for example, DiCarlo et al., 2013).

[0189] The present inventors have developed and described a method based on the use of two plasmids (see WO 2017 / 064439, Wasels et al., 2017 and accompanying publications). Figure 15 ) to modify bacteria, which is applicable to bacteria of the genus Clostridium and can also be used in the context of the present invention.

[0190] In a particular embodiment, the "first" plasmid of the tool allows expression of the Cas nuclease, and the "second" plasmid specific for the modification to be performed contains one or more gRNA expression cassettes (typically targeting different regions of the bacterial DNA), and a repair matrix that allows the portion of the bacterial DNA targeted by Cas to be replaced with a sequence of interest by a homologous recombination mechanism. The cas gene and / or gRNA expression cassette are placed under the control of a constitutive or inducible, preferably inducible, expression promoter known to those skilled in the art (e.g., described in application WO2017 / 064439 and incorporated herein by reference), preferably a different but inducible promoter.

[0191] The gRNA that can be used corresponds to the gRNA described above herein.

[0192] Specific methods involving CRISPR technology that can be used in the context of the present invention to transform and genetically modify, typically by homologous recombination, the bacteria described herein comprise the following steps:

[0193] a) introducing into said bacteria a nucleic acid or genetic tool as described by the inventors in the presence of an agent that induces expression of an anti-CRISPR protein, and

[0194] b) culturing the transformed bacteria obtained at the end of step a) on a medium without an inducer of expression of the anti-CRISPR protein (or under conditions not involving said inducer), generally allowing the expression of said DNA endonuclease / gRNA ribonucleoprotein complex, generally Cas / gRNA (in order to stop the production of said anti-CRISPR protein and allow the action of said endonuclease).

[0195] The inducer of expression of the anti-CRISPR protein is present in an amount sufficient to induce the expression. In the case of the Pbgal promoter, the inducer lactose is able to relieve the inhibition (transcriptional repression) of the expression of the anti-CRISPR protein associated with the expression of the BgaR protein.

[0196] The anti-CRISPR protein expression inducer is preferably used at a concentration between about 1 mM and about 1 M, preferably between about 10 mM and about 100 mM, for example about 40 mM.

[0197] In a preferred embodiment, said anti-CRISPR protein is capable of inhibiting, preferably neutralizing, the action of said nuclease, preferably during the introduction of the nucleic acid sequence of said genetic tool into the bacterial strain of interest.

[0198] In a specific embodiment, the method further comprises, during or after step b), a step of inducing expression of one or more inducible promoters controlling expression of nucleases and / or guide RNAs, when present in the genetic tool, so as to allow the genetic modification of interest to be made to the bacteria after the genetic tool has been introduced into the bacteria. Induction is performed using a substance capable of relieving the expression repression associated with the selected inducible promoter.

[0199] Thus, when present, the induction step can be performed by any culture method on a culture medium known to those skilled in the art that allows the expression of the endonuclease / gRNA ribonucleoprotein complex, after the introduction of the genetic tool according to the invention into the target bacteria. For example, it is performed by contacting the bacteria with a suitable substance present in sufficient quantity or by exposure to UV light. Such a substance is capable of relieving the expression inhibition associated with the selected inducible promoter. When the selected promoter is an anhydrotetracycline (aTc) inducible promoter selected from Pcm-2tetO1 and Pcm-tetO2 / 1, aTc is preferably present at a concentration of between about 1 ng / mL and about 5000 ng / mL, preferably between about 10 ng / mL and 1000 ng / mL, 10 ng / mL and 800 ng / mL, 10 ng / mL and 500 ng / mL, 100 ng / mL or 200 ng / mL to about 800 ng / mL or 1000 ng / mL or about 100 ng / mL or 200 ng / mL. Concentrations of between about 0 ng / mL to about 500 ng / mL, 600 ng / mL, or 700 ng / mL, for example, about 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL, 550 ng / mL, 600 ng / mL, 650 ng / mL, 700 ng / mL, 750 ng / mL, or 800 ng / mL are used.

[0200] In another specific embodiment, the method comprises an additional step c) to remove the nucleic acid containing the repair matrix (the bacterial cell is then considered to be "cleared" of the nucleic acid) and / or to remove the one or more guide RNAs or sequences encoding the one or more guide RNAs introduced using the genetic tools during step a).

[0201] In yet another specific embodiment, the method comprises, after step b) or step c), one or more additional steps of introducing, in the presence of an agent that induces expression of the anti-CRISPR protein, an nth nucleic acid, such as a third, fourth, fifth, etc. nucleic acid, which contains a repair matrix different from the one already introduced, and introducing an expression cassette for one or more guide RNAs that allows the sequence of interest contained in the different repair matrix to be integrated into the targeted region of the bacterial genome, each additional step being followed by a step of culturing the bacteria thus transformed on a medium that does not contain the agent that induces expression of the anti-CRISPR protein, which generally allows expression of the Cas / gRNA ribonucleoprotein complex.

[0202] In a specific embodiment of the method according to the present invention, the bacteria are transformed using, for example, nucleic acids or genetic tools as described above, using (e.g., encoding) an enzyme responsible for cleaving at least one chain of the target sequence of interest, wherein the enzyme in a specific embodiment is a nuclease, preferably a Cas type nuclease, preferably selected from the group consisting of a Cas9 enzyme and a MAD7 enzyme. In one embodiment, the target sequence of interest is an enzyme encoding resistance to one or more antibiotics, preferably one or more antibiotics belonging to the amide alcohol class, typically an amide alcohol-O-acetyltransferase, such as a chloramphenicol-O-acetyltransferase sequence, such as a catB gene, a sequence that controls transcription of the coding sequence, or a sequence flanking the coding sequence.

[0203] When used, the anti-CRISPR protein is typically an "anti-Cas" protein as described above. Advantageously, the anti-CRISPR protein is an "anti-Cas9" protein or an "anti-MAD7" protein.

[0204] Like the targeted DNA portion ("recognition sequence"), the editing / repair matrix itself may comprise one or more nucleic acid sequences or portions of nucleic acid sequences corresponding to natural and / or synthetic coding and / or non-coding sequences. The matrix may also comprise one or more "foreign" sequences, i.e., sequences that are not naturally present in the genomes of bacteria belonging to the phylum Firmicutes, in particular Clostridium, Bacillus, or Lactobacillus, or in the genomes of specific species of said genera. The matrix may also comprise a combination of sequences.

[0205] The genetic tools used in the context of the present invention allow the repair matrix to direct the incorporation of a nucleic acid of interest, typically a DNA sequence or portion of a sequence, comprising at least 1 base pair (bp), preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 50, 100, 1000, 10,000, 100,000 or 1,000,000 bp, typically between 1 bp and 20 kb, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 kb, or between 1 bp and 10 kb, preferably between 10 bp and 10 kb or between 1 kb and 10 kb, such as between 1 bp and 5 kb, between 2 kb and 5 kb or between 2.5 or 3 kb and 5 kb, into the bacterial genome.

[0206] In a particular embodiment, expression of the DNA sequence of interest allows bacteria belonging to the phylum Firmicutes, in particular to the genus Clostridium, Bacillus or Lactobacillus, to ferment (usually simultaneously) several different sugars, for example at least two different sugars, typically at least two different sugars among sugars containing 5 carbon atoms (e.g. glucose or mannose) and / or sugars containing 6 carbon atoms (e.g. xylose, arabinose or fructose), preferably at least three different sugars, for example selected from the group consisting of glucose, xylose and mannose, glucose, arabinose and mannose, and glucose, xylose and arabinose.

[0207] In another specific embodiment, the DNA sequence of interest encodes at least one product of interest, preferably a product that promotes solvent production in the modified bacteria, typically at least one protein of interest, such as an enzyme, a membrane protein such as a transporter, a protein for the maturation of other proteins (chaperone protein), a transcription factor or a combination thereof.

[0208] The elements of the genetic tool (nucleic acid or gRNA) are introduced into the bacteria by any direct or indirect method known to a person skilled in the art, such as by transformation, conjugation, microinjection, transfection, electroporation, etc., preferably by electroporation (Mermelstein et al., 1993).

[0209] In another embodiment, the method according to the invention is based on the use of group II introns, e.g. Technology / genetic tools or Genetic tools.

[0210] described The technology is based on the use of a reprogrammable class II intron (based on intron L1.ltrB from Lactococcus lactis), which enables rapid integration of the bacterial genome into the desired locus (Chen et al., 2005, Wang et al., 2013), usually to inactivate the targeted gene. The mechanism of recognition of the edited region and its insertion into the genome by back-splicing is based on the homology between the intron and the region in question and on the activity of the protein (ltrA).

[0211] described The technology is based on a similar approach, supplemented by the addition of a selection marker within the intron sequence (Heap et al., 2007). This marker allows selection for the integration of the intron into the genome, thus facilitating the generation of desired mutants. This genetic system also utilizes group I introns. In fact, the selection marker (called RAM, for marker activated by retrotransposition) is interrupted by this genetic element, preventing its expression from the plasmid (a more accurate description of the system: Zhong et al.). Splicing of this genetic element occurs before integration into the genome, allowing the generation of a chromosome with an active form of the resistance gene. An optimized version of the system contains FLP / FRT sites upstream and downstream of the gene, allowing the recombinase FRT to be used to remove the resistance gene (Heap et al., 2010).

[0212] In another embodiment, the method according to the invention is based on the use of an allelic exchange tool, and for example using Technology / genetic tools.

[0213] described The technology is based on the use of auxotrophic mutants (uracil auxotrophy in Clostridium acetobutylicum ATCC 824 by deletion of the pyrE gene, which also confers resistance to 5-fluoroorotic acid (5-FOA); Heap et al., 2012). The system uses an allelic exchange mechanism well known to those skilled in the art. After transformation with a pseudo-suicide vector (with very weak copies), its integration in the bacterial chromosome by a first allelic exchange event can be verified due to the resistance gene initially present on the plasmid. The integration step can be performed in two different ways, either within the pyrE locus or within another locus:

[0214] In the case of integration into the pyrE locus, the pyrE gene is also placed on the plasmid, but is not expressed (no functional promoter). A second recombination restores the functional pyrE gene, allowing selection by auxotrophy (minimal medium without uracil). Since the non-functional pyrE gene also has selectable properties (sensitivity to 5-FOA), further integrations can be envisioned on the same model by continuously alternating the pyrE state between functional and non-functional.

[0215] In the case of integration into another locus, targeting allows a genomic region (typically targeting an operon after another gene, preferably a strongly expressed gene) that expresses a counter-selectable marker after recombination. This second recombination is then selected by auxotrophy (minimal medium without uracil).

[0216] In the described method based on the use of group II introns and using, for example, Technology / genetic tools or Genetic tools or based on the use of allelic exchange tools and e.g. In embodiments of the technology / genetic tool, the targeted sequence is typically one of the sequences described herein.

[0217] In particularly advantageous cases, the nucleic acids and genetic tools according to the invention allow the introduction of both small and large sequences of interest into bacteria in one step, i.e. using a single nucleic acid (generally the "nucleic acid OPT" or the "second" or "nth" nucleic acid of the tools described herein) or in several steps, i.e. using several nucleic acids (generally the "second" and "nth" nucleic acids described herein), preferably in one step.

[0218] In a specific embodiment of the invention, the nucleic acids and genetic tools according to the invention make it possible to inhibit the targeted portion of the bacterial DNA or replace it with a shorter (e.g. with a sequence that has lost at least one base pair) and / or non-functional sequence. In a particularly preferred embodiment of the invention, the nucleic acids and genetic tools according to the invention make it possible to advantageously introduce into the bacterium, e.g. into the bacterial genome, a nucleic acid of interest comprising at least one base pair and up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 kb.

[0219] The present invention also relates to a transformed and / or genetically modified bacterium, typically a bacterium belonging to the phylum Firmicutes and belonging to, for example, the genus Clostridium, Bacillus or Lactobacillus, typically a solvent-producing bacterium, preferably a bacterium belonging to a species corresponding to one of the evolutionary branches described by the inventors herein or obtained using the methods described by the inventors herein, and any derivative bacteria, clones, mutants or genetically modified versions thereof, and uses thereof.

[0220] Examples of bacteria that are transformed and / or genetically modified according to the present invention are bacteria that no longer express an enzyme that confers resistance to one or more antibiotics, in particular bacteria that no longer express an amidol-O-acetyltransferase, for example bacteria that express the catB gene in the wild and that, after transformation and / or genetic modification according to the present invention, do not contain the catB gene or are unable to express the catB gene. Such bacteria are rendered sensitive to amidols, such as those described herein, in particular to chloramphenicol or thiamphenicol.

[0221] A specific example of a preferred genetically modified bacterium according to the present invention is the bacterium designated as Clostridium beijerinckii IFP962ΔcatB in the present specification, which was registered on December 6, 2018 at the Belgian Co-ordinated Collections of Micro-organisms ("BCCM", KLLedeganckstraat 35, B-9000 Gent-Belgium) under the deposit number LMG P-31151.

[0222] Another specific example of a preferred genetically modified bacterium according to the present invention is the bacterium indicated in the present specification as Clostridium beijerinckii strain IFP963ΔcatBΔpNF2, which was registered at the BCCM-LMG Collection on February 20, 2019 under the deposit number LMG P-31277.

[0223] The present description also relates to any derivative bacteria, clones, mutants or genetically modified versions of one of the bacteria, for example any derivative bacteria, clones, mutants or genetically modified versions that retain sensitivity to amide alcohols such as thiamphenicol and / or chloramphenicol, typically a bacterium lacking the catB gene of sequence SEQ ID NO: 18 and the plasmid pNF2.

[0224] According to a particular embodiment, the transformed and / or genetically modified bacteria according to the present invention, such as Clostridium beijerinckii IFP962ΔcatB or Clostridium beijerinckii IFP963ΔcatBΔpNF2, can still be transformed and preferably genetically modified. This can be done using nucleic acids such as the plasmids described in this specification, for example in the experimental part. An example of a nucleic acid that can be advantageously used is the plasmid pCas9 of sequence SEQ ID NO: 23 acr (described in the experimental part of this specification) or selected from pCas9 ind (SEQ ID NO: 22), pCas9 cond(SEQ ID NO: 133) and pMAD7 (SEQ ID NO: 134) plasmids.

[0225] A particular aspect of the present invention in fact relates to the use of the genetically modified bacteria described herein, preferably the bacterium Clostridium beijerinckii IFP962ΔcatB deposited under the number LMGP-31151 (also designated herein as Clostridium beijerinckii DSM 6423ΔcatB), even more preferably Clostridium beijerinckii IFP963ΔcatBΔpNF2 deposited under the number LMG P-31277, or a genetically modified version of one of these bacteria, for producing one or more solvents, preferably at least isopropanol, preferably on an industrial scale, due to the expression of one or more nucleic acids of interest deliberately introduced into its genome, e.g. using one of the nucleic acids, genetic tools or methods described herein.

[0226] The present invention also relates to a kit comprising (i) a nucleic acid as described herein, for example the "nucleic acid OPT" as described herein or a DNA fragment recognizing a target sequence in a bacterium belonging to the phylum Firmicutes, and (ii) at least one tool, preferably several tools, selected from: elements of the tool for genetic modification as described herein, making it possible to transform and generally genetically modify such a bacterium in order to produce improved variants of said bacterium; nucleic acids as gRNA; nucleic acids as repair substrates; "nucleic acid OPT"; at least one pair of primers, for example the primer pair described in the context of the present invention; and inducers allowing the expression of a protein encoded by said tool, for example a nuclease of the Cas9 or MAD7 type.

[0227] The genetic modification tools described herein for transforming and generally genetically modifying bacteria belonging to the phylum Firmicutes can, for example, be selected from "nucleic acid OPT", CRISPR tools, tools based on the use of group II introns and allelic exchange tools, as explained above.

[0228] In certain embodiments, the kits comprise some or all of the elements of the genetic tools described herein.

[0229] A specific kit for transforming and preferably genetically modifying a bacterium belonging to the phylum Firmicutes as described herein or for using such a bacterium to produce at least one solvent, for example a solvent mixture, comprises a nucleic acid containing or consisting of: i) all or part of the sequence SEQ ID NO: 126, and ii) sequences allowing the modification of the genetic material of the bacterium and / or the expression in said bacterium of DNA sequences that are partially or completely absent in the genetic material present in the wild-type version of said bacterium; and at least one inducer of an inducible promoter for the expression of the selected anti-CRISPR protein used in the genetic tool described herein.

[0230] The kit may also contain one or more inducers suitable for the selected inducible promoter optionally used in the genetic tool to control expression of the nuclease and / or one or more guide RNAs used.

[0231] Certain kits according to the invention allow for the expression of nucleases comprising a marker (or "tag").

[0232] The kit according to the present invention may further comprise one or more consumables such as culture medium, at least one competent bacterium (i.e., prepared for transformation) belonging to the phylum Firmicutes as described herein, such as Clostridium, Bacillus or Lactobacillus, at least one gRNA, a nuclease, one or more selection molecules or a set of instructions.

[0233] The present description also relates to the use of a kit according to the invention or one or more elements thereof for carrying out the methods described herein for transforming and ideally genetically modifying a bacterium belonging to the phylum Firmicutes, such as a bacterium of the genus Clostridium, Bacillus or Lactobacillus (for example the bacterium Clostridium beijerinckii IFP962ΔcatB deposited under the number LMG P-31151), preferably a bacterium that in the wild state has both a bacterial chromosome and at least one DNA molecule different from the chromosomal DNA (usually a natural plasmid), most preferably the bacterium Clostridium beijerinckii IFP963ΔcatBΔpNF2 deposited under the number LMG P-31277; and / or for using said bacteria to produce solvents or biofuels or mixtures thereof, preferably on an industrial scale.

[0234] The solvent that can be produced is usually acetone, butanol, ethanol, isopropanol or a mixture thereof, usually an ethanol / isopropanol, butanol / isopropanol or ethanol / butanol mixture, preferably an isopropanol / butanol mixture.

[0235] The use of the bacteria transformed according to the invention generally allows the production of at least 100 tons of acetone, at least 100 tons of ethanol, at least 1000 tons of isopropanol, at least 1800 tons of butanol or at least 40,000 tons of mixtures thereof per year on an industrial scale.

