Genetically modified Clostridium bacteria, their preparation and use

Abstract

The present invention relates to the genetic modification of Clostridium bacteria, in general solvent-producing bacteria of the genus Clostridium, in particular bacteria which, in the wild state, have a gene encoding an amide alcohol-O-acetyltransferase. The present invention therefore relates to methods, tools and kits which allow such genetic modifications, in particular the elimination or modification of sequences encoding or controlling the transcription of an amide alcohol-O-acetyltransferase, to the genetically modified bacteria obtained, and to their use, in particular preferably on an industrial scale, for the production of solvents.

Description

[0001] The present invention relates to the genetic modification of Clostridium bacteria, typically solventogenic Clostridium bacteria, in particular bacteria that, in the wild type, have a gene encoding an amido-ol-O-acetyltransferase. The present invention therefore relates to methods, tools and kits that allow for such genetic modifications, in particular the removal or modification of sequences encoding an amido-ol-O-acetyltransferase or sequences that control the transcription of sequences encoding an amido-ol-O-acetyltransferase, to the genetically modified bacteria obtained, and to their use, in particular, preferably for the production of solvents on an industrial scale. Background Art

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

[0003] The solventogenic Clostridium species display significant phenotypic similarities, making their classification difficult before the advent of modern sequencing technology (Rogers et al., 2006). Since the complete genomes of these bacteria have been sequenced, the genus can now be divided into four main species: C. acetobutylicum, C. saccharoperbutylacetonicum, C. saccharobutylicum, and C. beijerinckii. A recent publication, after comparative analysis of the complete genomes of 30 strains, divided these solventogenic Clostridia into four major clades ( Figure 1 ).

[0004] Specifically, these research groups separated the species C. acetobutylicum and C. beijerinckii and used C. acetobutylicum ATCC 824 (also designated DSM 792 or LMG 5710) and C. beijerinckii NCIMB 8052 as model strains for studying ABE-type fermentations.

[0005] There are few Clostridium strains naturally capable of IBE fermentation, and most belong to the species Clostridium beijerinckii (Zhang et al., 2018, Table 1). These strains are generally selected from C. butylicum LMD 27.6, C. aurantibutylicum 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, Clostridium species A1424, C. beijerinckii optinoii, and C. beijerinckii BGS1.

[0006] However, to date there are no strains of Clostridium bacteria that have been genetically modified to naturally produce isopropanol, in particular to naturally perform IBE fermentation, and in particular to be genetically modified to make them sensitive to antibiotics belonging to the amide alcohol type, such as chloramphenicol or thiamphenicol, preferably to optimize isopropanol production. Summary of the Invention

[0007] In the context of the present invention, the inventors describe for the first time a genetically modified Clostridium beijerinckii bacterium and tools allowing the genetic modification of Clostridium bacteria, which are generally solventogenic Clostridium bacteria that are naturally (i.e. in the wild type) capable of producing isopropanol, in particular naturally capable of performing IBE fermentation, in particular bacteria that contain in the wild type a gene that provides the bacterium with resistance to one or more antibiotics, in particular a gene encoding an amide alcohol-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase or thiamphenicol-O-acetyltransferase.

[0008] Preferred genetically modified bacteria according to the present invention are bacteria that do not express an enzyme that provides resistance to one or more antibiotics, in particular bacteria that do not express amide alcohol-O-acetyltransferase, for example bacteria that lack or are unable to express the catB gene.

[0009] The preferred genetically modified bacteria according to the present invention are bacteria identified in this specification as Clostridium beijerinckii DSM6423ΔcatB, which was registered on December 6, 2018 at the Belgian Coordinated Collections of Microorganisms ("BCCM", KLLedeganckstraat 35, B-9000 Gent-Belgium) under the deposit number LMG P-31151 (also identified as Clostridium beijerinckii IFP962ΔcatB). This specification also relates to any derivative bacteria, clones, mutants or genetically modified forms thereof.

[0010] A particular subject matter described by the inventors is a nucleic acid that recognizes (at least partially binds to) and preferably targets, i.e. recognizes and allows cleavage of at least one strand of the following sequences in the genome of a bacterium of interest: i) a sequence encoding an enzyme that allows the bacterium of interest to grow in a culture medium containing an antibiotic, typically an antibiotic belonging to the class of amide alcohols, preferably selected from chloramphenicol, thiamphenicol, chloramphenicol azido and florfenicol, typically an amide alcohol-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase or thiamphenicol-O-acetyltransferase; ii) a sequence that controls the transcription of the sequence encoding the enzyme; or iii) sequences flanking the sequence encoding the enzyme.

[0011] The inventors have also described the use of such nucleic acids for transforming and / or genetically modifying Clostridium bacteria, preferably Clostridium bacteria that are naturally capable of producing isopropanol, in particular Clostridium bacteria that are capable of performing IBE fermentation.

[0012] In particular, the inventors describe the use of a nucleic acid recognizing the catB gene of sequence SEQ ID NO: 18 in the genome of Clostridium beijerinckii DSM 6423 or a sequence at least 70% identical thereto for transforming and / or genetically modifying Clostridium beijerinckii DSM 6423 bacteria.

[0013] The bacterium capable of producing isopropyl alcohol in the wild type can be, for example, a bacterium selected from the group consisting of Clostridium beijerinckii, C. diolis, C. puniceum, C. butyricum, C. saccharoacetic acid and polybutanol Clostridium, C. botulinum, C. drakei, C. scatologenes, C. perfringens and C. tunisiense, preferably a bacterium selected from the group consisting of Clostridium beijerinckii, C. diolis, C. puniceum and C. saccharoacetic acid and polybutanol Clostridium. A particularly preferred bacterium naturally capable of producing isopropyl alcohol is Clostridium beijerinckii.

[0014] According to a specific aspect, the nucleic acid that recognizes and preferably targets i) a sequence encoding an amide alcohol-O-acetyltransferase, ii) a sequence that controls the transcription of this sequence or iii) sequences flanking this sequence, is used to transform a clade of Clostridium beijerinckii selected from the group consisting of DSM6423, LMG 7814, LMG 7815, NRRL B-593, NCCB 27006 and a clade that is at least 97% identical to strain DSM6423.

[0015] The present inventors have also described a method for transforming and preferably genetically modifying a Clostridium bacterium. This method comprises the step of transforming the bacterium by introducing into the bacterium a nucleic acid that recognizes and preferably targets i) a sequence encoding an enzyme of interest, preferably an amide alcohol-O-acetyltransferase, ii) a sequence that controls the transcription of the sequence encoding the enzyme, or iii) sequences flanking the sequence encoding the enzyme. This method is typically performed using genetic modification tools, such as those selected from CRISPR tools, group II intron-based tools, and allelic exchange tools. Bacteria transformed and genetically modified using this method are also described, an example of which is the bacterium Clostridium beijerinckii DSM 6423 ΔcatB.

[0016] Another aspect described by the inventors relates to the use of the genetically modified bacterium according to the invention, preferably the bacterium Clostridium beijerinckii DSM 6423 ΔcatB registered under the deposit number LMG P-31151, or a genetically modified form thereof, for producing a solvent, preferably isopropanol, or a mixture of solvents, preferably on an industrial scale.

[0017] Finally, the present description relates to a kit, in particular a kit comprising the following components: a nucleic acid as described herein and a genetic modification tool, in particular an element of a genetic tool selected from the group consisting of CRISPR tools, group II intron-based tools and allelic exchange tools, a nucleic acid serving as a guide RNA (gRNA), a nucleic acid serving as a repair template, at least one primer pair and an inducer allowing the expression of a protein encoded by the tool. DETAILED DESCRIPTION

[0018] Despite being used industrially for over a century, our understanding of Clostridium bacteria is limited by the difficulties encountered in genetically modifying them.

[0019] Different genetic tools have been designed in recent years to optimize strains of this genus, the latest generation being based on the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated protein (Cas) technology. This approach is based on the use of an enzyme called a nuclease (usually a Cas-type nuclease in the case of CRISPR / Cas genetic tools, such as the Cas9 protein from Streptococcus pyogenes), which performs double-stranded cuts in a DNA molecule (the target sequence of interest) under the guidance of an RNA molecule. The sequence of the guide RNA (gRNA) determines the cutting site of the nuclease, giving it an extremely high specificity ( Figure 17 ).

[0020] Since double-strand cuts in essential DNA molecules are lethal to an organism, the survival of the organism will depend on its ability to repair said cuts (see Cui & Bikard, 2016). In Clostridium bacteria, the repair of double-strand breaks relies on homologous recombination mechanisms, which require a complete copy of the cut sequence. By providing the bacteria with DNA fragments that allow this repair and at the same time modify the original sequence, it is possible to force the microorganism to integrate the desired changes into its genome. By modifying the target sequence or PAM site, the modifications that have been made must no longer allow the Cas9-gRNA ribonucleoprotein complex to target the genomic DNA ( Figure 18 ).

[0021] Different methods have been described to try to make such genetic tools that are functional in Clostridium bacteria. In fact, it is known that these microorganisms are difficult to genetically modify due to low frequencies of transformation and homologous recombination. Several methods are based on the use of Cas9 that is constitutively expressed in Clostridium beijerinckii and Clostridium ljungdahlii (Wang et al., 2015; Huang et al., 2016) or expressed under the control of an inducible promoter in Clostridium beijerinckii, Clostridium saccharoaceticum and Clostridium 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 performs single-stranded rather than double-stranded cutting within the genome (Xu et al., 2015; Li et al., 2016). This choice was due to the observation that under the experimental conditions tested, Cas9 was too toxic for use in Clostridium bacteria. Most of the above-mentioned tools rely on the use of a single plasmid. Finally, endogenous CRISPR / Cas systems can also be used when they are identified within the genome of a microorganism, as has been done in C. pasteurianum (Pyne et al., 2016).

[0022] Unless (as in the last case mentioned above) the endogenous machinery of the strain to be modified is used, the main disadvantage of tools based on CRISPR technology is the significant limitation on the size of the nucleic acid of interest (and therefore the number of coding sequences or genes) that can be inserted into the bacterial genome (according to Xu et al., 2015, a maximum of about 1.8 kb).

[0023] Based on the use of two different nucleic acids, usually two plasmids, this problem is significantly solved (see WO2017064439, Wasels et al., 2017 and Figure 3), the inventors have developed and described a more powerful genetic tool for modifying bacteria, which is suitable for bacteria, generally bacteria belonging to the Firmicutes phylum, and in particular Clostridium bacteria. In a particular embodiment, the first nucleic acid of this tool allows the expression of cas9, and the second nucleic acid specific for the modification to be performed contains one or more gRNA expression cassettes and a repair template that allows a portion of the bacterial DNA targeted by Cas9 to be replaced by a sequence of interest. By placing the cas9 and / or gRNA expression cassette under the control of an inducible promoter, the toxicity of the system is limited. The inventors have recently improved this tool so that it can significantly increase the transformation efficiency and thus obtain the genetically modified bacteria of interest in useful numbers and quantities (especially in the case of selecting robust strains for industrial-scale production) (see FR 18 / 54835). In this improved tool, at least one nucleic acid contains a sequence encoding an anti-CRISPR protein ("acr") placed under the control of an inducible promoter. This anti-CRISPR protein inhibits 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 phase of the bacteria.

[0024] In the context of the present specification, bacteria belonging to the phylum Firmicutes are understood to mean bacteria belonging to the classes Clostridia, Mollicutes, Bacilli or Togobacteria, preferably to the classes Clostridia or Bacilli.

