CN111041039B - Thermophilic anaerobic ethanol bacillus genome editing vector and application thereof - Google Patents
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Abstract
The invention belongs to the field of genetic engineering, and particularly relates to a thermophilic anaerobic ethanol bacillus genome editing vector and application thereof. The genome editing vector of the thermophilic anaerobic ethanol bacillus constructs a thermostable Cas9 expression element sequence and/or sgRNA expression element sequence into a shuttle expression vector of escherichia coli-thermophilic anaerobic ethanol bacillus; can realize the gene in-frame insertion or gene fragment deletion of the genome of the thermophilic anaerobic ethanol bacillus. The genome of the thermophilic anaerobic ethanol bacillus is edited by the vector to obtain a mutant engineering strain with gene deletion, and further, the genome editing method of the thermophilic anaerobic ethanol bacillus is provided.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a thermophilic anaerobic ethanol bacillus genome editing vector and application thereof.
Background
The thermophilic anaerobic ethanol bacillus is one of representative strains with higher ethanol yield in thermophilic anaerobic bacteria, is an obligate anaerobic and thermophilic gram-positive bacterium, and has the growth temperature of 65-79 ℃. The thermophilic anaerobic ethanol bacillus not only has higher ethanol yield, but also can decompose five-carbon sugar such as xylose and the like, has high fermentation temperature, can reduce the pollution of mixed bacteria, is favorable for recovering the product ethanol, and is considered as a potential strain for producing the cellulosic ethanol. The integrated bioprocessing technology (CBP) combines the production, hydrolysis and ethanol fermentation of cellulases and hemicellulases together, simplifying the processing process and reducing energy consumption, and is recognized as the most promising low-cost ethanol production route for lignocellulosic fuels. Thermophilic anaerobic ethanologen has the potential of meeting the requirement of CBP (cellulose-binding protein) for cellulose ethanol fermentation, but thermophilic anaerobic ethanologen has the problems of low ethanol yield, low ethanol tolerance and the like, so that thermophilic anaerobic ethanologen is greatly limited to be directly used for industrial production and needs to be genetically modified.
The thermophilic anaerobic ethanol bacillus is difficult to genetically manipulate, for example, the genetic transformation efficiency is low, the available vector is few, and the stability is poor, so that the thermophilic anaerobic ethanol bacillus lacks a simple and rapid genetic transformation method, and the research on the metabolic engineering of the thermophilic anaerobic ethanol bacillus is slow. Therefore, the editing of the genome inheritance of the thermophilic anaerobic ethanol bacillus becomes a hot spot of the current research, and is always paid extensive attention by researchers. Genome editing technology is an emerging molecular biology technology, and realizes insertion, deletion, replacement or modification aiming at specific target gene segments in a genome. The II-type CRISPR/Cas9 gene editing technology which is formed by modifying an adaptive immune system for cutting DNA guided by RNA has the advantages of simple operation, high efficiency and the like, but no report on the genome editing of thermophilic anaerobic ethanol bacillus of thermostable Cas9 nuclease exists at present.
Disclosure of Invention
In view of the above, the present invention aims to provide an editing vector for genome of ethanobacter thermophilus and an application thereof, wherein the editing vector provided by the present invention is suitable for editing genome of ethanobacter thermophilus, and a genome editing method based on the editing vector can edit genome of ethanobacter thermophilus to obtain mutant strain of ethanobacter thermophilus, thereby solving the current situations of difficult genetic manipulation and difficult gene editing of the existing ethanobacter thermophilus.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention provides a thermophilic anaerobic ethanol bacillus genome editing vector, wherein an expression element is constructed into an escherichia coli-thermophilic anaerobic ethanol bacillus shuttle expression vector by the editing vector, and the expression element comprises a thermostable Cas9 expression element and/or a sgRNA expression element.
In a specific embodiment, the shuttle expression vector of Escherichia coli-thermophilic anaerobic ethanol bacillus is a calicheamicin resistant shuttle expression vector pBlu-Htk or a chloramphenicol resistant shuttle expression vector pBlu-Cm.
In a specific embodiment, the editing vector is one or more of a Psly promoter expression thermostable Cas9 expression frame plasmid pBlu-Cm-ThermoCas9, a Ppat promoter expression sgRNA expression frame plasmid pBlu-Pat-sgRNA or an editing vector pBlu-Cas9-Pat-sgRNA containing both a Cas9 expression frame plasmid and a sgRNA expression frame plasmid.
In a specific embodiment, the invention further provides a method for performing gene editing on a genome of ethanobacter thermophilus, wherein the method for performing gene editing in ethanobacter thermophilus based on the vector for editing the genome of ethanobacter thermophilus specifically comprises the following steps:
(1) Designing upstream and downstream homology arm repair sequences aiming at a target gene by taking a thermophilic anaerobic ethanol bacillus genome as a template to obtain a target gene donor DNA sequence;
(2) Constructing a plasmid containing a donor DNA sequence and sgRNA expression;
(3) Transforming Cas9 expression frame plasmids into thermophilic anaerobic ethanol bacillus, transforming the plasmids constructed in the step (2) into thermophilic anaerobic ethanol bacillus, screening markers and selecting transformants to obtain gene editing mutant strains;
or connecting the Cas9 expression frame plasmid and the plasmid containing the donor DNA sequence and the sgRNA expression frame constructed in the step (2) into a single plasmid editing vector, and directly converting the single plasmid editing vector into the anoxybacter thermophilus to obtain a gene editing mutant strain.
Further, the objective gene described in the step (1) isTdkThe gene,adhEGenes andRSPa gene.
The Cas9 expression cassette plasmid in the step (3) is pBlu-Cm-ThermoCas9 or pBlu-Cas9-Pat-sgRNA.
The genome of the thermophilic anaerobic ethanol bacillus is derived from thermophilic anaerobic ethanol bacillus and is DSM 2246.
The invention also provides application of the thermophilic anaerobic ethanol bacillus genome editing vector in gene editing based on CRISPR/Cas9 in thermophilic anaerobic ethanol bacillus genome.
The invention has the beneficial effects that:
the invention provides a thermophilic anaerobic ethanol bacillus genome editing vector and application thereof, wherein the thermophilic anaerobic ethanol bacillus genome editing vector provided by the invention is constructed by taking Cas9 expression frame plasmids and/or sgRNA expression frame plasmids as elements into an escherichia coli-thermophilic anaerobic ethanol bacillus shuttle expression vector; can realize the genome of thermophilic anaerobic ethanol bacillus an in-frame insertion of a gene or a deletion of a gene fragment. The genome of the thermophilic anaerobic ethanol bacillus is edited by the vector to obtain a mutant engineering strain with gene deletion, and further, a genome editing method of the thermophilic anaerobic ethanol bacillus is provided.
Drawings
FIG. 1 is a schematic diagram of the constructed pBlu-Htk expression vector map and multiple cloning sites;
FIG. 2 isTdkConstruction of the genetically mutated strain;
FIG. 3 isTdkA gene fragment amplification electrophoretogram, wherein M is DNA marker, and the number 1~6 is the PCR amplification result of the selected transformant;
FIG. 4 is a drawingTdkA gene partial sequence sequencing result picture; wherein (a) is a wild typeTdkPartial sequence diagram of gene, (b) is mutantTdkA gene partial sequence diagram, (c) is a sequencing result;
FIG. 5 isadhEGene editing process and editing result graph; wherein A is a construction diagram of a simple particle editing vector pBlu-Htk-gE-donor-adhE-ThermoCas 9; b is transformation of the plasmid into thermophilic anaerobic ethanol bacillus; c is a transformant grown in an anaerobic tube; d isadhEGene editing and verifying an electrophoretogram, wherein M is a DNA marker, and 1-10 are PCR verification results of the selected transformant;
FIG. 6 is a drawingadhEA gene partial sequence sequencing result picture; wherein (a) is a wild typeadhEA gene part sequence; (b) Is a mutantadhEThe gene partial sequence (c) is the result of sequencing.
FIG. 7 isRSPGene editing and verifying electrophoretogram; wherein M is DNA marker, and the number 1~9 is the PCR verification result of the selected transformant.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative of the present invention and are not intended to limit the scope of the invention. One skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. The experimental starting materials used in the following examples are commercially available unless otherwise specified. The invention adopts the prior art in the field except for special notes.