[0236] The examples and figures given below are intended to more fully illustrate the present invention without limiting its scope. Sequence Listing <110> IFP New Energy <120> Optimized genetic tools for modifying bacteria <130> B3003PC00 <160> 134 <170> PatentIn version 3.5 <210> 1 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Primer ΔcatB-fwd <400> 1 tgttatggat tataagcggc tcgaggacgt caaaccatgt taatcattgc 50 <210> 2 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Primer ΔcatB-rev <400> 2 aatctatcac tgatagggac tcgagcaatt tcaccaaaga attcgctagc 50 <210> 3 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primer RH076 <400> 3 catataataa aaggaaacct cttgatcg 28 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer RH077 <400> 4 attgccagcc taacacttgg 20 <210> 5 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Primer RH001 <400> 5 atctccatgg acgcgtgacg tcgacataag gtaccaggaa ttagagcagc 50 <210> 6 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Primer RH002 <400> 6 tctatctcca gctctagacc attattattc ctccaagttt gct 43 <210> 7 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Primer RH003 <400> 7 ataatggtct agagctggag atagattattggtactaag 40 <210> 8 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Primer RH004 <400> 8 tatgaccatg attacgaatt cgagctcgaa gcgcttatta ttgcattagc 50 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primer pEX-fwd <400> 9 cagattgtac tgagagtgca cc 22 <210> 10 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primer pEX-rev <400> 10 gtgagcggat aacaatttca cac 23 <210> 11 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primer pEC750C-fwd <400> 11 caatattcca caatattata ttataagcta gc 32 <210> 12 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer M13-rev <400> 12 caggaaacag ctatgac 17 <210> 13 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primer RH010 <400> 13 cggatattgc attaccagta gc 22 <210> 14 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primer RH011 <400> 14 ttatcaatct cttacacatg gagc 24 <210> 15 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primer RH025 <400> 15 tagtatgccg ccattattac gaca 24 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer RH134 <400> 16 gtcgacgtgg aattgtgagc 20 <210> 17 <211> 3658 <212> DNA <213> Artificial sequence <220> <223> pEX-A258-ΔcatB <400> 17 ctcgagctgc agcaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact 60 agccttattt taacttgcta tttctagctc taaaactgtg gtctctcttt tcgttgatgg 120 tggaatgata agggtttgca ccttaatttc tcctattgag aaaatcgtct cttctcagac 180 gtcaaaccat gttaatcatt gcttttatca aaaataggat ccactctatc attgatagag 240 tttgaaactc tatcattgat agagtataat atctttgttc atgtacatca tgctatctgt 300 gagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa 360 gtggcaccga gtcggtgctt tttttgaagc ttgtctttac acttttgccc attaattttt 420 gagttcctta tttttaggga gcttttatta tttttatcat gaaaatttca taaaatactc 480 ataaactaag gatgtcttca taatcagatt agtactccat tttcaatcca tttaatctgg 540 gaatatgata tttataattac gtattattta agatatatta acgtgtaata taataccccg 600 caaatattaa ttatcacata catatcccc ctttattggg gcattttttg tacccattat 660 tttagtattg tgcagtactt aaataaaaaaa atgccgcaaa ttcattttta ttgaataatg 720 cggtatttct tctattcttt atttttatta ctctataaat aatgtaatca agacatgact 780 atctaaatat atgatatctt aattcataat tcgggcctcc taaaaatttt cgtaattcta 840 ttttagaagg cttttttccg tgacctagcc atttcaatct cctttttaca atgatattta 900 cgctttagtt tattatagca cattctgtaa taccgaacta ttcaattttc agagaccatt 960 ttttattgat tcataactta agaatactac gaattactct aatattttac tttttcttat 1020 ctcttgttat tttaacatcg gaattactac taatattaat ttttattttt ccatccgcat 1080 ttgctccaac attttttaa ctatactttc cttttgttaa taaattatgt tattgttgaa 1140 caatataaga aaagtgcgta acatttttta ttaaaaataa ttaggtattt ctatctgtgg 1200 ggtaccctcg aggtggcagc tctagagcta gcgaattctt tggtgaaatt gttatccgct 1260 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 1320 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 1380 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 1440 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 1500 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 1560 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 1620 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 1680 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 1740 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 1800 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 1860 tcgctccaag ctgggctgtg tgcacgacc cccgttcag cccgaccgct gcgccttatc 1920 cggtaactat cgtcttgagt ccaacccggt agacacgac ttatcgccac tggcagcagc 1980 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 2040 gtggcctaac tacggctaca ctagagaac agtatttggt atctgcgctc tgctgaagcc 2100 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 2160 cggtggtttt ttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaga 2220 tcctttgatc tttctacgg ggtctgacgc tcagtgac gaaaactcac gttaggt 2280 tttggtcatg agattatca aaaggatctt cacctagatc cttttaatt aaaaatgaag 2340 ttttaaatca atctaaagta tatatgagta aacttggtct gatacc aatgcttaat 2400 cagtgaggca cctactcag cgatctgtct atttcgttca tccatagttg cctgactccc 2460 cgtcgtgtag ataactacga tacgggagg cttaccatct ggccccagtg ctgcaatgat 2520 accgcgcgaa ccacgctcac cggctccaga tttatcagca ataaccagc cagccggaag 2580 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagcta ttaattgttg 2640 ccgggaagct agagtaagta gttcgccagt tatagtttg cgcaacgttg ttgccattgc 2700 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 2760 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 2820 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 2880 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 2940 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 3000 aatacgggat aataccgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 3060 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 3120 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 3180 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 3240 actcatactc ttccttttc aatattattg aagcatttat cagggttatt gtctcatgag 3300 cggatacata tttgaatgta tttagaaaaa taacaaata ggggttccgc gcacatttcc ccgaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa tagcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 3480 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 3540 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 3600 atcagagcag attgtactga gagtttggca attggtcgac ctcgagggcg cgcccgta 3658. <210> 18 <211> 660 <212> DNA <213> Organic strain(Clostridium beijerinckii) <400> 18 atgaatttta atttgataga tattaatcat tggagtaga agccatactt tgaacattat 120. ttaacaatg tgaaatgtac ttatagtatg actgccaata tagataac tgatttattg 180. 180. 180. 180. 180. 180. 180. 180. 180. 180. 180 gtggttaata agcataaaga attccgtatt tgttttgatc atgaaggtag tttaggatat tgggatagca tgaatccaag ctatactatt tttcataaag aaaacgaaac attttcaagt 300 atttggacgg aatataacaa aagtttttta cgtttttata gtgattatct tgacgatata 360 aaaaactatg gaaatatcat gaagtttact ccgaaatcaa atgaacctga caatacattt 420 tctgtatcaa gcattccttg ggtgagtttt acaggattta acttgaatgt gtataatgaa 480 ggaacatatt taattcctat ttttactgca ggaaagtatt tcaaacaaga aaataaaata 540 tttattccta tatcaataca agtacatcat gctatctgtg acggttatca tgctagtaga 600 tttattaatg aaatgcaaga attagcattt agttttcaag aatggttaga aaataaataa 660 [[ID=!13]]! <210> 19 <211> 160 <212> DNA <213> Artificial sequence <220> <223> gRNA expression cassette <400> 19 actctatcat tgatagagtt tgaaactcta tcattgatag agtataatat ctttgttcat 60 gtacatcatg ctatctgtga gttttagagc tagaaatagc aagttaaaat aaggctagtc 120 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 160 <210> 20 <211> 808 It should be noted that in the original text, the tag is written as <210> 19 in the following line, which seems a bit inconsistent in the format. I translated it according to the overall context. If there are any specific requirements or corrections regarding this, please let me know.<212> DNA <213> Artificial Sequence <220> <223> Editing Template <400> 20 gtctttacac ttttgcccat taatttttga gttccttatt tttagggagc ttttattatt 60 tttatcatga aaatttcata aaatactcat aaactaagga tgtcttcata atcagattag 120 tactccattt tcaatccatt taatctggga atatgatatt ttaattacgt attatttaag 180 atatattaac gtgtaatata ataccccgca aatattaatt atcacataca tatcccccct 240 ttattggggc attttttgta cccattattt tagtattgtg cagtacttaa ataaaaaaat 300 gccgcaaatt catttttatt gaataatgcg gtatttcttc tattctttat ttttattact 360 ctataaataa tgtaatcaag acatgactat ctaaatatat gatatcttaa ttcataattc 420 gggcctccta aaaattttcg taattctatt ttagaaggct tttttccgtg acctagccat 480 ttcaatctcc tttttacaat gatatttacg ctttagttta ttatagcaca ttctgtaata 540 ccgaactatt caattttcag agaccatttt ttattgattc ataacttaag aatactacga 600 attactctaa tattttactt tttcttatct cttgttattt taacatcgga attactacta 660 atattaattt ttatttttcc atccgcattt gctccaacat ttttttaact atactttcct 720 tttgttaata aattatgtta ttgttgaaca atataagaaa agtgcgtaac attttttatt 780 aaaaataatt aggtatttct atctgtgg 808 <210> 21 <211> 9954 <212> DNA <213> Artificial sequence <220> <223> pCas9ind-ΔcatB <400> 21 catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60 tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120 acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180 agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240 ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300 actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360 taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420 gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480 tatgattaaa tttagaggac atttttaat agaaggtgat ttaaacccag acaacagcga 540 tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600 aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660 aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac ttttggggaa 720 cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780 agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840 tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900 tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960 tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020 acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080 tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140 agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200 aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260 tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320 tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380 aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440 agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500 gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560 ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620 gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680 cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740 atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaaat 1800 tataaaagat agaattttt tagaatga agaaaatgaa gatatcttg aagatatagt 1860 attackacattg acacttttg aagatagaga atgatagaa gaagattaaaacatatgc 1920 acatcttttt gatgatagg tttagaagca acttaaaga agagatata caggttgggg 1980 acgtttgtca agaagctaa ttaatggtat tagagataa caatcaggaa agactattct 2040 cgattttctt aaatcagatg gatttgctaa tagaacttt atgcaattaa ttcatgatga 2100 ttctcttact ttcaagagg atattcaaa ggctcaagtt tctggacaag gcgatagctt 2160 acacgaacac attgctacc ttgcaggg acccgctatc aaaaaaggaa ttttacaacc 2220 agttaaagtt gtagatgaac ttgttaaagt tattgggaga cacaacctg agaatagt 2280 tatagaaatg gccagagaaa atcaacaac aaaaaagg ctagagagag 2340 aatgagaga attgagaag gaataaaga gctaggatca caatatta aagacatcc 2400 agttgaaaat actcaattgc aaatgaaa gttatatttg tattacttac aaatggaag 2460 agatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520 tattgttcct caatcatttt windows ttcaatcgat aaaaagtat windows 2580 agataaaat agaggaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640 gaactattgg agacaacttt taaatgcaaa gctattaca CAAAAAAT ttgacaattt 2700 aaaaaaaaagggaaaaaaaaaaaaaaaaaaaaaaaaaaaaggcha 2760 acttgttgag acacaaa taactaagca tgttgctcaa atacttgatt cagaatgaa 2820 tachaaat gatgaaatg aataatt cagagaagta aaagtaata cattaagtc 2880 aaaattagta tcagatttca gaaaggattt tcaatttac aaagttcgtg aataataa 2940 ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000 atatcctaaa ctagaaagcg aatttgttta tggagatt aaagtttatg atgtgcgcaa 3060 aatgatcgcg aaatccgaac aagaatcgg taaggctaca gcaaatatt tctttattag 3120 taatataatg aattttttta agacagaaat aactttggct aatggtgaa tcagaaaag 3180 accacttatc gaacaatg gagacacagg agaatagta tgggataag gagagattt 3240 tgctactgtt agaaaagtac taagtatgcc acagtaaat atcgtaaga aactgaagt 3300 tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360 tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420 ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480 aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540 cttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600 gtatagcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660 tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720 tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780 acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840 tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900 accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960 accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020 agagtatta gacgcaactt tatacatca atctattaca gggctttatg aaacaagaat 4080 tgatttaagt caactaggcg gagattaagt cgacaagta ttgttaaaaa taactctgta 4140 gattataaa ttagttctac agagttatttt ttgacccgg gtatattgat aaaataata 4200 atagtgggta tattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260 gtaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa gtttaacaa 4320 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380 ataagcgggc ttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440 ttgcccttt agaagggaa agctggcaag atttttcg taatacgct aaagtttta 4500 gatgtgcttt actaagtcat cgcgatggag caaagtaca tttaggtaca cggcctacag 4560 aaaaacagta tgaactctc gaaaatcaat tagccttttttgccaacaa ggttttcac 4620 tagagaatgc attatgca ctcagcgctg tggggcatttt tactttaggt tgcgtattgg 4680 aagatcaag gcatcaagtc gctaagaag aaagggaac acctactact gatagtagc 4740 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860 cttaaaagca gkataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920 tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160 gctcttcgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120 gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180 ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240 acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300 aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360 agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420 tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480 agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540 taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600 agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660 attccctaac aaacagaggt ataaattgt tgggagtatt ccttaccatt taagcacaca 6720 aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780 aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840 agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900 aaacagtgtc ttataaaac ttacccgcca taccacagat gttccagata atattggaa 6960 gctatacg tactttgttt caaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020 aaatcagtttt catcaagcaa tgaacacgc aaatcagtt aatttaagta ccgttactta 7080 tgagcaagta ttgtctattt ttatagtta tctattttt aacgggagga ataatttcta 7140 tgagtcccta ggcaggccctc cgccattatt ttttgaaca attgacaatt cattttcttat 7200 ttttattaa gtgagtca aaaggcataa cagtgctgaa tagaaagaa tttacagaaa 7260 agaaaattat agaatttagt atgattaattt attackcatttt atgaatgtttt aatgaatac 7320 aaaaaaaaat acttgttatg tattcaatta cgggttaaa tatagacaag ttgaaaaatt 7380 tataaaaaa ataagctccctc agctcttata tattaagcta ccaacttagt ataagcca 7440 aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaattt 7500 caatgtacc gaatacaag agaacatta actatatta ttcaatttat gagattatct 7560 taacagatat aaatgtaaat tgcaatagt aagatttaga agtttatagc ctttgtgtat 7620 tggaagcagt acgcaaggc tttttttt gataaaaatt agaagtatat ttttttc 7680 atattaattt tatgaaatg aaagggggtg agcaagtga cagaggaaag cagtatctta 7740 tcaataca aggtattagc atatcatta ttgacttag cagtaaacat tatgactttt 7800 atagtgcttg tagctaagta gtacgaaagg gggagcttta aaagctcct tggaatacat 7860 agaattcata attaattta tgaaagaag ggcgtatatg aaacttgta aaattgcaa 7920 agagtttatt aagatactg aaatatgcaa atacattcg ttgatgattc atgataaaac 7980 agtagcaacc tattgcagta atacaatga gtcaatgt ttacataag ggaagtcca 8040 atgtattaat tgttcaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100 tacagaggaa gacagaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160 aaagctcat gtaagaga gtaaaaga aaaataattt atttattaat ttatattga 8220 gagtgccgac acagtagca ctaaaaaata tatctgtggt gtagtgagcc gatacaaag 8280 gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340 acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400 taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460 aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataattttt 8520 tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580 ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640 tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700 tataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760 tatggattat aagcggctcg aggacgtcaa accatgttaa tcattgcttt tatcaaaaat 8820 aggatccact ctatcattga tagagtttga aactctatca ttgatagagt ataatatctt 8880 tgttcatgta catcatgcta tctgtgagtt ttagagctag aatagcaag ttaaaataag 8940 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagctttgtc 9000 tttacacttt tgcccattaa ttttgagtt ccttattttt agggagcttt tattattttt 9060 atcatgaaaa ttcataaaa tactcataaa ctaggatgt cttcataatc agattagtac 9120 tccattttca atccatttaa tctgggaata tgatatttta attacgtatt atttaagata 9180 tattaacgtg taatataata ccccgcaaat attattatc acatacatat ccccccttta 9240 ttggggcatt tttgtaccc attattttag tattgtgcag tacttaata aaaaaatgcc 9300 gcaaattcat ttttattgaa taatgcggta tttctctat tctttattt tattactcta 9360 taaataatgt atcagaca tgactatcta atatatgat atcttattc atattcggg 9420 cctcctaaaa atttcgtaa ttctatttta gaaggcttt ttccgtgacc tagccattc 9480 aatctccttt ttacaatgat atttacgctt tagtttatta tagcacattc tgtaataccg 9540 aactattcaa ttttcagaga ccatttttta ttgattcata acttaagaat actacgaatt 9600 actctatat tttactttt cttatctctt gttatttta catcggaatt actactaata 9660 ttaattttta tttccatc cgcatttgct ccaacatttt tttaactata ctttcctttt 9720 gttataaat tatgttattg ttgaacaata tagaaaagt gcgtaacatt ttttattaaa 9780 aataattagg tatttctatc tgtggggtac cctcgaggtg gcagctctag agctagcgaa 9840 ttctttggtg aaattgctcg agtccctatc agtgatagat tgaaactcta tcattgatag 9900 agtataatat ctttgttcat tagagcgata aacttgaatt tgagagggaa cttc 9954 <210> 22 <211> 8874 <212> DNA <213> Artificial Sequence <220> <223> pCas9ind <400> 22 catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60 tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120 acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180 agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240 ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300 actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360 taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420 gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480 tatgattaaa tttagaggac atttttaat agaaggtgat ttaaacccag acaacagcga 540 tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600 aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660 aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac ttttggggaa 720 cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780 agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840 tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900 tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960 tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020 acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080 tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140 agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200 aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260 tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320 tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380 aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440 agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500 gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560 ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620 gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680 cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740 atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaaat 1800 tataaaagat agaattttt tagaatga agaaaatgaa gatatcttg aagatatagt 1860 attackacattg acacttttg aagatagaga atgatagaa gaagattaaaacatatgc 1920 acatcttttt gatgatagg tttagaagca acttaaaga agagatata caggttgggg 1980 acgtttgtca agaagctaa ttaatggtat tagagataa caatcaggaa agactattct 2040 cgattttctt aaatcagatg gatttgctaa tagaacttt atgcaattaa ttcatgatga 2100 ttctcttact ttcaagagg atattcaaa ggctcaagtt tctggacaag gcgatagctt 2160 acacgaacac attgctacc ttgcaggg acccgctatc aaaaaaggaa ttttacaacc 2220 agttaaagtt gtagatgaac ttgttaaagt tattgggaga cacaacctg agaatagt 2280 tatagaaatg gccagagaaa atcaacaac aaaaaagg ctagagagag 2340 aatgagaga attgagaag gaataaaga gctaggatca caatatta aagacatcc 2400 agttgaaaat actcaattgc aaatgaaa gttatatttg tattacttac aaatggaag 2460 agatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520 tattgttcct caatcatttt windows ttcaatcgat aaaaagtat windows 2580 agataaaat agaggaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640 gaactattgg agacaacttt taaatgcaaa gctattaca CAAAAAAT ttgacaattt 2700 aaaaaaaaagggaaaaaaaaaaaaaaaaaaaaaaaaaaaaggcha 2760 acttgttgag acacaaa taactaagca