[0025] 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.

[0026] The “bacteria of the genus Bacillus” specifically means B. amyloliquefaciens, B. thuringiensis, B. coagulans, B. cereus, B. anthracis or B. subtilis.

[0027] "Clostridium" specifically means a Clostridium species of industrial interest, typically a solventogenic or acetogenic Clostridium. The expression "Clostridium" encompasses wild-type bacteria and 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.

[0028] By "industrially interesting Clostridium species" is meant a species capable of producing solvents and acids such as butyric acid or acetic acid by fermentation from sugars or monosaccharides, typically from sugars containing 5 carbon atoms, such as xylose, arabinose or fructose, from sugars containing 6 carbon atoms, such as glucose or mannose, from polysaccharides such as cellulose or hemicellulose, and / or from any other carbon source that can be assimilated and used by Clostridium bacteria, such as CO, CO2 and methanol. Examples of interesting solventogenic bacteria are Clostridium bacteria that produce acetone, butanol, ethanol and / or isopropanol, such as strains identified in the literature as "ABE strains" [strains that perform fermentations that allow the production of acetone, butanol and ethanol] and "IBE strains" [strains that perform fermentations that allow the production of isopropanol (by reduction of acetone), butanol and ethanol]. The solvent-producing Clostridium bacterium can, for example, be selected from the group consisting of Clostridium acetobutylicum, C. cellulolyticum, C. phytofermentans, C. beijerinckii, C. saccharobutylicum, C. saccharoaceticum, C. sporogenes, C. butyricum, C. aureobutyricum and C. tyrobutyricum, most preferably from the group consisting of Clostridium acetobutylicum, C. beijerinckii, C. butyricum, C. tyrobutyricum and C. cellulolyticum, even more preferably from the group consisting of Clostridium acetobutylicum and C. beijerinckii.

[0029] Clostridium bacteria that naturally produce isopropanol, typically possessing in their genome an adh gene encoding a primary / secondary alcohol dehydrogenase that reduces acetone to isopropanol, are genetically and functionally distinct from bacteria capable of naturally performing ABE fermentation.

[0030] Advantageously, in the context of the present invention, the inventors have succeeded in genetically modifying a naturally isopropanol-producing Clostridium bacterium, Clostridium beijerinckii DSM 6423, as well as the reference strain Clostridium acetobutylicum DSM 792.

[0031] Thus, the present inventors have described for the first time a genetically modified naturally (i.e., in the wild type) solventogenic Clostridium bacterium capable of producing isopropanol, in particular naturally capable of performing IBE fermentation, and tools, in particular genetic tools, that enable the production of such bacteria. The advantage of these tools is that they significantly facilitate the transformation and genetic modification of wild-type bacteria capable of producing isopropanol, in particular of performing IBE fermentation, in particular those carrying genes encoding enzymes responsible for antibiotic resistance.

[0032] Part of the work described in the experimental section was performed in a strain capable of IBE fermentation, Clostridium beijerinckii strain DSM6423, whose genomic and transcriptomic analysis has been recently described by the present inventors (Máté de Gerando et al., 2018).

[0033] Specifically, in the genome assembly of this strain, the inventors found that in addition to the chromosome, there were also mobile genetic elements (accession number PRJEB11626 - https: / / www.ebi.ac.uk / ena / data / view / PRJEB11626): two natural plasmids (pNF1 and pNF2) and a linear phage (Φ6423).

[0034] In a specific embodiment of the present invention, the inventors have succeeded in deleting the native plasmid pNF2 from the Clostridium beijerinckii strain DSM 6423.

[0035] In another specific embodiment, they succeeded in deleting the upp gene originally present in the chromosome of Clostridium beijerinckii strain DSM 6423. These experiments therefore demonstrate the possible use of the tools and more generally the technology described herein by the inventors for the genetic modification of bacteria capable of producing isopropanol in the wild type, in particular for IBE fermentation.

[0036] In a particularly advantageous embodiment, the inventors have been particularly successful in sensitizing bacteria that are natural carriers (carried in the wild type) of genes encoding enzymes responsible for resistance to amide antibiotics to these antibiotics.

[0037] Examples of amide antibiotics of interest in the context of the present invention are chloramphenicol, thiamphenicol, chloramphenicol azido and florfenicol (Schwarz S. et al., 2004), in particular chloramphenicol and thiamphenicol.

[0038] Thus, a first aspect of the present invention relates to a genetic tool that can be used to genetically transform and / or 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 solventogenic Clostridium bacterium that is naturally (i.e., in the wild type) capable 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 that is different from chromosomal DNA in the wild type.

[0039] According to a specific aspect, this genetic tool is composed of a nucleic acid (also referred to herein as "nucleic acid of interest") that recognizes (at least partially binds to) and preferably targets, i.e., recognizes and allows cutting of at least one chain of the following sequences in the genome of the bacterium of interest: i) a coding sequence for an enzyme that allows the bacterium of interest to grow in a culture medium containing an antibiotic to which it confers resistance, ii) a sequence that controls the transcription of a coding sequence for an enzyme that allows the bacterium of interest to grow in a culture medium containing an antibiotic to which it confers resistance, or iii) sequences flanking a coding sequence for an enzyme that allows the bacterium of interest to grow in a culture medium containing an antibiotic to which it confers resistance. In the context of the present invention, this nucleic acid of interest is typically used to delete the identified sequence from the genome of the bacterium or to modify its expression, such as to regulate / control 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. The identified sequence is also referred to herein as a "target sequence" or "targeted sequence."

[0040] In a specific embodiment, the nucleic acid of interest comprises at least one region complementary to the target sequence, which has 100% identity or at least 80% identity, preferably 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the targeted DNA region / portion / sequence within the bacterial genome, and is capable of hybridizing to all or part of the sequence complementary to the region / portion / sequence, typically hybridizing to a region 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 region complementary to 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.

[0041] In another specific embodiment, the nucleic acid of interest comprises at least two regions each complementary to a target sequence, which are 100% identical or at least 80% identical, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the targeted DNA region / portion / sequence within the bacterial genome. These regions are capable of hybridizing to all or part of a sequence complementary to the region / portion / sequence, typically hybridizing to a sequence comprising at least 1 nucleotide, preferably at least 100 nucleotides, typically between 100 and 1000 nucleotides as described above. The regions complementary to the target sequence present in the nucleic acid of interest can recognize, preferably target, the genetic modification tools described herein, e.g. genetic tools, Genetic tools or The 5' and 3' flanking regions of the targeted sequence described in the Type 2 Allelic Exchange Tool are selected.

[0042] Typically, the target sequence is a sequence encoding an amidol-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase or thiamphenicol-O-acetyltransferase, a sequence controlling the transcription of such a sequence, or sequences flanking such a sequence in the genome of a Clostridium bacterium of interest that is capable of growing in a culture medium containing one or more antibiotics belonging to the amidol class, such as chloramphenicol and / or thiamphenicol.

[0043] In a specific embodiment, the identified sequence is the sequence corresponding to the catB gene (CIBE_3859) encoding chloramphenicol-O-acetyltransferase from Clostridium beijerinckii DSM 6423, SEQ ID NO: 18, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to said 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 identified sequence can be at least 1 nucleotide comprising the sequence of SEQ ID NO: 18, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides, usually a sequence 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 of SEQ ID NO: 18.

[0044] Examples of amino acid sequences having at least 70% identity to the chloramphenicol-O-acetyltransferase encoded by the sequence SEQ ID NO: 18 correspond to the sequences identified in the NCBI database under the following entries: 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). ID 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).

[0045] 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_026887895.1AWK51568.1, WP_003359882.1, WP_091687918.1, WP_055668544.1 and KGK90159.1.

[0046] 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.

[0047] 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.

[0048] 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).

[0049] A specific sequence identical to SEQ ID NO: 18 is the sequence identified in the NCBI database under the entry WP_077843937.1.

[0050] In a specific embodiment, the target sequence is the sequence corresponding to the catQ gene encoding chloramphenicol-O-acetyltransferase from Clostridium perfringens, SEQ ID NO: 68, whose amino acid sequence corresponds to SEQ ID NO: 66 (WP_063843220.1), or a 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: 68.

[0051] In other words, the identified sequence can be at least 1 nucleotide comprising the sequence of SEQ ID NO: 68, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides, usually a sequence 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 of SEQ ID NO: 68.

[0052] In another specific embodiment, 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 Clostridium bacteria or artificially introduced into such bacteria.

[0053] As indicated above, according to another embodiment, 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).

[0054] Said nucleic acid of interest used as a genetic tool then recognizes and is therefore generally capable of binding to the sequence controlling the transcription of the coding sequence as described above.

[0055] According to another embodiment, the target sequence may be a sequence flanking a coding sequence as described above encoding an enzyme that allows the bacterium of interest to grow in a culture medium containing the antibiotic to which it confers resistance, for example, the catB gene of SEQ ID NO: 18, or a sequence having at least 70% identity thereto. Such flanking sequences typically comprise 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.

[0056] According to a specific embodiment, the target sequence corresponds to a pair of sequences on both sides of such a coding sequence, and each flanking sequence generally comprises at least 20 nucleotides, usually between 100 and 1000 nucleotides, and preferably between 200 and 800 nucleotides.

[0057] 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 containing, for example, modified linkages, modified bases and / or modified sugars), or is optimized so that the codons of the transcripts synthesized from the coding sequence are the codons most commonly found in the Clostridium bacterium in which it is intended to be used. In the case of Clostridium, the optimized codons are typically codons rich in adenine ("A") and thymine ("T") bases.

[0058] In the peptide sequences described herein, amino acids are represented by single-letter codes corresponding 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.

[0059] In the context of the present invention, the nucleic acid of interest used as a genetic tool to transform and / or genetically modify the bacterium of interest is a DNA fragment that i) recognizes the coding sequence of an enzyme of interest, ii) controls the transcription of the coding sequence of the enzyme of interest, or iii) flanks the coding sequence of the enzyme of interest in the genome of a Clostridium bacterium, in particular a solventogenic Clostridium bacterium that is naturally capable of producing isopropanol, in particular a solventogenic Clostridium bacterium that is naturally capable of performing IBE fermentation, preferably an amide alcohol-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase or thiamphenicol-O-acetyltransferase.

[0060] The bacteria capable of naturally producing isopropyl alcohol can be, for example, selected from Clostridium beijerinckii, Clostridium diolate, Clostridium violaceum, Clostridium butyricum, Clostridium saccharoacetic acid, Clostridium botulinum, Clostridium drakeshii, Clostridium faecalis, Clostridium perfringens and Clostridium tunisiense, preferably selected from Clostridium beijerinckii, Clostridium diolate, Clostridium violaceum and Clostridium saccharoacetic acid. A particularly preferred bacterium capable of producing isopropyl alcohol in the wild type is Clostridium beijerinckii.

[0061] According to a specific aspect, the Clostridium bacterium is a Clostridium beijerinckii bacterium, whose clade is selected from DSM 6423, LMG 7814, LMG 7815, NCCB 27006 and a clade that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to strain DSM 6423.

[0062] As indicated above, the nucleic acid of interest according to the present invention is capable of deleting a sequence recognized in the genome of the bacterium ("target sequence") 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, preferably an amide alcohol-O-acetyltransferase, in particular a functional protein, from said sequence.

