KR20190086984A - Transformed corynebacterium glutamicum having capability of producing squalene - Google Patents

Abstract

The present invention relates to a transformed Corynebacterium glutamicum strain having capability of producing squalene, and uses thereof. The present invention relates to a Corynebacterium strain transformed by using a transformed vector manufactured by genetic recombination involved in a metabolic pathway of the strain. By using the strain, it is possible to stably produce a large amount of squalene in high purity, thereby having economic feasibility.

Description

Transformed corynebacterium glutamicum having capability of producing squalene < RTI ID = 0.0 >

The present invention relates to the use of a transformed Corynebacterium glutamicum strain having squalene production ability, wherein a gene involved in the metabolic pathway of the strain is recombinant to produce a transformation vector, It is about four bacterium strains.

Squalene is a triterpenoid-based unsaturated hydrocarbon compound with 30 carbon atoms and 50 hydrogen atoms linked by double bonds. It is found in deep sea shark liver oil in nature and is a trace amount of a substance contained in vegetable oil. This substance has useful functions such as antioxidant function, anticancer effect, immunity enhancement function, and is useful substance for health functional foods, cosmetics, medicines and the like. Squalene was extracted from plants to replace undersea sharks in order to produce squalene. However, since plants contain a small amount of squalene, the production of squalene from plants is ineffective in terms of cost, and it is known that there is a possibility of contamination during plant cultivation by air heavy metals. In recent years, the beauty care industry has grown rapidly, and squalene consumption is expected to increase in the future. Therefore, it is necessary to replace the mass production of sustainable squalene in an economical and stable manner.

Corynebacterium glutamicum is capable of regulating metabolic pathways and metabolic pathways through genetic manipulation. It can be used to regulate existing metabolic pathways through synthetic biology / metabolism engineering techniques, It is known as an industrial strain that mass-produces metabolites. Therefore, there is a need for research on a method capable of producing a desired metabolite by recombinantly editing a gene involved in the metabolic pathway of Corynebacterium glutamicum, and in particular, a method for mass-producing squalene using the strain .

Korean Patent Application No. 10-2017-0099545 Korean Patent Application No. 10-2016-0041774

Photosynthetic conversion of CO 2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene) was investigated. and squalene) by engineered cyanobacteria. Biotechnol Biofuels 69: 202 Kang, M. K., Lee, J., Um, Y., Lee, T. S., Bott, M., Park, S. J., & Woo, H. M. (2014). Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization. Applied microbiology and biotechnology, 98 (13), 5991-6002.)

Thus, the present inventors have completed the present invention for Corynebacterium glutamicum transformed to have the ability to produce squalene as a metabolite.

Accordingly, an object of the present invention is to provide a method for producing squalene, which is transformed into a vector containing a gene involved in squalene production, which inhibits the expression of a part of the gene involved in the original metabolic process, .

Another object of the present invention is to provide a method for producing squalene by culturing the transformed Corynebacterium glutamicum.

It is still another object of the present invention to provide a culture composition composition for improving the squalene production ability of the transformed Corynebacterium glutamicum.

In order to solve the above-mentioned problems, the present invention provides a method for producing 1-deoxy-D-xylylulose 5- (4-hydroxyphenyl) -1,2,4-oxadiazole from pyruvate and D-glyceraldehyde 3-phosphate A gene encoding an enzyme that produces phosphate (1-deoxy-D-xylulose 5-phosphate, DXP); A gene encoding an enzyme that produces dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP); A gene encoding an enzyme that produces farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP); And a gene coding for an enzyme that produces squalene from fenicillin diphosphate (FPP). The present invention also provides a strain of Corynebacterium glutamicum,

In one embodiment of the present invention, 1-deoxy-D-xylulose 5-phosphate (1-deoxy-D-xylulose 5-phosphate) is obtained from pyruvate and D- glyceraldehyde 3- The gene coding for the enzyme producing deoxy-D-xylulose 5-phosphate (DXP) comprises the nucleotide sequence of SEQ ID NO: 1 and is derived from isopentenyl diphosphate (IPP) by dimethylallyl diphosphate The gene encoding the enzyme producing DMAPP contains the nucleotide sequence of SEQ ID NO: 2, and the gene encoding the enzyme producing farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP) 3, and the gene encoding an enzyme that produces squalene from panesyl diphosphate (FPP) comprises the nucleotide sequence of SEQ ID NO: 4.

Further, in one embodiment of the present invention, the enzyme that produces 1-deoxy-D-xylulose 5-phosphate (DXP) from (G3P) with pyruvate and D-glyceraldehyde 3- The enzyme producing deoxyxylulose-5-phosphate synthase (dxs) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is isopentenyl (Isopentenyl diphosphate delta isomerase, idi), and the enzyme producing panesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP) is geranyl transferase (ispA) , An enzyme that produces squalene from panesyl diphosphate (FPP) is a squalene synthase.

In another embodiment of the present invention, the Corynebacterium glutamicum of the present invention comprises a gene coding for the enzyme, a dxs gene, an idi gene, an ispA gene, and a sequence derived from a gene encoding a squalene synthase, trSQS CgCO). ≪ / RTI > (This is called a first vector.)

In another embodiment of the present invention, the dxs gene and the idi gene are derived from Corynebacterium glutamicum, the ispA gene is derived from E. coli, the squalene synthase gene is a Saccharomyce But are not limited to, those derived from Saccharomyces cerevisiae.

The Corynebacterium glutamicum of the present invention comprises a gene encoding the above-mentioned enzymes, and additionally, an enzyme encoding an enzyme producing geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP) ('GapA gene') encoding an enzyme that produces 1,3-bisphosphoglycerate (1,3BPG) from D-glyceraldehyde 3-phosphate (G3P) And a vector containing an sgRNA that inhibits the expression of a gene (" gdh gene ") encoding an enzyme producing glutamate from ketoglutarate (? -GK). (This is called a second vector.)

In one embodiment of the present invention, the gene encoding the enzyme producing geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP) comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7, The gene encoding the enzyme producing 1,3-bisphosphoglycerate (1,3BPG) from D-glyceraldehyde 3-phosphate (G3P) comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9, The gene encoding the enzyme producing glutamate from ketoglutarate (? -GK) includes, but is not limited to, the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

In another embodiment of the present invention, a gene encoding an enzyme that produces geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP), a gene encoding D-glyceraldehyde 3-phosphate (G3P) In order to inhibit the expression of a gene encoding an enzyme producing bisphosphoglycerate (1,3BPG) or a gene encoding an enzyme producing glutamate from alpha ketoglutarate (? KG) Regulatory system was used, for which the second vector contains a gene encoding the dCas9 protein.

That is, the Corynebacterium glutamicum strain of the present invention is transformed with a first vector and a second vector, and comprises the dxs gene, the idi gene, the ispA gene, and the sequence derived from squalene synthetase trSQS (CgCO) , The second vector is a gene encoding an enzyme that produces geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP), a gene encoding D-glyceraldehyde 3-phosphate (G3P) A gene encoding an enzyme that produces phosphoglycerate (1,3BPG) or an sgRNA and a dCas9 gene that inhibits the expression of a gene encoding an enzyme that produces glutamate from alpha ketoglutarate (? KG) .

The Corynebacterium glutamicum of the present invention transformed with the above vector is a strain having the accession number KCCM12189P.

In another aspect, the present invention relates to a method for producing squalene comprising culturing the strain.

In one embodiment of the present invention, the strain is cultivated from a nutrient-lowered culture medium, and may contain tryptone in the culture to increase the productivity of squalene.