tgttgctcaa atacttgatt cagaatgaa 2820 tachaaat gatgaaatg aataatt cagagaagta aaagtaata cattaagtc 2880 aaaattagta tcagatttca gaaaggattt tcaatttac aaagttcgtg aataataa 2940 ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000 atatcctaaa ctagaaagcg aatttgttta tggagatt aaagtttatg atgtgcgcaa 3060 aatgatcgcg aaatccgaac aagaatcgg taaggctaca gcaaatatt tctttattag 3120 taatataatg aattttttta agacagaaat aactttggct aatggtgaa tcagaaaag 3180 accacttatc gaacaatg gagacacagg agaatagta tgggataag gagagattt 3240 tgctactgtt agaaaagtac taagtatgcc acagtaaat atcgtaaga aactgaagt 3300 tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360 tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420 ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480 aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540 cttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600 gtatagcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660 tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720 tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780 acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840 tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900 accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960 accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020 agagtatta gacgcaactt tatacatca atctattaca gggctttatg aaacaagaat 4080 tgatttaagt caactaggcg gagattaagt cgacaagta ttgttaaaaa taactctgta 4140 gattataaa ttagttctac agagttatttt ttgacccgg gtatattgat aaaataata 4200 atagtgggta tattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260 gtaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa gtttaacaa 4320 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380 ataagcgggc ttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440 ttgcccttt agaagggaa agctggcaag atttttcg taatacgct aaagtttta 4500 gatgtgcttt actaagtcat cgcgatggag caaagtaca tttaggtaca cggcctacag 4560 aaaaacagta tgaactctc gaaaatcaat tagccttttttgccaacaa ggttttcac 4620 tagagaatgc attatgca ctcagcgctg tggggcatttt tactttaggt tgcgtattgg 4680 aagatcaag gcatcaagtc gctaagaag aaagggaac acctactact gatagtagc 4740 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860 cttaaaagca gkataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920 tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160 gctcttcgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120 gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180 ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240 acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300 aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360 agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420 tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480 agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540 taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600 agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660 attccctaac aaacagaggt ataaattgt tgggagtatt ccttaccatt taagcacaca 6720 aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780 aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840 agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900 aaacagtgtc ttataaaac ttacccgcca taccacagat gttccagata atattggaa 6960 gctatacg tactttgttt caaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020 aaatcagtttt catcaagcaa tgaacacgc aaatcagtt aatttaagta ccgttactta 7080 tgagcaagta ttgtctattt ttatagtta tctattttt aacgggagga ataatttcta 7140 tgagtcccta ggcaggccctc cgccattatt ttttgaaca attgacaatt cattttcttat 7200 ttttattaa gtgagtca aaaggcataa cagtgctgaa tagaaagaa tttacagaaa 7260 agaaaattat agaatttagt atgattaattt attackcatttt atgaatgtttt aatgaatac 7320 aaaaaaaaat acttgttatg tattcaatta cgggttaaa tatagacaag ttgaaaaatt 7380 tataaaaaa ataagctccctc agctcttata tattaagcta ccaacttagt ataagcca 7440 aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaattt 7500 caatgtacc gaatacaag agaacatta actatatta ttcaatttat gagattatct 7560 taacagatat aaatgtaaat tgcaatagt aagatttaga agtttatagc ctttgtgtat 7620 tggaagcagt acgcaaggc tttttttt gataaaaatt agaagtatat ttttttc 7680 atattaattt tatgaaatg aaagggggtg agcaagtga cagaggaaag cagtatctta 7740 tcaataca aggtattagc atatcatta ttgacttag cagtaaacat tatgactttt 7800 atagtgcttg tagctaagta gtacgaaagg gggagcttta aaagctcct tggaatacat 7860 agaattcata attaattta tgaaagaag ggcgtatatg aaacttgta aaattgcaa 7920 agagtttatt aagatactg aaatatgcaa atacattcg ttgatgattc atgataaaac 7980 agtagcaacc tattgcagta atacaatga gtcaatgt ttacataag ggaagtcca 8040 atgtattaat tgttcaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100 tacagaggaa gacagaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160 aaagctcat gtaagaga gtaaaaga aaaataattt atttattaat ttatattga 8220 gagtgccgac acagtagca ctaaaaaata tatctgtggt gtagtgagcc gatacaaag 8280 gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340 acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400 taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460 aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 8520 tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580 ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640 tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700 taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760 tatggattat aagcggctcg agtccctatc agtgatagat tgaaactcta tcattgatag 8820 agtataatat ctttgttcat tagagcgata aacttgaatt tgagagggaa cttc 8874 <210> 23 <211> 10534 <212> DNA <213> Artificial Sequence <220> <223> pCas9acr <400> 23 cgaattcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 60 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 120 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 180 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 240 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 300 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 360 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 420 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 480 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 540 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 600 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 660 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 720 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 780 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 840 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 900 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 960 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 1020 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 1080 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 1140 caatctaaag tatatatgag taaacttggt ctgacagtta ccaggtccac tgccgggcct 1200 cttgcgggat caaaagaaaa acgaaatgat acaccaatca gtgcaaaaaa agatataatg 1260 ggagataaga cggttcgtgt tcgtgctgac ttgcaccata tcataaaaat cgaaacagca 1320 aagaatggcg gaaacgtaaa agaagttatg gaaataagac ttagaagcaa acttaagagt 1380 gtgttgatag tgcagtatct taaaattttg tataatagga attgaagtta aattagatgc 1440 taaaaatttg taattaagaa ggagtgatta catgaacaaa aatataaaat attctcaaaa 1500 ctttttaacg agtgaaaaag tactcaacca aataataaaa caattgaatt taaaagaaac 1560 cgataccgtt tacgaaattg gaacaggtaa agggcattta acgacgaaac tggctaaaat 1620 aagtaaacag gtaacgtcta ttgaattaga cagtcatcta ttcaacttat cgtcagaaaa 1680 attaaaactg aatactcgtg tcactttaat tcaccaagat attctacagt ttcaattccc 1740 taacaaacag aggtataaaa ttgttgggag tattccttac catttaagca cacaaattat 1800 taaaaaagtg gtttttgaaa gccatgcgtc tgacatctat ctgattgttg aagaaggatt 1860 ctacaagcgt accttggata ttcaccgaac actagggttg ctcttgcaca ctcaagtctc 1920 gattcagcaa ttgcttaagc tgccagcgga atgctttcat cctaaccaa aagtaaacag 1980 tgtcttaata aaacttaccc gccataccac agatgttcca gataaatatt ggaagctata 2040 tacgtacttt gtttcaaaat gggtcaatcg agaatatcgt caactgttta ctaaaaaatca 2100 gtttcatcaa gcaatgaaac acgccaaagt aaacaattta agtaccgtta cttatgagca 2160 agtattgtct attttaata gttatctatt atttaacggg aggaataat tctatgagtc 2220 cctaggcagg cctccgccat tattttttg aacaattgac attcatttc ttatttta 2280 ttaagtgata gtcaaaggc atacagtgc tgaatagaaa gaatttaca gaaaagaaaa 2340 tattagaatt tagtattt attattactc atttatgaat gtttaattga atacaaaaa 2400 aaatacttgt tatgtattca attacggtt aaaataga caagttgaa aatttaataa 2460 aaaaataagt cctcagctct tatataa gctaccaact tagtatata gccaaaactt 2520 aaatgtgcta ccacacatc aagccgttag agaacctat ctatagcaat atttcaatg 2580 taccgacata taccgagaac attack tattcat tattgagatt attackacag 2640 ataataatgt aattgcaat aagtaagtt taagcttta tagccttgt gtattggaag 2700 cagtacgcaa aggctttttt atttgataaaattagaagt atatttattt ttcataatt 2760 aatttatgaa atgaaaggg ggtgagcaaa gtgacagagg aaagcagtat cttatcaaat 2820 aaaaggtat tagcaatc attattgact tagcagta acattgac tttagtg 2880 cttgtagcta agtagtacga aagggggagc tttaaaaagc tccttggaat acatagaatt 2940 cataaatta tttatgaaa gaagggcgta tatgaaaact tgtaaaaatt gcaagagtt 3000 tattaaagat actgaaat gcaaataca ttcgttgatg attcatgata aacagtagc 3060 aacctattgc agtaataca atgagtcaag atgtttacat aaagggaaag tccaatgtat 3120 taattgttca aagatgaacc gatatgtg gtgtgccata aaatgagat gttttacaga 3180 ggaagaacag aaaaagaac gtacatgcat taatattat gcaaggagct ttaaaaaagc 3240 tcatgtaag aagagtaaaaaaaaaata atttatttat taatttaata ttgagagtgc 3300 cgacacagta tgcactaaaa atatatctg tggtgtagtg agccgataca aaggatagt 3360 cactcgcatt ttcataatac atcttatgtt atgattgt gtcggtggga cttcacgacg 3420 aaaacccaca aaaaaaag agttcggggt agggttaagc atagttgagg caactaaca 3480 atcaagctag gatatgcagt agcagaccgt aaggtcgttg tttaggtg ttgtaataca 3540 tacgctatta agatgtaaaa atacggatac caatgaaggg aaagtataa tttttgtg 3600 tagtttgttt gttcatctat gggcaaacta cgtccaagc cgttttccaaa tctgctaaaa 3660 agtatatcct ttctaaaatc aaagtcaagt atgaatcat aaataagtt taattttga 3720 gttattatga tattatgttt ttctattaaa aaattaag tatatagaat agttattaataa 3780 tagtatatac ttaatgtgat aagtgtctga cagtgtcaca gaaaggatga ttgttatgga 3840 ttataagcgg ctcgagtccc tatcagtgat agattgaac tctatcattg atagagtata 3900 atatctttgt tcattagagc gataaacttg aatttgagag ggaacttcca tggataaaaa 3960 gtacagtatt ggtctagaca taggaacta ctctgttggg tggctgtta taacagatga 4020 atttaagtt ccatcaaaaa atttaaagt atttaggaac actgatagac attcaataaa 4080 aaaaaacttg ataggtgctt tattattcga ttcaggag actgctgaag ctacacgttt 4140 aaaaagaaca gctagacgta gatatacaag aaaaaaaat aggatatgtt atcttcaag 4200 aatttttagt atgaatgg haaagttga tgatcattc ttcacagac tagagaag 4260 tttcttagtt gagaagata agaagcatga agacaccct atttttggta atatcgtaga 4320 tgagtagca tatcatgaga agtatccaac tatcatcat tgaaaga aattagttga 4380 ttctacagat aaagctgatc tgattaat atatttagct ttagctcata tgattaatt 4440 tagaggat ttttatag aaggtgatt aaacccagac aacagcgatg tagataatt 4500 attackcaa ttagttcaa cttataatca attackcgaa gagaatccaa ttaatgcaag 4560 tggtgtagac gctaaggcta tattatcagc tagattatca aaatctagaa gattagaaa 4620 tctaatagct caacctctg gagaaaagaa aaatggactt ttgggaacc tatagctct 4680 ctcactcgga ctacaccaa attttaaag caatttgat ctgctgaag acgcaagtt 4740 acaactatca aggatacat acgatgatga tttagataat tgttagctc aaataggtga 4800 tcaatagct gatttgtttc ttgcagcaa aaacttaagt gatgcaattt tactatcaga 4860 tatacttaga gtaaatacag aaatacaaa ggctccttta tcagcagta tgattaacg 4920 attgatgag catcatcag attackacatt atttaaggca cttgtagac aacaattacc 4980 agaaaaatat aaagaattt tctttgatca atctaaaaat ggatatgctg gatatataga 5040 cggtggagca agtcaagaag agttttaata atttataag cctattttag aaaaatgga 5100 tggaactgaa gattacttg ttgaacttaa cagagagat tggaactga aaaagaac 5160 ttttgataat ggttcaatc ctcaccaat tcatttagga gatttacatg ctatactaag 5220 aagacaagaa gatttttatc cattcttta agatataga gaaaaaatttg aaaaaatttt 5280 aacttttaga ataccatatt atgtaggacc acttgcaagg ggaattca gatttgcatg 5340 gatgactga aatcagaag aaactatac cccgtggaat ttgagag tgtagata 5400 aggagctagt gctcaatcat tttagaag atgacaat tttgataaga atcttcctaa 5460 cgaaaaggtt ttgccaaagc atagccttct tttagagtat tttacagtttt atatgagct 5520 tactaagta aaatacgtta cagaggaat gagaaaacca gcattttgt ctggtgaaca 5580 aaagaaagca atagtagacc tattatta aaaaatagg aaggttaccg taaagcact 5640 windows tactcaaaaattgaatg ctttgaatgtgttgaatt caggagttga 5700 agatagattt aatgctcac ttggtacata tcacgatctc ttaaaaatta taaaagataa 5760 ggatttttta gatatgaag aaatgaga tattcttga gatatagtat taacattgac 5820 acttttgaa gatagagaaa tgatagaaga aagattaaa acattgcac atctttttga 5880 tgataggtt atgagcaac ttaaagaag agatataca ggttggggac gtttgtcaag 5940 aaagctatt atgtatta gagataaca atcaggaag actattctcg atttcttta 6000 atcagatgga ttgctaata gaactttat gcaattaatt catgatgatt ctcttacttt 6060 caagaggat attcaaagg ctcaagttc tggacaggc gatagctac acgacacat 6120 tgctaactt gcagggagcc ccgctatcaa aaaggaatt ttacaacag ttaaagttgt 6180 agatgaactt gttaagtta tgggagaca caacctgag atatagtta taggaatggc 6240 gagaaaaatstorm aaaaaaaaatctct wag wag wag 6300 tgaagaagg aaaagagc taggatcaca atattaaaa gaacatccag ttgaaaatac 6360 tcaattgcaa atgaaagt tatatttgta ttacttacaa atggaagg atatgtatgt 6420 tgatcaagaa ctcgatatta atagattaag tgactatgat gttgatcata ttgttcctca 6480 atcattttta aagatgatt caatcgaata caagtatta actagatcag aaaaatag 6540 aggaaagtca gataatgtac catctgaaga agttgttaaa aaaatgaaga actattggag 6600 acaactttta aatgcaaagc taattacaca aagaaaattt gacaatttaa caaaagcaga 6660 aagaggagga ttaagcgaat tagacaaagc tggatttata aaaagacaac ttgttgagac 6720 aagacaaata actaagcatg ttgctcaaat acttgattca agaatgaata caaaatatga 6780 tgaaaatgat aaattaatca gagaagtaaa agtaataaca ttaaagtcaa aattagtatc 6840 agatttcaga aagattatttc aattttacaa agttcgtgaa ataataatact atcatcatgc 6900 tcatgatgca tacttaaatg ctgttgtagg aactgctctt attaagaaat atcctaaact 6960 agaaagcgaa tttgtttatg gagattataa agtttatgat gtgcgcaaaa tgatcgcgaa 7020 atccgaacaa gaaatcggta aggctacagc aaaatatttc ttttatagta atataatgaa 7080 tttttttaag acagaaataa ctttggctaa tggtgaaatc agaaaaagac cacttatcga 7140 aacaaatgga gagacaggag aaatagtatg ggataaagga agagatttg ctactgttag 7200 aaaagtacta agtatgccac aagtaaatat cgtaaagaaa actgaagttc aaactggagg 7260 tttctctaag gatcaattt tacctaagg aattcagat aagttaattg CAggaaaaaa 7320 agattgggac ccaaaaaaat acggtggttt tgatagtcca acagttgcct atagtgttct 7380 tgtagtagcg aaagttgaga aaagtaagtc aaaaagttg aaagcgtaa aagaacttct 7440 tggtatcaca attatggaaa gatctcatt tgaaaaaaat ccaattgact ttttagaagc 7500 taagggttat aaagagtta aaaggattt atcataaa ctaccaagt atagtctatt 7560 tgaactcgaaaacggaaaaacgaatgct cgctagcgca ggagaacttc aaaaaggaaa 7620 tgaacttgcg ctgccatcaa agtatgtaaa ttcttatat ttagctctc attatgagaa 7680 attaaaagga tcaccagagg ataatgaca aaagcaacta ttgtagaac aacacaaca 7740 tttattagat gaaataatag aaaataatc tgaatttctct aaagagtta tacttgccga 7800 cgcaatcta taaggtgc ttcagcgta taaacac agagataac taagaga 7860 acagcagaa aacattatcc atctttttac attackactaat cttggtgcac cagctgcatt 7920 windowsttt gattaxa windows agatacaca tcttactag aagtattaga 7980 cgcaacttta atacatcaat ctattacagg gctttatgaa acagaattg atttaagtca 8040 actaggcgga gattaagtcg aaagtatt gttaaaaata actctgtaga attaaattt 8100 agttctacag agttatttt tgacccgggt atattgataa aaataataat agtgggtata 8160 attaagttgt taggaggtta gttagaatga tgtcagatt agataaagt aaagtgatta 8220 acagcgcatt agagctgctt aatgaggtcg gatcgaagg ttacaacc cgtaaactcg 8280 cccagaagct aggtgtagag cagcctacat tgttattggca tgtaaaaat aagcgggctt 8340 tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag 8400 aaggggaaag ctggcaagat ttttacgta ataacgctaa aagttttaga tgtgctttac 8460 taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa aacagtag 8520 aaactctcga aaatcatta gccttttt gccaacagg tttcacta gagaatgcat 8580 tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc 8640 atcaagtcgc taagagaa agggaacac ctactactga tagtatgccg ccattattac 8700 gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg 8760 aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtct taaaagcagc 8820 ataacctttt tccgtgatgg taacttcacg gtaaccaaga tgtcgagttg agctcttagt 8880 tcaactcact ttttaaggtg attgtttgca tgtcattata aaattcttct tcatcctcgt 8940 attcttgatt ccaaccgttt ttaaatgcag atatgaattt ttcaactatt gattcatttt 9000 cactttcaga aattacatac tcgtttccat cattattaac tctaataatt agctgtgtta 9060 tactattgct atccgtacca ctcaatttca ctgtgtaatc tttgttttt atttctctaa 9120 ttaagtcatt aatattcatt tcagccctcc tgtgaaattg ttatccgctc acaattccac 9180 gtcgactacc gcggattcta gattctgcag tatcttcatg gtattcattt tttaatatca 9240 ttttaccctc ccaatacatt taaataatt atgtattcat gaaacatgat tgtatatta 9300 agaaacataa ttccataataa atcatttttc aaaaatagttt ttacccataa ttaaatgtta 9360 atatgtaaat taatctttta gaatagttaa aaagttctaa aatatgttat aatgtttctt 9420 ataatcttat aaattttaat aactaatata taaagatatt tctttaaaat attcttatat 9480 ttagaagaat ttattttaaa ataaaaagct tttatgttga taaactgctt tgcaaagctc 9540 tcatgtaaat gtttaatata agactactat aaaattggct aattttatag gttaggaggt 9600 agaaatgcaa atattgtgga aaaagtatgt taaagaaaac tttgaaatga atgtagatga 9660 atgtggtata gaacaaggta taccaggatt aggatataac tatgaagtat tgaaaaatgc 9720 tgttatcat tacgtacta agggatatgg aacttttaaa tttaatggta aggtatataa 9780 cttaaaacaa ggtgatattt ttatactact aaaaggtatg caagttgagt atgtggcttc 9840 tattgatgat ccttgggaat actactggat aggatttagt ggttcaaatg ctaatgagta 9900 tttaaataga acttctatta ctaactcctg tgttgctaat tgtgaagaaa actcaaaaat 9960 tccacagata atattaaata tgtgcgaaat atcaaaaact tataatcctt caagatctga 10020 tgacatacta ttactaaaag aactttactc attatgtac gcacttatag aagaattccc 10080 aaaacctttt gaatacaaag ataaggaatt acacacatat attcaagatg ctcttaattt 10140 cattaattct aattacatgc atagcataac tgttcaagaa attgctgatt atgtgaactt aagtagaagt tatttatata aaatgttcat aaaaacctt ggaatttctc ctcaaagata tttaataac cttagaatgt acaaagccac ccttttatta aaaagcacta aacttcctat aggagaagtc gcaagtagtg taggttatag tgactccctg ttattttcaa aaactttttc aaaacatttt tcaatgtctc aaaacatttt aaaacattt aaaacattt 10500. 10500. 10500. 10500. 10500. 10500. 10500. 10500. 10500 cgtaattaa aacctctgtt atactttttg agct <210> 24 <211> 5754 <212> DNA <213> The snowstorm <220> <223> pEC750S-uppHR <400> 24 60. attachment attachment gcagcgctat gttcagatac atttagtgct catgcaacaa 120. snowflake snowflake snowflake snowflake snowflake snowflake snowflake ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa gattagata gatttcagat attgaaaaaa agtatacaca tgaaaaaa gcagctatgc 240 tgcaataag atcaatttat attagaaaaa agcagctatg ctgcaataa gatcaattta 300 tattagaaaa aagcagctat gctgcaata agatcaattt attagaaa aaagcagcta 360 tgctacaaat aagatcaatt tattagaa aaagtagct atgctgcaac atattaatt 420 tatacta gaagctaa tggggtatat aaataag ggctataaat actaaagca 480 aacttggagg aaataatg gtctagagct ggagatagat tattggtac tagtatta 540 gtaatctatt agaattaaaa gctactaca taagttttctg aatgaccca gatatttta 600 ctggggggaa tatagaaaat ggagagacga gatagaaaaattactt ggatattgct 660 gaaacagtttt tagagagagg aacctgtcta aggaaact atggttctat aattgttaaa 720 aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780 gatttgaata gttgcataag agaaagttg aaagttccaa gaggtactca ttatgagttg 840 tgtaggagtg tacatagtga agctaatgca ataataagcg cttcgagctc gattcgtaa 900 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 960 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 1020 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 1080 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 1140 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 1200 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 1260 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1320 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1380 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1440 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 1500 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 1560 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1620 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 1680 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 1740 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaga 1800 gttggtagct cttgatccgg siacaaacc accgctgta gcggtggtttt tttgtttgc 1860 aagcagcaga tcgcgcag aaaaaagga tccagag atccttgat ctttctacg 1920 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttgtcat gagatttaca 1980 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2040 atatatgagt aaacttggtc tgacagttac caaagctagc ttatactag tatatactta 2100 atgtgataag tgtctgacag ctgaccggtc taaagaggtc cgccaatgaa atcttaaat 2160 aaactaatt aagtttttt attackacac tatgatata aaataggtac ttcaat 2220 agtgaggagg atatatttga atacatacga aaattaat aaagtgaaa aaatacttcg 2280 gaaacattta aaaaatacc ttattggtac ttacatgttt ggatcaggag tgagagtgg 2340 actaaaacca atagtgatc ttgactttt agtcgtcgta tctgaaccat tgacagatca 2400 aagtaaaattag accttattac aaaaaattag acctattca aaaaaatag gagaaaag 2460 storm tattgaat windows tattcagca gaatggtac cgtggaatca 2520 tcctcccaaa caagaattta tttggaga atggttaca gagctttatg aaaaggata 2580 cattcctcag aaggaattta attcagattt aaccataatg ctttaccaag caaacgaaa 2640 aaaaaaga ataacggaa attatgactt agaggaatta ctacctgata ttccattttc 2700 tgatgtgaga agagccatta tggattcgtc agaggaatta atagatatt atcaggatga 2760 tgaaaccaac tctatattaa ctttatgccg tatgattta actaggaca cgggtaaaat 2820 cataccaaaa gatttgcgg gaatgcagt ggctgaatct tctcattag aacataggga 2880 gagaatttg ttagcagttc gtagttatct tggagaat attgaatgga ctaatgaaaa 2940 tgtaaattta actataaact atttaaaata cagattaaa aaaattgaaaaaattgaaa 3000 aaatggtgga aacacttttt tcaattttt tgttttatta ttatatattt gggaaatatt 3060 cattctaatt ggtaatcaga ttttagaagt tgttaacttc aggtttgtct gtaactaaaa 3120 actagtattt aacctaggat caaaaaaatt tccaataatc ccactctaag ccacaacac 3180 gcccttaaa atcccgcttt aatcccactt tgagacacat gtaatattac tttacgccct 3240 agtatagtga taattttta cattcaatgc cacgcaaaa aaaagggg cactataata 3300 aaagttcctt cggaactaac taaagtaaaaatttacttt acaacctccc CAAAAAAAAG 3360 aacaggtaca aagtacccta tatacaagc gtaaaaaaaa tgagggtaa aaaaaaaa 3420 taaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa being 3480 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 3540 taaaaaaaata taaaaattatt ttttatta agtttgaaa aaattttttt attattatata 3600 