[0063] Such nucleic acids of interest are typically in the form of expression cassettes (or "constructs"), for example nucleic acids comprising a transcription promoter operably linked (in the sense understood by those skilled in the art) to one or more (coding) sequences of interest, for example an operon comprising several coding sequences of interest whose expression products contribute to achieving the function of interest in the bacterium, or nucleic acids that additionally comprise an activation sequence and / or a transcription terminator; or in the form of circular or linear single-stranded or double-stranded vectors, such as plasmids, phages, cosmids, artificial or synthetic chromosomes, comprising one or more expression cassettes as defined above. Preferably, the vector is a plasmid.

[0064] The nucleic acid of interest, preferably an expression cassette or vector, can be constructed by conventional techniques known to those skilled in the art and can comprise one or more promoters, bacterial origins of replication (ORI sequences), termination sequences, selection genes such as antibiotic resistance genes, and sequences (e.g., "flanking regions") that allow targeted insertion of the expression cassette or vector. In addition, these expression cassettes and vectors can be integrated into the genome by techniques known to those skilled in the art.

[0065] The ORI sequence of interest may be selected from pIP404, pAMβ1, pCB102, 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 a Clostridial cell.

[0066] The termination sequence of interest may be selected from the adc, thl, bcs operons or any other terminator known to the skilled person that allows transcription termination in Clostridium.

[0067] The selection gene (resistance gene) of interest may be selected from ermB, catP, bla, tetA, tetM and / or any other gene that provides resistance to ampicillin, erythromycin, chloramphenicol, thiamphenicol, tetracycline or any other antibiotic known to the skilled person that can be used for selection of Clostridium bacteria.

[0068] In a specific embodiment in which the identified enzyme encoding sequence is a sequence that provides chloramphenicol and / or thiamphenicol resistance to the bacterium, the selection gene is not a chloramphenicol and / or thiamphenicol resistance gene, and preferably is not any of the catB, catQ, catD or catP genes.

[0069] In a specific embodiment, the nucleic acid of interest comprises one or more guide RNAs (gRNAs) that target a sequence encoding an enzyme of interest, particularly an amide-O-acetyltransferase, that controls the transcription of the enzyme, or that is on either side of the coding sequence of the enzyme ("target sequence", "targeted sequence" or "recognized sequence"), and / or a modification template (also referred to herein as "editing template"), for example, a template that can eliminate or modify all or a portion of the target sequence, preferably so as to inhibit or suppress the expression of the target sequence, typically comprising a target sequence located upstream of the target sequence as described above. The template comprises a sequence homologous to (corresponding to) a sequence upstream and downstream of the target sequence, typically the sequence (homologous to the sequences upstream and downstream of the target sequence) each comprises between 10 or 20 base pairs and 1000, 1500 or 2000 base pairs, for example, between 100, 200, 300, 400 or 500 base pairs and 1000, 1200, 1300, 1400 or 1500 base pairs, preferably between 100 and 1500 base pairs or between 100 and 1000 base pairs, even more preferably between 500 and 1000 base pairs or between 200 and 800 base pairs.

[0070] Nucleic acids of particular interest take the form of vectors comprising one or more expression cassettes, each encoding at least one guide RNA (gRNA).

[0071] The specific genetic tool according to the invention comprises several (at least two) nucleic acids of interest as described above, said nucleic acids of interest being different from each other.

[0072] In a specific embodiment, the nucleic acid of interest used as a genetic tool to transform and / or genetically modify a bacterium of interest, typically a bacterium of the genus Clostridium, is a nucleic acid that recognizes a coding sequence for an enzyme that provides the bacterium with resistance to one or more antibiotics, a sequence that controls the transcription of the coding sequence for the enzyme, or sequences on both sides of the coding sequence, and is capable of deleting the sequence in the genome of the bacterium or rendering it non-functional, in particular a nucleic acid that does not exhibit methylation at the level of the motif recognized by Dam- and Dcm-type methyltransferases (prepared from Escherichia coli bacteria exhibiting the dam-dcm- genotype).

[0073] When the bacterium of interest to be transformed and / or genetically modified is a Clostridium beijerinckii bacterium, in particular a Clostridium beijerinckii bacterium 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 exhibit methylation at the level of motifs recognized by Dam- and Dcm-type methyltransferases, typically a nucleic acid in which the adenosine ("A") of a GATC motif and / or the second cytidine "C" of a CCWGG motif (W can correspond to adenosine ("A") or thymidine ("T")) is demethylated.

[0074] Nucleic acids that do not exhibit methylation at motifs recognized by Dam- and Dcm-type methyltransferases can typically be prepared from Escherichia coli bacteria with the dam-dcm- genotype (e.g., Escherichia coli INV 110, Invitrogen). Such nucleic acids may contain additional methylation, for example, by EcoKI-type methyltransferases, which target adenine ("A") at AAC(N6)GTGC and GCAC(N6)GTT motifs (N can correspond to any base).

[0075] In a preferred embodiment, the targeted sequence corresponds to a gene encoding an amide alcohol-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase, such as the catB gene, a sequence controlling the transcription of the gene, or sequences flanking the gene.

[0076] Nucleic acids of particular interest for use as genetic tools in the context of the present invention 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 description, in particular versions of said sequences that do not exhibit methylation at the motifs recognized by Dam- and Dcm-type methyltransferases.

[0077] The present description also relates to the use of the nucleic acids of interest described herein for transforming and / or genetically modifying bacteria of interest, in particular solventogenic Clostridium bacteria capable of producing isopropanol in the wild type, in particular capable of performing IBE fermentation in the wild type.

[0078] Bacteria capable of producing isopropanol in the wild type, in particular capable of performing IBE fermentation in the wild type, can be, for example, bacteria selected from Clostridium beijerinckii, Clostridium diolate, Clostridium violaceum, Clostridium butyricum, Clostridium saccharoaceticum, Clostridium botulinum, Clostridium drakeshii, Clostridium faecalis, Clostridium perfringens and Clostridium tunisicum, preferably Clostridium beijerinckii, Clostridium diolate, Clostridium violaceum and Clostridium saccharoaceticum.

[0079] A particularly preferred bacterium which is (naturally) capable of producing isopropanol in the wild type, in particular capable of performing an IBE fermentation in the wild type, is the bacterium Clostridium beijerinckii.

[0080] The acetogenic bacteria of interest are bacteria that produce acids and / or solvents from CO and H. Acetogenic Clostridium bacteria can, for example, be selected from the group consisting of C. aceticum, C. thermoaceticum, C. ljungdahlii, C. autoethanogenum, C. difficile, C. faecalis, and C. carboxydivorans.

[0081] In a particular embodiment, the Clostridium bacterium of interest is an "ABE strain", preferably Clostridium acetobutylicum strain DSM 792 (also known as ATCC strain 824 or LMG 5710) or Clostridium beijerinckii strain NCIMB 8052.

[0082] In another specific embodiment, the Clostridium bacterium of interest is an "IBE strain," generally one of the Clostridium beijerinckii bacteria identified herein, such as a Clostridium beijerinckii or Clostridium aureobutyricum DSZM 793 bacterium whose clade is selected from DSM 6423, LMG 7814, LMG 7815, NRRL B-593, NCCB 27006 (Georges et al., 1983), and clades of such Clostridium beijerinckii or Clostridium aureobutyricum bacteria that exhibit at least 90%, 95%, 96%, 97%, 98%, or 99% identity to strain DSM 6423. Particularly preferred Clostridium beijerinckii bacteria or clades of Clostridium beijerinckii bacteria lack the pNF2 plasmid.

[0083] On the one hand, the corresponding genomes of the LMG 7814, LMG 7815, NRRL B-593 and NCCB 27006 clades, and on the other hand, the genome of DSZM 793, showing a sequence identity percentage of at least 97% with the genome of the DSM 6423 clade.

[0084] The present inventors have conducted fermentation experiments and confirmed that Clostridium beijerinckii bacteria of clades DSM 6423, LMG 7815 and NCCB 27006 are capable of producing isopropanol in the wild type (see Table 1).

[0085] [Table 1]

[0086]

[0087]

[0088] Overview of glucose fermentation experiments using the naturally occurring isopropanol-producing strains Clostridium beijerinckii DSM 6423, LMG 7815, and NCCB 27006

[0089] In a particularly preferred embodiment of the present invention, the Clostridium beijerinckii bacterium is a DSM 6423 clade bacterium.

[0090] In another embodiment of the present invention, the Clostridium beijerinckii bacterium is the Clostridium beijerinckii IFP963ΔcatBΔpNF2 strain (registered at the BCCM-LMG Collection under accession number LMG P-31277 on February 20, 2019, and also referred to herein as Clostridium beijerinckii DSM 6423ΔcatBΔpNF2).

[0091] According to a particular embodiment, the bacterium to be transformed and preferably genetically modified is a bacterium that has been exposed to a first transformation step and a first genetic modification step using a nucleic acid or genetic tool according to the invention, making it possible to delete at least one extrachromosomal DNA molecule (generally at least one plasmid) that is naturally present in the bacterium in the wild type.

[0092] Another aspect described by the inventors relates to a method for transforming and preferably also genetically modifying Clostridium bacteria using the genetic tools according to the present invention, typically using a nucleic acid of interest according to the present invention as described above. The method comprises the step of transforming the bacteria by introducing the nucleic acid of interest described herein into the bacteria. The method may also comprise the step of obtaining, recovering, selecting or isolating the transformed bacteria, i.e., bacteria having the desired recombinant / modified / optimized gene.

[0093] In a specific embodiment, the method for transforming and preferably genetically modifying Clostridium bacteria involves a genetic modification tool, for example selected from CRISPR, tools based on the use of group II introns (e.g. Tools or tools) and allelic exchange tools (e.g. The invention provides a genetic modification tool for the bacterium comprising the step of transforming the bacterium by introducing the nucleic acid of interest according to the present invention as described above into the bacterium.

[0094] In general, the present invention is advantageously implemented in the case where the genetic modification tool selected for transforming and preferably genetically modifying a bacterium of the genus Clostridium is intended for use on a bacterium that, in the wild type, carries a gene encoding an enzyme responsible for resistance to one or more antibiotics, such as Clostridium beijerinckii, and the implementation of the genetic tool comprises a step of transforming the bacterium with the help of a nucleic acid allowing the expression of a resistance marker for the antibiotic to which the bacterium is resistant in the wild type, and / or a step of selecting the transformed and / or genetically modified bacterium with the help of the antibiotic to which the bacterium is resistant in the wild type.

[0095] A modification that can be advantageously achieved by the present invention, for example using genetic modification tools selected from the group consisting of CRISPR tools, tools based on the use of class II introns and allelic exchange tools, consists in deleting the coding sequence of an enzyme that provides the bacterium with resistance to one or more antibiotics, or in rendering this sequence non-functional. Another modification that can be advantageously achieved by the present invention consists in genetically modifying a bacterium that has previously been modified by the present invention to make it sensitive to an antibiotic to which it is resistant in the wild type, in order to improve its performance, for example its performance in producing a solvent or solvent mixture of interest.

[0096] In a preferred embodiment, the method according to the present invention is based on the use (implementation) of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology / genetic tools, in particular CRISPR / Cas (CRISPR associated protein) genetic tools.