The Corynebacterium glutamicum of the present invention is transformed to regenerate squalene by regenerating a gene that regulates a metabolic pathway. In particular, a gene encoding an enzyme involved in squalene synthesis is introduced and, conversely, squalene synthesis The gene coding for the enzyme capable of inhibiting inhibits the expression and further has the property of further improving squalene production ability. The strain of the present invention can stably produce squalene having high purity and is suitable for mass production.

In particular, for the inhibition of gene expression, it is possible to produce a transformant having improved squalene production ability more easily by introducing a gene for regulating the gene of the genus Crisper, and further to provide a culture solution suitable for producing squalene from the transformant strain have.

Figure 1 schematically illustrates the squalene production pathway of the transformed Corynebacterium glutamicum strain. The names of genes involved in each process and the strains derived therefrom are described, and the expression of these genes can be regulated to produce squalene.
Figure 2 is a schematic representation of a first vector (left) and a second vector (right).
FIG. 3 shows the result of flame ionization detector gas chromatography confirmed that the substance produced by the transformant strain is squalene. The upper part shows the squalene standard (Sigma Aldrich, St. Louis, Missouri, United States), and the lower part shows all of the first and second vectors of the present invention and is produced from the transformed strain It is the result of confirming squalene.
FIG. 4 is a graph showing the relationship between the expression level of the squalene synthase gene (trSQS gene), the deoxygylulose 5-phosphate synthase gene ( dxs gene), the dimethylallyl diphosphate synthase gene ( idi gene), the phenesyl diphosphate synthase ( ispA gene) And a second vector containing no sgRNA. The results are shown in Fig.
FIG. 5 is a graph showing the expression of the squalene synthase gene (trSQS gene), the deoxygylulose 5-phosphate synthase expression gene ( dxs gene), the dimethylallyl diphosphate synthase expression gene ( idi gene), and the phenesyl diphosphate synthase expression The first vector containing the gene ( ispA gene) and the second vector including the vector to which the crystal gene expression inhibition technique was applied were confirmed to produce the squalene production of the strain transformed.
FIG. 6 is a graph showing the expression of the squalene synthetase gene (trSQS gene), the deoxygylulose 5-phosphate synthase expression gene ( dxs gene), the dimethylallyl diphosphate synthase expression gene ( idi (Control) containing a first vector having a phenesyl diphosphate synthase expression gene ( ispA gene) and a CGXII minimal (control) gene of a strain transformed with a first vector and a second vector as a chromosome gene expression inhibiting vector (N), CGXII minimal medium supplemented with tryptone, and 2YT medium supplemented with CGXII minimal medium.
Fig. 7 is a diagram showing a cleavage map of the first vector of the present invention.
FIG. 8 is a diagram showing a cleavage map of a vector into which sgRNA-idsA-r1 (a) and sgRNA-idsA-r3 (b) are inserted among the second vector of the present invention.
FIG. 9 is a diagram showing a cleavage map of a vector into which sgRNA-gapA-r1 (a) and sgRNA-gapA-r2 (b) are inserted, among the second vectors of the present invention.
Fig. 10 is a diagram showing a cleavage map of a vector into which sgRNA-gdh-r1 (a) and sgRNA-gdh-r2 (b) are inserted among the second vector of the present invention.

In order to develop a novel strain having improved squalene production ability,

D-xylulose 5-phosphate from D-glyceraldehyde 3-phosphate (G3P), pyruvate and D-glyceraldehyde 3-phosphate , A gene encoding an enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP), a gene encoding an enzyme producing DXP, dimethylallyl diphosphate (DMAPP) A first vector comprising a gene encoding an enzyme that produces farnesyl diphosphate (FPP) and a gene that encodes an enzyme that produces squalene from panesyl diphosphate (FPP); And

A gene encoding an enzyme that produces geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP), a gene encoding D-glyceraldehyde 3-phosphate (G3P), 1,3-bisphosphoglycerate 1,3BPG) or a vector comprising an sgRNA that inhibits the expression of a gene encoding an enzyme that produces glutamate from alpha ketoglutarate (alpha-KG) , The strain was transformed into Corynebacterium glutamicum, and the strain was confirmed to have a squalene-producing ability. The strain was deposited on December 19, 2017 with the deposit number KCCM 12189P at the Korean Society for Microbiological Care.

That is, the strain may be, but not limited to, transformed with a first vector and / or a second vector. The expression " first or second " is intended only to distinguish the types of vectors, and does not limit the order or method of transformation

The first vector is derived from pyruvate and D-glyceraldehyde 3-phosphate (G3P) by 1-deoxy-D-xylylulose 5-phosphate 5-phosphate synthase gene (dxs gene), an enzyme that produces D-xylulose 5-phosphate (DXP), isophentenyl isopentenyl diphosphate delta isomerase gene (idi gene), an enzyme that produces dimethylallyl diphosphate (DMAPP) from diphosphate (IPP), dimethylallyl diphosphate (DMAPP) ) From the geranyl transferase gene ('ispA gene'), an enzyme that produces farnesyl diphosphate (FPP), and squalene from panesyl diphosphate (FPP) And trSQS (CgCO), which is a sequence derived from a gene encoding an enzyme. The dxs gene comprises the nucleotide sequence of SEQ ID NO: 1 derived from Corynebacterium glutamicum, and the idi gene comprises the nucleotide sequence of SEQ ID NO: 2 derived from Corynebacterium glutamicum Wherein the ispA gene comprises the nucleotide sequence of SEQ ID NO: 3 derived from Escherichia coli, and the trSQS (CgCO) sequence comprises the nucleotide sequence of SEQ ID NO: 4 derived from Saccharomyces cerevisiae.

Also, the first vector may comprise the sequence of SEQ ID NO: 5.

In one embodiment of the present invention, Corynebacterium glutamicum transformed with only the first vector has squalene production ability. That is, in the transformed vector, Corynebacterium glutamicum transformed with the first vector containing the trSQS (CgCO) sequence and the dxs, idi and ispA genes and the second vector containing no sgRNA was cultured And confirmed by a flame ionization detector. As shown in Fig. 4, a squalene peak was confirmed. Therefore, Corynebacterium glutamicum transformed with only the first vector also has squalene production ability.

Among the genes inserted into the first vector, the dxs and idi genes are derived from Corynebacterium glutamicum, the ispA gene is derived from E. coli, the squalene synthase is saccharomyces cerevisiae It may be derived from Saccharomyces cerevisiae.

The second vector of the present invention comprises a gene (idsA gene) encoding an enzyme that produces geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP), D-glyceraldehyde 3-phosphate (G3P ) Encoding an enzyme that produces glutamate from a gene ('gapA gene') or alpha ketoglutarate (α-KG) encoding an enzyme that produces 1,3-bisphosphoglycerate (1,3BPG) Is a vector containing the sgRNA and the dCas9 gene which can inhibit the expression of the gene ('gdh gene').

The idsA gene comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7, the gapA gene comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9, the gdh gene comprises the nucleotide sequence of SEQ ID NO: SEQ ID NO: 10 or SEQ ID NO: 11.

The second vector may include a promoter derived from a gene coding for the enzyme. The second vector may be located near the promoter of each gene, and may include at least one of sequences targeting non-dominant strands. (SEQ ID NO: 12), idsA-r3 (SEQ ID NO: 13), gapA-r1 (SEQ ID NO: 14), gapA-r2 (SEQ ID NO: 15), gdh -r1 (SEQ ID NO: 16) and gdh-r2 (SEQ ID NO: 17).

In addition, the dCas9 gene contained in the second vector is a gene encoding a Cas9 protein having no nucleic acid degrading enzyme, and has the ability to bind to a specific DNA according to the sequence of the sgRNA, but has no ability to cut the target DNA. Thus, it directly regulates the transcription process without changing the sequence.