atctttgaag aaagaatat aaaaatgag cctttataa agcccatttt tttcatata 3660 cgtaatatga cgttctaatg ttttattgg tacttctaac attagagtaa ttctttatt 3720 tttaaagcct ttttctttaa gggcttttt ttttttctt atacattta attccctttt 3780 ttttgttgct tttcctttag cttttaattg ctcttgataa ttttttttac ctctaatatt 3840 ttctcttctc ttatattcct ttttagaaat tattattgtc atatattttt gttcttcttc 3900 tgtaatttct aataactcta taagagtttc attcttatac ttatattgct tatttttatc 3960 taaataacat ctttcagcac ttctagttgc tcttataact tctctttcac ttaaatgttg 4020 tctaaacata ctattaagtt ctaaaacatc atttaatgcc ttctcaatgt cttctgtaaa 4080 gctacaaaga taatatctat ataaaaataa tataagctct ctgtgtcctt ttaaatcata 4140 ttctcttagt tcacaaagtt ttattatgtc ttgtattctt ccataatata aacttctttc 4200 tctataaata taatttattt tgcttggtct accctttttc ctttcatatg gttttaattc 4260 aggtaaaaat ccattttgta tttctcttaa gtcataaata tattcgtact catctaatat 4320 attgactact gtttttgatt tagagtttat acttcctgga actcttaata ttctcgttgc 4380 atctaaggct tgtctatctg ctccaaagta ttttaattga ttatataaat attcttgaac 4440 cgctttccat aatggtaatg ctttactagg tactgcattt attatccata ttaaatacat 4500 tcctcttcca ctatctatta catagttttgg tataggata ctttgattta ataatttctt 4560 ttctaagtcc attaatacct ggtctttagt ttgccagtt ttataataat ccaagtctat 4620 aaacagtgta tttactctt tatatattc taatcgccta cacggcttat aaaagtatt 4680 tagagttata tagatattt catcactcat atctaaatct tttattcag cgtatttata 4740 gtgccattgg ctatatcctt tttatctat aacgctcctg gttatccacc ctttacttct 4800 actatgaata ttatctatat agttctttt attcagcttt atgcgtttc tcacttattc 4860 acctcccctt ctgtaaaact aagaaaatta tatcatattt tcaatatta ttaactattc 4920 ttaaactct aaaaaaat agagtaagtc cccaattgaa acttaatcta ttttttgt 4980 ttatttatt tattttatt aaaatatttt aaactaattt aaatgattct ttttatttt 5040 attackatttc attccataat attack attack attack attack attack attack attack tctcattg 5100 fatherttag atgatttact aatttagtt tttattatt aaatattaa tgtataattt 5160 ataaaaaaaaaaaaaaaaggagc tataatta tgatttattc caagatact aagatttaa 5220 tttttttcaa ttttaacaat actttttgta atattatgtt taaatttaat tgtatttttt 5280 tcatataata aagccgttga agtaaaccaa tccattttcc ttatgatgtt attattaaat 5340 ttaagtttta taataatatc tttattatat ttattgtttt taaaaaaact agtgaaattt 5400 ctagtgaaat ttccggcttt attaaactta tttttaggaa ttttattttc attttcatct 5460 ttacaggatt tgattatatc tttaaatatg ttttatcaaa tattatcttt ttctaaattt 5520 atatatattt ttattatatt tattattata tatattttat ttttaagttt ctttctaaca 5580 gctattaaaa agaaacttaa aaataaaaac acgtactcta aaccaataaa taaaactatt 5640 tttattattg ctgccttgat tggaatagtt tttagtaaaa ttaatttcaa tattccacaa 5700 tattatatta taagctagca ggcctcgaga tctccatgga cgcgtgacgt cgac 5754 <210> 25 <211> 884 <212> DNA <213> Artificial Sequence <220> <223> Repair Template <400> 25 ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60 gagaactaa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120 ctttagatat agtaaaaaca tttatatcag ctaaatttga aggatagg caccaaaaaa 180 gatataa gatttcagat attgaaaaaa agtatacaca taggaaaaaa gcagctatgc 240 tgcaatag atcaatttat attagaaaaa agcagctatg ctgcaataa gatcaattta 300 tattagaaaa aagcagctat gctgcaata agatcaattt attagaaa aaagcagcta 360 tgctacaaat aagatcaatt tattagaa aaagtagct atgctgcaac atattaatt 420 tatacta gaagctaa tggggtatat aaataag ggctataaat actaaagca 480 aacttggagg aaataatg gtctagagct ggagatagat tattggtac tagtatta 540 gtaatctatt agaattaaaa gctactaca taagttttctg aatgaccca gatatttta 600 ctggggggaa tatagaaaat ggagagacga gatagaaaaattactt ggatattgct 660 gaaacagtttt tagagagagg aacctgtcta aggaaact atggttctat aattgttaaa 720 aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780 gatttgaata gttgcataag agaaaagttg aaagttccaa gaggtactca ttatgagttg 840 tgtaggagtg tacatagtga agctaatgca ataataagcg cttc 884 <210> 26 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> Upstream fragment of upp gene <400> 26 ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60 gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120 ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180 gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240 tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300 tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360 tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420 tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480 aacttggagg aataataatg 500 <210> 27 <211> 377 <212> DNA <213> Artificial sequence <220> <223> Downstream fragment of upp gene <400> 27 gctggagata gattatttgg tactaagtaa ttagtaatct attagaatta aaagctatct 60 acataagttt ctgaatgacc caagataatt ttactggggg gaatatagaa aatggagaga 120 cgagataaga aaaattatta cttggatatt gctgaaacag ttttagagag aggaacctgt 180 ctaaggagaa actatggttc tataattgtt aaaaatgatg aaataatttc tactggatac 240 acaggagcac ctagaggtag aaaaaattgc atggatttga atagttgcat aagagaaaag 300 ttgaaagttc caagaggtac tcattatgag ttgtgtagga gtgtacatag tgaagctaat 360[[ID=?]] gcaataataa gcgcttc 377 <210> 28 <211> 2666 <212> DNA <213> Artificial sequence <220> <223> pEX - A2 - gRNA - upp <400> 28 ctcgagtatt tttgataaaa gcaatgatta acatggtttg acgtctgaga agagacgatt 60 ttctcaatag gagaaattaa ggtgcaaacc cttatcattc caccatgatc cacctgtagc 120 Note: There seems to be a problem with identifier in the original text, which is translated as in the English version but there is a '?' in the provided translation result. It should be corrected according to the actual situation. aagcatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 180 aaaagtggca ccgagtcggt gctttttttg ccatggacct gcttttgctc gcttggatcc 240 gaattcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 300 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 360 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 420 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 480 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 540 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 600 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 660 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 720 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 780 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 840 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 900 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 960 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1020 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 1080 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 1140 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1200 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1260 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1320 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1380 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1440 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1500 taccatctgg ccccagtgct gcaatgatac cgcgactccc acgctcaccg gctccagatt 1560 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1620 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1680 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1740 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1800 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1860 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1920 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1980 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 2040 cttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 2100 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 2160 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2220 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2280 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2340 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca 2400 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc 2460 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt 2520 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 2580 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccaat 2640 tgggtaccga gctcgcggcc gcaagc 2666 <210> 29 <211> 203 <212> DNA <213> Artificial sequence <220> <223> gRNA expression cassette <400> 29 tatttttgat aaaagcaatg attaacatgg tttgacgtct gagaagagac gattttctca 60 ataggagaaa ttaaggtgca aacccttatc attccaccat gatccacctg tagcaagcat 120 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 180 ggcaccgagt cggtgctttt ttt 203 <210> 30 <211> 100 <212> DNA <213> Artificial sequence <220> <223> constitutive promoter <400> 30 tatttttgat aaaagcaatg attaacatgg tttgacgtct gagaagagac gattttctca 60 ataggagaaa ttaaggtgca aacccttatc attccaccat 100 <210> 31 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Targeting the protospacer sequence of upp <400> 31 gatccacctg tagcaagcat 20 <210> 32 <211> 5954 <212> DNA <213> Artificial sequence <220> <223> pEC750S-Δupp <400> 32 ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60 gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120 ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180 gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240 tgcaatag atcaatttat attagaaaaa agcagctatg ctgcaataa gatcaattta 300 tattagaaaa aagcagctat gctgcaata agatcaattt attagaaa aaagcagcta 360 tgctacaaat aagatcaatt tattagaa aaagtagct atgctgcaac atattaatt 420 tatacta gaagctaa tggggtatat aaataag ggctataaat actaaagca 480 aacttggagg aaataatg gtctagagct ggagatagat tattggtac tagtatta 540 gtaatctatt agaattaaaa gctactaca taagttttctg aatgaccca gatatttta 600 ctggggggaa tatagaaaat ggagagacga gatagaaaaattactt ggatattgct 660 gaaacagtttt tagagagagg aacctgtcta aggaaact atggttctat aattgttaaa 720 aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780 gatttgaata gttgcataag agaaagttg aaagttccaa gaggtactca ttatgagttg 840 tgtaggagtg tacatagtga agctaatgca ataataagcg ctcgagctc gattcgtaa 900 tcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaatcc acacaacata 960 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 1020 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 1080 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 1140 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 1200 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 1260 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1320 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1380 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1440 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 1500 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 1560 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1620 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 1680 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 1740 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 1800 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggtttt ttttgtttgc 1860 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 1920 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 1980 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2040 atatatgagt aaacttggtc tgacagttac caaagctagc ttaatactag tatatactta 2100 atgtgataag tgtctgacag ctgaccggtc taaagaggtc cgccaatgaa atctataaat 2160 aaactaaatt aagtttattt attaacaac tatggatata aaataggtac taatcaaaat 2220 agtgaggagg atatatttga atacatacga acaattaat aaagtgaaaa aaatacttcg 2280 gaaacattta aaaaataacc ttattggtac ttacatgttt ggatcaggag ttgagagtgg 2340 actaaaacca aatagtgatc ttgacttttt agtcgtcgta tctgaaccat tgacagatca 2400 aagtaaaattag accttattac aaaaaattag acctattca aaaaaatag gagaaaag 2460 storm tattgaat windows tattcagca gaatggtac cgtggaatca 2520 tcctcccaaa caagaattta tttggaga atggttaca gagctttatg aaaaggata 2580 cattcctcag aaggaattta attcagattt aaccataatg ctttaccaag caaacgaaa 2640 aaaaaaga ataacggaa attatgactt agaggaatta ctacctgata ttccattttc 2700 tgatgtgaga agagccatta tggattcgtc agaggaatta atagatatt atcaggatga 2760 tgaaaccaac tctatattaa ctttatgccg tatgattta actaggaca cgggtaaaat 2820 cataccaaaa gatttgcgg gaatgcagt ggctgaatct tctcattag aacataggga 2880 gagaatttg ttagcagttc gtagttatct tggagaat attgaatgga ctaatgaaaa 2940 tgtaaattta actataaact atttaaaata cagattaaa aaaattgaaaaaattgaaa 3000 aaatggtgga aacacttttt tcaattttt tgttttatta ttatatattt gggaaatatt 3060 cattctaatt ggtaatcaga ttttagaagt tgttaacttc aggtttgtct gtaactaaaa 3120 actagtattt aacctaggat caaaaaaatt tccaataatc ccactctaag ccacaaacac 3180 gccctataaa atcccgcttt aatcccactt tgagacacat gtaatattac tttaacgccct 3240 agtatagtga taatttttta cattcaatgc cacgcaaaaa aataaagggg cactataata 3300 aaagttcctt cggactaac taaagtaaa aattatcttt acaacctccc caaaaaaaag 3360 aacaggtaca aagtacccta taatacaagc gtaaaaaaaaa tgagggtaaaa aataaaaaaa 3420 taaaaaaata aaaaaataaa aaaataaaaa aataaaaaaa taaaaaaata taaaaataaa 3480 aaaatataaa aataaaaaaa tataaaaata aaaaaataaa aaaatataaa aataaaaaaa 3540 taaaaaaata taaaaatatt ttttatttaa agtttgaaaa aaattttttt atattatata 3600 atctttgaag aaaagaatat aaaaaatgag cctttataaa agcccatttt ttttcatata 3660 cgtaatatga cgttctaatg tttttattgg tacttctaac attagagtaa tttctttatt 3720 tttaaagcct ttttctttaa gggcttttat tttttttctt aatacattta attcctcttt 3780 ttttgttgct tttcctttag cttttaattg ctcttgataa ttttttttac ctctaaatt 3840 ttctctctc tatatattcct ttttagaat tattattgtc atatattttt gttctcttc 3900 tgtaatttct aaactcta taagagtttc attcttatac tattattgct tatttttac 3960 taaataacat ctttcagcac ttctagttgc tcttataact tctcttcac ttaaatgttg 4020 tctaaacata ctattaagtt ctaaaaacatc atttaatgcc ttctcaatgt cttctgtaaa 4080 gctacaaga taatatctat aaaaata tataagctct ctgtgtcctt ttaatcata 4140 ttctcttagt tcacaagtt ttattatgtc ttgtattctt ccatatatata aacttctttc 4200 tctaaata taatttatt tgcttggtct accctttc cttcatatg gttttaattc 4260 aggtaaaaat ccatttgta tttctcttaa gtcataaata tattcgtact catctaatat 4320 attgactact gttttgatt tagagtttat acttcctgga actcttaata ttctcgttgc 4380 atctaaggct tgtctatctg ctccaagta ttttaattga tatatataat attcttgaac 4440 cgctttccat aatgtaatg ctttactagg tactgcattt attatccata ttaatacat 4500 tcctcttcca ctatctatta catagttttgg tataggata ctttgattta ataatttctt 4560 ttctaagtcc attaatacct ggtctttagt tttgccagtt ttataataat ccaagtctat 4620 aaacagtgta tttaactctt ttatattttc taatcgccta cacggcttat aaaaggtatt 4680 tagagttata tagattattt catcactcat atctaaatct tttaattcag cgtatttata 4740 gtgccattgg ctatatcctt tttatctat aacgctcctg gttatccacc ctttattct 4800 actatgaata ttatctatat agttctttt attcagcttt aatgcgtttc tcacttattc 4860 acctcccctt ctgtaaaact aagaaaatta tatcatattt tcaataatta ttaactattc 4920 ttaaactctt aataaaaaat agagtaagtc cccaattgaa acttaatcta tttttatgt 4980 tttaatttat tatttttatt aaaatatttt aaactaaatt aaatgattct ttttaatttt 5040 ttactatttc attccataat atattactat aattatttac aaataatatt tcttcatttg 5100 taatatttag atgatttact aattttagtt tttatatatt aaataattaa tgtataattt 5160 5220 tttttttcaa ttttaacaat actttttgta atattatgtt taaatttaat tgtatttttt 5280 tcatataata aagccgttga agtaaaccaa tccattttcc ttatgatgtt attattaaat 5340 ttaagtttta taatatatc tttattatat ttattgtttt taaaaaaact agtgaaattt 5400 ctagtgaaat ttccggcttt atttaactta ttttaggaa ttttttc atttcatct 5460 5520 tattatattt tattattatt tattattatt ttttagtttt ctttctaaca 5580 gctattaaaa agaacttaa aaaaaaac acgtactcta aaccaataa taaaactatt 5640 tttattg ctgccttgat tggaatagtt tttagtaaaa ttaatttca tattccacaa 5700 tattatta taagctagca cgcctcgagt atttttgata aaagcaatga ttaacatggt 5760 ttgacgtctg agagagacg atttctca taggagaat taaggtgcaa acccttatca 5820 ttccaccatg atccacctgt agcaagcatg ttttagagct agaatagca agttaaaata 5880 aggctagtcc gttatcaact tgaaaagtg gcaccgagtc gtgcttttt ttgccatgga 5940 cgcgtgacgt cgac 5954 <210> 33 <211> 5853 <212> DNA <213> Artificial sequence <220> <223> pEC750C-Δupp <400> 33 atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60 ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120 tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180 actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240 tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300 aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360 aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420 ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480 ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540 tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600 aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660 agcttttaat tgctcttgat aatttttt acctctaata tttctctc tcttatattc 720 ctttttagaa attattattg tcatatattttgttctct tctgtaattt ctaataactc 780 tataagagtt tcattcttat acttatattg cttatttta tctaaataac atctttcagc 840 acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaca tactattaag 900 ttctaaaaca tcatttaatg ccttctcaat gtctctgta aagctacaaa gataatatct 960 atataaaaat ataataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020 ttttattatg tcttgtattc ttccataata taaacttctt tcttattaa tataatttat 1080 ttgcttggt ctacccttt tccttcata tggttttaat tcaggtaaaa atccatttg 1140 tattctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200 tttagagttt atactcctg gaactcttaa tattctcgtt gcatctagg cttgtctatc 1260 tgctccaaag tattttaatt gattatataa atattctga accgctttcc ataatggtaa 1320 tgctttacta ggtactgcat ttattacca tattaatac attcctctc cactacttat 1380 tacatagtttt ggtataggaa tactttgatt aaaatattc tttctaagt ccattaatac 1440 ctggtcttta gttttgccag ttttaata atccaagtct ataacagtg tattactc 1500 ttttatattt tctaatcgcc tacacggctt aaaaggta tttagagtta tatagatatt 1560 ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620 ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680 atagttcttt ttcagct taatgcgtt tctcacttat tcacccccc ttctgtaaaa 1740 ctaagaaaat tattacatat ttcaataat tattaactat tcttaactc tattaaaaa 1800 atagagtaag tcccaatttg aaacttaatc tatttttt gttttaattt atttatttta 1860 ttaaaatatt ttaaactaa ttaaatgat ctttttaattt ttttactt tcattccata 1920 attattact attattatt acaatatta ttctcatt tgtatattt agatgattta 1980 ctaattttag ttttatta tattaatatt atgtataat tttattaaaaatcaagga 2040 gcttataaat tatgattatt tccaagata ctaaagattt atttttttc aattttaca 2100 atactttttg tatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160 gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagtttt tataataata 2220 tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280 ttattaaact tatttttagg aattttattt tcattttcat cttacagga tttgattata 2340 tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400 tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460 aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520 attggaatag tttttagtaa attaatttc aatattccac aatattatat tataagctag 2580 cacgcctcga gtatttttga taaaagcaat gattaacatg gtttgacgtc tgagaagaga 2640 cgatttctc aataggagaa attaaggtgc aaacccttat cattccacca tgatccacct 2700 gtagcaagca tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa 2760 cttgaaaaag tggcaccgag tcggtgcttt ttttgccatg gacgcgtgac gtcgacataa 2820 ggtaccagga attagagcag cgctatgttc agatacattt agtgctcatg caacaagaga 2880 acataataat gctaatatat taactatggg tcaaagggtt gttggagcag gtcttgcttt 2940 agatatagta aaaaacattta tatcagctaa atttgaagga gataggcacc aaaaaagaat 3000 agataagatt tcagatattg aaaaaagta tacacattag aaaaaagcag ctatgctgca 3060 aataagatca atttatatta gaaaaaagca gctatgctgc aataagatc aatttatatt 3120 agaaaaaagc agctatgctg caaataagat caatttat tagaaaaaag cagctatgct 3180 acaaataaga tcaatttata ttagaaaaaa gtagctatgc tgcaacaata ttaatttata 3240 ttactagaaa gctaaatggg gtatataaat ataaagggct ataaatacta aaagcaaact 3300 tggaggaata ataatggtct agagctggag atagattatt tggtactaag taattagtaa 3360 tctattagaa ttaaaagcta tctacataag tttctgaatg acccaagata attttactgg 3420 ggggaatata gaaaatggag agacgagata agaaaaaatta ttacttggat attgctgaaa 3480 cagttttaga gagaggaacc tgtctaagga gaaactatgg ttctataatt gttaaaaatg 3540 atgaaataat ttctactgga tacacaggag cacctagagg tagaaaaaat tgcatggatt 3600 tgaatagttg cataagagaa aagttgaaag ttccaagagg tactcattat gagttgtgta 3660 ggagtgtaca tagtgaagct aatgcaataa taagcgcttc gagctcgaat tcgtaatcat 3720 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 3780 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 3840 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 3900 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 3960 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4020 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4080 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4140 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4200 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4260 tgccgcttac cggatacctg tccgccttc tcccttcggg aagcgtggcg cttctcata 4320 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4380 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4440 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4500 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4560 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4620 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggtttttt gtttgcaagc 4680 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 4740 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga tttcaaaaa 4800 ggatcttcac ctagatcctt ttaaattaaa aatgaagtttt taaatcaatc taaagtatat 4860 atgagtaaac ttggtctgac agttaccaaa gctagcttaa tactagtata tacttaatgt 4920 gataagtgtc tgacagctga ccggtctaaa gaggtcccta gcgcctacgg ggaatttgta 4980 tcgataaggg gtacaaattc ccactaagcg ctcggccggg gatcgatccc cgggtacgta 5040 cccggcagtt tttctttttc ggcaagtgtt caagaagtta ttaagtcggg agtgcagtcg 5100 aagtgggcaa gttgaaaaat tcacaaaaat gtggtataat atctttgttc attagagcga 5160 taaacttgaa tttgagaggg aacttagatg gtatttgaaa aaattgataa aatagttgg 5220 aacagaaaag agtattttga ccactacttt gcaagtgtac cttgtaccta cagcatgacc 5280 gttaaagtgg atatcacaca aataaaggaa aagggaatga aactatatcc tgcaatgctt 5340 tattatattg caatgattgt aaaccgccat tcagagttta ggacggcaat caatcaagat 5400 ggtgaattgg ggatatatga tgagatgata ccaagctata caatatttca caatgatact 5460 gaaacattt ccagcctttg gactgagtgt aagtctgact ttaaatcatt tttagcagat 5520 tatgaaagtg atacgcaacg gtatggaaac aatcatagaa tggaaggaaa gccaaatgct 5580 ccggaaaaca