[0097] 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 CRISPR-associated protein 9 (Cas9 protein) from Streptococcus pyogenes), which make double-stranded cuts in a DNA molecule (the target sequence of interest) under the guidance of an RNA molecule. The sequence of the guide RNA (gRNA) determines the cutting site of the nuclease, providing it with very high specificity. Since double-stranded cuts within DNA molecules that are essential for the survival of the microorganism are in fact lethal to the organism, the survival of the organism depends on its ability to repair the cuts (see, for example, Cui & Bikard, 2016). In Clostridium bacteria, the repair of double-strand breaks relies on the mechanism of homologous recombination, which requires a complete copy of the cut sequence. By providing the bacterium with a DNA fragment that allows the repair to occur while modifying the original sequence, the microorganism can be forced to integrate the desired changes into its genome.

[0098] The present invention can be implemented using conventional CRISPR / Cas genetic tools in Clostridium bacteria, using a single plasmid containing a nuclease, gRNA, and a repair template as described by Wang et al. (2015). The CRISPR / Cas system contains two different essential elements, namely i) an endonuclease, in this case the CRISPR-associated nuclease Cas, and ii) a guide RNA. The guide RNA takes the form of a chimeric RNA, which is composed of a combination of bacterial CRISPR RNA (crRNA) and tracrRNA (trans-activating CRISPR RNA). The gRNA combines the targeting specificity of the crRNA corresponding to the "spacer sequence" that acts as a guide for the Cas protein with the conformational properties of the crRNA in a single transcript. When the gRNA and Cas protein are expressed simultaneously in a cell, the target genomic sequence can be permanently modified due to the provided repair template. Professionals and technicians can easily determine the sequence and structure of the gRNA based on the chromosomal region or mobile genetic element to be targeted using well-known techniques (see, for example, DiCarlo et al., 2013).

[0099] The introduction of the elements of the genetic tool (nucleic acids or gRNA) into the bacteria is carried out by any direct or indirect method known to the skilled person, for example by transformation, conjugation, microinjection, transfection, electroporation, etc., preferably by electroporation (Mermelstein et al., 1993).

[0100] The present inventors have recently developed and described a genetic tool for modifying bacteria based on the use of two plasmids, which is suitable for Clostridium bacteria and can be used in the context of the present invention (see WO 2017 / 064439, Wasels et al., 2017, and related publications in this specification). Figure 15 ).

[0101] In a particular embodiment, the "first" plasmid of this 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 template 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, preferably different but inducible by the same inducer (e.g., described in application WO2017 / 064439 and incorporated herein by reference).

[0102] The gRNA can be a natural RNA, a synthetic RNA or an RNA produced by recombinant technology. These gRNAs can be prepared by any method known to professionals, such as chemical synthesis, in vivo transcription or amplification technology. When multiple gRNAs are used, the expression of each gRNA can be controlled by a different promoter. Preferably, the promoter for all gRNAs is the same. In a specific embodiment, the same promoter can be used to express several, for example, only some, or in other words all or some of the gRNAs intended to be expressed.

[0103] In another specific embodiment suitable for use in the context of the present invention, ii) at least one of the "first" and "second" nucleic acids further encodes one or more guide RNAs (gRNAs), or the genetic tool further comprises one or more guide RNAs, each guide RNA comprising an RNA structure that binds to the Cas enzyme and a sequence complementary to the targeted part of the bacterial DNA, and 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 the genetic tool further comprises a "third" nucleic acid encoding an anti-CRISPR protein under the control of an inducible promoter, which is preferably different from the promoter controlling the expression of Cas and / or RNA and can be inducible by another inducing agent.

[0104] In a preferred embodiment, the anti-CRISPR protein is able to inhibit, preferably neutralize, the action of the nuclease, preferably at the stage of introducing the nucleic acid sequence of the genetic tool into the bacterial strain of interest.

[0105] A specific method involving CRISPR technology that can be implemented in the context of the present invention to transform and, typically, genetically modify Clostridium bacteria by homologous recombination comprises the following steps:

[0106] a) introducing the CRISPR genetic tool described by the inventors into said bacteria in the presence of an agent that induces expression of an anti-CRISPR protein, and

[0107] b) culturing the transformed bacteria obtained at the end of step a) on a medium that does not contain an agent for inducing the expression of an anti-CRISPR protein (or under conditions that do not involve said agent), generally allowing the expression of the Cas / gRNA ribonucleoprotein complex.

[0108] In a specific embodiment, the method further comprises, during or after step b), a step of inducing an inducible promoter controlling the expression of Cas and / or guide RNA (if such promoter is present in the genetic tool) so as to allow the genetic modification of interest of the bacterium after the genetic tool is introduced into the bacterium. The induction is performed using a substance that allows the release of the expression inhibition associated with the selected inducible promoter.

[0109] In another specific embodiment, the method further comprises an additional step c) of removing the nucleic acid containing the repair template (the bacterial cell is then considered to be "cleared" of the nucleic acid) and / or removing the guide RNA or sequence encoding the guide RNA introduced in step a) using the genetic tool.

[0110] In yet another specific embodiment, the method comprises, after step b) or step c), one or more additional steps of introducing an nth, e.g., third, fourth, fifth, etc., nucleic acid in the presence of an agent that induces the expression of an anti-CRISPR protein, said nucleic acid comprising a repair template different from the one already introduced and one or more guide RNA expression cassettes that allow the sequence of interest contained in said different repair template 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 in a medium that does not contain said agent for inducing the expression of an anti-CRISPR protein, typically allowing the expression of the Cas / gRNA ribonucleoprotein complex.

[0111] In a particular embodiment of the method according to the invention, the bacteria are transformed using a CRISPR tool or method, e.g., as described above, which uses (e.g., encodes) an enzyme responsible for cleaving at least one strand of a target sequence of interest, wherein in a particular embodiment, the enzyme is a nuclease, preferably a Cas-type nuclease, preferably selected from the group consisting of a Cas9 enzyme and a MAD7 enzyme. Preferably, the target sequence of interest is an enzyme that provides the bacteria with resistance to one or more antibiotics, preferably one or more antibiotics belonging to the class of amides, typically an amide-O-acetyltransferase, e.g., a coding sequence for chloramphenicol-O-acetyltransferase, e.g., the catB gene, a sequence that controls the transcription of the coding sequence, or sequences flanking the coding sequence.

[0112] Examples of Cas9 proteins suitable for use in the present invention include, but are not limited to, Cas9 proteins from Streptococcus pyogenes (see SEQ ID NO: 1 of application WO 2017 / 064439 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).

[0113] The MAD7 nuclease, also known as "Cas12" or "Cpf1" (whose amino acid sequence corresponds to SEQ ID NO: 72), can also be advantageously used in the context of the present invention in combination with gRNAs known to those skilled in the art that are capable of binding to such nucleases (see Garcia-Doval et al., 2017 and Stella S. et al., 2017).

[0114] According to a specific aspect, the sequence encoding the MAD7 nuclease is a sequence optimized for easy expression in a Clostridium strain, preferably the sequence SEQ ID NO: 71.

[0115] When used, the CRISPR protein is typically an "anti-Cas" protein, i.e., a protein that is capable of inhibiting or preventing / neutralizing the action of Cas, and / or a protein that is capable of inhibiting or preventing / neutralizing the action of a CRISPR / Cas system, such as a type II CRISPR / Cas system when the nuclease is a Cas9 nuclease.

[0116] 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. Such proteins are generally capable of significantly limiting, and ideally preventing, the action of Cas9, for example by binding to the Cas9 enzyme.

[0117] Another anti-CRISPR protein that can be used advantageously is an “anti-MAD7” protein, such as the AcrVA1 protein (Marino et al., 2018).

[0118] Similar to the targeted DNA portion ("recognition sequence"), the editing / repair template 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 template may also comprise one or more "foreign" sequences, i.e., sequences that are not naturally present in the genome of bacteria belonging to the genus Clostridium or the genome of a particular species of said genus. The template may also comprise a combination of sequences.

[0119] The genetic tools used in the present invention allow the repair template 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, 1,000, 10,000, 100 000 or 1 000 000 bp, typically between 1 bp and 20 kb, for example 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, for example between 1 bp and 5 kb, between 2 kb and 5 kb or between 2.5 or 3 kb and 5 kb.

[0120] In a particular embodiment, expression of the DNA sequence of interest allows the Clostridium bacterium to ferment (usually simultaneously) several different sugars, such as at least two different sugars, typically at least two different sugars among sugars containing 5 carbon atoms (such as glucose or mannose) and / or sugars containing 6 carbon atoms (such as xylose, arabinose or fructose), preferably at least three different sugars, for example selected from glucose, xylose and mannose; glucose, arabinose and mannose; and glucose, xylose and arabinose.

[0121] In another specific embodiment, the DNA sequence of interest encodes at least one product of interest, preferably a product that promotes solvent production in Clostridium bacteria, typically at least one protein of interest, such as an enzyme, a membrane protein such as a transporter, a maturation protein for other proteins (chaperone protein), a transcription factor or a combination thereof.

[0122] In another embodiment, the method according to the invention is based on the use of group II introns and implements e.g. Genetic techniques / tools or Genetic tools.

[0123] described The technology relies on the use of reprogrammable class II introns (based on the L1.ltrB intron of Lactococcus lactis), which can be rapidly integrated into the bacterial genome at the desired locus (Chen et al., 2005, Wang et al., 2013), usually with the goal of inactivating the targeted gene. The mechanism of recognition of the edited region and insertion into the genome by backsplicing is based on the homology between the intron and the region in question and on the activity of the protein (ltrA) on the other hand.

[0124] The technology is based on a similar approach, supplemented by the addition of a selection marker to the intron sequence (Heap et al., 2007). This marker allows selection for the integration of the intron into the genome, thus facilitating the acquisition of desired mutants. This genetic system also utilizes class I introns. In fact, the selection marker (called a retrotransposition-activated marker or RAM) is interrupted by this genetic element, which prevents its expression from the plasmid (a more precise description of the system: Zhong et al.). Splicing of this genetic element occurs before integration into the genome, resulting in a chromosome with an activated form of the resistance gene. An optimized version of the system includes FLP / FRT sites upstream and downstream of the gene, allowing the removal of the resistance gene using FRT recombinase (Heap et al., 2010).

[0125] In another embodiment, the method according to the invention is based on the use of an allelic exchange tool and performing e.g. Genetic techniques / tools.

[0126] The technology is based on the use of auxotrophic mutants (for uracil auxotrophy, in Clostridium acetobutylicum ATCC 824, this is achieved by deletion of the pyrE gene, which also confers resistance to 5-fluoroorotic acid (A-5-FO); Heap et al., 2012). The system uses an allelic exchange mechanism well known to those skilled in the art. Following transformation with a pseudosuicide (very low copy) vector, the latter is integrated into the bacterial chromosome via a first allelic exchange event, resulting in selection for the resistance gene originally present on the plasmid. This integration step can be performed in two different ways: within the pyrE locus or within another locus:

[0127] In the case of integration at the pyrE locus, the pyrE gene is also placed on the plasmid, but is not expressed (no functional promoter). A second recombination restores a functional pyrE gene, which can then be selected by auxotrophy (minimal medium without uracil). Because the nonfunctional pyrE gene also possesses selectable properties (sensitivity to A–5–FO), additional integrations can occur in the same model, sequentially alternating the pyrE state between functional and nonfunctional.

[0128] In the case of integration at another locus, targeting allows a genomic region (usually in an operon following another gene, preferably a highly expressed gene) in which a counterselectable marker is expressed after recombination. This second recombination is then selected by auxotrophy (minimal medium without uracil).