The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., russell, david W., molecular Cloning: A Laboratory Manual,3rd edition,2001, NY, cold spring harbor); natural transformation methods described herein are described in Shaw AJ, hogsett DA, lynd LR. Natural company in Thermoanaerobacterium and Thermoanaerobacterium Specifiers. Applied and Environmental Microbiology, 2010, 76 (14): 4713-4719;
the original starting strain, ethanobacterium thermoanaerobacter thermophilus, used in the examples was DSM 2246, purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, germany collection of microorganisms and cell cultures). Thermostable kanamycin resistance gene fragment derived from plasmid pTZeco013_1049del (GenBank: KY 110710.1), thermostable replicon sequence fragment derived from plasmid pMU131 (Shaw AJ, hogsett DA, lynd LR. Natural company in Thermoanaerobacter and Thermoanaerobacter bacteria Specifics, applied and Environmental Microbiology, 2010, 76 (14): 4713-4719), thermostable Cas9 nuclease gene sequence (Genbank accession No.: NZ _ LUCR 01000150) derived from Bacillus stearothermophilus strain thermophilus strain GS27, the primers used were synthesized by Kinsys Biotech, inc., and gene synthesis and DNA sequencing were accomplished by Biotechnology (Shanghai) Plien.
Example 1: cas9 nuclease-mediated construction of genome editing vector of ethanobacterium thermoanaerobium
(1) Construction of the shuttle expression vector pBlu-Htk for resistance to Caravamycin:
and synthesizing a thermostable replicon sequence fragment, a thermostable kanamycin resistance gene fragment, a promoter sequence, an SD sequence, a multiple cloning site sequence and a terminator sequence into a calicheamicin resistance gene through a whole gene, wherein the sequence is shown as SEQ ID NO: shown at 45. A calicheamicin resistance gene synthesized by whole genes is taken as a template, pBlu-Htk-N and pBlu-Htk-C are taken as primers, a 3814bp DNA fragment is amplified, and the sequences of the primers pBlu-Htk-N and pBlu-Htk-C are shown as SEQ ID NO: 1~2.
The Escherichia coli replicon sequence and the kalamycin resistance gene fragment are respectively amplified by taking pBluescript II SK (+) plasmid (GenBank: X52328.1) as a template and pBlue-N and pBlue-C as primers, and the sequences of the pBlue-N and pBlue-C are shown as SEQ ID NO: 3~4. The amplified fragments are connected to construct an escherichia coli-thermophilic anaerobic ethanol bacillus shuttle expression vector pBlu-Htk, and the nucleotide sequence of the expression vector is shown as SEQ ID NO. 46. The PCR reaction system is as follows: 5 × PrimeSTAR Buffer (Mg) 2+ plus) 10. Mu.L, 4. Mu.L of 10 mM dNTPs, 0.25. Mu.L of primer, 1. Mu.L of template DNA, 0.5. Mu.L of PrimeSTAR HS DNA polymerase, and 34. Mu.L of water; the PCR reaction conditions are as follows: 10s at 98 ℃; 15s at 57 ℃, 3.5min at 72 ℃,29 cycles; finally, 10min at 72 ℃ and 10min at 4 ℃.
FIG. 1 is a schematic diagram of the constructed pBlu-Htk expression vector map and multiple cloning sites;
(2) Construction of the chloramphenicol resistant shuttle expression vector pBlu-Cm:
pBlu-Htk plasmid is used as a template, and the nucleotide sequence is shown as SEQ ID NO:5 and the nucleotide sequence of the primer pBlu-cm-N is shown as SEQ ID NO:6, amplifying a linear fragment by using a primer pBlu-cm-C; the chloramphenicol resistance gene sequence derived from plasmid pHK (GenBank: KY 792637.1) is taken as a template, and the nucleotide sequence is shown as SEQ ID NO:7~8 shows CM-N and CM-C as primer, PCR amplifies 651bp chloramphenicol gene fragment, connects the linear fragment and chloramphenicol gene fragment to construct chloramphenicol resistant Escherichia coli-thermophilic anaerobic ethanol bacillus shuttle expression vector pBlu-Cm, its nucleotide sequence is shown in SEQ ID NO: 47.
(3) Construction of a Psly promoter expression thermostable Cas9 expression plasmid
Using a thermostable Cas9 nuclease (ThermoCas 9) gene sequence as a template, wherein the nucleotide sequence is shown as SEQ ID NO: cas-xho shown in 9 to 10 and Cas-xba are used as primers, and a 3264bp DNA fragment is amplified by PCR. The PCR reaction system is as follows: 5 × PrimeSTAR Buffer (Mg) 2+ plus) 10 μL，10 mM dNTPs 4 μ L, primer Cas-xho 0.25 μ L, primer Cas-xba 0.25 μ L, template DNA 1 μ L, primeSTAR HS DNA polymerase 0.5 μ L, water 34 μ L; the PCR reaction conditions were: firstly, the temperature is 98 ℃ for 10s; 15s at 57 ℃, 3.5min at 72 ℃,29 cycles; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10min. And (3) connecting the PCR product with the shuttle plasmid pBlu-Cm constructed in the step (2), transforming the connecting solution, selecting the plasmid for verification, and constructing the plasmid pBlu-ThermoCas9.
The method is characterized in that a thermophilic anaerobic ethanol bacillus genome (a thermophilic anaerobic ethanol bacillus DSM 2246 genome is extracted by adopting a molecular cloning standard method, DSM 2246 is purchased from German microorganism culture collection DSMZ) is taken as a template, and the nucleotide sequence is shown as SEQ ID NO: sly-N and Sly-C shown in 11 to 12 are used as primers to amplify a Psly promoter sequence (the sequence is shown as SEQ ID NO: 48); the plasmid pBlu-ThermoCas9 is taken as a template, and the nucleotide sequence is shown as SEQ ID NO: casEcoRI-N and CasEcoRV-C shown in 13 to 14 are used as primers to amplify linear fragments, the amplified Psly promoter fragment is connected with the linear fragments to construct a Psly promoter expression thermostable Cas9 expression frame plasmid pBlu-Cm-ThermoCas9, and the nucleotide sequence of the Psly promoter expression frame plasmid is shown in SEQ ID NO: 49.
(4) Construction of sgRNA expression plasmid for Pcat promoter expression
With thymidine kinase (Tdk) A gene (GenBank: AEYS 01000001.1) is used as an editing target, a target DNA sequence is designed, a single-stranded guide RNA (sgRNA) is synthesized from the target DNA sequence, crRNA and tracrRNA by a whole-gene synthesis method, and the nucleotide sequence of the synthesized sgRNA is shown as SEQ ID NO. 50. The nucleotide sequence of the template is shown as SEQ ID NO: sgRNA-N and sgRNA-C shown in 15 to 16 are used as primers for amplifying an sgRNA expression frame sequence by a PCR method, and the PCR reaction system is as follows: 5 XPrimeSTAR Buffer (Mg) 2+ plus) 10. Mu.L, 10 mM dNTPs 4. Mu.L, primer sgRNA-N0.25. Mu.L, primer sgRNA-C0.25. Mu.L, template DNA 1. Mu.L, primeSTAR HS DNA polymerase 0.5. Mu.L, water 34. Mu.L; the PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 10s; 15s at 57 ℃,1 min at 72 ℃,29 cycles; finally, 10min at 72 ℃ and 10min at 4 ℃. The sgRNA expression cassette sequence of the PCR product was ligated to the plasmid pBlu-Htk constructed in example 1, the ligation solution was transformed, and the plasmid was selected for verification, thereby constructing a sgRNA expression cassette plasmid pBlu-Htk-sgRNA.
The genome of the thermophilic anaerobic ethanol bacillus is taken as a template, and the nucleotide sequence is shown as SEQ ID NO: pat-N and Pat-C shown in 17 to 18 are used as primer amplification Pat promoter sequences, and the nucleotide sequences of the Pat promoters are shown as SEQ ID NO. 51; the constructed plasmid pBlu-Htk-sgRNA is used as a template, and the nucleotide sequence is shown as SEQ ID NO: p1 and P2 shown in 19-20 are primers to amplify a linear fragment, the amplified Pat promoter fragment is connected with the linear fragment to construct a Pcat promoter expression sgRNA expression cassette plasmid pBlu-Pat-sgRNA, and the nucleotide sequence of the plasmid is shown in SEQ ID NO: 52.