The second vector may preferably be selected from the sequence of SEQ ID NO: 18 to SEQ ID NO: 23, but is not limited thereto.

In one embodiment of the present invention, the transformed Corynebacterium glutamicum including both the first vector and the second vector has improved squalene production ability as compared with the strain transformed with only the first vector. The second vector contains the sequence selected from the sequence of SEQ ID NO: 6 to SEQ ID NO: 11, and the squalene production efficiency may be different when the gene expression is inhibited. However, when the first vector and the second vector were simultaneously transformed, it was confirmed that they had a higher squalene production ability than the case where only the first vector was transformed. (Fig. 5)

The genes in all vectors disclosed in the specification of the present invention are operably linked to each other. The meaning of being operable means that the target gene can be normally expressed

In the present specification, the term "target gene" may mean a gene inserted into a vector to be expressed in a strain so that the function of the gene can be exerted. The strain may be a gene having the first vector and / It may be transformed into four bacterium glutamicum (Accession No: ATCC 13032). In the parent strain, only the first vector may be introduced, and both the first vector and the second vector may be introduced.

The transformed strain may be a strain having the accession number KCCM12189P. The KCCM12189P strain of the present invention contains a strain transformed with the first vector, pBbEB1c-trSQS (CgCO) -dxs-idi-ispA, and the second vector, pCoryne-sgRNA target-r, with the parent strain Corynebacterium glutamicum It means.

In another aspect, the present invention is a method for producing squalene, comprising culturing the transformed Corynebacterium glutamicum strain.

The culture method may be performed by a method commonly used in the art, more preferably by shake culture using a baffled-flask or by using a bioreactor. When using the biorecerer, the culture can be performed at a temperature of 29 to 31 ° C and 120 to 900 rpm for 12 to 60 hours. In one embodiment of the present invention, the amount of squalene produced is measured after culturing at 30 ° C. and 900 rpm .

In another aspect, the present invention relates to a culture composition for culturing the transformed Corynebacterium glutamicum strain.

The culture medium is based on a CgXII minimal medium. To control the amount of nitrogen, a culture solution composition in which the amount of ammonium sulfate and urea is limited, a culture solution composition containing tryptone in a medium, or a culture solution further containing yeast extract and sodium chloride Composition.

Ammonium sulfate 0.001 to 0.1 g / L and urea 0.01 to 1.0 g / L are used, and more preferably 0.01 g / L of ammonium sulfate and 0.5 g / L of urea are used. The above range is for adjusting the nitrogen content in the culture liquid. When the concentration is below the above range, there is a problem that the nitrogen concentration for culturing the strain is too small. If the concentration is above the above range, there is no influence by the nitrogen amount control.

In the case of the tryptone, it may be contained in the culture medium in an amount of 10 to 20 g / L, more preferably 15 g / L.

In addition, the yeast extract may contain 1 g / L to 20 g / L of sodium chloride and 1 g / L to 10 g / L of sodium chloride, and more preferably 10 g / L of yeast extract and 5 g / L of sodium chloride, .

The composition of the culture solution is a composition when the squalene production amount of the transformed Corynebacterium glutamicum of the present invention is at a maximum, and the amount of squalene produced may vary depending on the components in the culture solution.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

≪ Example 1 >

Screening of genes for squalene production

In order to produce squalene using the isoprenoid metabolic pathway, a new method for the production of squalene using the isoprenoid metabolic pathway was developed. 2016) Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria Biotechnol Biofuels 69:. depending on the contents described in 202), dxs gene, idi Gene , ispA The metabolic pathway to squalene using the gene was applied to Corynebacterium glutamicum. The dxs Gene , idi The gene is derived from Corynebacterium glutamicum, and the ispA gene is derived from E. coli.

Further, the gene coding for the production of an enzyme that produces squalene from FPP are saccharide in my process to be derived from the celebrity busy (Saccharomyces cerevisiae), Corey the gene 72bp is removed from the DNA sequence 3 'of the ERG9 gene Corynebacterium After codon optimization in accordance with glutamicum, the squalene synthesis sequence trSQS (CgCO) was customized in Genscript. Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme. Archives of biochemistry and biophysics, 304 (1), 133-143 (1993) .)

≪ Example 2 >

A squalene synthesis gene expression vector ('first vector') and a transformant strain

The CoryneBrick vector pBbEb1c-rfp (Kang, MK, Lee, J., Um, Y., Lee, TS, Bott, M., Park, SJ, The rfp portion of the Corynebacterium glutamicum and its application to xylose utilization (Applied microbiology and biotechnology, 98 (13), 5991-6002.) was removed using EcoRI-BamHI restriction enzyme and the squalene synthase gene (trSQS gene ) Was inserted to prepare vector pBbEB1c-trSQS (CgCO) for producing squalene.

The pBbEB1c-trSQS (CgCO) -dxs vector in which the DNA sequence of the dxs gene of Corynebacterium glutamicum was inserted into the prepared pBbEB1c-trSQS (CgCO) using the BglII-BamHI-XhoI restriction enzyme, Corynebacterium glue Tamikum dxs Gene, the DNA sequence is inserted pBbEB1c-trSQS the idi gene (CgCO) -dxs-idi vector, Corynebacterium glutamicum dxs, idi gene and the E. coli of the ispA pBbEB1c-trSQS (CgCO) DNA sequence is inserted in the gene The -dxs-idi-ispA vector was constructed as shown in FIG.

Transformation of the vector was confirmed by PCR (5 '→ 3' primer sequence: forward-GAG CTC GAT ATC AAA TTA CG / reverse-GAG CTA AGG AAG CTA AAA TG).

≪ Example 3 >

A vector for suppressing gene expression in squalene producing strains ('second vector') and a gene inhibitor system design

A gene expressing an enzyme producing geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP), a gene expressing an enzyme producing 1,3-bisphosphoglycerate (1) from D-glyceraldehyde 3-phosphate (G3P) , 3BPG), an sgRNA which inhibits a gene expressing an enzyme producing glutamate from alpha ketoglutarate (? -GK), is located near the promoter of each gene, and a non-template strand Were selected for each of the 20 bp protospace genes. (5 '→ 3'idsA-r1 (SEQ ID NO: 12): CTC ATC ACA CTA AGA TAC CC, idsA-r3 AGA CGT CCT TCA ATC GAA TA, gdh-r1: TAA ATG CTC ACG TTC AAT TA, gdh-r2 : GAG GGC ACC ATG TAC ATC CT, gapA-r1: CAA CCT GTC CTC AAC TTT AG, gapA-r2: CGC ACC AAA CCT ACT CAC AT)

A primer containing the corresponding sequence was prepared and subjected to PCR using the plasmid pgRNA-bacteria (Addgene Plasmid # 44251) as a template to prepare pgRNA-target-r. The constructed sgRNA unit was inserted into the pCoryne-sgRNA-dCas9 vector, which is a gene expression control vector, using EcoRI and BamHI restriction enzymes to construct a second vector.

PCoryne-sgRNA-idsA-r1-dCas9, pCoryne-sgRNA-dCas9 containing sgRNA for inhibiting the expression of a gene encoding an enzyme producing geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP) (s) to inhibit the expression of a gene encoding an enzyme that produces 1,3-bisphosphoglycerate (1,3BPG) from D-glyceryl aldehyde 3-phosphate (G3P) PCoryne-sgRNA-gapA-r1-dCas9, pCoryne-sgRNA-gapA-r2-dCas9 vector, and sgRNA for inhibiting the expression of a gene encoding an enzyme producing glutamate from alpha ketoglutarate (? PCoryne-sgRNA-gdh-r1-dCas9 and pCoryne-sgRNA-gdh-r2-dCas9 vectors were constructed. Transformation was confirmed by PCR (5 '→ 3' primer sequence: forward-CCC GAA CAA AAA CTC ATC TC / reverse-GCC GCC ATT ATT ACG ACA AG).