[0129] In the described method based on the use of group II introns and Genetic techniques / tools or Genetic tools or based on the use of allelic exchange tools and implementation of e.g. In an embodiment of the genetic technique / tool, the targeted sequences are preferably sequences flanking the coding sequence of the enzyme of interest, preferably the amide alcohol-O-acetyltransferase as explained above.

[0130] Another subject matter of the present invention relates to transformed and / or genetically modified bacteria obtained using the methods described by the inventors herein, generally bacteria of the genus Clostridium belonging to the species described by the inventors or corresponding to one of the evolutionary branches described by the inventors, and any derivative bacteria, clones, mutants or genetically modified forms thereof.

[0131] Typical bacteria of the present invention that are transformed and / or genetically modified in this way are bacteria that no longer express an enzyme that confers resistance to one or more antibiotics, in particular bacteria that no longer express an amide-O-acetyltransferase, for example bacteria that express the catB gene in the wild type but lack the catB gene or are unable to express the catB gene after transformation and / or genetic modification according to the present invention. The bacteria transformed and / or genetically modified according to the present invention are rendered sensitive to amide alcohols, for example, the amide alcohols described herein, in particular, chloramphenicol or thiamphenicol.

[0132] A specific example of a preferred genetically modified bacterium according to the present invention is a bacterium identified in this specification as Clostridium beijerinckii DSM 6423ΔcatB, which was registered at the Belgian Co-ordinated Collections of Micro-organisms ("BCCM", KLLedeganckstraat 35, B-9000 Gent-Belgium) on December 6, 2018 under the deposit number LMG P-31151. The present specification also relates to any derivative bacteria, clones, mutants or genetically modified forms of the bacteria that retain sensitivity to amide alcohols such as thiamphenicol and / or chloramphenicol.

[0133] According to a particular embodiment, the bacteria transformed and / or genetically modified according to the invention, such as Clostridium beijerinckii DSM 6423 ΔcatB bacteria, which do not express an enzyme that confers resistance to one or more antibiotics, in particular an amide-O-acetyltransferase, such as chloramphenicol-O-acetyltransferase, can still be transformed and preferably genetically modified. This can be done using nucleic acids, such as plasmids, as described in this specification, for example in the experimental section. 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).

[0134] Indeed, a particular aspect of the present invention relates to the use of a genetically modified bacterium according to the invention, preferably the Clostridium beijerinckii DSM 6423 ΔcatB bacterium deposited under number LMG P-31151, or a genetically modified form thereof, for producing one or more solvents, preferably at least isopropanol, preferably on an industrial scale, by expression of a nucleic acid of interest autonomously introduced into its genome, e.g. using one of the genetic tools or methods described herein.

[0135] The present invention also relates to a kit comprising: (i) a nucleic acid of interest according to the present invention, generally a DNA fragment, which identifies a coding sequence for an enzyme of interest in a bacterium of the genus Clostridium, in particular a bacterium capable of IBE fermentation as described herein, or a sequence controlling the transcription of said coding sequence; and (ii) at least one means, preferably several means, chosen from elements of a genetic modification tool for transforming and generally genetically modifying a bacterium of the genus Clostridium in order to produce an improved variant of said bacterium, a nucleic acid serving as a gRNA, a nucleic acid serving as a repair template, at least one primer pair, for example the primer pairs described in the context of the present invention, and an inducer allowing the expression of a protein encoded by said tool, for example a Cas9 or MAD7 type nuclease.

[0136] Said genetic modification tools for transforming and generally genetically modifying Clostridium bacteria may be selected from the group consisting of, for example, the CRISPR tools, group II intron-based tools and allelic exchange tools explained above.

[0137] The kit may also contain one or more inducers customized for the selected inducible promoter optionally used within the genetic tool to control expression of the nuclease and / or one or more guide RNAs used.

[0138] The specific kit according to the invention allows the expression of a nuclease comprising a tag.

[0139] The kit according to the present invention may further comprise one or more consumables, such as culture medium, at least one competent Clostridium bacterium (ie, prepared for transformation), at least one gRNA, a nuclease, one or more selection molecules, or instructional materials.

[0140] The present description also relates to the use of a kit according to the present invention or one or more elements thereof for carrying out the methods described herein for transformation and, ideally, genetic modification of Clostridium bacteria, and / or for producing solvents or biofuels or mixtures thereof using Clostridium bacteria, preferably Clostridium bacteria that naturally produce isopropanol, preferably on an industrial scale.

[0141] 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.

[0142] 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.