(5) Construction of editing vector containing Cas9 expression plasmid and sgRNA expression plasmid simultaneously
The Cas9 expression frame plasmid pBlu-Cm-ThermoCas9 is taken as a template, and the nucleotide sequence is shown as SEQ ID NO:11 and the nucleotide sequence of Sly-N shown in SEQ ID NO:1, pBlu-Htk-N is used as a primer to amplify a Cas9 expression frame sequence; and the nucleotide sequence is shown as SEQ ID NO:29 and the nucleotide sequence of the primer k-N is shown as SEQ ID NO:17, amplifying a DNA sequence by taking an sgRNA expression frame plasmid pBlu-Pat-sgRNA as a template; and connecting the two DNA fragments to construct an editing vector pBlu-Cas9-Pat-sgRNA simultaneously containing a Cas9 expression plasmid and a sgRNA expression plasmid. The nucleotide sequence is shown in SEQ ID NO. 53.
Example 2: thermophilic anaerobic ethanol bacillus Tdk gene fragment deletion and insertion mutation
According toTdkGene (GenBank: AEYS 01000001.1) sequence design, thermophilic anaerobic ethanol bacillusTdkThe gene homologous arm amplification primers Tdk-N and Tdk-C have nucleotide sequences shown in SEQ ID NO:23 to 24, using the genome of the thermophilic anaerobic ethanol bacillus as a template to amplify a Tdk homologous arm fragment, wherein a PCR reaction system is as follows: 5 × PrimeSTAR Buffer (Mg) 2+ plus) 10. Mu.L, 4. Mu.L of 10 mM dNTPs, 0.25. Mu.L of each primer, 1. Mu.L of template DNA, 0.5. Mu.L of PrimeSTAR HS DNA polymerase, and 34. Mu.L of water; the PCR reaction conditions are as follows: firstly, 10s at 98 ℃; 15s at 57 ℃, 2.5 min at 72 ℃,29 cycles; finally, 10min at 72 ℃ and 10min at 4 ℃. The PCR amplified fragment was inserted into pBluescript II SK (+) plasmid (Biotechnology engineering (Shanghai) GmbH), and PCR amplification was performed by inverse PCR method using Tdk-289ERv-N and Tdk-289ERv-C as amplification primers, and the nuclei thereofThe nucleotide sequence is shown as SEQ ID NO:25 to 26 inclusive; obtaining linear fragment, phosphorylating, connecting, self-cyclizing to obtain an inserted enzyme cutting site, and simultaneously deleting a small segment of DNA sequence to obtainTdkDonor DNA sequence with Tdk-N and Tdk-C as primer pairTdkAnd amplifying the donor DNA sequence to obtain a PCR amplification product fragment.
(1) Two plasmid editing strategy: the sgRNA expression cassette plasmid pBlu-Htk-sgRNA constructed in example 1 was used as a template, and the nucleotide sequence was as shown in SEQ ID NO: k-N and k-C shown in 29 to 30 are primers for PCR amplification of linear fragments; connecting the PCR amplification product fragment with the linear fragment to obtain a product containingTdkDonor DNA sequence and plasmid pBlu-Htk-sgRNA-donor-Tdk for sgRNA expression.
The thermophilic anaerobic ethanol bacillus adopts DSMZ medium 122 culture medium, and anaerobic culture is carried out at 65 ℃. The Cas9 expression cassette plasmid pBlu-Cm-ThermoCas9 constructed in the example 1 is transformed into the ethanobacterium thermoanaerobicum by a natural transformation method, then the constructed plasmid pBlu-Htk-sgRNA-donor-Tdk is transformed into the ethanobacterium thermoanaerobicum, and screening is carried out in a kalamycin culture medium to obtain the strainTdkThe gene mutant strain, FIG. 2 isTdkConstruction of the genetically mutated strain; as can be seen in FIG. 2, the dual plasmid editing vector plasmids pBlu-Htk-sgRNA-donor-Tdk and pBlu-Cm-ThermoCas9 were constructed, respectively, plasmid pBlu-Cm-ThermoCas9 was transformed first, and pBlu-Htk-sgRNA-donor-Tdk was transformed, and transformants grown in a kanamycin anaerobic medium. Transformants were picked for colony PCR validation.
The nucleotide sequence is shown as SEQ ID NO:31 and a homologous arm upstream primer Tdk-yanzh-N shown in SEQ ID NO:32 of the strain with Tdk gene mutation by using a downstream primer Tdk-yanzh-C of the homologous armTdkAnd (5) gene editing verification. FIG. 3 isTdkA gene fragment amplification electrophoresis picture, wherein M is DNA marker, and the number 1~6 is a selected transformant; as shown in FIG. 3, all of the transformants No. 1~5 were non-mutated transformants, and the transformant No. 6TdkThe gene is subjected to deletion mutation, and the gene is,Tdkthe gene is reduced by 297bp. Performing PCR amplification on the mutant strain No. 6, and performing PCR amplification on the amplified mutant strainTdkThe gene partial sequence is connected to pMDT-18 vector, and the sample is sent for sequencing. FIG. 4 isTdkPartial sequence of geneA sequencing result graph; in the figure, (a) is a wild typeTdkPartial sequence diagram of gene, (b) is mutantTdkA gene partial sequence diagram, (c) is a sequencing result; as shown in FIG. 4, compared with the wild typeTdkGene in mutant formTdkA297 bp fragment is deleted from a partial gene sequence, and a sequence is inserted:GATATCTCTGTTCTCGAGc, comprising Xho I (CTCGAG) and EcoRV (GATATC) cleavage sites.
(2) Single plasmid editing strategy
Taking the plasmid pBlu-Htk-sgRNA-donor-Tdk constructed in the step (1) as a template, wherein the nucleotide sequence is shown as SEQ ID NO: k-N and Tdk-C shown in 29 to 30 are used as primers to amplify linear fragments; and the Cas9 expression frame plasmid pBlu-Cm-ThermoCas9 constructed in example 1 is used as a template, and the nucleotide sequence is shown as SEQ ID NO:2 and the nucleotide sequence of pBlue-Htk-C shown in SEQ ID NO:11 is used as a primer to amplify a Cas9 expression frame sequence, and the linear fragment is connected with the Cas9 expression frame sequence to construct a single plasmid editing vector pBlu-Htk-sgRNA-donor-Tdk-ThermoCas9. Transforming it into thermophilic anaerobic ethanol bacillus, screening in carramycin culture medium to obtainTdkA genetically mutated strain.
The embodiment provides two editing methods of double-plasmid editing and single-plasmid editing, a double-plasmid editing strategy needs to be implemented by two steps of transformation of thermophilic anaerobic ethanol bacillus, the constructed plasmid has a small molecular weight, and is more convenient for construction of the plasmid, but the steps are relatively complex; the plasmid constructed in the single particle editing method has larger molecular weight, but only needs to be transformed into the thermophilic anaerobic ethanol bacillus in one step; the mutant strain can be successfully edited by the two editing methods provided by the invention.
Example 3: thermophilic anaerobic ethanol bacillusadhEDeletion and insertion mutation of gene fragment
According toadhEGene (GenBank: AEYS 01000007.1) sequence design, thermophilic anaerobic ethanol bacillusadhEThe gene homologous arm amplification primers adhE-N and a dhE-C have nucleotide sequences shown in SEQ ID NO:21 to 22, the genome of the thermophilic anaerobic ethanol bacillus is used as a template to amplify the homologous arm fragment, and the PCR reaction system and conditions are the same as those in the embodiment 2. Will PCThe R amplified fragment was inserted into pBluescript II SK (+) plasmid (Biotechnology engineering (Shanghai) Co., ltd.), and PCR amplification was performed by the reverse PCR method using E231-nde-N and E231-nde-C primers whose nucleotide sequences are shown in SEQ ID NO:27 to 28; obtaining linear fragment, phosphorylating, connecting, self-cyclizing to obtain an inserted enzyme cutting site, and simultaneously deleting a small segment of DNA sequence to obtainadhEAnd (3) a donor DNA sequence, and performing amplification on the adhE donor DNA sequence by using adhE-N and a dhE-C as primers to obtain a PCR amplification product fragment.