<Example 4>

Corynebacterium & lt ; / RTI &gt; transformed by the first vector The ability of squalene production of Glutamicum strain

The transformed strains were cultured for a certain period of time to confirm the production of squalene. Culture conditions of the flask culture were inoculated into 50 mL of a CgXII minimal medium containing 2% glucose in a 250 mL baffled flask with an OD 600 of 1 at the initial incubation, and cultured at 30 rpm in an incubator at 120 rpm. 25 μg / mL of kanamycin antibiotics and 7.5 μg / mL of chloramphenicol antibiotics were added to the culture, and 1 mM IPTG and 100 nM aTc were added to the vector for gene expression.

To determine whether the transformant was producing squalene, the cultured strain was disrupted using glass beads, and squalene was extracted with chloroform: methanol (2: 1) as an extraction solvent. The extracted squalene was analyzed by GC-FID after addition of 20 μg / mL 1-phenyl octadecene as an internal standard.

a first vector in which the trSQS gene is inserted or a first vector in which the trSQS gene and the dxs gene are inserted or a trSQS gene and a dxs gene and a first vector in which the idi gene is inserted and a second vector not containing the sgRNA, No peak of squalene was observed in the strain. However, the trSQS gene and dxs gene, was gene, ispa Squalene peaks were confirmed in the transformed strains having the first vector inserted with the gene and the second vector not including the sgRNA (Fig. 3B). Squalene synthetase gene containing (trSQS gene), deoxy-5-xylene trehalose phosphate synthase gene (dxs gene) dimethyl allyl diphosphate synthase gene (idi gene) Ipanema chamber diphosphate synthase (ispA gene) with the rule The strain transformed with the first vector and the second vector containing no sgRNA was cultured and the strain cultured for 12 hours, 24 hours, 36 hours, 48 hours, and 60 hours after the start of the culture was collected to confirm the amount of squalene produced . As a result, as shown in FIG. 4, it was confirmed that the squalene production was almost constant in each hour, and 4.5 mg / g DCW squalene was produced.

&Lt; Example 5 >

Corynebacterium strains transformed by a first vector and a second vector Glue squalene production capacity of Tamiflu Qom strains

A second vector (pCoryne-sgRNA-target-r), which is an expression vector of the Crisper gene regulation system, and a second vector (pBbEB1c-trSQS (CgCO) -dxs-idi-ispA) The amount of squalene produced in the strain of glutamicum was determined as follows. For culturing, 1 mL of CgXII minimal medium containing 2% glucose was added to a flower-48-micro well plate at a temperature of 30 ° C and 900 rpm, and the culture was inoculated at an initial OD 600 of 1. In culture, 25 μg / mL of kanamycin antibiotics and 7.5 μg / mL of chloramphenicol antibiotics were added, and inducer 1 mM IPTG and 100 nM aTc for gene expression were added. The squalene production amount of the strain cultured for 48 hours after the start of the culture was measured.

As a result, trSQS gene, dxs gene, idi gene ispA The squalene production amount of the existing squalene producing strain having the first vector having all of the genes and the second vector having no sgRNA was measured to be 2.65 ± 0.034 mg / gDCW (FIG. 5, control). On the other hand, it was confirmed that squalene production was increased in the strain transformed with the second vector using the Crispell gene inhibition technique (FIG. 5). In other words, the amount of squalene produced by the strain using the Crisp gene inhibition technique together with the first vector having both the trSQS gene, the dxs gene and the idi gene ispA gene was 3.47 ± 0.16 in the strain containing the pCoryne-sgRNA-idsA-r1-dCas9 vector mg / gDCW in the strain containing the pCoryne-sgRNAgdh-r1-dCas9 vector and 4.28 ± 0.17 mg / gDCW in the strain containing the pCoryne-sgRNAgapA-r1-dCas9 vector.

&Lt; Example 6 >

Optimal medium composition

In order to maximize the production of squalene in the transformed Corynebacterium glutamicum strains producing squalene, a medium optimization technique was employed to apply various media conditions. Cultivation of the strain was carried out using a bioretector. The culture conditions were 30 ° C and 900 rpm. The culture conditions were as follows: a flower-48-micro well plate was prepared on the basis of a CgXII minimal medium containing 2% glucose and 0.01 g / L of ammonium sulfate and 0.5 1 mL of medium (N) with reduced nitrogen content in g / L, 1 mL of medium supplemented with 15 g / L tryptone in a CgXII minimal medium containing 2% glucose, or 2YT containing 10 g / L yeast extract, 16 g / L tryptone, 5 g / L NaCl) was added to the medium and the OD 600 was adjusted to 1 for the first incubation. In culture, 25 μg / mL of kanamycin antibiotics and 7.5 μg / mL of chloramphenicol antibiotics were added, and inducer 1 mM IPTG and 100 nM aTc for gene expression were added.

The squalene production amount of the strain cultured for 48 hours after the start of the culture was measured. In the case of applying the medium with low nitrogen content of CGXII minimal medium (CgXII N), squalene production was increased in all strains. In particular, strains containing the pCoryne-sgRNA-idsA-r1-dCas9 vector, Squalene production was improved in medium supplemented with tryptone in the minimal medium and in 2YT medium. [Figure 6]

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Korea Microorganism Conservation Center (overseas) KCCM12189P 20171219