[0143] The following examples and figures are intended to more fully illustrate the present invention but are not intended to limit the scope thereof. Sequence Listing <110> IFP Energies nouvelles <120> Genetically modified Clostridium bacteria, their preparation and use <130> B2903PC00 <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 aagacacgac 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 gtctgacgc tcagtgaac gaaaacctcac gttaggt 2280 tttggtcatg agattatca aaaggatctt cacctagatc cttttaaatt 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 <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 <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 atcaaaaaaaaaaaaatt 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<\(0000806\)><211> 8874<\(0000807\)><212> DNA<\(0000808\)><213> Artificial sequence<\(0000809\)><220><\(0000810\)><223> pCas9ind<\(0000811\)><400> 22<\(0000812\)>catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60<\(0000813\)>tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120<\(0000814\)>acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180<\(0000815\)>agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240<\(0000816\)>ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300<\(0000817\)>actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360<\(0000818\)>taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420It should be noted that the <\(0000806\)> and <\(0000807\)> and other tags are kept as they are because they are likely specific identifiers in a particular context and may not have a direct equivalent in a simple translation. If there is more context or specific requirements regarding these tags, further adjustment might be needed.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 atcaaaaaaaaaaaaatt 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 tattaagat actgaaat gcaaataca ttcgttgatg attcatgata aacagtagc 3060 aacctattgc agtaaataca atgagtcaag atgtttacat aaagggaag 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 atgagcac ttaaagaag agatataca ggttggggac gtttgtcaag 5940 aaagctatt atgtatta gagataaca atcaggaag actattctcg atttcttta 6000 atcagatgga ttgctaata gaactttat gcaattaatt catgatgatt ctcttacttt 6060 caagaggat attcaaagg ctcaagttc tggacaggc gatagctac acgaacacat 6120 tgctaactt gcagggagcc ccgctatcaa aaaggaatt ttacaacag ttaaagttgt 6180 agatgaactt gttaagtta tgggagaca caacctgag atatagtta taggaatggc 6240 gagaaaaatstorm aaaaaaaattct 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 aaaaaggaaaa 7620 tgaacttgcg ctgccatcaa agtatgtaaa ttcttatat ttagctctc attatgagaa 7680 attaaagga tcaccagagg ataatgaca aaagcaacta ttgtagaac aacacaaca 7740 tttattagat gaaataatag aaaataatc tgaatttctct aaagagtta tacttgccga 7800 cgcaatcta taaggtgc ttcaagcgta 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 tgtaaaaaat 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 aaaatagttt 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 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 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 aagtaaaaaactataac 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 tgtaattta actataact atttaaatta cagattaaa aaatttaaa aaaattgaa 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 attack 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 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 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 ctttaaaagt 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 cttcgagctc gattcgtaa 900 tcatggtcat 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 tctaaatac 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 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 ctagaatag 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<(0001604)>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 aatttgtta 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 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<00​ 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 atcaaaaaaaaaaaaatt 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 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> 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 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 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[[ID=3D]] 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[[ID=2,9]] <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 Note: There seems to be a typo in the original text where "215" is followed by a "?" which is translated as "?" in the English version as it's not clear what it should be. If this is an error, please correct the original text for a more accurate translation.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 ​​​​​​​​​​​​​​​​​​​ 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<00023三9>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<0三46>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 Note: There seems to be a typo in the original text where is likely meant to be and <0三46> is likely meant to be . These have been left as is in the translation as per the instruction to preserve all 7 - digit tags exactly. 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 40 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 20 25 30 Asn Ile Glu Ile Thr Asn Leu Leu His Glu Ile Lys Leu Lys Asp Leu 35 40 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Leu 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 Glu Asn Glu 85 90 95 Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Ser Arg Phe 100 105 110 Tyr Thr Ala Tyr Leu Asp Asp Ile Lys Asn His Gly Asn Ile Met Lys ​​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 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 <212> PRT<C <213> Artificial sequence <220> <223> Desnuesiella massiliensis<0002<C <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 <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 <400> 63 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<​​​​​​​​​​​​​​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 Ala Ser Asn Tyr Glu Thr Trp Leu Gly Glu Lys <210> 67 <211> 218 <212> PRT <213> Synthetic Sequence <220> <223> Clostridium sp. BL8 <400> 67 Met Lys Phe Asn Leu Ile Asp Ile Asp Gln Trp Asp Arg Lys Pro Tyr 1 5 10 15 Phe Glu His Tyr Phe Asn Ser Val Lys Cys Thr Tyr Ser Ile Thr Ala 20 25 30 Asn Ile Glu Ile Thr Asn Leu Leu Lys Asp Ile Lys Ile Thr Lys Leu 35 40 45 Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Ile Asn Asn 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 Dear Asn Dear Gln Dear 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 ttatactgtc 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 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> 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 cgggggtgga 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 aatacttcc ttcatatgcc tattacgata 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 ttagatatga cagtgaaaaa aatttattct gctttacatt tgactacaat 3360 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 <213> Artificial Sequence <220> <223> MAD-7 <400> Amino acids 1 to 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 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 His Glue House 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 gaaaaggatg ctttgcgatg gtggcaaact gtctgttagg aggttattct caaaggattg 240. ttgaggataa tccgtataac taactattac acattcttaa cattgctggt ttgtatcggt ttgtagtagc ttgtagtagc 300. ttgtagtagc aagtgtattt gttttatatt ctatgaacct at <210> 75 <211> 1368 <212> PRT <213> Antibiotic(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 Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Light Arg Thr Ala Arg Arg Arg Tire Thr Arg Arg Light Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp 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 Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu 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 Leu 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 His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys 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 Lys Asp Leu Ile Ile Lys 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 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His 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 Ile Leu Ala 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 atggggagc 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 aaagacttaa aacatatgct 1920 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt 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 gcaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300 cagacaggcg gattctccaa ggagtcatt ttaccaaaa gaattcgga caagcttatt 3360 gctcgtaaaa aagactggga tccaaaaaaa tatgtggtt ttgatagtcc aacggtagct 3420 tattcagtcc tagtggttgc taggtgga aaagggaat cgagaagtt aaatccgtt 3480 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540 tttttagag ctaaggata taaggatt aaaaagact 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 ttaaatattt 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 ctgaatgga tcaaattgca gtaatttcaa atatggagac taatgaaaag cttggagtag gacatgatga tatgagacct aaattttcag tgttagatcc tacatatact tttacagtac ctaaaaatca aacagcagcg ggaacagctg acattatgag tcacaccttt gaatcttact ttagtggtgt tgaaggtgct tatgtgcagg acggtatagc agaagcaatc 720. 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 tatttaaaaa atcttattaa 1170 <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<​​​​​​​​​​​​​​​​​​​ <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 tctttgaaga 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 aatttattt 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 aggctattaa aaatacgaga gatacttta attcattggg tattccttca aagcttagag 5340 aagttggaat aggaagat aaactagaac taatggcaa gcaagctgtt agaattctg 5400 gaggaacaat aggaagtttta agaccataa atgcagagga tgttcttgag atatttaaaa 5460 aatcttatta atagaactg tagaggtatt tttataattt aaagatgtt aaagagtgag 5520 gagtaattt gttctaacgc ctcactctt tcattttatg attaatgta tgctgattta 5580 cgctaactta atcctaaat aaacctaa tgttaatatt ttgtaacaa tggataaag 5640 cgtaaaaata ttattgtaat aatttaagt agttttaaaa tatatataat gtagaagcat 5700 tcctacatta tattatta ataataatct aaacaggagg gggttaaagtg gttgatttca 5760 aatctgtgta aacctaccgg ggtttggggg tagccattat attcatgac tccaagaag 5820 cagtagcta gcaaagaat aaaactcaa gcagagagaa atttagaca ttcaactata 5880 aaaaaaat accccaaa gcattaatat cttggggaggt attttttatt ttgaagtatt 5940 ctgttcagct aaatattctt ctaagtaat acctctgttc ataatttctt gtgaggcagg 6000 aagaccgata tatcttacat gccatggctc aaaattatac ttgttatgt tttctttatc 6060 cttaggatat cttattatga accatattt accacaattt tgttgaagcc attataga 6120 atttgtattc ataaatccat catctaaaga agagtattcg gttgatagta agtccattgc 6180 caatccagtt tgatgctcac tgtaccagg ttcagctaca tatttatcag cttcggcttt 6240 tccgtctcgt gctacttttt cattatataa ttttgctga tacgaataag gtctaacc 6300 tgaaacagct agaagtgtaa gaccatcctt tgatgctgca ttaaacatat ttcaagtcc 6360 tgttgcagct tcgctctcca ttgatttac attaggatca gaactacta taaatttac 6420 gttaggagtt ctcaaatttt gaggtatata gtttcctgat aatttacttt gcttgtttac 6480 aagtaggatg ttctgtttct ttacctcggg tttcttggct tgttttttag 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 ३०० 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 cggaaaaaga gttggtagct cttgatccgg caaacaaacc 960 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1020 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1080 cgttaaggga ttttggtcat gagattatca aaaaggatct 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 tctttgaaga 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 aatttattt 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 aactaaatta aatgattctt tttaattttt tactatttca 4080 ttccataata tattactata attatttaca aataatattt cttcattgt aatatttaga 4140 tgattacta attttagttt ttatatatta aataattaat gtataattta tataaaaaaat 4200 caaaggagct tataaattat gattatttcc aaagatacta aagatttaat ttttttcaat 4260 tttaacaata cttttgtaa tattatgttt aaatttaatt gtattttttt catataataa 4320 agccgttgaa gtaaaccaat ccattttcct tatgatgtta ttattaaatt taagtttat 4380 aataatatct ttattattatt tattgtttt aaaaaaaacta gtgaaatttc tagtgaaatt 4440 tccggcttta ttaaacttat ttttaggaat tttattttca ttttcatctt tacaggattt 4500 gattatatct ttaaatatgt tttatcaaat attatctttt tctaaattta tatatatttt 4560 tattatattt attattatat atattttatt tttaagtttc tttctaacag ctattaaaaa 4620 gaaacttaaa aataaaaaca cgtactctaa accaataaat aaaactattt ttattattgc 4680 tgccttgatt ggaatagttt ttagtaaaat taatttcaat attccacaat attatattat 4740 aagctagcac gcctcgagac tctatcattg atagagtttg aaacctatc attgatagag 4800 tataattct ttgttcatgc ttattacgac ataacacagt tttagagcta gaaatagcaa 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 attttaatt ctctaatcgt taaatctgag caatttgtaa ataaggtttc tatagtatgt ttatttgttt taaggctagt tgaaaccgtc ttcgcgttat ttttagatgc ttcttcttta 5580. ttaaaattt tattaaacaa cgaaaattc accccctcaa tttatttata taatagtagt ttgcatgaaa tttcgttgtt tattcatatt agatgcttgt attack 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 6240 tccgtctcgt gctacttttt cattatataa tttttgctga tacgaataag gtctataacc 6300 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 <locus_tag> 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 tgcattcta cctcctaacc tataaaatta gccaatttta 960 tagtagtctt attackaca tttacatgag agctttgcaa agcagtttat air 1020 gctttttatt ttaaaataa ttctctaaa tataagaata ttttaaagaa atatctttat 1080 atattagtta ttaaattta windowata agaacatta windows tagacttt 1140 taactattct aaagattaa tttacatatt aacatttaat tatgggtaa actattttg 1200 aaaaatgatt tatggaat tatgttctt aaatacaa tcatgtttca tgaatacata 1260 attattttaa atgtattggg agggtaaat gatattaaa atgaatacc atgagatac 1320 tgcagaatct agaatccgcg gtagtcgacg tggaattgtg agcggataac aatttcacag 1380 gaggggctga attacatta atgattaat tagagaata aaaaaaaaaaag attacacagt 1440 gaattgagt ggtacggata gcaatagt aacacagcta attattagag ttaataatga 1500 tggaaacgag tatgtaattt ctgaaagtga aaatgaatca atagttgaa aattcatatc 1560 tgcatttaa aacggttgga atcagaata cgaggatgaa gagaattttt atatgacat 1620 gcaaacaatc accttaaaaa gtgagttgaa ctaa 1654 <210> 82 <211> 4984 <212> DNA <213> Artificial Sequence <220> <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 agggatttg gtcatgagat tatchcaaag gatcttcacc aggattttt taaattaaaa 1320 atgaagtttt aaatcaatct aaagtatata tgagtaact tggtctgaca gttaccaag 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 aaaaaaatttttttattattattattttt gagaaaaga 2820 atataaaaaa tgagccttta taaaagccca ttttttca tatacgtaat atgacgttct 2880 aatgttttta ttggtacttc taacattaga gtaatttctt tattttttaa gccttttttct 2940 ttaagggctt ttattttt tcttaataca ttatattcct ctttttttgt tgctttttcct 3000 ttagctttta attgctcttg atatttttt ttacctctaa tattttctt tctcttatat 3060 tcctttttag aaattattat tgtcatatat tttgttctt cttctgtaat ttctaataac 3120 tctataagag ttcattctt atacttatat tgcttatttt tatctaaata acatctttca 3180 gcacttctag ttgctcttat aacttctt tcacttaat gttgtctaaa catactatta 3240 agttctaaaa catcatttaa tgccttctca atgtctctg taagctaca agatatatat 3300 ctatataaa atataag ctctctgtgt ccttttaaat catattctct tagttcacaa 3360 agttttatta tgtcttgtat tcttccataa tataaacttc tttctctata atataattt 3420 attttgcttg gtctaccctt tttcctttca tatggtttta attcaggtaa aaatccattt 3480. tgtatttctc ttagtcata fathertattcg tactcatcta fathertattgac tactgttttt gatttaggt ttatacttcc tggaactctt aatattctcg ttgcatctaa ggcttgtcta tctgctccaa agtattttaa ttgattatat aaatattctt gaaccgcttt ccataatggt aatgctttac taggtactgc atttattatc catattaat acattcctct tccactatct attacatagt ttggtatagg aatactttga ttaaataat tcttttctaa gtccattaat acctggtctt tagttttgcc agttttataa taatccaagt ctaaacag tgtatttaac tcttttatat tttctaatcg cctacacggc tttaaaagg tatttaggt father ttttcatcac tcatatctaa atcttttaat tcagcgtatt tatagtgcca ttggctatat ccttttttat ctataacgct cctggttatc caccctttac ttctactatg aatattatct atatagttct ttttattcag ctttaatgcg tttctcactt attcacctcc ccttctgtaa aactaagaaa attatcat attttcaat attachment attcttaac tcttaataa aatagagta agtccccaat tgaaacttaa tctatttttt atgttttaat ttattatttt tattaaata ttttaaacta aattaaatga ttctttttaa ttttttacta tttcattcca 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 tgattggaat agtttttagt aaaattaatt tcaatattcc acaatattat attataagct 4920 agcacgcctc gagatctcca tggacgcgtg acgtcgactc tagaggatcc ccgggtaccg 4980 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> Primer <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<​​​​​ 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 tctaaatac 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 gaagcttcaa aaaaagcacc gactcggtgc cactttttca agttgataac 2640 ggactagcct tattttaact tgctatttct agctctaaaa cctagctaat atggagaaat 2700 catgaacaaa gatattatac tctatcaatg atagagtttc aaactctatc 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 gtttttcttt ttcggcaagt gttcaagaag 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 <400> 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 aaaaaatg agcctttata aaagcccatt tttttcata tacgtatatat gacgttctaa 540 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 tctaaatac 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 gaactcttta 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 father father father acaaataata tttcttcatt tgtaataattt 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 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 3540 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3600 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 3660 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3720 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3780 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3840 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3900 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3960 atctaaagta tatatgagta aacttggtct gacagttacc aaagctagct tatactagt 4020 atatacttaa tgtgataagt gtctgacagc tgaccggtct aaagaggtc ctagcgccta 4080 cgggggaattt gtatcgataa ggggtacaaa ttcccactaa gcgctcggcc ggggatcgat 4140 ccccgggtac gtacccggca gtttttcttt ttcggcaagt gttcaagaag 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 acaaaattat acttcctttg gcaattcaag ttcatcacgc 4860 agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag 4920 ttaacttcag gtttgtctgt aactaaaaac tagtatttaa cctagg 4966 <210> 104 <211> 4966 <212> DNA <213> Artificial sequence <220> <223> pGRNA-glcG <400> 104 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 aatttgtta 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 ttcatttccg 4860 gcagtaggat ccccagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 4920 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttg 4966 <210> 105 <211> 4938 <212> DNA <213> Artificial sequence <220> <223> pGRNA-bdhB <400> 105 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 aaaaaaaaaaaaata aaaaaaaaaaaaaaaaaaaaaa 420 tttttattt aaagtttgaa aaaaattttt ttttatta taatctttga agaaaagaat 480 aaaaaatg agcctttata aaagcccatt tttttcata tacgtatatat gacgttctaa 540 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 tctaaatac 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 tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860 ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920 atatattact ataattattt acaataata tttcttcatt tgtaatattt agatgattta 1980 ctaatttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040 gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100 atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160 gaagtaaacc aatccatttt ccttatgatg ttatattaa atttaagttt tataataata 2220 tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280 ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340 tctttaaata tgttttatca aatattatct ttttctaaat ttatatat ttttattata 2400 tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460 aaaaataaaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520 attggaatag tttttagtaa attaatttc aatattccac aatattatat tataagctag 2580 cacgcctcga gtatattgat aaaaataata atagtgggta taattaagtt gttaggaggt 2640 tagttagagc ttattacgac ataacacagt tttagagcta gaatagcaa gttaaaataa 2700 ggctagtccg tttcaactt gaaaaagtgg caccgagtcg gtgcttttt tgaagcttgt 2760 cgactctaga ggatccccgg gtaccgagct cgaattcgta atcatggtca tagctgtttc 2820 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 2880 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 2940 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 3000 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 3060 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 3120 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 3180 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 3240 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 3300 cgtttcccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 3360 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 3420 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 3480 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 3540 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 3600 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 3660 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 3720 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 3780 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 3840 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 