(1) Two plasmid editing strategy: the sgRNA expression cassette plasmid pBlu-Htk-sgRNA constructed in example 1 was used as a template, and the nucleotide sequence was as shown in SEQ ID NO:33 to 34 gRNA-E-N and gRNA-E-C are used as primers, linear fragments are amplified by a PCR method, and phosphorylation connection is carried out to obtain the gRNA-E-N and gRNA-E-CadhEThe sgRNA expression cassette plasmid pBlu-Htk-gE whose gene is the subject of editing.
The adhE donor DNA sequence was amplified by PCR method, and the nucleotide sequence was shown as SEQ ID NO: performing PCR amplification on DNA product fragments by using adhE-N and adhE-C shown in 21-22 as primers; the sgRNA expression cassette plasmid pBlu-Htk-gE with adhE gene as editing object is used as template, and the nucleotide sequence is shown as SEQ ID NO: taking k-N and k-C shown in 29-30 as primers, and carrying out PCR amplification on linear fragments; ligating the product fragments with linear fragments to obtain fragments containingadhEThe donor DNA sequence and plasmid pBlu-Htk-gE-donor-adhE of the sgRNA expression cassette. The thermophilic anaerobic ethanol bacillus adopts DSMZ medium 122 culture medium, and anaerobic culture is carried out at 65 ℃. The Cas9 expression frame plasmid pBlu-Cm-ThermoCas9 constructed in the embodiment 1 is transformed into thermophilic anaerobic ethanol bacillus by a natural transformation method, then transformed into a plasmid pBlu-Htk-gE-donor-adhE, and screened in a kalamycin culture medium to obtain the plasmid pBlu-Cm-ThermoCas9adhEA genetically mutated strain.
(2) Single plasmid editing strategy
Taking the plasmid pBlu-Htk-gE-donor-adhE constructed in the step (1) as a template, wherein the nucleotide sequence is shown as SEQ ID NO:29 and the nucleotide sequence is shown in SEQ ID NO:22 is a linear segment amplified by a primer; the linear fragment was ligated with Cas9 expression cassette plasmid pBlu-Cm-ThermoCas9 of example 1 to construct a single plasmid editing vector pBlu-Htk-gE-donor-adhE-ThermoCas9. And transforming the strain into thermophilic anaerobic ethanol bacillus, and screening in a kalamycin culture medium to obtain the strain 2 with the adhE gene mutation. Selecting a transformant, and adopting an upstream primer of a homology arm, wherein the nucleotide sequence is shown as SEQ ID NO:35 and a downstream primer of the homologous arm, wherein the nucleotide sequence of the primer E-yanzh-N is shown as SEQ ID NO:36 of primer E-yanzh-C pairadhEIn genetically mutated strainsadhECarrying out colony PCR verification on the gene; FIG. 5 isadhEGene editing process and editing result graph; wherein A is a construction diagram of a simple particle editing vector pBlu-Htk-gE-donor-adhE-ThermoCas 9; b is transformation of the plasmid into thermophilic anaerobic ethanol bacillus; c is a schematic diagram of transformant extraction; d isadhEGene editing and verifying an electrophoretogram, wherein M in the graph D is a DNA marker, and the numbers 1-10 are selected transformants; as shown in D in FIG. 5, transformants No. 6 and No. 8 were completely mutant, the adhE gene had deletion mutation,adhEthe gene is reduced by 232bp; no. 7 and No. 9 are wild type and mutant type mixture; no. 1-5 and No. 10 were not mutated. For mutant No. 6adhESubjecting the gene to PCR amplificationadhEThe gene partial sequence is connected to pMDT-18 vector for sequencing. FIG. 6 isadhEA gene partial sequence sequencing result graph; wherein (a) is a wild typeadhEA gene part sequence; (b) Is a mutantadhEThe gene partial sequence (c) is the result of sequencing. As can be seen in FIG. 6, mutant formadhEThe gene fragment is deleted and one is inserted at the same timeNde I, enzyme cutting site sequence CATATG.
Example 4: thermophilic anaerobic ethanol bacillusRSPDeletion mutation of gene fragment
According toRSPGene (GenBank: AEYS 01000003.1) sequence, designing thermophilic anaerobic ethanol bacillusRSPThe nucleotide sequences of the gene homologous arm amplification primers RSP-N and RSP-C are shown in SEQ ID NO:37 to 38, using the genome of the thermophilic anaerobic ethanol bacillus as a template to amplify RSP homologous arm fragments, wherein a PCR reaction system is as follows: 5 × PrimeSTAR Buffer (Mg) 2+ plus) 10. Mu.L, 4. Mu.L of 10 mM dNTPs, 0.25. Mu.L of each primer, 1. Mu.L of template DNA, 0.5. Mu.L of PrimeSTAR HS DNA polymerase, and 34. Mu.L of water; the PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 10s; 15s at 57 ℃, 2.5 min at 72 ℃,29 cycles; most preferablyThen, 10min at 72 ℃ and 10min at 4 ℃. The PCR-amplified fragment was inserted into pBluescript II SK (+) plasmid (Biotechnology engineering (Shanghai) Co., ltd.). And then PCR amplification is carried out by an inverse PCR method, the RSP donor DNA sequences are Rspzx-N and Rspzx-C, and the nucleotide sequences are shown as SEQ ID NO:39 to 40; and (3) carrying out PCR amplification to obtain linear fragment phosphorylation, then carrying out connection self-cyclization, and simultaneously deleting a DNA fragment with the size of 578bp to obtain an RSP donor DNA sequence, wherein the nucleotide sequence is shown as SEQ ID NO: shown at 41.
The editing vector pBlu-Cas9-Pat-sgRNA constructed in example 1 and containing both Cas9 expression plasmid and sgRNA expression plasmid is used as a template, and the nucleotide sequence is shown as SEQ ID NO: and (4) amplifying linear fragments by using gRSP-N and gRSP-C shown in 41-42 as primers through a PCR (polymerase chain reaction) method, and carrying out phosphorylation connection to obtain an editing plasmid pBlu-Cas9-Pat-gRSP taking an RSP gene as an editing object.