<110> Sungkyunkwan university <120> Transformed corynebacterium glutamicum having capability of          producing squalene <130> PN1712-441 <160> 23 <170> KoPatentin 3.0 <210> 1 <211> 1911 <212> DNA <213> Corynebacterium glutamicum <400> 1 atgggcattc tgaacagtat ttcaacacct gctgacttaa aggcccttaa tgatgaggat 60 ttggacgctc ttgccaaaga aatccgaact ttcctggtcg ataaagtcgc agcaactggt 120 ggccacttag gtccaaattt gggcgtagtg gaattaacca tcggtcttca tcgagttttc 180 gattcgcctc aagacccgat catctttgat acttctcacc agtcctatgt gcataagatc 240 ctgacgggtc gcgctaaaga ttttgattct ttgcgtcaaa aagatggcct ttctggttac 300 acctgccgtg ctgaaagtga gcacgattgg actgagtctt cgcatgcttc ggcggccttg 360 tcttatgcgg atggtttgtc taaagccaag cagttggatg gcgataccac gcatagtgtg 420 gttgctgtcg ttggtgatgg cgctctaact ggcggcatgt gtgggaagc actgaacaat 480 attgctgctg gtaaagaccg caaagttgtt gtcgtagtca atgacaatgg ccggagttat 540 tctccaacca ttggcggatt tgcggaaaac cttgcgggcc ttcgcatgca gcctttctat 600 gatcgcttca tggaaaaggg caagacgtcc ctgaaatcca tggggtgggt aggggagcgt 660 acttttgaag cgctccatgc atttaaagaa ggtgtgaaga gcaccgtcat tcccaccgaa 720 atgttccctg aactgggcat gaaatacgtg ggtccggttg atggacataa ccaaaaagct 780 gtcgacaatg cgctgaaata cgctcatgat tatgatggcc ccatcatcgt gcacatggtc 840 accgaaaagg gtcgtggtta cgcgcctgct gagcaggatt tggacgaatt gatgcactcc 900 acgggcgtca tcgatccgct cacaggagct cctaaatctg catcaaagcc cggttggacc 960 tctgtgttca gcgatgagct ggtcaagatt ggtgcgcaga atgaaaacgt tgttgccatc 1020 accgccgcga tggcaggtcc taccggtctg tccaagttcg aagccaattt ccccaaccga 1080 ttctttgatg tcggcattgc tgagcagcac gcggtaactt ctgccgcagg cctcgcattg 1140 ggtggaaaac accctgtggt ggctatttac tccacgttct tgaaccgcgc ttttgatcag 1200 ctgctcatgg atgtgggcat gctcaaccag cctgttactt tggtgcttga tcgctcaggt 1260 gtcacgggtt cggatggagc gagccacaat ggcgtctggg atatggcgct gacctcgatc 1320 gttccaggcg tgcaggtggc ggcaccacgt gatgaggatt ccttgcgtga gctgctcaat 1380 gaggctattt ccatcgatga tggccccaca gttgtgcgtt tccccaaggg cgacttgcca 1440 actccaattg ttgctatcga caccttggaa gacggcgtgg atgtcctcgc atatgaagac 1500 gccactgacg ttgaatcaac cgacgatgcg ccatcagttc tcatcattgc ggtaggcgag 1560 cgcgcaactg ttgcacttga cgttgcttcc aggattaaac agcacggcgt gaacgtcacg 1620 gttgttgacc cccgctggat tgtccccatc ccgcagtcct tggtcgcgct gtctgatgat 1680 catgacctcg tgatcaccat cgaagacggc gtcatccacg gcggcgtggg ctccttgctc 1740 tctgatgcgc ttaacgcctc tgaggtggat acccctcgcc gacaaatcgc cgtgccccag 1800 aagtacctgg atcacgcgtc ccgcaatgaa gtgctcgccg attatggcct cgacgccgac 1860 ggcattgaaa ccactgttgt tggatggctg gattccctgt tcggggaata a 1911 <210> 2 <211> 588 <212> DNA <213> Corynebacterium glutamicum <400> 2 gtgtctaagc ttaggggcat gactactgag gttgaactgg ttgttttagc tgattccgag 60 ggcaatccta ttggtactgc gccgaaagct acggtgcaca ctaaggacac gcctctgcat 120 ttcgcgtttt ccacctatat tttgaacccg cgtggggagc tgttggtgac gcgtcgtgca 180 ttgtcgaaga agacatggcc tggtgtgtgg acgaactcta tgtgtgggca ccctggtccg 240 gatgagacaa acgcggatgc gattcgtcgc aggggtgtcg atgagttggg gctggaggta 300 gattctttct tggatattca agagattctg cctgattacc agtaccgtgc tgtcgacgcg 360 tccggcattg tggagtggga gttgtgcccg gtccacctcg tgcgtttagc ggtgggggaa 420 tttgtggagc cactggatga tgaggtggag gagttcgagt gggcggaacc gcagaagctt 480 ttcgacgctg ttgatgccac accatttgtg ttttctccat ggctagtgga tcagcttagc 540 gcccctgagc tgcgccaagc catcctggaa gcgtttgacg cagagtaa 588 <210> 3 <211> 900 <212> DNA <213> Escherichia coli <400> 3 atggactttc cgcagcaact cgaagcctgc gttaagcagg ccaaccaggc gctgagccgt 60 tttatcgccc cactgccctt tcagaacact cccgtggtcg aaaccatgca gtatggcgca 120 ttattaggtg gtaagcgcct gcgacctttc ctggtttatg ccaccggtca tatgttcggc 180 gttagcacaa acacgctgga cgcacccgct gccgccgttg agtgtatcca cgcttactca 240 ttaattcatg atgatttacc ggcaatggat gatgacgatc tgcgtcgcgg tttgccaacc 300 tgccatgtga agtttggcga agcaaacgcg attctcgctg gcgacgcttt acaaacgctg 360 gcgttctcga ttttaagcga tgccgatatg ccggaagtgt cggaccgcga cagaatttcg 420 atgatttctg aactggcgag cgccagtggt attgccggaa tgtgcggtgg tcaggcatta 480 gatttagacg cggaaggcaa acacgtacct ctggacgcgc ttgagcgtat tcatcgtcat 540 aaaaccggcg cattgattcg cgccgccgtt cgccttggtg cattaagcgc cggagataaa 600 ggacgtcgtg ctctgccggt actcgacaag tatgcagaga gcatcggcct tgccttccag 660 gttcaggatg acatcctgga tgtggtggga gatactgcaa cgttgggaaa acgccagggt 720 gccgaccagc aacttggtaa aagtacctac cctgcacttc tgggtcttga gcaagcccgg 780 aagaaagccc gggatctgat cgacgatgcc cgtcagtcgc tgaaacaact ggctgaacag 840 tcactcgata cctcggcact ggaagcgcta gcggactaca tcatccagcg taataaataa 900                                                                          900 <210> 4 <211> 1263 <212> DNA <213> Saccharomyces cerevisiae <400> 4 atgggcaagc tgctgcagct ggcactgcac ccagtggaaa tgaaggcagc actgaagctg 60 aagttctgcc gcaccccact gttctccatc tacgatcagt ccacctcccc atacctgctg 120 cactgcttcg aactgctgaa cctgacctcc cgctccttcg cagcagtgat ccgcgaactg 180 cacccagaac tgcgcaactg cgtgaccctg ttctacctga tcctgcgcgc actggatacc 240 atcgaagatg atatgtccat cgaacacgat ctgaagatcg atctgctgcg ccacttccac 300 gaaaagctgc tgctgaccaa gtggtccttc gatggcaacg caccagatgt gaaggatcgc 360 gcagtgctga ccgatttcga atccatcctg atcgagttcc acaagctgaa gccagaatac 420 caggaagtga tcaaggaaat caccgaaaag atgggcaacg gcatggcaga ttacatcctg 480 gt; tacgtggcag gcctggtggg cgatggcctg acccgcctga tcgtgatcgc aaagttcgca 600 aacgaatccc tgtactccaa cgaacagctg tacgaatcca tgggcctgtt cctgcagaag 660 accaacatca tccgcgatta caacgaagac ctggtggatg gccgctcctt ctggccaaag 720 gaaatctggt cccagtacgc accacagctg aaggatttca tgaagccaga aaacgaacag 780 ctgggcctgg attgcatcaa ccacctggtg ctgaacgcac tgtcccacgt gatcgatgtg 840 ctgacctacc tggcaggcat ccacgaacag tccaccttcc agttctgcgc aatcccacag 900 gtgatggcaa tcgcaaccct ggcactggtg ttcaacaacc gcgaagtgct gcacggcaac 960 gtgaagatcc gcaagggcac cacctgctac ctgatcctga agtcccgcac cctgcgcggc 1020 tgcgtggaaa tcttcgatta ctacctgcgc gatatcaagt ccaagctggc agtgcaggac 1080 ccaaacttcc tgaagctgaa catccagatt tccaagatcg aacagttcat ggaagaaatg 1140 taccaggata agctgccacc aaacgtgaag ccaaacgaaa ccccaatctt cctgaaggtg 1200 aaggaacgct cccgctacga tgatgaactg gtgccaaccc agcaggaaga agaatacaag 1260 taa 1263 <210> 5 <211> 10885 <212> DNA <213> Artificial Sequence <220> PBbEB1c-trSQS (CgCO) -dxs-idi-ispA vector <400> 5 gcgtcgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa 60 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat 120 gt; aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg 240 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc 300 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc 360 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac 420 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat 480 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac 540 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag 600 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc 660 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg 720 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg 780 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc 840 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga 900 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat 960 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg 1020 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc 1080 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 1140 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt 1200 gatctggttt gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca 1260 gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct 1320 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa 1380 atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg tggaattgtg 1440 agcggataac aatttcagaa ttcaaaagat cttaatacgg acgaggttaa atagaaggag 1500 gtaatagatg ggcaagctgc tgcagctggc actgcaccca gtggaaatga aggcagcact 1560 gaagctgaag ttctgccgca ccccactgtt