3900 tcctttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 3960 ctgacagtta ccaaagctag cttaatacta gtatatactt aatgtgataa gtgtctgaca 4020 gctgaccggt ctaaagaggt ccctagcgcc tacggggaat ttgtatcgat aaggggtaca 4080 aattcccact aagcgctcgg ccggggatcg atccccgggt acgtacccgg cagtttttct 4140 ttttcggcaa gtgttcaaga agttattaag tcgggagtgc agtcgaagtg ggcaagttga 4200 aaaattcaca aaaatgtggt ataatatctt tgttcattag agcgataaac ttgaatttga 4260 gagggaactt agatggtatt tgaaaaaatt gataaaaata gttggaacag aaaagagtat 4320 tttgaccact actttgcaag tgtaccttgt acctacagca tgaccgttaa agtggatatc 4380 acacaaataa aggaaaaggg aatgaaacta tatcctgcaa tgctttatta tattgcaatg 4440 attgtaaacc gccattcaga gtttaggacg gcaatcaatc aagatggtga attggggata 4500 tatgatgaga tgataccaag ctatacaata tttcacaatg atactgaaac attttccagc 4560 ctttggactg agtgtaagtc tgactttaaa tcatttttag cagattatga aagtgatacg 4620 caacggtatg gaaacaatca tagaatggaa ggaaagccaa atgctccgga aaacattttt 4680 aatgtatcta tgataccgtg gtcaaccttc gatggcttta atctgaattt gcagaaagga 4740 tatgattatt tgattcctat ttttactatg gggaaatatt ataaagaaga taacaaaatt 4800 atacttcctt tggcaattca agttcatcac gcagtatgtg acggatttca catttgccgt 4860 tttgtaaacg aattgcagga attgataaat agttaacttc aggtttgtct gtaactaaaa 4920 actagtattt aacctagg 4938 <210> 106 <211> 4790 <212> DNA <213> Artificial Sequence <220> <223> pEC750C <400> 106 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 aaaaaaaaaaaaaaaaaaaaaaaaata aaaaaaaaaaaaaaa 360 aaaaaaaaaaaaata aaaaaaaaaaaaaaaaaaaaaa 420 tttttattt aaagtttgaa aaaaattttt ttttatta taatctttga agaaaagaat 480 aaaaaatg agcctttata aaagcccatt tttttcata tacgtatatat gacgttctaa 540 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 tctaaatac 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 tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800 atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860 ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920 atatattact ataattattt acaataata tttcttcatt tgtaatattt agatgattta 1980 ctaatttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040 gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100 atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160 gaagtaaacc aatccatttt ccttatgatg ttatattaa atttaagttt tataataata 2220 tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280 ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340 tctttaaata tgttttatca aatattatct ttttctaaat ttatatat ttttattata 2400 tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460 aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520 attggaatag tttttagtaa attaatttc aatattccac aatattatat tataagctag 2580 cacgcctcga gatctccatg gacgcgtgac gtcgactcta gaggatcccc gggtaccgag 2640 ctcgaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 2700 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 2760 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagcgggaa acctgtcgtg 2820 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 2880 ttccgcttc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 2940 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 3000 catgtgagca aaaggccagc aaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 3060 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 3120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 3180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 3240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 3300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 3360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 3420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 3480 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 3540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 3600 ttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatcctttt 3660 gatctttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 3720 catgagatta tcaaaaagga tcttcaccta gatcctttta attaaaaat gaagttttaa 3780 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaaagct agcttaatac 3840 tagtatac ttaatgtgat aagtgtctga cagctgaccg gtctaaagag gtccctagcg 3900 cctacgggga atttgtatcg aaggggta caattccca ctaagcgctc ggccggggat 3960 cgatccccgg gtacgtaccc ggcagtttttt cttttcggc aagtgttcaa gaagttatta 4020 agtcgggagt gcagtcgaag tgggcaagtt gaaaaattca caaaatgtg gtataatatc 4080 tttgttcatt agagcgataa acttgaattt gagagggaac ttagatgta tttgaaaaaa 4140 ttgataaaaa tagttggaac agaaagagt attttgacca ctactttgca agtgtacctt 4200 gtacctacag catgaccgtt aaagtgata tcacacaat aaaggaaag ggatgaaac 4260 tatatcctgc atgctttat tatattgcaa tgattgtaaa ccgccattca gagtttagga 4320 cggcaatcaa tcaagatggt gattgggga tatatgatga gatgatacca agctatacaa 4380 tattcacaa tgatactgaa acatttcca gccttttggac tgagtgtaag tctgacttta 4440 aatcatttt agcagattat gaagtgata cgcaacggta tggaacaat catagaatgg 4500 aaggaaagcc aaatgctccg gaaacattt ttaatgtatc tatgataccg tggtcaacct 4560 tcgatggctt taatctgaat ttgcagaag gatatgatta ttgattcct atttttacta 4620 tggggaaata ttataaagaa gataacaaaa ttatacttcc tttggcaatt caagttcatc 4680 acgcagtatg tgacggattt cacatttgcc gttttgtaaa cgaattgcag gaattgataa 4740 atagttaact tcaggtttgt ctgtaactaa aaactagtat ttaacctagg 4790 <210> 107 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 107 acttgggtcg accacgataa aacaaggttt taagg 35 <210> 108 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 108 taccagggat ccgtattaat gtaactatga tatcaattct tg 42 <210> 109 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 109 atgcatggtc ccaatgaata ggtttacact tactttagtt ttatgg 46 <210> 110 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 110 atgcgagtta acaacttcta aaatctgatt accaattag 39 <210> 111 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 111 atgcatggat cccaatgaat aggtttacac ttactttagt tttatgg 47 <210> 112 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 112 atgcgagagc tcaacttcta aaatctgatt accaattag 39 <210> 113 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 113 atgcatggat ccgtctgaca gttaccaggt cc 32 <210> 114 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 114 atgcgagagc tccaattgtt caaaaaaata atggcggag 39 <210> 115 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 115 atgcatggat cccggcagtt tttctttttc gg 32 <210> 116 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 116 atgcgagagc tcggttaaat actagttttt agttacagac 40 <210> 117 <211> 2686 <212> DNA <213> Artificial Sequence [[ID=三十一]]<220> <223> pUC19 <400> 117 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240 atgcctgcag gtcgactcta gaggatcccc gggtaccgag ctcgaattca ctggccgtcg 300 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 360 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 420 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 480 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 540 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 600 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 660 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 720 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cactttttcgg ggaaatgtgc 780 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 840 aataaccctg ataaatgctt aataatatt gaaaaaggaa gagtatgagt attcaacatt 900 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 960 aaacgctggt gaagtaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 1020 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 1080 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 1140 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 1200 tcacagaaaa gcatcttacg gatggcatga cattaagaga attatgcagt gctgccataa 1260 1320 taaccgcttt tttgcacaac atggggatc atgtaactcg ccttgatcgt tgggaaccgg 1380 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1440 caacgttgcg caaactatta actggcgaac tacttactct agcttccccgg caaattaa 1500 tagactggat ggagggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1560 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1620 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1680 1740 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1800 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1860 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1920 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1980 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 2040 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 2100 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 2160 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 2220 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 2280 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2340 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2400 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2460 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2520 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2580 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2640 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga 2686 <210> 118 <211> 4282 <212> DNA <213> Artificial Sequence <220> <223> pNF2 <400> 118 ctggagagga ttgtccttat acttatcata agcatgaagg acttgttatt cctagataga 60 gaattaatta tgttaaagag atataataaa ctcattataa ttataatttt tagtataatt 120 attattgcaa ttttttcgta taaatatcta ataatgccaa aagagcatag aatagaaatt 180 tcaacattat caaacataga agtttttaaa tttaatagtt tttcaaagtt tagtaacgaa 240 aaaatgtata ctattaatga tagtgataag ttaataaaat tcaaaacact atttaataat 300 ttagataaat caaaagatat aaaaaagatt agtattccgg aaagtgaaaa tttaaatgca 360 tttaaatttt ctgcacatat aaaacttaac tttaactatg ttaataaaga tagccaaata 420 actgaaggtg cttttcttat gtatattttg gtagacaatt tagaagggaa gtcatatatg 480 acttttttag gacaagattc aagctatata ttagatagta atgaaactaa cattttaaga 540 gaaatatta tgaattcaga gattaattaa tttatgaatt cataaatatt atctaagcac 600 gataaaacaa ggttttaagg ataagaaaag tcatgagatt tatagtaaat cttgtgactt 660 ttttattga atagtagaga gagttcggaa gtataacacg ctatattctt gatattttta 720 gaatagcaag cattggattt gtcctgacac tttcccaaaa attaaggagt tattccttaa 780 accaaaaaga ttaatgtggg aacaaattta gtgtatccat ttttgaaggg cgcacttata 840 caccaccaaa atggtgtgtg cgaaatcttt aaaaaagatt tatcaaaaag cttttttaaa 900 gctgggacat ttagaaaatc aataatgttt tttgcccaat acgctagtct taaaatctgc 960 aaggttgata actatttagt cccaggtatt agaatggggc atatatatac aaagtatata 1020 tatgcgtaaa tatatgtggg actgtgggaa caaaattgcg tgctaaaatt gtattgaaaa 1080 ggtaatgaaa aggtcatgct ttggtattgc taacgtatag aaaaggtaat gaaaagctca 1140 tggttctata aaaaagatgt accacgaaa atataggct ttgcctattt ccccatgtaa 1200 tatgggggca gttttctctt atgctttc ttaacatatt gaataatac aaaatgcagc 1260 tttgtgggaa taaaaatatt tttgtttt ttcttagt tagacaaat tttaatcttt 1320 tttgtgctat aacagatta aaatttgtgg gaacattag aaatattgtt gtcacaaata 1380 aaaaggagag tgggaacaat tgcttaaaaaacgcagaaa ttagattag agttacaaaa 1440 gagcaaaag attatta gaaaattgca aaagctgaa atatgagtat gagtgaattt 1500 attattgtga ccacagaata tttagccaga aaaaagatg aaataatgaa atcaaagac 1560 atgatcgaga gagagctgc gagactgaa gaaaaatta tgagctaa aagaaacta 1620 aaaaaaca ggtaatatag attacagttt taagcttgtt ttccctatag actagagtaa 1680 atatataaat atacctgtca agggcttata agcccctta gggggtgcgt agcaccttg 1740 acaggtatat ttatattt taggtgcca ttaagggaaa caagctttaa atgccttta 1800 aaggcatttt aaaataata aaaaaaagat gttttttacc atcttttta accccgaaa 1860 gggagttctt tcttttcttg atactatacg taactatttc gatttgccct gaacctaatc 1920 aaagctagat aaattcagta ttagggcata aaaaaacttg ctttttcggg tggaaatctg 1980 tataatttaa attgcttaga taaaaattac caattccata cgaaaggagc aagttttaca 2040 taaggttaaa gccttatgtg aattctcatt taattacatg aataataata acacagaaag 2100 tgaagaatta aaagagcaaa gtcaactatt gcttgacaaa tgcacaaaaa agaaaaagaa 2160 aaatcctaaa tttagtagtt atatagaacc attagtaagc aagaaattat ctgaaagaat 2220 aaaggaatgt ggtgactttt tgcagatgtt atctgattta aaccttgaaa attcgaaact 2280 gcatagagca agtttttgtg gtaacagatt ttgtcctatg tgtagctggc gtattgcttg 2340 taaggatagt ttggaaatat ctattctcat ggagcattta cgcaaagagg aaagcaaaga 2400 atttatcttt ttgaccttaa caactccaaa tgtgaaaggt gcggaccttg ataattccat 2460 aaaagcatac aataaagcat ttaaaaagtt aatggaacgc aaagaggtca agagcatagt 2520 aaaaggctac ataagaaagc tagaagtaac ctataatttg gacaagagtt ccaaatcata 2580 taatacttat caccacatt tccatgtggt actagcagtc atagaagtt actttaaaaa 2640 gcaaaatcta tataaacc atcatagatg gcttagttg tggcaaggt caactggtga 2700 ttattcgata actcaagttg atgtaagaa ggctaaaatt aacgatta aagaggttta 2760 tgagcttgct aagtattcgg ctaggattc cgactattta atcaatagag aagtgtttac 2820 ggtattctac aaatctttaa agggtaaca ggtacttgta tttagtggat tatttaaga 2880 cgctcataaa atgtataaga atggagct agatctgtat aagaagttgg atactatcga 2940 attgcttat atggtaagtt atactggct taaaaagaag tatgatactt caatattag 3000 agaatttact gaggagaa agcagaatt caataaaat ttaatcgaag atgtgaat 3060 tgagtaggtg ggattatatc tcaccttttttgtctt tcatgttgaa attttgacgc 3120 ttaatgcatg aagtattgac aagtttaaa attacggtttt ttaatcctta gttgattagc 3180 aggattatgg ccggaatgct ccgtccagtc ctgttaagga attaaaatttc cctaaaaccc 3240 ttggctatga ttatagcga gatcgtcaa ttaaaaattt ataggtgct atgaaagtcg 3300 attaataatt aatttaaaa tgcaatatga aacataatta caagaatttg acttttaata 3360 caagaattga tatcatagtt acattaatac atttattttg aagggggaaa atgttttatg 3420 aaaagactac ttaaactacc tattttatca ttattaggat tatttttaat tggatcaact 3480 ccaacattag ctttaactaa agataataat caaaatttag atactatgaa agtaaactta 3540 tatactgaaa cagtagatgt gtttgataaa gatgcattta aaaaacatt tactaataaa 3600 gatataaaat ttctagagga ttctttgaat gcaaaaataa attattcagg taaatctgtt 3660 acagtaacaa tgaaaaacaa aattaagcca tctactaaac aagggcttgt tttatatgta 3720 aatggaaaat cagttaatgt tgattcagat ggcagtataa aagtacctaa agatactaag 3780 aaaatttcta attaaataa agataaatca atgatggatg gatcaatgat ggataaatca 3840 ttacatgatg agaattgtgt agtatcagat agtttttata atgctgatgt tataatata 3900 aattcaaaag aagcagaagc tgtatttaaa gtaagttctg gtgaattatt agctaaaatg 3960 gatgaaaaag aagatgatta catacaaaag aactcatcta aaattctagc agctgcttat 4020 cataagggat atggggacaa gtactatgaa ggagattggg ttcattgcaa taggtttaat 4080 ggtcaactta cagatgatgt tcactataat tggagaactg gaagtgtttc agaaaaagca 4140 gctgcaatga gaaattttta tggcagtgat tgtcatatag cattagttca agcaggtagt 4200 ggatgtacaa gtataggttc atgcgaatgc aatacagatc aaatagctgc gtattgttca 4260 ggtttcgtaa aagataaaaa ta 4282 <210> 119 <211> 5473 <212> DNA [[ID=*]]<213> Artificial sequence <*20> <223> pNF3<00045*0><400> 119​​​​​​​​​​​atagtaaatc ttgtgacttt tttgaa tagtagagag agttcggaag tataacacgc 360 tatattcttg atattttg atagcaagc attggattg tcctgacact ttcccaaaaa 420 ttaggagtt attccttaaa ccaaaagat taatgtggga acaatttag tgtatccatt 480 tttgaagggc gcacttatac accaccaaa tggtgtgtgc gaatcttta aaaaagattt 540 atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600 cgctagtctt aaatctgca aggttgataa ctatttagtc ccaggtatta gatggggca 660 tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720 gctaaaattg tattgaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780 aaagtaatg aaagctcat gggttcttaa aaagatgta cccacgaaaa tataggctt 840 tgcctatttc cccatgtaat atggggcag tttcttttgctttct taacatattg 900 aaaataca aaatgcagct ttgtgggaat aaaatatttt ttgttttt tcttatagtt 960 agacaaaatt ttaatcttt ttgtgctata acagattaa atttgtggg aacattaaga 1020 atattgttg tcaataa aaaggagt gggaacaatt gctaaaaa acggcagaat 1080 window gttacaaag agcaaaag atttattag aaattgcaa aagctgaaaa 1140 tatgagtag agtgaattta ttattgtgac cacagaat tagccagaa aaaagatga 1200 aaataatgaaa tcaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260 gaagctaaaaaaactaa aaaaacag gtaatataga ttacagtttt aagcttgttt 1320 tccctataga ctagagtaaa tataata tacctgtcaa gggcttataa gccccttag 1380 ggggtgcgta gcaccttga caggtatatt tatatattt agggtgccat taagggaaac 1440 aagctttaaa atgccttaa agcatttta aaaaata aaaaagatg gttttacca 1500 tcttttta ctcccgaaag ggagttttt ctttcttga tactatacgt aactatttcg 1560 atttgccctg aacctaatca aagctagata attcagtat taggcataa aaaaacttgc 1620 ttttcggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680 gaaaggagca agttcat agagttaag ccttatgtga attctcatttt attackatga 1740 aataataa cacagaaagt gaaattaa aagagcaaag tcaactattg cttgacaaat 1800 gcaacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860 agaaattatc tgaaagaata aaagaatgtg gtgacttttt ccagatgtta tctgatttaa 1920 accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980 gtagctggcg tattgcttgt areatagtt tggaaatatc tattctcatg gagcatttac 2040 gcaaagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100 cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160 aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220 aaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280 atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340 ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400 acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460 tcatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520 ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580 agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640 atgatactc aaatattaga gaattaactg aggaaaaaa gcagaaattc aataaaaatt 2700 taatcgaaga tgtggaatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760 catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820 taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880 ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940 ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000 aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatccccggg 3060 taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 3120 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 3180 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga 3240 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 3300 gtacaatctg ctctgatgcc ccatagttaa gccagccccg acacccgcca acacccgctg 3360 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3420 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 3480 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 3540 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 3600 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 3660 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 3720 tttgccttcc tgtttttgct caccagaaa cgctggtgaa agtaaaagat gctgaagatc 3780 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 3840 gttttcgcc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 3900 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 3960 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 4020 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 4080 tgacaacgat cggaggaccg aaggagctaa ccgcttttt gcacaacatg ggggatcatg 4140 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 4200 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 4260 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 4320 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 4380 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 4440 4500 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 4560 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 4620 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 4680 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 4740 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 4800 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 4860 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 4920 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 4980 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 5040 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 5100 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 5160 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 5220 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 5280 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 5340 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 5400 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 5460 aggaagcgga aga 5473 <210> 120 <211> 9128 <212> DNA <213> Artificial Sequence <220> <223> pMTL007S-E1 <400> 120 gatcgggccc cctgcagggt gtagtagcct gtgaaataag taaggaaaaa aaagaagtaa 60 gtgttatata tgatgattat tttgtagatg tagataggat aatagaatcc atagaaaata 120 taggttatac agttatataa aaattacttt aaaaattaat aaaaacatgg taaaatataa 180 atcgtataaa gttgtgtaat ttttaagctt gagctcataa caatttcaca caggaaacag 240 ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 300 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 360 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 420 gctaataaag atcttgtaca atctgtagga gaacctatgg gaacgaaacg aaagcgatgc 480 cgagaatctg aatttaccaa gacttaacac taactggggga taccctaaac aagaatgcct 540 aatagaaagg aggaaaaagg ctatagcact agagcttgaa aatcttgcaa gggtacggag 600 tactcgtagt agtctgagaa gggtaacgcc ctttacatgg caaaggggta cagttatgt 660 gtactaaaaat taaaaattga ttagggagga aaacctcaaa atgaaaccaa caatggcaat 720 tttagaaaga atcagtaaaa attcacaaga aaatatagac gaagttttta caagacttta 780 tcgttatctt ttacgtccag atatttatta cgtggcgacg cgtgcgactc atagaattat 840 ttcctcccgt taaataatag ataactatta aaaatagaca atacttgctc ataagtaacg 900 gtacttaaat tgtttacttt ggcgtgtttc attgcttgat gaaactgatt tttagtaaac 960 agttgacgat attctcgatt gacccatttt gaaacaaagt acgtatatag cttccaatat 1020 ttatctggaa catctgtggt atggcgggta agttttatta agacactgtt tacttttggt 1080 ttaggatgaa agcattccgc tggcagctta agcaattgct gaatcgagac ttgagtgtgc 1140 aagagcaacc ctagtgttcg gtgaatatcc aaggtacgct tgtagaatcc ttcttcaaca 1200 atcagataga tgtcagacgc atggctttca aaaacccactt ttttaataat ttgtgtgctt 1260 aaatggtaag gaatactccc aacaatttta tacctctgtt tgttagggaa ttgaaactgt 1320 agaatatctt ggtgaattaa agtgacacga gtattcagtt ttaatttttc tgacgataag 1380 ttgaatagat ...