tttttaatgt atctatgata ccgtggtcaa ccttcgatgg ctttaatctg 5640 aatttgcaga aaggatatga ttatttgatt cctattttta ctatggggaa atattataaa 5700 gaagataaca aaattatact tcctttggca attcaagttc atcacgcagt atgtgacgga 5760 tttcacattt gccgttttgt aaacgaattg caggaattga taaatagtta acttcaggtt 5820 tgtctgtaac taaaaactag tatttaacct agg 5853 <210> 34 <211> 4966 <212> DNA <213> Artificial sequence <220> <223> pGRNA-pNF2 <400> 34 agctcggtac ccggggatcc tctagagtcg acgtcacgcg tccatggaga tctcgaggcg 60 tgctagctta taatataata ttgtggaata ttgaaattaa ttttactaaa aactattcca 120 atcaaggcag caataataaa aatagtttta tttattggtt tagagtacgt gtttttattt 180 ttaagtttct ttttaatagc tgttagaaag aaacttaaaa ataaaatata tataataata 240 aatataataa aaatatatat aaatttagaa aaagataata tttgataaaa catatttaaa 300 gatataatca aatcctgtaa agatgaaaat gaaaataaaa ttcctaaaaa taagtttaat 360 aaagccggaa atttcactag aaatttcact agttttttta aaaacaataa atataataaa 420 gatattatta taaaacttaa atttaataat aacatcataa ggaaaatgga ttggtttact 480 tcaacggctt tattatatga aaaaaataca attaaattta aacataatat tacaaaaagt 540 attgttaaaa ttgaaaaaaa ttaaatcttt agtatctttg gaaataatca taatttataa 600 gctcctttga ttttttat aaattataca ttaattattt aatatataaa aactaaaatt 660 agtaaatcat ctaaatatta caaatgaaga aatattatt gtaaataatt atagtaatat 720 attatggaat gaaatagtaa aaaattaaaa agaatcattt aatttagttt aaaatatttt 780 aataaaaata ataaattaaa acataaaaaa tagattaagt ttcaattggg gacttactct 840 attttttat aagagtttaa gaatagttaa taattattga aaatatgata taattttctt 900 agttttacag aaggggaggt gaataagtga gaaacgcatt aaagctgaat aaaaagaact 960 atatagataa tattcatagt agaagtaaag ggtggataac caggagcgtt atagataaaa 1020 aggattatag ccaatggcac tataaatacg ctgaattaaa agatttagat atgagtgatg 1080 aaaatatcta tataactcta aatacctttt ataagccgtg taggcgatta gaaaatataa 1140 aagagttaaa tacactgttt atagacttgg attattaataa aactggcaaa actaaagacc 1200 aggtattaat ggacttagaa aagaattatt ttaatcaaag tattcctata ccaaactatg 1260 taatagatag tggaagagga atgtatttaa tatggataat aaatgcagta cctagtaaag 1320 cattaccatt atggaaagcg gttcaagaat atttatataa tcaattaaaa tactttggag 1380 cagatagaca agccttagat gcaacgagaa tattaagagt tccaggaagt ataaactcta 1440 aatcaaaaac agtagtcaat atattagatg agtacgaata tatttatgac ttaagagaaa 1500 tacaaaatgg atttttacct gaattaaaac catatgaaag gaaaaagggt agaccaagca 1560 aataaatta tatttataga gaaagaagtt tatattatgg aagaatacaa gacatataa 1620 aactttgtga actaagagaa tatgatttaa aaggacacag agagcttata ttatttttat 1680 atagatatta tctttgtagc tttacagaag acattgagaa ggcattaaat gatgttttag 1740 aacttaatag tatgtttaga caacatttaa gtgaaagaga agttataaga gcaactagaa 1800 gtgctgaaag atgttattta gataaaaata agcaatataa gtataagaat gaaactctta 1860 tagagttat agaaattaca gaagaagaac aaaaatatat gacaataata atttctaaaa 1920 aggaataataa gagagaagaa aatattagag gtaaaaaaaaa ttatcaagag caattaaaag 1980 ctaaaaggaaa agcaacaaaa aaagaggaat taaatgtatt aagaaaaaaa ataaaaagccc 2040 ttaaagaaaa aggctttaaa aataaagaaa ttactctaat gttagaagta ccaataaaaa 2100 cattagaacg tcatattacg tatatgaaaa aaaatgggct tttataaagg ctcattttt 2160 atattctttt cttcaaagat tatataatat aaaaaaaattt ttttcaaact ttaaataaaa 2220 aatattttta tattttttta tttttttatt tttatatttt tttattttt tatttttata 2280 2340 tttatttttt tatttttta tttttttatt tttaccctca ttttttttac gcttgtatta 2400 tagggtactt tgtacctgtt cttttttttg gggaggttgt aaagataatt ttttacttta 2460 gttagttccg aaggacttt tattatagtg cccctttatt ttttgcgtg gcattgaatg 2520 taaaaaatta tcactatact agggcgtaaa gtaatattac atgtgtctca aagtgggatt 2580 aaagcgggat tttatagggc gtgtttgtgg cttagagtgg gattattgga aatttttttg 2640 atcctaggtt aataactagt ttttagttac agacaaacct gaagttaact attatcaat 2700 tcctgcaatt cgtttacaaa acggcaaatg tgaaatccgt cacatactgc gtgatgaact 2760 tgaattgcca aaggaagtat aattttgtta tcttctttat aatatttccc catagtaaaa 2820 ataggaatca aataatcata tcctttctgc aaattcagat taaagccatc gaaggttgac 2880 cacggtatca tagatacatt aaaaatgttt tccggagcat ttggctttcc ttccattcta 2940 tgattgtttc cataccgttg cgtatcactt tcataatctg ctaaaaatga tttaaagtca 3000 gacttacact cagtccaaag gctggaaaat gtttcagtat cattgtgaaa tattgtatag 3060 cttggtatca tctcatcata tatccccaat tcaccatctt gattgattgc cgtcctaaac 3120 tctgaatggc ggtttacaat cattgcaata tataaagca ttgcaggata tagttcatt 3180 cccttttcct ttatttgtgt gatatccact ttaacggtca tgctgtaggt acaaggtaca 3240 cttgcaaagt agtggtcaaa atactctttt ctgttccaac tatttttatc aatttttca 3300 aataccatct aagttccctc tcaaattcaa gtttatcgct ctaatgaaca aagatattat 3360 accacatttt tgtgaatttt tcaacttgcc cacttcgact gcactcccga cttaataact 3420 tcttgaacac ttgccgaaaa agaaaaactg ccgggtacgt acccggggat cgatccccgg 3480 ccgagcgctt agtgggaatt tgtacccctt atcgatacaa attccccgta ggcgctaggg 3540 acctctttag accggtcagc tgtcagacac ttatcacatt aagtatatac tagtattaag 3600 ctagctttgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 3660 tcatttttaa tttaaaagga tctaggtgaa gatcctttt gataatctca tgaccaaaat 3720 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3780 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3840 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3900 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 3960 cttcaagaac tctgtagcac cgctcatata cctcgctctg ctaatcctgt taccagtggc 4020 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4080 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4140 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4200 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4260 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4320 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4380 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4440 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4500 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 4560 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 4620 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 4680 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 4740 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg agctcactct 4800 atcattgata gagtttgaaa ctctatcatt gatagagtat aatatctttg ttcatttaag 4860 ccatctacta aacaagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 4920 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttg 4966 <210> 35 <211> 400 <212> DNA <213> Artificial sequence <220> <223> Upstream fragment of catB gene <400> 35 gtctttacac ttttgcccat taatttttga gttccttatt tttagggagc ttttattatt 60 tttatcatga aaatttcata aaatactcat aaactaagga tgtcttcata atcagattag 120 tactccattt tcaatccatt taatctggga atatgatatt ttaattacgt attatttaag 180 atatattaac gtgtaatata ataccccgca aatattaatt atcacataca tatcccccct 240 [[ID=3,0]]ttattggggc attttttgta cccattattt tagtattgtg cagtacttaa ataaaaaaat 300 gccgcaaatt catttttatt gaataatgcg gtatttcttc tattctttat ttttattact 360 ctataaataa tgtaatcaag acatgactat ctaaatatat 400 <210> 36 <211> 400 <212> DNA <213> Artificial sequence <220> <223> Downstream fragment of catB gene <400> 36 aattcataat tcgggcctcc taaaaatttt cgtaattcta ttttagaagg cttttttccg 60 tgacctagcc atttcaatct cctttttaca atgatattta cgctttagtt tattatagca 120 cattctgtaa taccgaacta ttcaattttc agagaccatt ttttattgat tcataactta 180 agaatactac gaattactct aatattttac tttttcttat ctcttgttat tttaacatcg 240 gaattactac taatattaat ttttattttt ccatccgcat ttgctccaac atttttttaa 300 ctatactttc cttttgttaa taaattatgt tattgttgaa caatataaga aaagtgcgta 360 acatttttta ttaaaaataa ttaggtattt ctatctgtgg 400 <210> 37 <211> 218 <212> PRT <213> Clostridium beijerinckii <400> 37 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Lys 50 55 60 His Lys Glu Phe Arg Ile Cys Asp His Glu Gly Ser Leu Gly Tyr Trp 65 70 75 80 Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu Thr 85 90 95 Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe Tyr 100 105 110 Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys Phe 115 120 125 Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser Ile 130 135 140 Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu Gly 145 150 155 160 Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln Glu 165 170 175 Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile Cys 180 185 190 Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu Ala 195 200 205 Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 38 <211> 9113 <212> DNA <213> Artificial sequence <220> <223> pCas9ind - gRNA_catB <400> 38 catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60 tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120 acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180 agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240 ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300 actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360 taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420 gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480 tatgattaaa tttagaggac atttttaat agaaggtgat ttaaacccag acaacagcga 540 tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600 aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660 aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac ttttggggaa 720 cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780 agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840 tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900 tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960 tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020 acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080 tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140 agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200 aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260 tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320 tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380 aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440 agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500 gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560 ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620 gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680 cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740 atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaaat 1800 tataaaagat agaattttt tagaatga agaaaatgaa gatatcttg aagatatagt 1860 attackacattg acacttttg aagatagaga atgatagaa gaagattaaaacatatgc 1920 acatcttttt gatgatagg tttagaagca acttaaaga agagatata caggttgggg 1980 acgtttgtca agaagctaa ttaatggtat tagagataa caatcaggaa agactattct 2040 cgattttctt aaatcagatg gatttgctaa tagaacttt atgcaattaa ttcatgatga 2100 ttctcttact ttcaagagg atattcaaa ggctcaagtt tctggacaag gcgatagctt 2160 acacgaacac attgctacc ttgcaggg acccgctatc aaaaaaggaa ttttacaacc 2220 agttaaagtt gtagatgaac ttgttaaagt tattgggaga cacaacctg agaatagt 2280 tatagaaatg gccagagaaa atcaacaac aaaaaagg ctagagagag 2340 aatgagaga attgagaag gaataaaga gctaggatca caatatta aagacatcc 2400 agttgaaaat actcaattgc aaatgaaa gttatatttg tattacttac aaatggaag 2460 agatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520 tattgttcct caatcatttt windows ttcaatcgat aaaaagtat windows 2580 agataaaat agaggaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640 gaactattgg agacaacttt taaatgcaaa gctattaca CAAAAAAT ttgacaattt 2700 aaaaaaaaagggaaaaaaaaaaaaaaaaaaaaaaaaaaaaggcha 2760 acttgttgag acacaaa taactaagca tgttgctcaa atacttgatt cagaatgaa 2820 tachaaat gatgaaatg aataatt cagagaagta aaagtaata cattaagtc 2880 aaaattagta tcagatttca gaaaggattt tcaatttac aaagttcgtg aataataa 2940 ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000 atatcctaaa ctagaaagcg aatttgttta tggagatt aaagtttatg atgtgcgcaa 3060 aatgatcgcg aaatccgaac aagaatcgg taaggctaca gcaaatatt tctttattag 3120 taatataatg aattttttta agacagaaat aactttggct aatggtgaa tcagaaaag 3180 accacttatc gaacaatg gagacacagg agaatagta tgggataag gagagattt 3240 tgctactgtt agaaaagtac taagtatgcc acagtaaat atcgtaaga aactgaagt 3300 tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360 tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420 ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480 aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540 cttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600 gtatagcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660 tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720 tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780 acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840 tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900 accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960 accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020 agagtatta gacgcaactt tatacatca atctattaca gggctttatg aaacaagaat 4080 tgatttaagt caactaggcg gagattaagt cgacaagta ttgttaaaaa taactctgta 4140 gattataaa ttagttctac agagttatttt ttgacccgg gtatattgat aaaataata 4200 atagtgggta tattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260 gtaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa gtttaacaa 4320 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380 ataagcgggc ttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440 ttgcccttt agaagggaa agctggcaag atttttcg taatacgct aaagtttta 4500 gatgtgcttt actaagtcat cgcgatggag caaagtaca tttaggtaca cggcctacag 4560 aaaaacagta tgaactctc gaaaatcaat tagccttttttgccaacaa ggttttcac 4620 tagagaatgc attatgca ctcagcgctg tggggcatttt tactttaggt tgcgtattgg 4680 aagatcaag gcatcaagtc gctaagaag aaagggaac acctactact gatagtagc 4740 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860 cttaaaagca gkataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920 tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160 gctcttcgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120 gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180 ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240 acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300 aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360 agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420 tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480 agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540 taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600 agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660 attccctaac aaacagaggt ataaattgt tgggagtatt ccttaccatt taagcacaca 6720 aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780 aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840 agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900 aaacagtgtc ttataaaac ttacccgcca taccacagat gttccagata atattggaa 6960 gctatacg tactttgttt caaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020 aaatcagtttt catcaagcaa tgaacacgc aaatcagtt aatttaagta ccgttactta 7080 tgagcaagta ttgtctattt ttatagtta tctattttt aacgggagga ataatttcta 7140 tgagtcccta ggcaggccctc cgccattatt ttttgaaca attgacaatt cattttcttat 7200 ttttattaa gtgagtca aaaggcataa cagtgctgaa tagaaagaa tttacagaaa 7260 agaaaattat agaatttagt atgattaattt attackcatttt atgaatgtttt aatgaatac 7320 aaaaaaaaat acttgttatg tattcaatta cgggttaaa tatagacaag ttgaaaaatt 7380 tataaaaaa ataagctccctc agctcttata tattaagcta ccaacttagt ataagcca 7440 aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaattt 7500 caatgtacc gaatacaag agaacatta actatatta ttcaatttat gagattatct 7560 taacagatat aaatgtaaat tgcaatagt aagatttaga agtttatagc ctttgtgtat 7620 tggaagcagt acgcaaggc tttttttt gataaaaatt agaagtatat ttttttc 7680 atattaattt tatgaaatg aaagggggtg agcaagtga cagaggaaag cagtatctta 7740 tcaataca aggtattagc atatcatta ttgacttag cagtaaacat tatgactttt 7800 atagtgcttg tagctaagta gtacgaaagg gggagcttta aaagctcct tggaatacat 7860 agaattcata attaattta tgaaagaag ggcgtatatg aaacttgta aaattgcaa 7920 agagtttatt aagatactg aaatatgcaa atacattcg ttgatgattc atgataaaac 7980 agtagcaacc tattgcagta atacaatga gtcaatgt ttacataag ggaagtcca 8040 atgtattaat tgttcaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100 tacagaggaa gacagaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160 aaagctcat gtaagaga gtaaaaga aaaataattt atttattaat ttatattga 8220 gagtgccgac acagtagca ctaaaaaata tatctgtggt gtagtgagcc gatacaaag 8280 gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340 acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400 taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460 aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 8520 tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580 ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640 tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700 taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760 tatggattat aagcggctcg aggacgtcaa accatgttaa tcattgcttt tatcaaaaat 8820 aggatccact ctatcattga tagagtttga aactctatca ttgatagagt ataatatctt 8880 tgttcatgta catcatgcta tctgtgagtt ttagagctag aaatagcaag ttaaaataag 8940 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttgtc 9000 tttacacttt tgcccctcga gtccctatca gtgatagatt gaaactctat cattgataga 9060 gtataatatc tttgttcatt agagcgataa acttgaattt gagagggaac ttc 9113 <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer pNF2 <400> 39 gggcgcactt atacaccacc 20 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer pNF2 <400> 40 tgctacgcac cccctaaagg 20 <210> 41 <211> 50 <212> DNA <213> Artificial sequence <220> <223> ΔcatB_gRNA_rev <400> 41 aatctatcac tgatagggac tcgaggggca aaagtgtaaa gacaagcttc 50 <210> 42 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer pCas9ind_fwd <400> 42 agctcttgat ccggcaaaca 20 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer pCas9ind_rev <400> 43 gcaaccctag tgttcggtga 20 <210> 44 <211> 219 <212> PRT <213> Clostridium butyricum <400> 44 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45<00​​​​​​​​​​​​​​​​​​​ Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 45 <211> 219 <212> PRT <213> Clostridium beijerinckii <400> 45 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ile Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile<0035 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 46 <211> 219 <212> PRT <213> Clostridium beijerinckii <400> 46 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Glu Glu Phe Arg Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Gly Asn Lys Val Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 47 <211> 219 <212> PRT <213> Clostridium beijerinckii <400> 47 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Ser Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 48 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Clostridium sp. 2-1 <400> 48 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Gln Pro Asp Asn Thr Phe Ser Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 49 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Clostridium diolis <400> 49 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 50 <211> 219 <212> PRT <213> Clostridium beijerinckii <400> 50 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ile Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Gln Pro Asp Asn Thr Phe Ser Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Asn Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Lys Glu Trp Leu Glu Asn Lys 210 215 <210> 51 <211> 221 <212> PRT <213> Clostridium beijerinckii <400> 51 Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys Tyr Ile 210 215 220 <210> 52 <211> 219 <212> PRT <213> Clostridium beijerinckii <400> 52 Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Arg Glu Trp Leu Glu Asn Lys 210 215 <210> 53 <211> 219 <212> PRT <213> Clostridium saccharoperbutylacetonicum <400> 53 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Thr Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Ile Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Ile Phe Pro Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 54 <211> 219 <212> PRT <213> See also photo(Clostridium saccharoperbutylacetonicum) <400> 54 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Thr Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn With Glu Is Thr Asp With Tyr Glu With Lys With Lys Asn 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 Lys Glu Phe Arg Ile Cys Phe Asp is Lys Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe Tyr Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Ile Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Ile Phe Pro Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys 210 215 <210> 55 <211> 219 <212> PRT <213> Clostridium beijerinckii <400> 55 Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Arg Ser Asp Asn Thr Phe Pro Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Arg Glu Trp Leu Glu Asn Lys 210 215 <210> 56 <211> 221 <212> PRT <213> Clostridium beijerinckii <400> 56 Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Asn Arg Lys Pro Phe 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile 35 40 45 Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Glu Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe 100 105 110 Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Cys Asn Glu 145 150 155 160 Gly Thr Tyr Leu Thr Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln 165 170 175 Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ser Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys Tyr Ile 210 215 220 <210> 57 <211> 219 <212> PRT <213> Clostridium beijerinckii <400> 57 Met Asn Phe Asn Leu Ile Asp Ile Lys His Trp Ser Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Arg Leu Lys Asn Ile 35 40 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Thr Cys Phe Asp His Ser Gly Ser Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Pro Arg Phe 100 105 110 Tyr Ser Asp Tyr Phe Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys 115 120 125 Phe Thr Pro Lys Leu Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu 145 