ctccatctac gatcagtcca cctccccata 1620 cctgctgcac tgcttcgaac tgctgaacct gacctcccgc tccttcgcag cagtgatccg 1680 cgaactgcac ccagaactgc gcaactgcgt gaccctgttc tacctgatcc tgcgcgcact 1740 ggataccatc gaagatgata tgtccatcga acacgatctg aagatcgatc tgctgcgcca 1800 cttccacgaa aagctgctgc tgaccaagtg gtccttcgat ggcaacgcac cagatgtgaa 1860 ggatcgcgca gtgctgaccg atttcgaatc catcctgatc gagttccaca agctgaagcc 1920 agaataccag gaagtgatca aggaaatcac cgaaaagatg ggcaacggca tggcagatta 1980 catcctggat gaaaactaca acctgaacgg cctgcagacc gtgcacgatt acgatgtgta 2040 ctgccactac gtggcaggcc tggtgggcga tggcctgacc cgcctgatcg tgatcgcaaa 2100 gttcgcaaac gaatccctgt actccaacga acagctgtac gaatccatgg gcctgttcct 2160 gcagaagacc aacatcatcc gcgattacaa cgaagacctg gtggatggcc gctccttctg 2220 gccaaaggaa atctggtccc agtacgcacc acagctgaag gatttcatga agccagaaaa 2280 cgaacagctg ggcctggatt gcatcaacca cctggtgctg aacgcactgt cccacgtgat 2340 cgatgtgctg acctacctgg caggcatcca cgaacagtcc accttccagt tctgcgcaat 2400 cccacaggtg atggcaatcg caaccctggc actggtgttc aacaaccgcg aagtgctgca 2460 cggcaacgtg aagatccgca agggcaccac ctgctacctg atcctgaagt cccgcaccct 2520 gcgcggctgc gtggaaatct tcgattacta cctgcgcgat atcaagtcca agctggcagt 2580 gcaggaccca aacttcctga agctgaacat ccagatttcc aagatcgaac agttcatgga 2640 agaaatgtac caggataagc tgccaccaaa cgtgaagcca aacgaaaccc caatcttcct 2700 gaaggtgaag gaacgctccc gctacgatga tgaactggtg ccaacccagc aggaagaaga 2760 atacaagtaa ggatctatta aagaggagaa tactagatgg gcattctgaa cagtatttca 2820 acacctgctg acttaaaggc ccttaatgat gaggatttgg acgctcttgc caaagaaatc 2880 cgaactttcc tggtcgataa agtcgcagca actggtggcc acttaggtcc aaatttgggc 2940 gtagtggaat taaccatcgg tcttcatcga gttttcgatt cgcctcaaga cccgatcatc 3000 tttgatactt ctcaccagtc ctatgtgcat aagatcctga cgggtcgcgc taaagatttt 3060 gattctttgc gtcaaaaaga tggcctttct ggttacacct gccgtgctga aagtgagcac 3120 gattggactg agtcttcgca tgcttcggcg gccttgtctt atgcggatgg tttgtctaaa 3180 gccaagcagt tggatggcga taccacgcat agtgtggttg ctgtcgttgg tgatggcgct 3240 ctaactggcg gcatgtgttg ggaagcactg aacaatattg ctgctggtaa agaccgcaaa 3300 gttgttgtcg tagtcaatga caatggccgg agttattctc caaccattgg cggatttgcg 3360 gaaaaccttg cgggccttcg catgcagcct ttctatgatc gcttcatgga aaagggcaag 3420 acgtccctga aatccatggg gtgggtaggg gagcgtactt ttgaagcgct ccatgcattt 3480 aaagaaggtg tgaagagcac cgtcattccc accgaaatgt tccctgaact gggcatgaaa 3540 tacgtgggtc cggttgatgg acataaccaa aaagctgtcg acaatgcgct gaaatacgct 3600 catgattatg atggccccat catcgtgcac atggtcaccg aaaagggtcg tggttacgcg 3660 cctgctgagc aggatttgga cgaattgatg cactccacgg gcgtcatcga tccgctcaca 3720 ggagctccta aatctgcatc aaagcccggt tggacctctg tgttcagcga tgagctggtc 3780 aagattggtg cgcagaatga aaacgttgtt gccatcaccg ccgcgatggc aggtcctacc 3840 ggtctgtcca agttcgaagc caatttcccc aaccgattct ttgatgtcgg cattgctgag 3900 cagcacgcgg taacttctgc cgcaggcctc gcattgggtg gaaaacaccc tgtggtggct 3960 atttactcca cgttcttgaa ccgcgctttt gatcagctgc tcatggatgt gggcatgctc 4020 aaccagcctg ttactttggt gcttgatcgc tcaggtgtca cgggttcgga tggagcgagc 4080 cacaatggcg tctgggatat ggcgctgacc tcgatcgttc caggcgtgca ggtggcggca 4140 ccacgtgatg aggattcctt gcgtgagctg ctcaatgagg ctatttccat cgatgatggc 4200 cccacagttg tgcgtttccc caagggcgac ttgccaactc caattgttgc tatcgacacc 4260 ttggaagacg gcgtggatgt cctcgcatat gaagacgcca ctgacgttga atcaaccgac 4320 gatgcgccat cagttctcat cattgcggta ggcgagcgcg caactgttgc acttgacgtt 4380 gcttccagga ttaaacagca cggcgtgaac gtcacggttg ttgacccccg ctggattgtc 4440 cccatcccgc agtccttggt cgcgctgtct gatgatcatg acctcgtgat caccatcgaa 4500 gcggcgtca tccacggcgg cgtgggctcc ttgctctctg atgcgcttaa cgcctctgag 4560 gtggataccc ctcgccgaca aatcgccgtg ccccagaagt acctggatca cgcgtcccgc 4620 aatgaagtgc tcgccgatta tggcctcgac gccgacggca ttgaaaccac tgttgttgga 4680 tggctggatt ccctgttcgg ggaataagga tctggatcta aagaggagaa atactaggtg 4740 tctaagctta ggggcatgac tactgaggtt gaactggttg ttttagctga ttccgagggc 4800 aatcctattg gtactgcgcc gaaagctacg gtgcacacta aggacacgcc tctgcatttc 4860 gcgttttcca cctatatttt gaacccgcgt ggggagctgt tggtgacgcg tcgtgcattg 4920 tcgaagaaga catggcctgg tgtgtggacg aactctatgt gtgggcaccc tggtccggat 4980 gagacaaacg cggatgcgat tcgtcgcagg ggtgtcgatg agttggggct ggaggtagat 5040 tctttcttgg atattcaaga gattctgcct gattaccagt accgtgctgt cgacgcgtcc 5100 ggcattgtgg agtgggagtt gtgcccggtc cacctcgtgc gtttagcggt gggggaattt 5160 gtggagccac tggatgatga ggtggaggag ttcgagtggg cggaaccgca gaagcttttc 5220 gacgctgttg atgccacacc atttgtgttt tctccatggc tagtggatca gcttagcgcc 5280 cctgagctgc gccaagccat cctggaagcg tttgacgcag agtaaggatc taagaaggag 5340 gtaaacaatg gactttccgc agcaactcga agcctgcgtt aagcaggcca accaggcgct 5400 gagccgtttt atcgccccac tgccctttca gaacactccc gtggtcgaaa ccatgcagta 5460 tggcgcatta ttaggtggta agcgcctgcg acctttcctg gtttatgcca ccggtcatat 5520 gttcggcgtt agcacaaaca cgctggacgc acccgctgcc gccgttgagt gtatccacgc 5580 ttactcatta attcatgatg atttaccggc aatggatgat gacgatctgc gtcgcggttt 5640 gccaacctgc catgtgaagt ttggcgaagc aaacgcgatt ctcgctggcg acgctttaca 5700 aacgctggcg ttctcgattt taagcgatgc cgatatgccg gaagtgtcgg accgcgacag 5760 aatttcgatg atttctgaac tggcgagcgc cagtggtatt gccggaatgt gcggtggtca 5820 ggcattagat ttagacgcgg aaggcaaaca cgtacctctg gacgcgcttg agcgtattca 5880 tcgtcataaa accggcgcat tgattcgcgc cgccgttcgc cttggtgcat taagcgccgg 5940 agataaagga cgtcgtgctc tgccggtact cgacaagtat gcagagagca tcggccttgc 6000 cttccaggtt caggatgaca tcctggatgt ggtgggagat actgcaacgt tgggaaaacg 6060 ccagggtgcc gaccagcaac ttggtaaaag tacctaccct gcacttctgg gtcttgagca 6120 agcccggaag aaagcccggg atctgatcga cgatgcccgt cagtcgctga aacaactggc 6180 tgaacagtca ctcgatacct cggcactgga agcgctagcg gactacatca tccagcgtaa 6240 taaataagga tccaaactcg agtaaggatc tccaggcatc aaataaaacg aaaggctcag 6300 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt 6360 cacactggct caccttcggg tgggcctttc tgcgtttata cctagggcgt tcggctgcgg 6420 cgagcggtat cagcagttat tggtgccctt cgaaatgacc gaccaagcga cgcccaacct 6480 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt 6540 tttccgggac gccaacaaca agacccatca tagtttgccc ccgcgacatt gaccataaat 6600 tcatcgcaca aaatatcgaa cggggtttat gccgctttta gtgggtgcga agaatagtct 6660 gctcattacc cgcgaacacc gccgcattca gatcacgctt agtagcgtcc ccatgagtag 6720 gcagaaccgc gtccaagtcc acatcatcca taacgatcat gcacggggtg gaatccacac 6780 ccagacttgc cagcacctca ttagcgacac gttgcgcagc ggccacgtcc ttagccttat 6840 ccacgcaatc gagaacgtac tgcctaaccg cgaaatcaga ctgaatcagt ttccaatcat 6900 cgggcttcac caaagcaaca gcaacgcggg ttgattcgac ccgttccggt gcttccagac 6960 cggcgagctt gtacagttct tcttccattt cacgacgtac atcagcgtct atgtaatcaa 7020 tgcccaaagc acgcttagcc ccacgtgacc aggacgaacg caggttttta gaaccaacct 7080 catactcacg ccaccgagcc accaaaacag cgtccatatc ctcgccggcg tcgctttgat 7140 cggccaacat atccaacatc tgaaacggcg tgtacgaccc cttagacgcg gttttagtag 7200 cggagccagt cagttcctga gacatgccct tagcgaggta ggttgccatt ttcgcagcgt 7260 ctccacccca ggtagacacc tgatcaagtt tgaccccgtg ctcacgcagt ggcgcgtcca 7320 taccggcctt aaccacacca gcagaccagc gggaaaacat ggaatcctca aacgccttga 7380 gttcatcgtc agacagtgga cgatccaaga acaacagcat gttgcggtgc aagtgccaac 7440 cgttcgccca agagtctgtg acctcatagt cactataggt gtgctccacc ccgtaccgtg 7500 cacgttcttt cttccactga gatgttttca ccatcgaaga gtacgcagtc ttaatacccg 7560 cttcaacctg cgcaaatgac tgtgagcggt tgtgtcgaac agtgcccaca aacatcatga 7620 gcgcgccacc cgccgccaag tgattcttag tagcaatagc cagctcaatg cggcgttcgc 7680 ccatgacttc caattcagcc agaggtgacc cccagcgaga gtgagagttt tgcagaccct 7740 caaactgcga agcaccgtta gacgaccagg acaccgcaac agcttcgtcc ctgcgccacc 7800 tatggcaccc cgccagagcc ttactattgg tgatcttgta catgacgttt tgcctacgcc 7860 acgccctagc gcgagtgacc ttagaaccct cattgacctg cggttcctta gaggtgttca 7920 cttctatttc agtgttactc agtgttacct agacccgatg ttgtgcgggg ttgcgcagtg 7980 cgagtttgtg cgggtgttgt gcccgttgtc ttagctagtg ctatggttgt caattgaaac 8040 