Will be provided withRSPConstructing a donor DNA sequence, a Cas9 expression frame sequence and a sgRNA expression frame into a plasmid, and constructing a single plasmid editing vector: pBlu-Cas9-Pat-gRSP-donor-RSP. The method specifically comprises the following steps:
the nucleotide sequence is shown as SEQ ID NO:11 and the nucleotide sequence of Sly-N shown in SEQ ID NO:29 is used as a primer, and a linear fragment is amplified by taking the plasmid pBlu-Cas9-Pat-gRSP as a template; the nucleotide sequence is shown as SEQ ID NO:37 and nucleotide sequence of the primer RSP-N is shown as SEQ ID NO:38 to obtain a RSP donor sequence, connecting the two fragments, and constructing a single plasmid editing vector pBlu-Cas9-Pat-gRSP-donor-RSP. Transforming it into thermophilic anaerobic ethanol bacillus, screening in carramycin culture medium to obtainRSPA genetically mutated strain. And (3) selecting a transformant, and adopting a homology arm upstream primer, wherein the nucleotide sequence is shown as SEQ ID NO:43 and downstream primers of homologous arms, wherein the nucleotide sequence of the primers RSP-yzh-N is shown as SEQ ID NO:44, performing colony PCR verification by using a primer RSP-yzh-C; to pairRSPAmplification of a partial fragment of the gene, FIG. 7RSPGene editing and verifying electrophoretogram, wherein M is DNA marker, and the number 1~9 is selected transformant; as shown in figure 7 of the drawings,RSPthe gene is mutated, and complete mutation types of No. 1, no. 4 and No. 8 in 9 transformants,RSPthe gene reduction is 578bp,nos. 2, 3, 5, 6, 7 and 9 are wild type and mutant type mixtures.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> university of Jiangsu
<120> thermophilic anaerobic ethanol bacillus genome editing vector and application thereof
<160> 53
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agaacatgtg agcaaaaggc c 21
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gagtatggac agttgcggat g 21
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
caaccctatc tcggtctatt c 21
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aattgacaaa gttttctatt tg 22
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ttattatttc cttcctcttt tctac 25
<210> 6
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgacattaag aaaggtggtt tttatg 26
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgaacttta ataaaattga tttag 25
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tcataaaagc cagtcattag gcc 23
<210> 9
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gtgctcgaga gatacaaaat cggccttg 28
<210> 10
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gggtctagat caatcacgag ttgattgtaa c 31
<210> 11
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tttgaattcg tgagataccc ttggaacag 29
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tttgatatca cagtcaatcc tcctccttg 29
<210> 13
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ggtgaattct tataaaagcc agtcattagg c 31
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
agatacaaaa tcggccttga tatc 24
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gtgctcgagt agtacctaga tttagatgtc 30
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ccctctagat gcgcaaggat gggg 24
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tttgaattcg cgaaattttc agctgtagag 30
<210> 18
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggggatatct ttaattttat cactataata ttg 33
<210> 19
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tttgaattcg agtatggaca gttgc 25
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atgtggcaat cctgctactc g 21
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
agaaaaaaca gaagtgaaag aaac 24
<210> 22
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
aatggatcct atgcatgaag cattgtgcat c 31
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gtacaagaaa agaagaattg c 21
<210> 24
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttgtctagac aaacagtaag caaggagcag 30
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
atccatcaat tgcgataacc tctg 24
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
atcatgccat gaagttcctc gtac 24
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
atgatttcag caatttttgt tgcc 24
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
atgagagtac gagcaggaag aattc 25
<210> 29
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aatggatcca tcctcgagca taaaaaccac c 31
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tataggccta gcgccaagaa atagtagtac 30
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
agaacgaagg atattccctg c 21
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ggttttcttc atctctacac c 21
<210> 33
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gtgtataaag cctacacttt tataataaaa cagg 34
<210> 34
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
agtcaaagat tgtcatagtt cccctgagaa atc 33
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tgcctaactt attacaagaa ag 22
<210> 36
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ggtggaactc tgaaccattt c 21
<210> 37
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
aaaactgcag agacaaatta ggtccttcta tag 33
<210> 38
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ccgctcgagt cctccatatt cctttggata tg 32
<210> 39
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
aatagccatt gatactatag tctt 24
<210> 40
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cgcggatcca gattaaatga agaagagctc 30
<210> 41
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gaaatacgct cctacacttt tataataaaa cagg 34
<210> 42
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ttctagagaa tgtcatagtt cccctgagaa atc 33
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gttaagtaat ttaagtgaac c 21
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
agagctcttc ttcatttaat c 21
<210> 45
<211> 3814
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
aattgacaaa gttttctatt tgtgttaaca ttgtttatat aatagtgaac agtgttaaga 60
ttaaatgtga ggtgtttgta tggatattaa tgattataaa gagaagggac tttatttatt 120
aagtagtatg gatgatttta ttaaaattaa tgatttgttt atgggtaaag ttgtttctcc 180
tggctatgtt gcttcggttt ttggtgtttc caggtctact gttacacaat ggattcaaag 240
acgtaaaatt agagctttta agtataaagg taaggaaggt gactatatgg ttatacctat 300
tgctgatatt attgattaca aaagattgag taataatgat tttatttatg ataagttagt 360
gaggtgattt attttatgtt tgacgatagc tatgttgtta atgagtgttc gtctaatgtt 420
agtgaaaatg atagagattt ttgtagtttg gttggtcgtt ttatgattat taatggtata 480
gataagttgg ttattaagat taatagaaaa tttaatagga aatctttaag tttagatttt 540
agtgttgatt tattcccttc tatcaaagtt tctgaattag ttttttttga tgagtttaac 600
aaaacgtgtg gtttttattt ttcttttaat tcttttacaa tttttaaggc ttttagagat 660
gttcataatc ataataaaat atcattttat tttgcataat ttcgggtctg ggccgcagac 720
caggcccagt gctaacaata ttaattttta atgttaggaa ttgtttaatt cttaattgtg 780
tttttaaagg tagaataatt acccattcgc cctttagcca acaaaaatta aggaggtata 840
aacatggata aaatggattt gattcttcaa gatgaaagac tgggtgagat atttaaagat 900
atagatttaa cagataatga aaagagatat cttaaatggt tatggaaatg ggattatgaa 960
acacgtgata cttttgtatc aatttttttg aagctaaaaa atggtggaaa atgatttttt 1020
tcttatcttg atatattaga aaaaagcgta ctcacgaagt aagaatttgt aaaaaaagaa 1080
ggggggattt ttttggatga gagtttgtac aagcagattt taagtaatat tattattact 1140
cgtgattatt gtaaaaatgt tttagataat ataaagttca atgaaaaaat aattgattat 1200