150 155 160 Gly Thr Tyr Leu Ile Pro Ile Phe Thr Thr Gly Lys Tyr Phe Lys Gln 165 170 175 Glu Asn Lys Met Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile 180 185 190 Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu 195 200 205 Ala Phe Ser Phe Gln Asp Trp Leu Glu Asn Lys 210 215 <210> 58 <211> 219 <212> PRT <213> Clostridium botulinum <400> 58 Met Lys Phe Asn Leu Ile Asp Ile Glu His Trp Asn Arg Lys Pro Tyr 1 5 10 15 Phe Glu Tyr Tyr Leu His Ser Val Arg Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asn Leu Leu His Glu Ile Lys Leu Lys Lys Leu 35 4x0 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn 50 55 60 His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Gly Asn Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Asp Tyr Asp Glu Ser Phe Ser Cys Phe 100 105 110 Tyr Asn Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Ala Ile Met Lys 115 120 125 Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Val Ser Ser 130 135 140 Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asn 145 150 155 160 Gly Thr Tyr Leu Val Pro Ile Phe Thr Met Gly Lys Tyr Phe Glu Gln 165 170 175 Asn Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Ile Ser Arg Phe Ile Asn Glu Val Gln Glu Leu 195 200 205 Ala Leu Asn Ser Gln Thr Trp Leu Lys His Lys 210 215 <210> 59 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Anaerocolumna aminovalerica <400> 59 Met Lys Phe Asn Leu Ile Asp Ile Glu Asn Trp Asn Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Met Thr Ala [[ID=2​​​​​​​​​​​​​​​​​​​​​​​​​Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Ser 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asp 145 150 155 160 Gly Lys Tyr Leu Leu Pro Ile Phe Thr Thr Gly Lys Tyr Phe Glu Gln 165 170 175[[ID=ll]] Asn Ser Lys Ile Phe Ile Pro Met Ser Val Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Ile Ser Arg Phe Ile Asn Glu Val Gln Glu Val 195 200 205 Ile Leu Asn Tyr Gln Thr Trp Leu Gly Asp Lys 210 215 <210> 60 <211> 219[[ID=z7]] <212> PRT <21l> 人工序列 <220> ]><223> Desnuesiella massiliensis <400> 60 Met Lys Phe Asn Leu Ile Asp Ile Glu His Trp Asn Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asn Leu Leu His Asp Ile Lys Leu Lys Lys Leu 35 40 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn 50 55 60 His Glu Glu Phe Arg Thr Cys Phe Tyr Glu Asn Gly Asn Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu 85 90 95 Thr Phe Ser Glu Ile Trp Ser Glu Tyr Asp Glu Ser Phe Ser Cys Phe 100 105 110 Tyr Ser Lys Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asp Ile Met Arg 115 120 125 Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asp 145 150 155 160 Gly Arg Tyr Leu Val Pro Ile Phe Thr Ile Gly Lys Tyr Phe Glu Gln 165 170 175 Asn Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu 195 200 205 Ala Leu Asn Ser Gln Thr Trp Leu Arg His Lys 210 215 <210> 61 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Clostridium sp. HMP27 <400> 61 Met Lys Phe Asn Leu Ile Asp Thr Glu His Trp Asn Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Ile Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asn Leu Leu His Asp Ile Lys Gln Lys Lys Leu 35 40 45 Lys Leu Tyr Pro Thr Phe Ile Tyr Ile Ile Ala Thr Val Val Asn Thr 50 55 60 His Lys Glu Phe Arg Thr Cys Phe Asp Glu Ser Gly Asn Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Lys Ser Phe Ser Cys Phe 100 105 110 Tyr Ser Lys Tyr Leu His Asp Ile Lys Asn Tyr Gly Asp Ile Met Ser 115 120 125 Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp 145 150 155 160 Gly Thr Tyr Leu Val Pro Ile Phe Thr Ile Gly Lys Tyr Phe Lys Gln 165 170 175 Ala Asp Lys Ile Leu Ile Pro Ile Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu 195 200 205 Ile Leu Asn Tyr Gln Thr Trp Leu Lys His Lys 210 215 <210> 62 <211> 219 <212> PRT <213> Artificial Sequence [[ID=...]]<220> <223> Clostridium drakei <400> 62 Met Lys Phe Asn Leu Ile Asp Ile Glu Asn Trp Asn Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu 35 40 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Ala Val Ile Asn Arg 50 55 60 His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Arg Lys Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Ser Pro Ser Tyr Thr Val Phe His Lys Glu Asp Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Pro Arg Phe 100 105 110 Tyr Asp Asn Tyr Leu Asp Asp Ile Lys Ser Tyr Gly Asp Val Leu Lys 115 120 125 Phe Met Pro Lys Pro Asp Glu Pro Gly Asn Thr Phe Asn Val Ser Ser 130 135 140 Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp 145 150 155 160 Ala Thr Tyr Leu Ile Pro Ile Phe Thr Met Gly Lys Phe Phe His Gln 165 170 175 Asp Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Val Gln Glu Leu 195 200 205 Ser Ser Asn Phe Glu Thr Trp Leu Asp Glu Lys 210 215 <210> 63 <211> 219 <212> PRT <213> Clostridium scatologenes <4​​​​​​​​​​​​​​​​​​​​​​​​​Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Pro Arg Phe 100 105 110 Tyr Asp Asn Tyr Leu Asp Asp Ile Lys Ser Tyr Gly Asp Val Leu Lys 115 120 125 Phe Met Pro Lys Pro Asp Glu Pro Gly Asn Thr Phe Asn Val Ser Ser 130 135 140 Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp 145 150 155 160 Ala Thr Tyr Leu Ile Pro Ile Phe Thr Met Gly Lys Phe Phe His Gln 165 170 175 Asp Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Val Gln Glu Leu 195 200 205 Ser Ser Asn Phe Glu Thr Trp Leu Gly Glu Lys 210 215 <210> 64 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Clostridium tunisiense <400> 64 Met Lys Phe Asn Leu Ile Asp Thr Glu His Trp Asp Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Phe Asn Ser Val Lys Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asn Leu Leu Asn His Ile Arg Leu Lys Lys Leu 35 40 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn 50 55 60 His Glu Glu Phe Arg Ile Cys Phe Asp Glu Asn Asn Asn Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Ser Pro Asn Tyr Thr Ile Phe His Glu Asp Asn Lys 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Glu Glu Ser Phe Ser Gly Phe 100 105 110 Tyr Asn Lys Tyr Leu Glu Asp Ile Lys Thr Tyr Gly His Ile Met Ser 115 120 125 Phe Glu Pro Lys Leu Asn Glu Ser Thr Asn Thr Phe Pro Ile Ser Cys 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Gln Asp Asp 145 150 155 160 Gly Thr Tyr Leu Thr Pro Ile Phe Thr Leu Gly Lys Tyr Phe Glu Gln 165 170 175 Asn Asn Lys Thr Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu 195 200 205 Ala Ser Asp Phe Gln Ile Trp Leu Thr Tyr Lys 210 215 <210> 65 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Lachnospiraceae <400> 65 Met Lys Phe Asn Leu Ile Asp Ile Glu Asp Trp Asn Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu 35 40 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Thr Val Val Asn Arg 50 55 60 His Lys Glu Phe Arg Thr Cys Phe Asp Gln Lys Gly Lys Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Asp Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Asn Phe Pro Arg Phe 100 105 110 Tyr Tyr Asn Tyr Leu Glu Asp Ile Arg Asn Tyr Ser Asp Val Leu Asn 115 120 125 Phe Met Pro Lys Thr Gly Glu Pro Ala Asn Thr Ile Asn Val Ser Ser 130 135 140 Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp 145 150 155 160 Ala Thr Tyr Leu Ile Pro Ile Phe Thr Leu Gly Lys Tyr Phe Gln Gln 165 170 175 Asp Asn Lys Ile Leu Leu Pro Met Ser Val Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Ala Gln Glu Leu 195 200 205 Ala Ser Asn Tyr Glu Thr Trp Leu Gly Glu Lys 210 215 <210> 66 <211> 219 <212> PRT <213> Clostridium perfringens <400> 66 Met Lys Phe Asn Leu Ile Asp Ile Glu Asp Trp Asn Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala 20 25 30 Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu 35 40 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Thr Val Val Asn Arg 50 55 60 His Lys Glu Phe Arg Thr Cys Phe Asp Gln Lys Gly Lys Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Asp Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Asn Phe Pro Arg Phe 100 105 110 Tyr Tyr Asn Tyr Leu Glu Asp Ile Arg Asn Tyr Ser Asp Val Leu Asn 115 120 125 Phe Met Pro Lys Thr Gly Glu Pro Ala Asn Thr Ile Asn Val Ser Ser 130 135 140 Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp 145 150 155 160 Ala Thr Tyr Leu Ile Pro Ile Phe Thr Leu Gly Lys Tyr Phe Gln Gln 165 170 175 Asp Asn Lys Ile Leu Leu Pro Met Ser Val Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Ile Ser Arg Phe Phe Asn Glu Ala Gln Glu Leu 195 200 205 Ala Ser Asn Tyr Glu Thr Trp Leu Gly Glu Lys 210 215 <210> 67 <211> 218 <212> PRT<0​​​​​​​​​​​​​​​​​​​​​​50 55 60 His Glu Glu Phe Arg Thr Cys Phe Asp Glu Asn Asn Asn Leu Gly Tyr 65 70 75 80 Trp Asp Ser Met Ser Pro Asn Tyr Thr Ile Phe His Glu Glu Thr Lys 85 90 95 Thr Phe Ser Asn Ile Trp Thr Glu Tyr Asp Lys Ser Phe Ser Gly Phe 100 105 110 Tyr Asn Lys Tyr Val Glu Asp Asn Lys Asn Tyr Gly Asn Ile Met Asn 115 120 125 Phe Asp Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys 130 135 140 Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Gln Asp His 145 150 155 160 Gly Thr Tyr Leu Thr Pro Ile Phe Thr Leu Gly Lys Tyr Phe Glu Glu 165 170 175 Asn Asn Lys Val Phe Ile Pro Met Ser Ile Gln Val His His Ala Val 180 185 190 Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu 195 200 205 Ala Ser Asn Ser Gln Ser Trp Leu Lys His 210 215 <210> 68 <211> 660 <212> DNA <213> Clostridium perfringens <400> 68 atgaaattta atttgataga tattgaggat tggaatagaa agccatactt tgagcattat 60 ttaaatgcgg ttaggtgcac ttacagtatg actgcaaata tagagataac tggtttactg 120 cgtgaaatta aacttaaggg cctgaaactg taccctacgc ttatttatat catcacaact 180 gtggttaacc gtcacaagga gttccgcacc tgttttgatc aaaaaggtaa gttaggatac 240 tgggatagta tgaacccaag ttactgtc tttcataagg ataacgaaac tttttcaagt 300 atttggacag agtatgacga gaacttccca cgtttttact ataattacct tgaggatatt 360 agaaactata gcgacgtttt gaatttcatg cctaagacag gtgaacctgc tatacaatt 420 aatgtgtcca gcattccttg ggtgaatttt accggattca acctgaatat atacaatgat 480 gcaacatatc taatccctat ttttactttg ggtaagtatt ttcagcagga tataaaatt 540 ttattaccta tgtctgtaca ggtgcatcat gcggtttgcg acggttatca tataagcaga 600 ttttttaatg aggcacagga attagcgtca aattatgaga catggttagg agaaaaataa 660 <210> 69 <211> 624 <212> DNA <213> Clostridium difficile <400> 69 atggtatttg aaaaaattga tgaaaatagt tggacagaa aagagtattt tgaccactac 60 ttgcaagtg taccttgtac atacagcatg accgttaaag tggatatcac acaataag 120 gaaaagggaa tgaaactata tcctgcaatg ctttatta ttgcaatgat tgtaaaccgc 180 cattcagagt ttaggacggc aatcaatcaa gatggtgaat tgggatata tgatgagatg 240 ataccaagct atacaatatt tcaatgat actgaaact ttcagcct tggactgag 300 tgtaagtctg actttaaatc atttttagca gatttagaa gtgaacgca acggtatgga 360 aacaatcata gatggaagg aaagccaat gctccggaaa acatttttaa tgtatctatg 420 ataccgtggt caacttcga tggctttaat ctgaatttgc agaaaggata tgattatttg 480 attcctattt ttactatggg gaatattatt aaagata acaaattat acttcctttg 540 gcaattcaag ttcatcacgc agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa 600 ttgcaggaat tgataaatag ttaa 624 <210> 70 <211> 624 <212> DNA <213> Clostridium perfringens <400> 70 atggtatttg aaaaaattga taaaaatagt tggaacagaa aagagtattt tgaccactac 60 tttgcaagtg taccttgtac atacagcatg accgttaaag tggatatcac acaaataaag 120 gaaaagggaa tgaaactata tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc 180 cattcagagt ttaggacggc aatcaatcaa gatggtgaat tggggatata tgatgagatg 240 ataccaagct atacaatatt tcacaatgat actgaaacat tttccagcct ttggactgag 300 tgtaagtctg actttaaatc atttttagca gattatgaaa gtgatacgca acggtatgga 360 aacaatcata gaatggaagg aaagccaaat gctccggaaa acatttttaa tgtatctatg 420 ataccgtggt caaccttcga tggctttaat ctgaatttgc agaaaggata tgattatttg 480 attcctattt ttactatggg gaaatattat aaagaagata acaaaattat acttcctttg 540 gcaattcaag ttcatcacgc agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa 600 ttgcaggaat tgataaatag ttaa 624 <210> 71 <211> 3897 <212> DNA <213> Artificial sequence <220> <223> Optimized MAD7 <400> 71 ctcgagtccc tatcagtgat agattgaaac tctatcattg atagagtata atatctttgt 60 tcattagagc gataaacttg aatttgagag ggaacttaga tgaacaacgg cacaaataat 120 tttcagaact tcatagggat atcaagtttg cagaaaacgt taagaaatgc tttaataccc 180 acggaaacca cgcaacagtt catagttaag aacggaataa ttaaagaaga tgagttaaga 240 ggcgagaaca gacagatttt aaaagatata atggatgact actacagagg attcatatct 300 gagactttaa gttctattga tgacatagat tggactagct tattcgaaaa aatggaaatt 360 cagttaaaaa atggtgataa taaagatacc ttaattaagg aacagacaga gtatagaaaa 420 gcaatacata aaaaatttgc gaacgacgat agatttaaga acatgtttag cgccaaatta 480 attagtgaca tattacctga atttgttata cacaacaata attattcggc atcagagaaa 540 gaggaaaaa cccaggtgat aaaattgttt tcgagatttg cgactagctt taaagattac 600 ttcaagaaca gagcaaattg cttttcagcg gacgatattt catcaagcag ctgccataga 660 atagttaacg acaatgcaga gatattcttt tcaaatgcgt tagtttacag aagaatagta 720 aaatcgttaa gcaatgacga tataaacaaa atttcgggcg atatgaaaga ttcattaaaa 780 gaaatgagtt tagaagaaat atattcttac gagaagtatg gggaatttat tacccaggaa 840 ggcattagct tctataatga tatatgtggg aaagtgaatt cttttatgaa cttatattgt 900 cagaaaaata aagaaaacaa aaattttac aaacttcaga aacttcacaa acagattcta 960 tgcattgcgg acactagcta tgaggttccg tataaatttg aaagtgacga ggaagtgtac 1020 caatcagtta acggcttcct tgataacatt agcagcaaac atatagttga aagattaaga 1080 aaaataggcg ataactataa cggctacaac ttagataaaa tttatatagt gtccaaattt 1140 tacgagagcg ttagccaaaa aacctacaga gactgggaaa caattaatac cgccttagaa 1200 attcattaca samaaatatt gccgggtaac ggtaaaagta aagccgacaa agtaaaaaaa 1260 gcggttaaga atgatttaca gaaatccata accgaaataa atgaactagt gtcaaactat 1320 aagttatgca gtgacgacaa cataaaagcg gagacttata tacatgagat tagccatata 1380 ttgaataact ttgaagcaca ggaattgaaa tacaatccgg aaattcacct agttgaatcc 1440 gagttaaaag cgagtgagct taaaaacgtg ttagacgtga taatgaatgc gtttcattgg 1500 tgttcggttt ttatgactga ggaacttgtt gataaagaca acaattttta tgcggaatta 1560 gaggagattt acgatgaaat ttatccagta attagtttat acaacttagt tagaaactac 1620 gttacccaga aaccgtacag cacgaaaaag attaaattga actttggaat accgacgtta 1680 gcagacggtt ggtcaaagtc caaagagtat tctaataacg ctataatatt aatgagagac 1740 aatttatatt atttaggcat atttaatgcg aagaataaac cggacaagaa gattatagag 1800 ggtaatacgt cagaaaataa gggtgactac aaaaagatga tttataattt gttaccgggt 1860 cccaacaaaa tgataccgaa agttttcttg agcagcaaga cggggtgga aacgtataaa 1920 ccgagcgcct atatactaga ggggtataaa cagaataaac atataaagtc ttcaaaagac 1980 tttgatataa ctttctgtca tgatttaata gactacttca aaaactgtat tgcaattcat 2040 cccgagtgga aaaacttcgg ttttgatttt agcgacacca gtacttatga agacatttcc 2100 gggttttata gagaggtaga gttacaaggt tacaagattg attggacata cattagcgaa 2160 aaagacattg atttattaca ggaaaaaaggt caattatatt tattccagat atataacaaa 2220 gatttttcga aaaaatcaac cgggaatgac aaccttcaca ccatgtactt aaaaaatctt 2280 ttctcagaag aaaatcttaa ggatatagtt ttaaaactta acggcgaagc ggaaatattc 2340 ttcaggaaga gcagcataaa gaacccaata attcataaaa aaggctcgat tttagttaac 2400 agaacctacg aagcagaaga aaaagaccag tttggcaaca ttcaaattgt gagaaaaaat 2460 attccggaaa acatttatca ggagttatac aaatacttca acgataaaag cgacaaagag 2520 ttatctgatg aagcagccaa attaaagaat gtagtgggac accacgaggc agcgacgaat 2580 atagttaagg actatagata cacgtatgat aaatacttcc ttcatatgcc tattacgata 2640 aatttcaaag ccaataaaac gggttttatt aattagga tattacagta tatagctaaa gaaaaagact tacatgtgat aggcattgat agaggcgaga gaaacttaat atacgtgtcc gtgattgata cttgtggtaa tatagttgaa cagaaagct ttaacattgt aaacggctac 2880. snowfall snowfall snowfall snowfall snowfall snowfall aaagaaattg gtaaaattaa agagataaaa gagggctact tagcttagt aatacacgag 3000. 3000. 3000. 3000. 3000. 3000. 3000. 3000 tttaaaaaag ggagatttaa ggttgaaaga caagtttacc agaaatttga aaccatgtta father taaactattt agtatttaa throwing ttaccgaga tggcggttta ttaaaaggtt atcagttaac attackatcct throw aaaacgtggg tcatcagtgc ggctgcattt tttatgtgcc tgctgcatac acgagcaaaa ttgatccgac caccggcttt 3240 gtgaatat ttaatttaa agacttaaca gtggacgcaa aaagagaatt cattaaaaaa tttgactcaa ttagatga cagtgaaaaa aatttattct gctttacatt tgactacaat aactttatta cgcaaaacac ggttatgagc aaatcatcgt ggagtgtgta tacatacggc 3420 gtgagaataa aaagaagatt tgtgaacggc agattctcaa acgaaagtga taccattgac 3480 ataaccaaag atatggagaa aacgttggaa atgacggaca ttaactggag agatggccac 3540 gatcttagac aagacattat agattatgaa attgttcagc acatattcga aattttcaga 3600 ttaacagtgc aaatgagaaa ctccttgtct gaattagagg acagagatta cgatagatta 3660 atttcacctg tattaaacga aaataacatt ttttatgaca gcgcgaaagc gggggatgca 3720 cttcctaagg atgccgatgc aaatggtgcg tattgtattg cattaaaagg gttatatgaa 3780 attaaacaaa ttaccgaaaa ttggaaagaa gatggtaaat tttcgagaga taaattaaaa 3840 ataagcaata aagattggtt cgactttata cagaataaga gatatttata agtcgac 3897 <210> 72 <211> 1263 <212> PRT [[ID=2,4]]<213> Artificial Sequence <220> <223> MAD7 <400> 72 Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile Ser Ser 1 5 10 15 Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr Thr Gln 20 25 30 Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu Arg Gly 35 40 45 Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr Arg Gly 50 55 60 Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp Thr Ser 65 70 75 80 Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn Lys Asp 85 90 95 Thr Leu Ile Lys Glu Gln Thr Glu Tyr Arg Lys Ala Ile His Lys Lys 100 105 110 Phe Ala Asn Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys Leu Ile 115 120 125 Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr Ser Ala 130 135 140 Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser Arg Phe 145 150 155 160 Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys Phe Ser 165 170 175 Only Asp Asp Ile Ser Ser Ser Cys His Arg Ile Val Asn Asn Asn 180 185 190 Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile Val Lys 195 200 205 Ser Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Met Lys Asp 210 215 220 Ser Leu Lys Glue Meets Ser Leu Glu Glu Ile Tyr Ser Tyr Glu Lys Tyr 225 230 235 240 Gly Glu Phe with Thr Gln Glu Gly with Phe Tyr Asn and Cys 245 250 255 Gly Lys Val Is Phe Met With Leu Tyr Cys Gln Lys Asn Lys Glu 260 265 270 Asn Lys Asn Your Tyr Lys Your Gln Lys Your His Lys Gln And Your Cys 275 280 285 Has Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser Asp Glu 290,295,300 Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser Ser Lys 305 310 315 320 His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn Gly Tyr 325 330 335 Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser Val Ser 340 345 350 Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu Glu Ile 355 360 365 His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala Asp Lys 370 375 380 Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr Glu Ile 385 390 395 400 Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Ser Asp Asp Asn Ile Lys 405 410 415 Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn Phe Glu 420 425 430 Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu Ser Glu 435 440 445 Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met Asn Ala 450 455 460 Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp Lys Asp 465 470 475 480 Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile Tyr Pro 485 490 495 Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln Lys Pro 500 505 510 Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr Leu Ala 515 520 525 Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile Ile Leu 530 535 540 Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys Asn Lys 545 550 555 560 Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys Gly Asp 565 570 575 Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys Met Ile 580 585 590 Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr Lys Pro 595 600 605 Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Ile Lys Ser 610 615 620 Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp Tyr Phe 625 630 635 640 Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly Phe Asp 645 650 655 Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr Arg Glu 660 665 670 Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser Glu Lys 675 680 685 Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe Gln Ile 690 695 700 Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn Leu His 705 710 715 720 Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys Asp Ile 725 730 735 Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser Ser 740 745 750 Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val Asn Arg 755 760 765 Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln Ile Val 770 775 780 Arg Lys Asn Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys Tyr Phe 785 790 795 800 Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys Leu Lys 805 810 815 Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys Asp Tyr 820 825 830 Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr Ile Asn 835 840 845 Phe Lys Ala Asn Lys Thr Gly Phe Ile Asn Asp Arg Ile Leu Gln Tyr 850 855 860 Ile Ala Lys Glu Lys Asp Leu His Val Ile Gly Ile Asp Arg Gly Glu 865 870 875 880 Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn Ile Val 885 890 895 Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln Ile Lys 900 905 910 Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu Trp Lys 915 920 925 Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser Leu Val 930 935 940 Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile Ile Ala 945 950 955 960 Met Glu Asp Leu Ser Tyr Gly Phe Lys Gly Arg Phe Lys Val Glu 965,970,975 Arg Gln Val Tyr Gln Lys Phe Glu Thr Met With Lys Leu Asn 980,985,990 Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly Leu Leu 995 1000 1005 Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn Val 1010 1015 1020 Gly His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr 1025 1030 1035 Ser Lys Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe 1040 1045 1050 