cccttcgggt tatgtggccc ccgtgcatat gagttggtag ctcgcacggg ggtttgtctt 8100 gtctagggac tattaatttt tagtggtgtt tggtggccgc ctagcttggc tatgcgtgcc 8160 agcttacccg tactcaatgt taaagatttg catcgacatg ggagggttac gtgtccgata 8220 cctagggggg gtatccgcga ctaggtgccc cggtgctcac tgtctgtacc ggcggggcaa 8280 gccccacacc ccgcatggac agggtggctc cgccccctgc acccccagca atctgcatgt 8340 acatgtttta cacattagca cgacatgact gcatgtgcat gcactgcatg cagactaggt 8400 aaatatgagt atgtacgact agtaacagga gcactgcaca taatgaatga gttgcaggac 8460 aatgtttgct acgcatgcgc atgacatatc gcaggaaagc tactagagtc ttaaagcatg 8520 gcaaccaagg cacagctaga acagcaacta caagaagctc aacaggcact acaggcgcag 8580 caagcgcagg cacaagccac catcgaagca ctagaagcgc aggcaaaggc taagcccgtc 8640 gtggtcaccg cacgcgttcc tttggcacta cgtgaggaca tgaagcgcgc aggcatgcag 8700 aacggtgaaa acctccaaga gttcatgatc gccgcgttta ccgagcggct agaaaagctc 8760 accaccaccg acaacgagga aaacaatgtc taacccacta gttctctttg cccaccgtga 8820 cccggtaaat gacgtgacgt tcgagtgcat tgagcacgcc acctacgaca cactttcaca 8880 cgctaaagac cagatcaccg cccaaatgca agccctagac gaagaagccg ccctactgcc 8940 ctaatgggtg tttcatgggt gtttccctag tgtttcatgg tgttttcacc taagctaggg 9000 aattgcgcga gaagtctcgc aaaaatcagc aacccccgga accacacagt tcacgggggt 9060 tcttctatgc cagaaatcag aaaggggaac cagtgaacga ccccgaatgg ctggatgatc 9120 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaaaaggat ctaggtgaag 9180 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 9240 tcagaccccg tatgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 9300 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 9360 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 9420 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 9480 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 9540 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 9600 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 9660 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 9720 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 9780 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 9840 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 9900 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 9960 attttggtca tgactagtgc ttggattctc accaataaaa aacgcccggc ggcaaccgag 10020 cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca acaggagtcc 10080 aagcgagctc gatatcaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt 10140 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca 10200 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga 10260 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg 10320 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt 10380 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac 10440 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac 10500 tatcccatat caccagctca ccgtctttca ttgccatacg aaattccgga tgagcattca 10560 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg 10620 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg 10680 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc 10740 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa 10800 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat 10860 caacgtctca ttttcgccag atatc 10885 <210> 6 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-idsA-r1 <400> 6 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtc tcatcacact 60 aagatacccg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 7 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-idsA-r3 <400> 7 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagta gacgtccttc 60 aatcgaatag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 8 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-gapA-r1 <400> 8 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtc aacctgtcct 60 caactttagg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 9 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-gapA-r2 <400> 9 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtc gcaccaaacc 60 tactcacatg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 10 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-gdh-r1 <400> 10 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtt aaatgctcac 60 gttcaattag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 11 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-gdh-r2 <400> 11 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtg agggcaccat 60 gtacatcctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 12 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> iapA-r1 promotor <400> 12 ctcatcacac taagataccc 20 <210> 13 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> iapA-r3 promotor <400> 13 agacgtcctt caatcgaata 20 <210> 14 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> gapA-r1 promotor <400> 14 caacctgtcc tcaactttag 20 <210> 15 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> gapA-r2 promotor <400> 15 cgcaccaaac ctactcacat 20 <210> 16 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> gdh-r1 promotor <400> 16 taaatgctca cgttcaatta 20 <210> 17 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> gdh-r2 promotor <400> 17 gagggcacca tgtacatcct 20 <210> 18 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-idsA-r1-dCas9 vector <400> 18 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtctca tcacactaag atacccgttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gt; caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 ggggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060                                                                        12060 <210> 19 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-idsA-r3-dCas9 vector <400> 19 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtagac gtccttcaat cgaatagttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gt; caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 ggggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060                                                                        12060 <210> 20 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-gapA-r1-dCas9 vector <400> 20 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtcaac ctgtcctcaa ctttaggttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gt; caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 ggggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060                                                                        12060 <210> 21 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-gapA-r2-dCas9 vector <400> 21 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtcgca ccaaacctac tcacatgttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gt; caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 ggggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060                                                                        12060 <210> 22 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-gdh-r1-dCas9 vector <400> 22 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagttaaa tgctcacgtt caattagttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gt; caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 ggggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060                                                                        12060 <210> 23 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-gdh-r2-dCas9 vector <400> 23 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtgagg gcaccatgta catcctgttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gt; caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 ggggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060                                                                        12060