tatgttatgt tacaaaatga tgtttttatt gattttacta ataaaataaa ttcaataagg 1260
gattgtaata aatattggta tttggatgtt tataaaaagc agaaaataaa ggattttaaa 1320
aagactaatt tgtgtaaaga taagttctgt aataattgta agaaagttaa acaggcttca 1380
agaatgcaaa aatatattcc tgaattacag aaatacaaag atggcttata tcattttata 1440
tttactgttg aaaatgtgcc aggtagtgaa ttaagagata ctattgatag gttgtttaag 1500
tcttttaagt catttacaag gtatttaagt ggtaatctta aaataaaagg tgttaatttt 1560
gataaatggg gttataaagg ctgtgtaagg tctttagagg taacttatag tatgattgat 1620
aatcatatta tgtatcatcc acacttgcat gttgcgatga tattagatcc tttttacgat 1680
ggttttaatg ttgaaaggat gcatataatt aataagttta gttatagcta tggtgtttta 1740
aaaaggttgt ttactgatga tgaattatta attcaaaaaa tttggtattt attgtttaat 1800
aatattgagg ttaacatggc caatataaat aatttagagg atggttattc ttgtttagtt 1860
aataagttta gtgattatga ttatgcggag ctgtttaagt atatttgtaa aaatactgat 1920
gaacaaggtt tacttatgac ttatgatatt tttaaagatt tatattttgc attacataat 1980
gttcatcaga tacaaggcta tggttgttta tataatataa gagatgatac tcaattagat 2040
ttaaaggttg atgacattta taatgatttg attgatttat tacaagttac agaaaatcct 2100
atacagtcta tggaaactgt acaggattta ttaaaggata ctgaatatac aataataagc 2160
cgtaagcgta tatttaagta tctaacacaa ttatatcata aggattgata tttataccgt 2220
ctgtcggact catgcggagg gggacttgag ggggtctccc ctcgcattgt acgacagacg 2280
gtattattat tatacaaatt ttttttatgt aatttttttt gtgtaatttt tttatacaaa 2340
taatatttca attcgtcgac ctgcagaagc ctggctgcag gtcgataaac ccagcgaacc 2400
atttgaggtg ataggtaaga ttataccgag gtatgaaaac gagaattgga cctttacaga 2460
attactctat gaagcgccat atttaaaaag ctaccaagac gaagaggatg aagaggatga 2520
ggaggcagat tgccttgaat atattgacaa tactgataag ataatatatc ttttatatag 2580
aagatatcgc cgtatgtaag gatttcaggg ggcaaggcat aggcagcgcg cttatcaata 2640
tatctataga atgggcaaag cataaaaact tgcatggact aatgcttgaa acccaggaca 2700
ataaccttat agcttgtaaa ttctatcata attgtggttt caaaatcggc tccgtcgata 2760
ctatgttata cgccaacttt caaaacaact ttgaaaaagc tgttttctgg tatttaaggt 2820
tttagaatgc aaggaacagt gaattggagt tcgtcttgtt ataattagct tcttggggta 2880
tctttaaata ctgtagaaaa gaggaaggaa ataataaatg aaaggaccaa taataatgac 2940
tagagaagaa agaatgaaga ttgttcatga aattaaggaa cgaatattgg ataaatatgg 3000
ggatgatgtt aaggcaattg gtgtttatgg ctctcttggt cgtcagactg atgggcccta 3060
ttcggatatt gagatgatgt gtgttctgtc aacagaggga gtagagttca gctatgaatg 3120
gacaaccggt gagtggaagg cggaagtgaa tttttatagc gaagagattc tactagatta 3180
tgcatctcgg gtggaaccgg attggccgct tacacatggt cgatttttct ctattttgcc 3240
gatttatgat ccaggtggat actttgagaa agtgtaccaa actgctaaat cggtagaagc 3300
ccaaaagttc cacgatgcga tctgtgccct tatcgtagaa gagctgtttg aatatgcagg 3360
caaatggcgt aatattcgtg tgcaaggacc gacaacattt ctaccatcct tgactgtaca 3420
ggtggcaatg gcaggtgcca tgttgattgg tctgcatcat cgcatctgtt atacgacgag 3480
cgcttcggtc ttaactgaag cagttaagca accagatctt cctccaggtt atgtccaact 3540
gtgccagctc gtaatgtctg gtcaactttc cgaccctgag aaacttctgg aatcgctaga 3600
gaatttctgg aatggggttc aggagtgggc ggaacgacac ggatatatag tggatgtgtc 3660
aaaacgcata ccattttgat gacattaaga aaggtggttt ttatgctcga ggagctcggt 3720
accggatcct ctagatagta cctagattta gatgtctaaa aagcttttta gacatctaat 3780
cttttctgaa gtacatccgc aactgtccat actc 3814
<210> 46
<211> 5756
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg aattgacaaa gttttctatt tgtgttaaca ttgtttatat aatagtgaac 180
agtgttaaga ttaaatgtga ggtgtttgta tggatattaa tgattataaa gagaagggac 240
tttatttatt aagtagtatg gatgatttta ttaaaattaa tgatttgttt atgggtaaag 300
ttgtttctcc tggctatgtt gcttcggttt ttggtgtttc caggtctact gttacacaat 360
ggattcaaag acgtaaaatt agagctttta agtataaagg taaggaaggt gactatatgg 420
ttatacctat tgctgatatt attgattaca aaagattgag taataatgat tttatttatg 480
ataagttagt gaggtgattt attttatgtt tgacgatagc tatgttgtta atgagtgttc 540
gtctaatgtt agtgaaaatg atagagattt ttgtagtttg gttggtcgtt ttatgattat 600
taatggtata gataagttgg ttattaagat taatagaaaa tttaatagga aatctttaag 660
tttagatttt agtgttgatt tattcccttc tatcaaagtt tctgaattag ttttttttga 720
tgagtttaac aaaacgtgtg gtttttattt ttcttttaat tcttttacaa tttttaaggc 780
ttttagagat gttcataatc ataataaaat atcattttat tttgcataat ttcgggtctg 840
ggccgcagac caggcccagt gctaacaata ttaattttta atgttaggaa ttgtttaatt 900
cttaattgtg tttttaaagg tagaataatt acccattcgc cctttagcca acaaaaatta 960
aggaggtata aacatggata aaatggattt gattcttcaa gatgaaagac tgggtgagat 1020
atttaaagat atagatttaa cagataatga aaagagatat cttaaatggt tatggaaatg 1080
ggattatgaa acacgtgata cttttgtatc aatttttttg aagctaaaaa atggtggaaa 1140
atgatttttt tcttatcttg atatattaga aaaaagcgta ctcacgaagt aagaatttgt 1200
aaaaaaagaa ggggggattt ttttggatga gagtttgtac aagcagattt taagtaatat 1260
tattattact cgtgattatt gtaaaaatgt tttagataat ataaagttca atgaaaaaat 1320
aattgattat tatgttatgt tacaaaatga tgtttttatt gattttacta ataaaataaa 1380
ttcaataagg gattgtaata aatattggta tttggatgtt tataaaaagc agaaaataaa 1440
ggattttaaa aagactaatt tgtgtaaaga taagttctgt aataattgta agaaagttaa 1500
acaggcttca agaatgcaaa aatatattcc tgaattacag aaatacaaag atggcttata 1560
tcattttata tttactgttg aaaatgtgcc aggtagtgaa ttaagagata ctattgatag 1620
gttgtttaag tcttttaagt catttacaag gtatttaagt ggtaatctta aaataaaagg 1680
tgttaatttt gataaatggg gttataaagg ctgtgtaagg tctttagagg taacttatag 1740
tatgattgat aatcatatta tgtatcatcc acacttgcat gttgcgatga tattagatcc 1800
tttttacgat ggttttaatg ttgaaaggat gcatataatt aataagttta gttatagcta 1860
tggtgtttta aaaaggttgt ttactgatga tgaattatta attcaaaaaa tttggtattt 1920
attgtttaat aatattgagg ttaacatggc caatataaat aatttagagg atggttattc 1980
ttgtttagtt aataagttta gtgattatga ttatgcggag ctgtttaagt atatttgtaa 2040
aaatactgat gaacaaggtt tacttatgac ttatgatatt tttaaagatt tatattttgc 2100
attacataat gttcatcaga tacaaggcta tggttgttta tataatataa gagatgatac 2160
tcaattagat ttaaaggttg atgacattta taatgatttg attgatttat tacaagttac 2220
agaaaatcct atacagtcta tggaaactgt acaggattta ttaaaggata ctgaatatac 2280
aataataagc cgtaagcgta tatttaagta tctaacacaa ttatatcata aggattgata 2340
tttataccgt ctgtcggact catgcggagg gggacttgag ggggtctccc ctcgcattgt 2400
acgacagacg gtattattat tatacaaatt ttttttatgt aatttttttt gtgtaatttt 2460
tttatacaaa taatatttca attcgtcgac ctgcagaagc ctggctgcag gtcgataaac 2520
ccagcgaacc atttgaggtg ataggtaaga ttataccgag gtatgaaaac gagaattgga 2580
cctttacaga attactctat gaagcgccat atttaaaaag ctaccaagac gaagaggatg 2640
aagaggatga ggaggcagat tgccttgaat atattgacaa tactgataag ataatatatc 2700
ttttatatag aagatatcgc cgtatgtaag gatttcaggg ggcaaggcat aggcagcgcg 2760
cttatcaata tatctataga atgggcaaag cataaaaact tgcatggact aatgcttgaa 2820
acccaggaca ataaccttat agcttgtaaa ttctatcata attgtggttt caaaatcggc 2880
tccgtcgata ctatgttata cgccaacttt caaaacaact ttgaaaaagc tgttttctgg 2940
tatttaaggt tttagaatgc aaggaacagt gaattggagt tcgtcttgtt ataattagct 3000
tcttggggta tctttaaata ctgtagaaaa gaggaaggaa ataataaatg aaaggaccaa 3060
taataatgac tagagaagaa agaatgaaga ttgttcatga aattaaggaa cgaatattgg 3120
ataaatatgg ggatgatgtt aaggcaattg gtgtttatgg ctctcttggt cgtcagactg 3180
atgggcccta ttcggatatt gagatgatgt gtgttctgtc aacagaggga gtagagttca 3240
gctatgaatg gacaaccggt gagtggaagg cggaagtgaa tttttatagc gaagagattc 3300
tactagatta tgcatctcgg gtggaaccgg attggccgct tacacatggt cgatttttct 3360
ctattttgcc gatttatgat ccaggtggat actttgagaa agtgtaccaa actgctaaat 3420
cggtagaagc ccaaaagttc cacgatgcga tctgtgccct tatcgtagaa gagctgtttg 3480
aatatgcagg caaatggcgt aatattcgtg tgcaaggacc gacaacattt ctaccatcct 3540