Lys Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe 1055 1060 1065 Asp Served With Arg Tyr Asp Served With Glu Lys Asn Leu Phe Cys Phe Thr 1070 1075 1080 Phe Asp Tyr Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys 1085 1090 1095 Ser Ser Trp Ser Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg 1100 1105 1110 Phe Val Asn Gly Arg Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile 1115 1120 1125 Thr Lys Asp Met Glu Lys Thr Leu Glu Met Thr Asp Ile Asn Trp 1130 1135 1140 Arg Asp Gly His Asp Leu Arg Gln Asp Ile Ile Asp Tyr Glu Ile 1145 1150 1155 Val Gln His Ile Phe Glu Ile Phe Arg Leu Thr Val Gln Met Arg 1160 1165 1170 Asn Ser Leu Ser Glu Leu Glu Asp Arg Asp Tyr Asp Arg Leu Ile 1175 1180 1185 Ser Pro Val Leu Asn Glu Asn Asn Ile Phe Tyr Asp Ser Ala Lys 1190 1195 1200 Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr 1205 1210 1215 Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln Ile Thr Glu 1220 1225 1230 Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu Lys Ile 1235 1240 1245 Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr Leu 1250 1255 1260 <210> 73 <211> 363 <212> DNA <213> Artificial sequence <220> <223> CatB promoter <400> 73 taaaaaatgt tacgcacttt tcttatattg ttcaacaata acataattta ttaacaaaag 60 gaaagtatag ttaaaaaaat gttggagcaa atgcggatgg aaaaataaaa attaatatta 120 gtagtaattc cgatgttaaa ataacaagag ataagaaaaa gtaaaatatt agagtaattc 180 gtagtattct taagttatga atcaataaaa aatggtctct gaaaattgaa tagttcggta 240 ttacagaatg tgctataata aactaaagcg taaatatcat tgtaaaaagg agattgaaat 300 ggctaggtca cggaaaaaag ccttctaaaa tagaattacg aaaattttta ggaggcccga 360 att 363 <210> 74 <211> 322 <212> DNA <213> Artificial sequence <220> <223> CATQ promoter <400> 74 ctgcgtacac atccagacat cgctttagag tatggtgaat taaagatgga gcgggcttat 60 cgattctcag aggatattga aggctactgc actggtaagg atgcatttgt aaagcaacta 120 gaaaaggatg ctttgcgatg gtggcaact gtctgttagg aggttattct haaggattg 180 CAagaagcag ttgaggataa tccgtataac taactattac acatcttta cattgctggt 240 ttgtatcggt agaataacac gattaacaa aggatatatt ttgtagtagc aagtgtattt 300 gtttatatt ctatgaacct at 322 <210> 75 <211> 1368 <212> PRT <213> Streptococcus pyogenes (Streptococcus pyogenes) <400> 75 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Asn Leu Ile 35 40 45 Gly Ala Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Leu To Asn Ala Lys With Thr Gln Arg Lys 885,890,895 Phe Asp Asp With Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu With Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Asp Leu Ile Ile Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Leu Glu Gln Lys Gly Asn Leu Glu Wing Pro Ser Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser is Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Leu Valley Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 76 <211> 4107 <212> DNA <213> Streptococcus pyogenes <400> 76 atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860 ttaacattga ccttatttga agatagggag atgattgagg aagactta aacatatgct 1920 cacctctttg atgataggt gatgaacag cttaaacgtc gccgttatac tggttgggga 1980 cgtttgtctc gaaattgat taatgtatt agggataagc aatctggcaa aacaatatta 2040 gattttttga aatcagatgg tttgccaat cgcaattta tgcagctgat ccatgatgat 2100 agtttgacat ttaagaga cattcaaaa gcacaagtgt ctggacaag cgatagttta 2160 catgaacata ttgcaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280 attgaaatgg cacgtgaaaa tcagacaact caaagggcc agaaaaattc gcgagagcgt 2340 atgaacgaa tcgagaagg tatcaagaa taggagtc agatcttta agagcatcct 2400 gttgaaaata ctcaattgca aaatgaaag ctctatctct attatctcca aaatggaaga 2460 gatagtatg tggaccaag attagatatt atcgtttaa gtgatttaga tgtcgatcac 2520 attgttccac aaagttttcct taagacgat tcaatagaca aataggtctt aacgcgttct 2580 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300 cagacaggcg gattctccaa ggagtcatt ttaccaaaa gaattcgga caagcttatt 3360 gctcgtaaaa aagactggga tccaaaaaaa tatgtggtt ttgatagtcc aacggtagct 3420 tattcagtcc tagtggttgc taggtgga aaaggaat cgagaagtt aaatccgtt 3480 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540 ttttagaag ctaaggata taaggatt aaaaaagact taatcattaa actacctaaa 3600 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660 caaaaaggaa atgagctggc tctgccaagc aaattgtga attttta tttagctagt 3720 cattatgaaa agttgaaggg tagtccagaa gatacgaac aaaaacatt gtttgtggag 3780 cagcataagc attatttaga tgagattatt gagcaatca gtgaattttc taagcgtgtt 3840 attttagcag atgccaattt agataagtt cttagtgcat atacaaca tagagacaa 3900 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960 cccgctgctt ttataattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080 gatttgagtc agctaggagg tgactga 4107 <210> 77 <211> 1170 <212> DNA <213> Artificial Sequence <220> <223> bdhA <400> 77 atgctaagtt ttgattattc aataccaact aaagtttttt ttggaaaagg aaaaatagac 60 gtaattggag aagaaattaa gaaatatggc tcaagagtgc ttatagttta tggcggagga 120 agtataaaaa ggaacggtat atatgataga gcaacagcta tattaaaaga aaacaatata 180 gctttctatg aactttcagg agtagagcca aatcctagga taacaacagt aaaaaaaggc 240 atagaaatat gtagagaaaa taatgtggat ttagtattag caataggggg aggaagtgca 300 atagactgtt ctaaggtaat tgcagctgga gtttattatg atggcgatac atgggacatg 360 gttaaagatc catctaaaat aactaaagtt cttccaattg caagtatact tactctttca 420 gcaacagggt ctgaaatgga tcaaattgca gtaatttcaa atatggagac taatgaaaag 480 cttggagtag gacatgatga tatgagacct aaattttcag tgttagatcc tacatatact tttacagtac ctaaaaatca aacagcagcg ggaacagctg acattatgag tcacaccttt gaatcttact ttagtggtgt tgaaggtgct tatgtgcagg acggtatagc agaagcaatc ttagaacat gtataagta tggaaaaata gcaatggaga agactgatga ttacgaggct agagctaatt tgatgtgggc ttcaagttta gctaaatg gtctattatc acttggtaag gatagaaaat ggagttgtca tcctatggaa cacgagttaa gtgcatatta tgatataaca catggtgtag gacttgcaat tttaacacct aattggatgg aatattct aaatgacgat acacttcata aatttgtttc ttatggaata aatgtttggg aactatgaaa tagcacgaga ggctattaaa aatacgagag aatactttaa ttcattgggt attccttcaa agcttagaga agttggaata ggaaaagata aactagaact aatggcaaag caagctgtta gaaattctgg aggaacaata ggaagtttaa gaccaata tgcagaggat gttcttgaga tatttaaaa atctttattaa <210> 78 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> bdhB <400> 78 gtggttgatt tcgaatattc aataccaact agaatttttt tcggtaaaga taagataaat 60 gtacttggaa gagagcttaa aaaatatggt tctaaagtgc ttatagttta tggtggagga 120 agtataaaga gaaatggaat atatgataaa gctgtaagta tacttgaaaa aaacagtatt 180 aaattttatg aacttgcagg agtagagcca aatccaagag taactacagt tgaaaaagga 240 gttaaaatat gtagagaaaa tggagttgaa gtagtactag ctataggtgg aggaagtgca 300 atagattgcg caaaggttat agcagcagca tgtgaatatg atggaaatcc atgggatatt 360 gtgttagatg gctcaaaaat aaaaagggtg cttcctatag ctagtatatt aaccattgct 420 gcaacaggat cagaaatgga tacgtgggca gtaataaata atatggatac aaacgaaaaa 480 ctaattgcgg cacatccaga tatggctcct aagttttcta tattagatcc aacgtatacg 540 tataccgtac ctaccaatca aacagcagca ggaacagctg atattatgag tcatatattt 600 gaggtgtatt ttagtaatac aaaaacagca tatttgcagg atagaatggc agaagcgtta 660 ttaagaactt gtattaaata tggaggaata gctcttgaga agccggatga ttatgaggca 720 agagccaatc taatgtgggc ttcaagtctt gcgataaatg gacttttaac atatggtaaa 780 gacactaatt ggagtgtaca cttaatggaa catgaattaa gtgcttatta cgacataaca 840 cacggcgtag ggcttgcaat tttaacacct aattggatgg agtatatttt aaataatgat 900 acagtgtaca agtttgttga atatggtgta aatgtttggg gaatagacaa agaaaaaaat 960 cactatgaca tagcacatca agcaatacaa aaaacaagag attactttgt aaatgtacta 1020 ggtttaccat ctagactgag agatgttgga attgaagaag aaaaattgga cataatggca 1080 aaggaatcag taaagcttac aggaggaacc ataggaaacc taagaccagt aaacgcctcc 1140 gaagtcctac aaatattcaa aaaatctgtg taa 1173 <210> 79 <211> 65,60 <212> DNA <213> Artificial sequence <220> <223> pGRNA-ΔbdhB <400> 79 gatccccggg taccgagctc gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 60 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 120 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 180 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 240 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 300 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 360 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 420 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 480 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 540 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 600 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 660 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 720 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 780 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 840 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 900 ctgctgaagc cagttacctt cggaaaaga gttggtagct cttgatccgg caacaaacc 960 accgctgta gcggtggtttt tttgttgc aagcagcaga ttacgcgcag aaaaaaagga 1020 tctcaagaag atcctttgat ctttctacg gggtctgacg ctcagtggaa cgaaaactca 1080 cgttaaggga ttttgtcat gagattatca aaaggatct tcacctagat ccttttaaat 1140 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1200 caaagctagc ttaatactag tatatactta atgtgataag tgtctgacag ctgaccggtc 1260 taaagaggtc cctagcgcct acgggaatt tgtatcgata aggggtacaa attcccacta 1320 agcgctcggc cgggatcga tcccgggta cgtacccggc agttttctt ttcggcaag 1380 tgttcaagaa gttattaagt cgggagtgca gtcgaagtgg gcaagttga aaattcacaa 1440 aaatgtgta taatatcttt gttcattaga gcgataact tgaatttgag aggaactta 1500 gatggtattt gaaaaaatttg aaaaatag ttggacaga aaagagtatt ttgaccacta 1560 ctttgcaagt gtaccttgta cctacagcat gaccgttaaa gtggatatca cacataaa 1620 ggaaaaggga atgaaactat atcctgcaat gctttattat attgcaatga ttgtaaccg 1680 ccattcagag tttaggacgg caatcaatca agatggtgaa ttggggatat atgatgagat 1740 gataccaagc tatacatat ttcacaatga tactgaaca ttttccagcc ttggactga 1800 gtgtaagtct gactttaaat cattttagc agatttagaa agtgatagcc aacggtatgg 1860 aaacaatcat agaatggaag gaagccaaa tgctccggaa aacatttta atgtatctat 1920 gataccgtgg tcaccttcg atggctttaa tctgaatttg cagaaaggat atgattttt 1980 gattcctatt tttactatgg ggaatatta windows aaaaatta tactctcctttt 2040 ggcaattcaa gttcatcacg cagtatgtga cggatttcac atttgccgtt ttgtaaacga 2100 attgcaggaa ttgataata gttaacttca ggtttgtctg taactaaaa ctagtattta 2160 acctaggatc aaaaaaattt ccaataatcc cactctaagc cacaacacg ccctataaaa 2220 tcccgcttta atccccacttt gagacacatg taatattact ttaccccta gtatagtgat 2280 aattttttac attcaatgcc acgcaaaaaaaaggggc actataataa aagttccttc 2340 ggaactaact aaagtaaaa attatcttta caacctcccc aaaaaaaga acaggtaca 2400 agtaccctat atacaagcg taaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2460 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2520 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaat 2580 aaaaatatttt tttattaaa gtttgaaaa aattttta tattatataa tctttgaga 2640 aaagaata aaaaatgagc ctttataaa gcccatttttt ttcatatac gtataatgac 2700 gttctaatgt ttttggt acttctaaca ttagagtaat ttcttttt ttaaagcctt 2760 tttctttaag ggcttttt ttttctta atacatttta ttcctttttt ttgttgctt 2820 ttcctttagc ttttaattgc tcttgataat ttttttacc tctatattt tctctctct 2880 tatattcctt tttagaaatt attattgtca tatattttg ttctctct gtaatttcta 2940 aaactctat agagtttca ttcttatact tatattgctt attttct aaataacatc 3000 tttcagcact tctagttgct cttataactt ctcttcact taaatgttgt ctaaacatac 3060 tattaagttc taaaacatca tttaatgcct tctcaatgtc ttctgtaag ctacaagat 3120 ataatctata taaaaataat ataagctctc tgtgtcctttt taaatcatat tctcttagtt 3180 cacaagtttt tattatgtct tgtattctc cataatataa acttctttct ctataaatat 3240 aatttatttt gcttggtcta ccctttttcc ttcatatgg ttttaattca ggtaaaaatc 3300 cattttgtat ttctcttaag tcataaatat atcgtactc atctaata ttgactactg 3360 ttttgattt agagttatta cttcctggaa ctcttattat tctcgttgca tctaggctt 3420 gtctatctgc tccaaagtat tttattgat tataataata ttcttgacc gctttccata 3480 atggtaatgc tttactaggt actgcattta ttatccatat taaatacatt cctcttccac 3540 tatctattac atagtttggt taggaatac tttgatttaa atattcttt tctaagtcca 3600 ttaatacctg gtctttagtt ttgccagtttt tataataatc caagtctata aacagtgtat 3660 ttactcttt tatattttct aatcgcctac acggcttata aaaggtattt agagttatat 3720 agatatttc atcactcata tctaaatctt ttaattcagc gtatttatag tgccattggc 3780 tatatcctttt ttatctata acgctcctgg ttatccaccc tttactctta ctatgaatat 3840 tatctatata gttcttttta ttcagcttta atgcgtttct cacttattca cctccccttc 3900 tgtaaaacta agaaaattat atcatatttt caatattat taactattct taaactctta 3960 aaaaaata gagtaagtcc ccaattgaaa cttaatctat tttttagtt ttaatttatt 4020 atttttatta aaatatttta aactaatta atgattctt tttaatttt tactatttca 4080 ttcataata tattactata attackttaca ataatattt cttcattgt atattaga 4140 tgatttacta attttagttttttattattattaataattaat gtataattta tataaaaaat 4200 caaaggagct tataaattat gattatttcc aagatacta aagatttaat tttttcaat 4260 tttaacaata ctttttgtaa tattatgttt aaatttaatt gtatttttt catataataa 4320 agccgttgaa gtaaaccaat ccattttcct tatgatgtta tattaaatt taagttttat 4380 aaataatct tattattatt tattgttttt aaaaaaacta gtgaaatttc tagtgaattt 4440 4500 gattatatct ttaaatatgt tttatcaat attatcttt tctaaattta tatatatttt 4560 tattatattt attattatat tattttatt tttaagtttc ttctacag ctattaaaaa 4620 gaaacttaa aaaaaaca cgtactctaa accaataat aaactattt ttattattgc 4680 tgccttgatt ggaatagttt ttagtaaaat taatttcaat attccacaat attattat 4740 aagctagcac gcctcgagac tctatcattg atagagtttg aaacttatc attgatagag 4800 tataatatct ttgttcatgc ttattacgac atacacagt tttagagcta gaatagcaa 4860 gttaaaataa ggctagtccg ttcaactt gaaaagtgg caccgagtcg gtgcttttt 4920 tgaagcttct cgagatctcc atggacgcgt gacgtcgact cttaagaca tgtataaagt 4980 atggaaaaat agcaatggag aagactgatg attackagggc tegagctat ttgatgtggg 5040 cttcaagttt agcttaaat ggtcttatt cacttggtaa ggatagaaaa tggattgtc 5100 atcctatgga acacgagtta agtgcatatt atgatataac acatggtgta ggacttgcaa 5160 ttttaacacc taattggatg gatatattc taaatgacga tacactcat aaatttgttt 5220 cttatggaat aaatgttttgg ggaatagaca agaaaaga taactatgaa atagcacgag 5280 agctattaa aaatacgaga gatacttta attcattggg tattccttca aagcttagag 5340 aagttggaat aggaagat aaactagaac taatggcaa gcaagctgtt agaattctg 5400 gaggaacaat aggagttta agaccaata atgcagagga tgttcttgag atatttaaaa 5460 aatcttatta atagaactg tagagtatt tttataattt aaagatgtt aaagagtgag 5520 gagtaattt gttctaacgc ctcactctt tcattttatg attaatgta tgctgattta 5580 cgctaactta atcctaaat aaaccta tgttaatatt ttgtaacaa tggataaag 5640 cgtaaaaata ttattgtaat attttaagt agttttaaaa tatatataat gtagaagcat 5700 tcctacatta tattatta ataataatct aaacaggagg gggttaaagtg gttgatttca 5760 aatctgtgta aacctaccgg ggtttggggg tagccattat attcatgac tccaagaag 5820 cagtatgcta gcaaagaaat aaaactcaaa gcagagaga aatttagaca ttcaactata aataaaaaat accccccaaa gcattaatat cttggggagt attttttatt ttgaagtatt ctgttcagct aatattctt ctaaggtaat acctctgttc aatattctt gtgaggcagg aagaccgata tatcttacat gccatggctc aaaattatac tttgttatgt tttctttatc cttaggatat cttattatga aaccatattt accacaattt tgttgaagcc atttataaga atttgtattc ataaatccat catctaaaga agtagtattcg gttgatagta agtccattgc caatccagtt tgatgctcac ttgtaccagg ttcagctaca tatttatcag cttcggcttt tccgtctcgt gctacttttt cattatta tttttgctga cattatag gtctatacc tgaaacagct agaagtgtaa gaccatcctt tgatgctgca ttaaacatat tttcaagtcc tgttgcagct tcgctctcca tttgatttac attack 6420. tgttgcagct tcgctctcca tttgatttac gttaggagtt ctcaaatttt gaggtatata gtttcctgat aatttacttt gcttgtttac 6480. aagtaggatg ttctgtttct ttacctcggg tttcttggct tgtttttag gtgtagaaac 6540 tttctttttg ggttcgtttg 6560 <210> 80 <211> 6560 <212> DNA <213> Artificial sequence <220> <223> pGRNA_ΔbdhA_ΔbdhB <400> 80 gatccccggg taccgagctc gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 60 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 120 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 180 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 240 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 300 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 360 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 420 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 480 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 540 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 600 tttctcctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 660 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 720 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 780 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 840 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 900 ctgctgaagc cagttacctt cggaaaaga gttggtagct cttgatccgg caacaaacc 960 accgctgta gcggtggtttt tttgttgc aagcagcaga ttacgcgcag aaaaaaagga 1020 tctcaagaag atcctttgat ctttctacg gggtctgacg ctcagtggaa cgaaaactca 1080 cgttaaggga ttttgtcat gagattatca aaaggatct tcacctagat ccttttaaat 1140 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1200 caaagctagc ttaatactag tatatactta atgtgataag tgtctgacag ctgaccggtc 1260 taaagaggtc cctagcgcct acgggaatt tgtatcgata aggggtacaa attcccacta 1320 agcgctcggc cgggatcga tcccgggta cgtacccggc agttttctt ttcggcaag 1380 tgttcaagaa gttattaagt cgggagtgca gtcgaagtgg gcaagttga aaattcacaa 1440 aaatgtgta taatatcttt gttcattaga gcgataact tgaatttgag aggaactta 1500 gatggtattt gaaaaaatttg aaaaatag ttggacaga aaagagtatt ttgaccacta 1560 ctttgcaagt gtaccttgta cctacagcat gaccgttaaa gtggatatca cacataaa 1620 ggaaaaggga atgaaactat atcctgcaat gctttattat attgcaatga ttgtaaccg 1680 ccattcagag tttaggacgg caatcaatca agatggtgaa ttggggatat atgatgagat 1740 gataccaagc tatacatat ttcacaatga tactgaaca ttttccagcc ttggactga 1800 gtgtaagtct gactttaaat cattttagc agatttagaa agtgatagcc aacggtatgg 1860 aaacaatcat agaatggaag gaagccaaa tgctccggaa aacatttta atgtatctat 1920 gataccgtgg tcaccttcg atggctttaa tctgaatttg cagaaaggat atgattttt 1980 gattcctatt tttactatgg ggaatatta windows aaaaatta tactctcctttt 2040 ggcaattcaa gttcatcacg cagtatgtga cggatttcac atttgccgtt ttgtaaacga 2100 attgcaggaa ttgataata gttaacttca ggtttgtctg taactaaaa ctagtattta 2160 acctaggatc aaaaaaattt ccaataatcc cactctaagc cacaacacg ccctataaaa 2220 tcccgcttta atccccacttt gagacacatg taatattact ttaccccta gtatagtgat 2280 aattttttac attcaatgcc acgcaaaaaaaaggggc actataataa aagttccttc 2340 ggaactaact aaagtaaaa attatcttta caacctcccc aaaaaaaga acaggtaca 2400 agtaccctat atacaagcg taaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2460 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2520 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaat 2580 aaaaatatttt tttattaaa gtttgaaaa aattttta tattatataa tctttgaga 2640 aaagaata aaaaatgagc ctttataaa gcccatttttt ttcatatac gtataatgac 2700 gttctaatgt ttttggt acttctaaca ttagagtaat ttcttttt ttaaagcctt 2760 ttctttaag ggctttttttttttctata atacatttta ttccttttttttgttgctt 2820 ttcctttagc ttttaattgc tcttgataat ttttttacc tctatattt tctctctct 2880 tatattcctt tttagaaatt attattgtca tatattttg ttctctct gtaatttcta 2940 aaactctat agagtttca ttcttatact tatattgctt attttct aaataacatc 3000 tttcagcact tctagttgct cttataactt ctcttcact taaatgttgt ctaaacatac 3060 tattaagttc taaaacatca tttaatgcct tctcaatgtc ttctgtaag ctacaagat 3120 ataatctata taaaaataat ataagctctc tgtgtcctttt taaatcatat tctcttagtt 3180 cacaagtttt tattatgtct tgtattctc cataatataa acttctttct ctataaatat 3240 aatttatttt gcttggtcta ccctttttcc ttcatatgg ttttaattca ggtaaaaatc 3300 cattttgtat ttctcttaag tcataaatat atcgtactc atctaata ttgactactg 3360 ttttgattt agagttatta cttcctggaa ctcttattat tctcgttgca tctaggctt 3420 gtctatctgc tccaaagtat tttattgat tataataata ttcttgacc gctttccata 3480 atggtaatgc tttactaggt actgcattta ttatccatat taaatacatt cctcttccac 3540 tatctattac atagtttggt taggaatac tttgatttaa atattcttt tctaagtcca 3600 ttaatacctg gtctttagtt ttgccagtttt tataataatc caagtctata aacagtgtat 3660 ttactcttt tatattttct aatcgcctac acggcttata aaaggtattt agagttatat 3720 agatatttc atcactcata tctaaatctt ttaattcagc gtatttatag tgccattggc 3780 tatatcctttt ttatctata acgctcctgg ttatccaccc tttactctta ctatgaatat 3840 tatctatata gttcttttta ttcagcttta atgcgtttct cacttattca cctccccttc 3900 tgtaaaacta agaaaattat atcatatttt caatattat taactattct taaactctta 3960 aaaaaata gagtaagtcc ccaattgaaa cttaatctat tttttagtt ttaatttatt 4020 atttttatta aaatatttta aactaatta atgattctt tttaatttt tactatttca 4080 ttcataata tattactata attackttaca ataatattt cttcattgt atattaga 4140 tgatttacta attttagttttttattattattaataattaat gtataattta tataaaaaat 4200 caaaggagct tataaattat gattatttcc aagatacta aagatttaat tttttcaat 4260 tttaacaata ctttttgtaa tattatgttt aaatttaatt gtatttttt catataataa 4320 agccgttgaa gtaaaccaat ccattttcct tatgatgtta ttattaaatt taagttttat 4380 aaataatct tattattatt tattgttttt aaaaaaacta gtgaaatttc tagtgaattt 4440 4500 gattatatct ttaaatatgt tttatcaat attatcttt tctaaattta tatatatttt 4560 tattatattt attattatat tattttatt tttaagtttc ttctacag ctattaaaaa 4620 gaaacttaa aaaaaaca cgtactctaa accaataat aaactattt ttattattgc 4680 tgccttgatt ggaatagttt ttagtaaaat taatttcaat attccacaat attattat 4740 aagctagcac gcctcgagac tctatcattg atagagtttg aaacttatc attgatagag 4800 tataatatct ttgttcatgc ttattacgac atacacagt tttagagcta gaatagcaa 4860 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgcttttt 4920 tgaagcttct cgagatctcc atggacgcgt gacgtcgacc ttctaatctc ctctactatt 4980 ttagggttag ctacattagc taataggta atagctacag ttgtctttga attctcacct 5040 aaagtaagtt cttccacttt aaaatcagtg cttctaattt ttttctttaa aagggctaca 5100 tttgtggtta aagattcagt gaagccctct ctaggacctc ttattacagt ttcaacagtt 5160 ggttctgtta tagctctttc agggggtttt ccaatactta tataattgc tttactttca 5220 ccatctagga ataatgctat acttcctttt aaaatggaca atataacatc atccatgctt 5280 ttatatacat tttatcatt aacagcaaaa attgattttg tatattcaaa tatgtttaaa 5340 tggggatggt tattgtaatc ttcttctata agtttttta taacagagga ttctattaca 5400 tcagattgga taagattatt tatgtagaca atcattgcag aaaaatttct attattagct 5460 atttaaatt ctctaatcgt taaatctgag caatttgtaa ataaggtttc tatagtatgt 5520 ttatttgttt taaggctagt tgaaaccgtc ttcgcgttat ttttagatgc ttcttcttta 5580 ttaaaaattt tattaaacaa cgaaaattc accccctcaa tttatttata taatagtagt ttgcatgaaa tttcgttgtt tattcatatt agatgcttgt attachment aatataagt agacaaacta taaatctatt actaggaggt aagaagtatg ctaagtttta aatctgtgta