Claims (22)

D-xylulose 5-phosphate from D-glyceraldehyde 3-phosphate (G3P), pyruvate and D-glyceraldehyde 3-phosphate , &Lt; / RTI &gt;DXP);
A gene encoding an enzyme that produces dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP);
A gene encoding an enzyme that produces farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP); And
A Corynebacterium glutamicum strain comprising a gene encoding an enzyme that produces squalene from panesyl diphosphate (FPP). The gene encoding an enzyme that produces 1-deoxy-D-xylulose 5-phosphate (DXP) from (G3P) with pyruvate and D-glyceraldehyde 3-phosphate, Comprising the nucleotide sequence of SEQ ID NO: 1,
The gene encoding the enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP) comprises the nucleotide sequence of SEQ ID NO: 2,
The gene encoding the enzyme producing panesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP) comprises the nucleotide sequence of SEQ ID NO: 3,
The gene encoding squalene-producing enzyme from panesyl diphosphate (FPP) comprises the nucleotide sequence of SEQ. ID. NO. 4, a strain of Corynebacterium glutamicum. The method according to claim 1, wherein the enzyme that produces 1-deoxy-D-xylulose 5-phosphate (DXP) from (G3P) with pyruvate and D-glyceraldehyde 3-phosphate is deoxycycyl Is a deoxyxylulose-5-phosphate synthase (dxs)
The enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP) is isopentenyl diphosphate delta isomerase (idi)
The enzyme that produces panesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP) is geranyl transferase (ispA)
The enzyme producing squalene from panesyl diphosphate (FPP) is the Corynebacterium glutamicum strain, a squalene synthase. 4. The method according to claim 3, wherein the deoxyxylulose-5-phosphate synthase (dxs) gene and the isopentenyl diphosphate delta isomerase (idi) gene Is a strain of Corynebacterium glutamicum, which is derived from Corynebacterium glutamicum. The Corynebacterium glutamicum strain according to claim 3, wherein the geranyl transferase (ispA) gene is derived from E. coli. The Corynebacterium glutamicum strain according to claim 3, wherein the squalene synthase gene is derived from Saccharomyces cerevisiae. The Corynebacterium glutamicum strain of claim 1, wherein the strain is transformed with a vector comprising a dxs gene, an idi gene, an ispA gene, and a sequence derived from a squalene synthetase, trSQS (CgCO). The method according to claim 1,
A gene encoding an enzyme producing geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP), a gene encoding D-glyceraldehyde 3-phosphate (G3P), 1,3-bisphosphoglycerate (1 , 3BPG) or a vector containing an sgRNA that inhibits the expression of a gene encoding an enzyme that produces glutamate from alpha ketoglutarate (? -GK) Glutamicum strain. 9. The Corynebacterium glutamicum strain according to claim 8, wherein the sgRNA is prepared from at least one primer selected from the primers of SEQ ID NOS: 12 to 17. 9. The method according to claim 8, wherein the gene comprising sgRNA that inhibits expression of an enzyme that produces geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP) comprises a sequence of SEQ ID NO: 6 or SEQ ID NO: 7 And the gene comprising sgRNA which inhibits the expression of the enzyme producing 1,3-bisphosphoglycerate (1,3BPG) from the D-glyceraldehyde 3-phosphate (G3P) is SEQ ID NO: 8 or SEQ ID NO: SEQ ID NO: 10 or SEQ ID NO: 11, wherein the gene comprising sgRNA comprising SEQ ID NO: 9 and inhibiting the expression of an enzyme producing glutamate from alpha ketoglutarate (? -GK) Corynebacterium glutamicum strain. 9. The Corynebacterium glutamicum strain of claim 8, wherein said vector comprises a gene encoding a dCas9 protein. 9. The Corynebacterium glutamicum strain according to claim 8, wherein the inhibition of the expression of the gene is by a crystal gene regulatory system. The method according to claim 1,
The strain was transformed with a first vector and a second vector,
Wherein said first vector comprises a dxs gene, an idi gene, an ispA gene and a sequence derived from squalene synthetase trSQS (CgCO)
The second vector is a gene encoding an enzyme that produces geranyl geranyl pyrophosphate (GGPP) from dimethylallyl diphosphate (DMAPP), a gene encoding D-glyceraldehyde 3-phosphate (G3P) A gene encoding an enzyme that produces polyglycerate (1,3BPG), or an sgRNA and a dCas9 gene that inhibits the expression of a gene encoding an enzyme that produces glutamate from alpha ketoglutarate (? KG) , Corynebacterium glutamicum strain. 14. The method of claim 13,
Wherein said first vector is the sequence of SEQ ID NO: 5. &lt; RTI ID = 0.0 &gt; 5. &lt; / RTI &gt; 14. The method of claim 13,
Wherein said second vector comprises at least one of SEQ ID NO: 6 to SEQ ID NO: 11. 14. The method of claim 13,
Wherein the second vector is one of the sequences of SEQ ID NO: 18 to SEQ ID NO: 23. The strain according to claim 1, wherein the strain is a Corynebacterium glutamicum strain having the accession number KCCM12189P. 18. A method for producing squalene, comprising culturing the strain of any one of claims 1 to 17. The culture medium composition according to claim 18, wherein as the culture medium composition for use in the culture, 0.001 to 0.01 g / L of ammonium sulfate and 0.01 to 0.5 g / L of urea are used as the minimum medium component of CgXII. 18. A culture medium composition for use in the above culture according to claim 18, wherein tryptone is added to the minimum medium composition of CgXII. The culture composition according to claim 20, wherein the tryptone is contained in an amount of 10 to 20 g / L.
The culture liquid composition according to claim 20, wherein the culture medium further comprises 1 g / L to 20 g / L of yeast extract and 1 g / L to 10 g / L of sodium chloride.

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