tgactgtaca ggtggcaatg gcaggtgcca tgttgattgg tctgcatcat cgcatctgtt 3600
atacgacgag cgcttcggtc ttaactgaag cagttaagca accagatctt cctccaggtt 3660
atgtccaact gtgccagctc gtaatgtctg gtcaactttc cgaccctgag aaacttctgg 3720
aatcgctaga gaatttctgg aatggggttc aggagtgggc ggaacgacac ggatatatag 3780
tggatgtgtc aaaacgcata ccattttgat gacattaaga aaggtggttt ttatgctcga 3840
ggagctcggt accggatcct ctagatagta cctagattta gatgtctaaa aagcttttta 3900
gacatctaat cttttctgaa gtacatccgc aactgtccat actcagaaca tgtgagcaaa 3960
aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 4020
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 4080
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 4140
gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 4200
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 4260
tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 4320
gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 4380
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 4440
cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 4500
agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 4560
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 4620
ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 4680
aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 4740
tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 4800
agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 4860
gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 4920
accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 4980
tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 5040
tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 5100
acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 5160
atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 5220
aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 5280
tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 5340
agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 5400
gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 5460
ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 5520
atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 5580
tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 5640
tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 5700
tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac 5756
<210> 47
<211> 5645
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg aattgacaaa gttttctatt tgtgttaaca ttgtttatat aatagtgaac 180
agtgttaaga ttaaatgtga ggtgtttgta tggatattaa tgattataaa gagaagggac 240
tttatttatt aagtagtatg gatgatttta ttaaaattaa tgatttgttt atgggtaaag 300
ttgtttctcc tggctatgtt gcttcggttt ttggtgtttc caggtctact gttacacaat 360
ggattcaaag acgtaaaatt agagctttta agtataaagg taaggaaggt gactatatgg 420
ttatacctat tgctgatatt attgattaca aaagattgag taataatgat tttatttatg 480
ataagttagt gaggtgattt attttatgtt tgacgatagc tatgttgtta atgagtgttc 540
gtctaatgtt agtgaaaatg atagagattt ttgtagtttg gttggtcgtt ttatgattat 600
taatggtata gataagttgg ttattaagat taatagaaaa tttaatagga aatctttaag 660
tttagatttt agtgttgatt tattcccttc tatcaaagtt tctgaattag ttttttttga 720
tgagtttaac aaaacgtgtg gtttttattt ttcttttaat tcttttacaa tttttaaggc 780
ttttagagat gttcataatc ataataaaat atcattttat tttgcataat ttcgggtctg 840
ggccgcagac caggcccagt gctaacaata ttaattttta atgttaggaa ttgtttaatt 900
cttaattgtg tttttaaagg tagaataatt acccattcgc cctttagcca acaaaaatta 960
aggaggtata aacatggata aaatggattt gattcttcaa gatgaaagac tgggtgagat 1020
atttaaagat atagatttaa cagataatga aaagagatat cttaaatggt tatggaaatg 1080
ggattatgaa acacgtgata cttttgtatc aatttttttg aagctaaaaa atggtggaaa 1140
atgatttttt tcttatcttg atatattaga aaaaagcgta ctcacgaagt aagaatttgt 1200
aaaaaaagaa ggggggattt ttttggatga gagtttgtac aagcagattt taagtaatat 1260
tattattact cgtgattatt gtaaaaatgt tttagataat ataaagttca atgaaaaaat 1320
aattgattat tatgttatgt tacaaaatga tgtttttatt gattttacta ataaaataaa 1380
ttcaataagg gattgtaata aatattggta tttggatgtt tataaaaagc agaaaataaa 1440
ggattttaaa aagactaatt tgtgtaaaga taagttctgt aataattgta agaaagttaa 1500
acaggcttca agaatgcaaa aatatattcc tgaattacag aaatacaaag atggcttata 1560
tcattttata tttactgttg aaaatgtgcc aggtagtgaa ttaagagata ctattgatag 1620
gttgtttaag tcttttaagt catttacaag gtatttaagt ggtaatctta aaataaaagg 1680
tgttaatttt gataaatggg gttataaagg ctgtgtaagg tctttagagg taacttatag 1740
tatgattgat aatcatatta tgtatcatcc acacttgcat gttgcgatga tattagatcc 1800
tttttacgat ggttttaatg ttgaaaggat gcatataatt aataagttta gttatagcta 1860
tggtgtttta aaaaggttgt ttactgatga tgaattatta attcaaaaaa tttggtattt 1920
attgtttaat aatattgagg ttaacatggc caatataaat aatttagagg atggttattc 1980
ttgtttagtt aataagttta gtgattatga ttatgcggag ctgtttaagt atatttgtaa 2040
aaatactgat gaacaaggtt tacttatgac ttatgatatt tttaaagatt tatattttgc 2100
attacataat gttcatcaga tacaaggcta tggttgttta tataatataa gagatgatac 2160
tcaattagat ttaaaggttg atgacattta taatgatttg attgatttat tacaagttac 2220
agaaaatcct atacagtcta tggaaactgt acaggattta ttaaaggata ctgaatatac 2280
aataataagc cgtaagcgta tatttaagta tctaacacaa ttatatcata aggattgata 2340
tttataccgt ctgtcggact catgcggagg gggacttgag ggggtctccc ctcgcattgt 2400
acgacagacg gtattattat tatacaaatt ttttttatgt aatttttttt gtgtaatttt 2460
tttatacaaa taatatttca attcgtcgac ctgcagaagc ctggctgcag gtcgataaac 2520
ccagcgaacc atttgaggtg ataggtaaga ttataccgag gtatgaaaac gagaattgga 2580
cctttacaga attactctat gaagcgccat atttaaaaag ctaccaagac gaagaggatg 2640
aagaggatga ggaggcagat tgccttgaat atattgacaa tactgataag ataatatatc 2700
ttttatatag aagatatcgc cgtatgtaag gatttcaggg ggcaaggcat aggcagcgcg 2760
cttatcaata tatctataga atgggcaaag cataaaaact tgcatggact aatgcttgaa 2820
acccaggaca ataaccttat agcttgtaaa ttctatcata attgtggttt caaaatcggc 2880
tccgtcgata ctatgttata cgccaacttt caaaacaact ttgaaaaagc tgttttctgg 2940
tatttaaggt tttagaatgc aaggaacagt gaattggagt tcgtcttgtt ataattagct 3000
tcttggggta tctttaaata ctgtagaaaa gaggaaggaa ataataaatg aactttaata 3060
aaattgattt agacaattgg aagagaaaag agatatttaa tcattatttg aaccaacaaa 3120
cgacttttag tataaccaca gaaattgata ttagtgtttt ataccgaaac ataaaacaag 3180
aaggatataa attttaccct gcatttattt tcttagtgac aagggtgata aactcaaata 3240
cagcttttag aactggttac aatagcgacg gagagttagg ttattgggat aagttagagc 3300
cactttatac aatttttgat ggtgtatcta aaacattctc tggtatttgg actcctgtaa 3360
agaatgactt caaagagttt tatgatttat acctttctga tgtagagaaa tataatggtt 3420
cggggaaatt gtttcccaaa acacctatac ctgaaaatgc tttttctctt tctattattc 3480
catggacttc atttactggg tttaacttaa atatcaataa taatagtaat taccttctac 3540
ccattattac agcaggaaaa ttcattaata aaggtaattc aatatattta ccgctatctt 3600
tacaggtaca tcattctgtt tgtgatggtt atcatgcagg attgtttatg aactctattc 3660
aggaattgtc agataggcct aatgactggc ttttataatg acattaagaa aggtggtttt 3720
tatgctcgag gagctcggta ccggatcctc tagatagtac ctagatttag atgtctaaaa 3780
agctttttag acatctaatc ttttctgaag tacatccgca actgtccata ctcagaacat 3840
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 3900
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 3960
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4020
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4080
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 4140
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 4200
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 4260
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4320