Claims

1. A nucleic acid that recognizes the catB gene of SEQ ID NO: 18 in the genome of a bacterium of C. beijerinckii that is capable of producing isopropanol in the wild type, characterized in that It is the plasmid pCas9ind–ΔcatB of sequence SEQ ID NO: 21 or the plasmid pCas9ind–gRNA_catB of sequence SEQ ID NO:

38.

2. The nucleic acid according to claim 1, characterized in that The nucleic acid comprises a guide RNA (gRNA) and / or a modification template.

3. Use of a nucleic acid according to claim 1 or 2 for transforming and / or genetically modifying Clostridium beijerinckii DSM 6423 bacteria which are capable of producing isopropanol in the wild type.

4. Use according to claim 3, wherein the nucleic acid does not show methylation at the motif recognized by Dam- and Dcm-type methyltransferases.

5. A method for transforming Clostridium beijerinckii DSM 6423 bacteria capable of producing isopropanol in the wild type using genetic modification tools, characterized in that The method comprises introducing into the bacteria The step of transforming the bacteria with the nucleic acid according to claim 1 or 2.

6. A method for genetically modifying Clostridium beijerinckii DSM 6423 bacteria capable of producing isopropanol in the wild type using a genetic modification tool, characterized in that The method comprises introducing into the bacteria The step of transforming the bacteria with the nucleic acid according to claim 1 or 2.

7. The method according to claim 5 or 6, characterized in that The bacteria are transformed with the CRISPR tool using an enzyme responsible for cleaving at least one strand of a target sequence encoding or controlling transcription of an amide alcohol-O-acetyltransferase.

8. A genetically modified Clostridium beijerinckii bacterium obtained by the method according to any one of claims 5 to 7.

9. A Clostridium beijerinckii DSM6423ΔcatB bacterium deposited under the number LMG P-31151.

10. Use of the genetically modified Clostridium beijerinckii bacterium according to claim 8 or the Clostridium beijerinckii DSM6423ΔcatB bacterium deposited under number LMG P-31151 according to claim 9 for the production of isopropanol.

11. Use of the genetically modified Clostridium beijerinckii bacterium according to claim 8 or the Clostridium beijerinckii DSM6423 ΔcatB bacterium deposited under number LMG P-31151 according to claim 9 for the production of isopropanol on an industrial scale.

12. A kit comprising (i) a nucleic acid according to claim 1 or 2 and (ii) at least one tool selected from the group consisting of an element of a genetic modification tool, a nucleic acid as a gRNA, a nucleic acid as a repair template, at least one primer pair and an inducer allowing expression of a protein encoded by the tool.

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