aacctaccgg ggtttgggcg tagccattat attcatgaac tccaagaaag cagtatgcta gcaaagaaat aaaactcaaa gcagagaga aatttagaca ttcaactata aataaaaaat accccccaaa gcattaatat cttggggagt attttttatt ttgaagtatt ctgttcagct aatattctt ctaaggtaat acctctgttc aatattctt gtgaggcagg aagaccgata tatcttacat gccatggctc aaaattatac tttgttatgt tttctttatc cttaggatat cttattatga aaccatattt accacaattt tgttgaagcc atttataaga atttgtattc ataaatccat catctaaaga agtagtattcg gttgatagta agtccattgc caatccagtt tgatgctcac ttgtaccagg ttcagctaca tatttatcag cttcggcttt tccgtctcgt gctacttttt cattatta tttttgctga cattatag gtctatacc tgaaacagct agaagtgtaa gaccatcctt tgatgctgca ttaaacatat tttcaagtcc 6360 tgttgcagct tcgctctcca tttgatttac attaggatca gaactactaa taaatttaac 6420 gttaggagtt ctcaaatttt gaggtatata gtttcctgat aatttacttt gcttgtttac 6480 aagtaggatg ttctgtttct ttacctcggg tttcttggct tgttttttag gtgtagaaac 6540 tttctttttg ggttcgtttg 6560 <210> 81 <211> 1654 <212> DNA <213> Artificial sequence <220> <223> bgaR acrIIA4 expression cassette <400> 81 aaaaagtata acagaggttt taatttacgc ctctgttata ctttttattt ttgaaatttt 60 tttgttttaa agctgtattt taaatttata tacttggttt atttacttga ttatttctgt 120 aatttagtgg agacattgaa aaatgttttg aaaaagtttt tgaaaataac agggagtcac 180 tataacctac actacttgcg acttctccta taggaagttt agtgcttttt aataaaaggg 240 tggctttgta cattctaagg tttattaaat atctttgagg agaaattcca aggtttttta 300 tgaacattt atataaataa cttctactta agttcacata atcagcaatt tcttgaacag 360 ttatgctatg catgtaatta gaattaatga aattaagagc atcttgaata tatgtgtgta 420 attccttatc tttgtattca aaaggttttg ggaattcttc tataagtgcg tacaataatg 480 agtaaagttc ttttagtaat agtatgtcat cagatcttga aggattataa gtttttgata 540 tttcgcacat attaatatt atctgtggaa ttttgagtt ttcttcacaa ttgcaacac 600 aggagttagt aatagaagtt ctatttaaat actcattagc atttgaacca ctaaatccta 660 tccagtagta ttcccaagga tcatcaatag aagccacata ctcaacttgc atacctttta 720 gtagtataaa aatatcacct tgttttaagt tatatacctt accattaaat ttaaaagttc 780 catatccctt agttacgtaa tgaataacag cattttcaa tacttcatag ttatatccta 840 atcctggtat accttgttct ataccacatt catctacatt catttcaaag ttttctttaa 900 catacttttt ccacaatatt tgcatttcta cctcctaacc tataaaatta gccaatttta 960 tagtagtct atattaaaca tttacatgag agctttgcaa agcagtttat caacataaaa 1020 gctttttatt ttaaaataaa ttcttctaaa tataagaata ttttaaagaa atatctttat 1080 atattagtta ttaaaattta taagattata agaaacatta taacatattt tagaactttt 1140 taactattct aaaagattaa tttacatatt aacatttaat tatgggtaaa aactattttg 1200 aaaaatgatt tatatggaat tatgtttctt aaatatacaa tcatgtttca tgaatacata 1260 attattttaa atgtattggg agggtaaaat gatattaaaa aatgaatacc atgaagatac 1320 tgcagaatct agaatccgcg gtagtcgacg tggaattgtg agcggataac aatttcacag 1380 gagggctgaa atgaatatta atgacttaat tagagaaata aaaaacaaag attacacagt 1440 gaaattgagt ggtacggata gcaatagtat aacacagcta attattagag ttaataatga 1500 tggaaacgag tatgtaattt ctgaaagtga aaatgaatca atagttgaaa aattcatatc 1560 tgcatttaaa aacggttgga atcaagaata cgaggatgaa gaagaatttt ataatgacat 1620 gcaaacaatc accttaaaaa gtgagttgaa ctaa 1654 <210> 82 <211> 4984 <212> DNA​​​​​<223> pGRNAind <400> 82 caagcttcaa aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 60 tattttaact tgctatttct agctctaaaa cagagaccgc tagcgatatc cccgggagat 120 ctggtctcaa tgaacaaaga tattatactc tatcaatgat agagtttcaa actctatcaa 180 tgatagagtg agctcgaatt cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 240 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 300 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 360 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 420 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 480 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 540 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 600 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 660 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 720 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 780 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 840 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 900 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 960 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 1020 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 1080 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 1140 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 1200 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 1260 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 1320 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaaag 1380 ctagcttaat actagtatat acttaatgtg ataagtgtct gandagctgac cggtctaaag 1440 aggtccctag cgcctacgggg gatttgtat cgatagggg tacaatttcc cactaagcgc 1500 tcggccgggg atcgatcccc gggtacgtac ccggcagtttt ttctttcg gcaagtgttc 1560 aagaagttat taagtcggga gtgcagtcga agtggggcaag ttgaaaaatt cacaaaatg 1620 tggtataata tctttgttca ttagagcgat aaacttgaat tgagaggga acttagatgg 1680 tatttgaaa attgaaa atagttgga acagaaaga gtattttgac cactactttg 1740 caagtgtacc ttgtacctac agcatgaccg ttaagtgga tatcacacaa AAaggaaa 1800 agggaatgaa actatatcct gcaatgcttt attatattgc aatgattgta aaccgccatt 1860 cagagtttag gacggcaatc aatcagatg gtgaattggg gatatgat gagatgat 1920 caagctatac atattcac atgatactg aacattttc cagccttttgg actgagtgta 1980 agtctgactt taaatcattt tagcagatt atgaagtga tacgcaacgg tatggaaca 2040 atcatagaat ggaaggaag ccaatgctc cggaaacat ttttaatgta tctatgatac 2100 cgtggtcaac cttcgatggc tttaatctga atttgcagaa aggatatgat tattgattc 2160 ctatttttac tatggggaaa tattataag agataacaaattatactt cctttggcaa 2220 ttcaagttca tcacgcagta tgtgacggat ttcacattg ccgttttgta aacgaattgc 2280 aggaattgat aaatagttaa cttcaggttt gtctgtact aaaaactagt atttaccta 2340 ggatcaaaa aatttccaat aatcccactc taagccacaa acacgcccta taaatcccg 2400 ctttaatccc actttgagac acatgtaata ttactttacg ccctagtata gtgataattt 2460 tttacattca atgccacgca aaaaaaay ggggcactat ataaaagtt ccttcggac 2520 taactaagt aaaaaattt ctttacaacc tcccaaaa aaagaacagg tactaactac 2580 cctataatac aagcgtaaaaaatgagggg taaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2640 taaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2700 Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2760 tattttttatttttaagttg aaaaaaatttttttttattattattttt gagaaaaga 2820 atataaaaaa tgagccttta taaaagccca ttttttttca tatacgtaat atgacgttct 2880 aatgttttta ttggtacttc taacattaga gtaatttctt tatttttaaa gccttttct 2940 ttaagggctt ttatttttt tcttaataca tttaattcct ctttttttgt tgcttttcct 3000 ttagctttta attgctcttg ataatttttt ttacccttaa tattttctct tctctttat 3060 tcctttttag aaattattat tgtcatatat ttttgttctt cttctgtaat ttctaataac 3120 tctataagag tttcattctt atacttatat tgcttatttt tatctaaata acatctttca 3180 gcactttag ttgctcttat aacttctctt tcacttaaat gttgtctaaa catactatta 3240 agttctaaaa catcatttaa tgccttctca atgtcttctg taaagctaca aagataatat 3300 ctatataaaaa samaataag ctctctgtgt ccttttaaat catattctct tagttcacaa 3360 agttttatta tgtcttgtat tcttccataa tataaacttc tttctctata aatataattt 3420 attttgcttg gtctaccctt tttcctttca tatggtttta attcaggtaa aaatccattt 3480 tgtatttctc ttaagtcata aatatattcg tactcatcta atatattgac tactgttttt 3540 gatttagagt ttacttcc tggaactctt aatattctcg ttgcatctaa ggcttgcta 3600 tctgctccaa agtattttaa ttgattatat aatattctt gaaccgcttt ccataatggt 3660 aatgctttac taggtactgc atttattatc catattaaat acattcctct tccactatct 3720 attacatagt ttggtatagg aatactttga ttaaaataat tctttctctaa gtccattaat 3780 acctggtctt tagttttgcc agttttataa taatccaagt ctataaacag tgtatttaac 3840 tcttttatat tttctaatcg cctacacggc ttataaaagg tatttagagt tatatagata 3900 ttttcatcac tcatatctaa atcttttaat tcagcgtatt tatagtgcca ttggctatat 3960 ccttttttat ctataacgct cctggttatc caccctttac ttctactatg aattatct 4020 atatagttct ttttattcag cttaatgcg tttctcactt attcacctcc ccttctgtaa 4080 aactaagaaa attatatcat atttcaata attattaact attcttaaac tcttaataaa 4140 aatagagta agtccccaat tgaaacttaa tctatttttt atgttttaat ttattatttt 4200 tattaaaata ttttaaacta attaaatga ttctttttaa ttttttacta tttcattcca 4260 father ctather ttacaaata tatttcttca tttgtaatat ttagatgatt tactatttt agtttttata tattatta tattattatta aaaatcaaag gagcttataa attachment tttccaaaga tactaaagat ttaatttttt tcaattttaa caatactttt tgtaatatta tgtttaatt tattgtatt tttttcatat aataaagccg ttgaagtaa ccaatccatt ttccttatga tgttattatt aaatttaagt tttataata tatctttatt atatttattg tttttaaaaa aactagtgaa atttctagtg aaatttccgg ctttattaaa cttattttta ggaattttat tttcattttc atctttacag gattttgatta tatctttaaa tatgttttat caaatattat ctttttctaa atttatatat atttttatta 4800. gtttctttct aacagctatt aaaaagaaac ttaaaaataa aaacacgtac tctaaaccaa taaataaac tatttttatt attgctgcct tgattggat agtttttagt aaattaatt tcaatattcc acaatattat attataagct agcacgcctc gagatctcca tggacgcgtg acgtcgactc tagaggatcc ccgggtaccg agct 4984 <210> 83 <211> 200 <212> DNA <213> Artificial sequence <220> <223> gRNA expression cassette <400> 83 gagctcactc tatcattgat agagtttgaa actctatcat tgatagagta taatatcttt 60 gttcattgag accagatctc ccggggatat cgctagcggt ctctgtttta gagctagaaa 120 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 180 tttttttgaa gcttgagctc 200 <210> 84 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 84 tcatgatttc tccatattag ctag 24 <210> 85 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 85 aaacctagct aatatggaga aatc 24 <210> 86 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 86 tcatgttaca cttggaacag gcgt 24 <210> 87 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 87 aaacacgcct gttccaagtg taac 24 <210> 88 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 88 tcatttccgg cagtaggatc ccca 24 <210> 89 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 89 aaactgggga tcctactgcc ggaa 24 <210> 90 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 90 tcatgcttat tacgacataa caca 24 <210> 91 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 91 aaactgtgtt atgtcgtaat aagc 24 <210> 92 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 92 atgcatggat ccaaacgaac ccaaaaagaa agtttc 36 <210> 93 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 93 ggttgatttc aaatctgtgt aaacctaccg 30 <210> 94 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 94 acacagattt gaaatcaacc actttaaccc 30 <210> 95 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 95 atgcatgtcg actcttaaga acatgtataa agtatgg 37 <210> 96 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 96 atgcatggat ccaaacgaac ccaaaaagaa agtttc 36 <210> 97 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 97 gctaagtttt aaatctgtgt aaacctaccg 30 <210> 98 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 98 acacagattt aaaacttagc atacttctta cc 32 <210> 99 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 99 atgcatgtcg accttctaat ctcctctact attttag 37 <210> 100 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 100 acacattgaa gggagctttt 20 <210> 101 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 101 ggcaacaaca tcaggccttt 20 <210> 102 <211> 4966 <212> DNA <213> Artificial sequence <220> <223> pGRNA-xylB <400> 102 atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60 ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120 tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180 actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240 tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300 aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360 aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420 ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480 ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540 tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600 aagggctttt atttttttc taatacatt taatttctct ttttgttg cttttccttt 660 agcttttaat tgctcttgat aatttttt acctctaata tttctctc tcttatattc 720 ctttttagaa attattattg tcatatattttgttctct tctgtaattt ctaataactc 780 tataagagtt tcattcttat acttatattg cttatttta tctaaataac atctttcagc 840 acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaca tactattaag 900 ttctaaaaca tcatttaatg ccttctcaat gtctctgta aagctacaaa gataatatct 960 atataaaaat ataataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020 ttttattatg tcttgtattc ttccataata taaacttctt tcttattaa tataatttat 1080 ttgcttggt ctacccttt tccttcata tggttttaat tcaggtaaaa atccatttg 1140 tattctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200 tttagagttt atactcctg gaactcttaa tattctcgtt gcatctagg cttgtctatc 1260 tgctccaaag tattttaatt gattatataa atattctga accgctttcc ataatggtaa 1320 tgctttacta ggtactgcat ttattacca tattaatac attcctctc cactacttat 1380 tacatagtttt ggtataggaa tactttgatt aaaataatc tttctaagt ccattaatac 1440 ctggtcttta gttttgccag ttttaata atccaagtct ataacagtg tattactc 1500 ttttatattt tctaatcgcc tacacggctt aaaaggta tttagagtta tatagatatt 1560 ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620 ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680 atagttcttt ttcagct taatgcgtt tctcacttat tcacccccc ttctgtaaaa 1740 ctaagaaaat tattacatat ttcaataat tattaactat tcttaactc tattaaaaa 1800 atagagtaag tcccaatttg aaacttaatc tatttttt gttttaattt atttatttta 1860 ttaaaatatt ttaaactaa ttaaatgattttttaattt ttttactt tcattccata 1920 attattact attattatt acaatata tttctcatt tgtatattt agatgattta 1980 ctaattttag ttttatta tattaatatt atgtataat tttattaaaaatcaagga 2040 gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100 atactttttg tatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160 gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagtttt tataataata 2220 tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280 ttattaaact tatttttagg aattttattt tcattttcat cttacagga tttgattata 2340 tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400 tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460 aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520 attggaatag tttttagtaa attaatttc aatattccac aatattatat tataagctag 2580 cacgcctcga gaagcttcaa aaaaagcacc gactcggtgc cactttttca agttgataac 2640 ggactagcct tattttaact tgctatttct agctctaaaa cctagctaat atggagaaat 2700 catgaacaaa gatattatac tctatcaatg atagagtttc aaacctatc aatgatagag 2760 tctcgagatc tccatggacg cgtgacgtcg actctagagg atccccgggt accgagctcg 2820 aattcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 2880 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 2940 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 3000 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 3060 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 3120 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 3180 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 3240 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 3300 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 3360 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3420 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3480 ctgggctgtg tgcacgacc cccgttcag cccgaccgct gcgccttatc cggtaactat 3540 cgtcttgagt ccaacccggt agacacgac ttatcgccac tggcagcagc cactggtaac 3600 aggattagca gagcgaggta tgtaggcggt gctacagagt tctgaagtg gtggcctaac 3660 tacggctaca ctagagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3720 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3780 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaga tcctttgatc 3840 tttctacgg gtctgacgc tcagtggaac gaaaaccc gttaaggat ttggtcatg 3900 agatttaca aaaggatctt cacctagatc cttttaaattt aaaaatgaag ttttaatca 3960 atctaagta tatactgagta aacttggtct gandagttacc aaagctagct taatactagt 4020 atatacttaa tgtgataagt gtctgacagc tgaccggtct aaagaggtcc ctagcgccta 4080 cggggaattt gtatcgataa ggggtacaa ttcccactaa gcgctcggcc ggggatcgat 4140 ccccgggtac gtacccggca gttttcttt ttcggcaagt gttcagaag ttattaagtc 4200 gggagtgcag tcgaagtggg caagttgaaa aattcacaaa aatgtggtat aatatctttg 4260 ttcattagag cgataaactt gaatttgaga gggaacttag atggtatttg aaaaaattga 4320 taaaaatagt tggaacagaa aagagtattt tgaccactac tttgcaagtg taccttgtac 4380 ctacagcatg accgttaaag tggatatcac acaaataaag gaaaagggaa tgaaactata 4440 tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc cattcagagt ttaggacggc 4500 aatcaatcaa gatggtgaat tggggatata tgatgagatg ataccaagct atacaatatt 4560 tcacaatgat actgaaacat tttccagcct ttggactgag tgtaagtctg actttaaatc 4620 atttttagca gattatgaaa gtgatacgca acggtatgga aacaatcata gaatggaagg 4680 aaagccaaat gctccggaaa acatttttaa tgtatctatg ataccgtggt caaccttcga 4740 tggctttaat ctgaatttgc agaaaggata tgattatttg attcctattt ttactatggg 4800 gaaatattat aaagaagata acaaattat acttcctttg gcaattcaag ttcatcacgc 4860 agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag 4920 ttaacttcag gtttgtctgt aactaaaaac tagtatttaa cctagg 4966 <210> 103 <211> 4966 <212> DNA <213> Artificial Sequence <220> <223> pGRNA-xylR <40T> 103 atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60 ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120 tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180 actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240 tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300 aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360 aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420 ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480 ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540 It should be noted that there may be some inaccuracies in the translation as the original text seems to have some unclear or potentially incorrect notations. For example, "<40T>" in the translation of line might need further clarification in the original text.tgttttatt ggtacttcta acattagagt aatttcttta ttttaagc ctttttcttt 600 aagggctttt atttttttc taatacatt taatttctct ttttgttg cttttccttt 660 agcttttaat tgctcttgat aatttttt acctctaata tttctctc tcttatattc 720 ctttttagaa attattattg tcatatattttgttctct tctgtaattt ctaataactc 780 tataagagtt tcattcttat acttatattg cttatttta tctaaataac atctttcagc 840 acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaca tactattaag 900 ttctaaaaca tcatttaatg ccttctcaat gtctctgta aagctacaaa gataatatct 960 atataaaaat ataataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020 ttttattatg tcttgtattc ttccataata taaacttctt tcttattaa tataatttat 1080 ttgcttggt ctacccttt tccttcata tggttttaat tcaggtaaaa atccatttg 1140 tattctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200 tttagagttt atactcctg gaactcttaa tattctcgtt gcatctagg cttgtctatc 1260 tgctccaaag tattttaatt gattatataa atattctga accgctttcc ataatggtaa 1320 tgctttacta ggtactgcat ttattacca tattaatac attcctctc cactacttat 1380 tacatagtttt ggtataggaa tactttgatt aaaataatc tttctaagt ccattaatac 1440 ctggtcttta gttttgccag ttttaata atccaagtct ataacagtg tattactc 1500 ttttatattt tctaatcgcc tacacggctt aaaaggta tttagagtta tatagatatt 1560 ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620 ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680 atagttcttt ttcagct taatgcgtt tctcacttat tcacccccc ttctgtaaaa 1740 ctaagaaaat tattacatat ttcaataat tattaactat tcttaactc tattaaaaa 1800 atagagtaag tcccaatttg aaacttaatc tatttttt gttttaattt atttatttta 1860 ttaaaatatt ttaaactaa ttaaatgattttttaattt ttttactt tcattccata 1920 attattact attattatt acaatata tttctcatt tgtatattt agatgattta 1980 ctaattttag ttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040 gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100 atactttttg tatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160 gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagtttt tataataata 2220 tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280 ttattaaact tatttttagg aattttattt tcattttcat cttacagga tttgattata 2340 tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400 tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460 aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520 attggaatag tttttagtaa attaatttc aatattccac aatattatat tataagctag 2580 cacgcctcga gactctatca ttgatagagt ttgaaactct atcattgata gagtataata 2640 tctttgttca tgttacactt ggaacaggcg tgttttagag ctagaaatag caagttaaaa 2700 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgaagct 2760 tctcgagatc tccatggacg cgtgacgtcg actctagagg atccccgggt accgagctcg 2820 aattcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 2880 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 2940 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 3000 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 3060 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 3120 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 3180 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 3240 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 3300 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 3360 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3420 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3480 ctgggctgtg tgcacgacc cccgttcag cccgaccgct gcgccttatc cggtaactat 3540 cgtcttgagt ccaacccggt agacacgac ttatcgccac tggcagcagc cactggtaac 3600 aggattagca gagcgaggta tgtaggcggt gctacagagt tctgaagtg gtggcctaac 3660 tacggctaca ctagagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3720 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3780 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaga tcctttgatc 3840 tttctacgg gtctgacgc tcagtggaac gaaaaccc gttaaggat ttggtcatg 3900 agatttaca aaaggatctt cacctagatc cttttaaattt aaaaatgaag ttttaatca 3960 atctaagta tatactgagta aacttggtct gandagttacc aaagctagct taatactagt 4020 atatacttaa tgtgataagt gtctgacagc tgaccggtct aaagaggtcc ctagcgccta 4080 cggggaattt gtatcgataa ggggtacaa ttcccactaa gcgctcggcc ggggatcgat 4140 ccccgggtac gt...

Claims

1. A Clostridium beijerinckii, which is a Clostridium beijerinckii (C. beijerinckii) IFP963ΔcatBΔpNF2 bacterium registered with the BCCM-Belgian Coordinating Collection of Microorganisms under the deposit number LMG P-31277 on February 20, 2019.

Images