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4380
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 4440
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 4500
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 4560
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 4620
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 4680
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 4740
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 4800
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 4860
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 4920
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 4980
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 5040
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 5100
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 5160
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 5220
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 5280
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 5340
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 5400
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 5460
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 5520
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 5580
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 5640
gccac 5645
<210> 48
<211> 285
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gtgagatacc cttggaacag aatgtttgaa atagaagttt tacctaaaat aatattacaa 60
cagggttcgt aatgttaaat taatattaca gaaatattac aaattggtaa catttattga 120
ctcattaaat acctgatgct ataatataat caggttgttt atccaaaaca tactacatgc 180
tctaaaatgt acttacatag gtaaaaaatt tttgaaaggc attatgcctt tcaaaacaaa 240
aaatataaaa attaaaatac aaatacaagg aggaggattg actgt 285
<210> 49
<211> 9179
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg aattgacaaa gttttctatt tgtgttaaca ttgtttatat aatagtgaac 180
agtgttaaga ttaaatgtga ggtgtttgta tggatattaa tgattataaa gagaagggac 240
tttatttatt aagtagtatg gatgatttta ttaaaattaa tgatttgttt atgggtaaag 300
ttgtttctcc tggctatgtt gcttcggttt ttggtgtttc caggtctact gttacacaat 360
ggattcaaag acgtaaaatt agagctttta agtataaagg taaggaaggt gactatatgg 420
ttatacctat tgctgatatt attgattaca aaagattgag taataatgat tttatttatg 480
ataagttagt gaggtgattt attttatgtt tgacgatagc tatgttgtta atgagtgttc 540
gtctaatgtt agtgaaaatg atagagattt ttgtagtttg gttggtcgtt ttatgattat 600
taatggtata gataagttgg ttattaagat taatagaaaa tttaatagga aatctttaag 660
tttagatttt agtgttgatt tattcccttc tatcaaagtt tctgaattag ttttttttga 720
tgagtttaac aaaacgtgtg gtttttattt ttcttttaat tcttttacaa tttttaaggc 780
ttttagagat gttcataatc ataataaaat atcattttat tttgcataat ttcgggtctg 840
ggccgcagac caggcccagt gctaacaata ttaattttta atgttaggaa ttgtttaatt 900
cttaattgtg tttttaaagg tagaataatt acccattcgc cctttagcca acaaaaatta 960
aggaggtata aacatggata aaatggattt gattcttcaa gatgaaagac tgggtgagat 1020
atttaaagat atagatttaa cagataatga aaagagatat cttaaatggt tatggaaatg 1080
ggattatgaa acacgtgata cttttgtatc aatttttttg aagctaaaaa atggtggaaa 1140
atgatttttt tcttatcttg atatattaga aaaaagcgta ctcacgaagt aagaatttgt 1200
aaaaaaagaa ggggggattt ttttggatga gagtttgtac aagcagattt taagtaatat 1260
tattattact cgtgattatt gtaaaaatgt tttagataat ataaagttca atgaaaaaat 1320
aattgattat tatgttatgt tacaaaatga tgtttttatt gattttacta ataaaataaa 1380
ttcaataagg gattgtaata aatattggta tttggatgtt tataaaaagc agaaaataaa 1440
ggattttaaa aagactaatt tgtgtaaaga taagttctgt aataattgta agaaagttaa 1500
acaggcttca agaatgcaaa aatatattcc tgaattacag aaatacaaag atggcttata 1560
tcattttata tttactgttg aaaatgtgcc aggtagtgaa ttaagagata ctattgatag 1620
gttgtttaag tcttttaagt catttacaag gtatttaagt ggtaatctta aaataaaagg 1680
tgttaatttt gataaatggg gttataaagg ctgtgtaagg tctttagagg taacttatag 1740
tatgattgat aatcatatta tgtatcatcc acacttgcat gttgcgatga tattagatcc 1800
tttttacgat ggttttaatg ttgaaaggat gcatataatt aataagttta gttatagcta 1860
tggtgtttta aaaaggttgt ttactgatga tgaattatta attcaaaaaa tttggtattt 1920
attgtttaat aatattgagg ttaacatggc caatataaat aatttagagg atggttattc 1980
ttgtttagtt aataagttta gtgattatga ttatgcggag ctgtttaagt atatttgtaa 2040
aaatactgat gaacaaggtt tacttatgac ttatgatatt tttaaagatt tatattttgc 2100
attacataat gttcatcaga tacaaggcta tggttgttta tataatataa gagatgatac 2160
tcaattagat ttaaaggttg atgacattta taatgatttg attgatttat tacaagttac 2220
agaaaatcct atacagtcta tggaaactgt acaggattta ttaaaggata ctgaatatac 2280
aataataagc cgtaagcgta tatttaagta tctaacacaa ttatatcata aggattgata 2340
tttataccgt ctgtcggact catgcggagg gggacttgag ggggtctccc ctcgcattgt 2400
acgacagacg gtattattat tatacaaatt ttttttatgt aatttttttt gtgtaatttt 2460
tttatacaaa taatatttca attcgtcgac ctgcagaagc ctggctgcag gtcgataaac 2520
ccagcgaacc atttgaggtg ataggtaaga ttataccgag gtatgaaaac gagaattgga 2580
cctttacaga attactctat gaagcgccat atttaaaaag ctaccaagac gaagaggatg 2640
aagaggatga ggaggcagat tgccttgaat atattgacaa tactgataag ataatatatc 2700
ttttatatag aagatatcgc cgtatgtaag gatttcaggg ggcaaggcat aggcagcgcg 2760
cttatcaata tatctataga atgggcaaag cataaaaact tgcatggact aatgcttgaa 2820
acccaggaca ataaccttat agcttgtaaa ttctatcata attgtggttt caaaatcggc 2880
tccgtcgata ctatgttata cgccaacttt caaaacaact ttgaaaaagc tgttttctgg 2940
tatttaaggt tttagaatgc aaggaacagt gaattggagt tcgtcttgtt ataattagct 3000
tcttggggta tctttaaata ctgtagaaaa gaggaaggaa ataataaatg aactttaata 3060
aaattgattt agacaattgg aagagaaaag agatatttaa tcattatttg aaccaacaaa 3120
cgacttttag tataaccaca gaaattgata ttagtgtttt ataccgaaac ataaaacaag 3180
aaggatataa attttaccct gcatttattt tcttagtgac aagggtgata aactcaaata 3240
cagcttttag aactggttac aatagcgacg gagagttagg ttattgggat aagttagagc 3300
cactttatac aatttttgat ggtgtatcta aaacattctc tggtatttgg actcctgtaa 3360
agaatgactt caaagagttt tatgatttat acctttctga tgtagagaaa tataatggtt 3420
cggggaaatt gtttcccaaa acacctatac ctgaaaatgc tttttctctt tctattattc 3480
catggacttc atttactggg tttaacttaa atatcaataa taatagtaat taccttctac 3540
ccattattac agcaggaaaa ttcattaata aaggtaattc aatatattta ccgctatctt 3600
tacaggtaca tcattctgtt tgtgatggtt atcatgcagg attgtttatg aactctattc 3660
aggaattgtc agataggcct aatgactggc ttttataaga attcgtgaga tacccttgga 3720
acagaatgtt tgaaatagaa gttttaccta aaataatatt acaacagggt tcgtaatgtt 3780
aaattaatat tacagaaata ttacaaattg gtaacattta ttgactcatt aaatacctga 3840
tgctataata taatcaggtt gtttatccaa aacatactac atgctctaaa atgtacttac 3900
ataggtaaaa aatttttgaa aggcattatg cctttcaaaa caaaaaatat aaaaattaaa 3960
atacaaatac aaggaggagg attgactgtg atcattaaga aaggtggttt ttatgctcga 4020
gagatacaaa atcggccttg atatcggcat tacctctgta ggttgggcag tcatgaactt 4080
ggatattccc cgcatcgaag atttgggcgt ccgcattttt gacagagccg aaaatccgca 4140
gacgggagaa tccctagctc ttccccgacg cctcgcccgc tccgcccggc gtcgattgcg 4200
ccgccgcaag catcggctgg agcgcattcg ccgtctggtc attcgcgaag gaattttaac 4260
gaaagaggaa ctggacaaac tattcgagga aaaacacgaa atcgacgtct ggcagctgcg 4320
tgttgaggca ttggatcgaa aattaaacaa cgacgagctg gctcgcgtcc tccttcatct 4380
agccaagcgg cgcggcttca aatccaaccg caaaagcgag cgcagcaaca aagaaaacag 4440
cacgatgctc aaacatatcg aagaaaaccg ggccattctc tcaagctacc ggaccgtcgg 4500
cgaaatgatt gtgaaagatc caaagtttgc gctccataag cgcaataaag gagaaaatta 4560
tacaaacaca atcgcccgcg atgacttaga acgcgagatc cgactcattt tctccaaaca 4620
gcgtgagttt ggaaacatga gttgcacgga agagtttgag aatgaatata tcaccatttg 4680
ggcttcccag cgtcctgtcg cttccaaaga tgacatcgag aaaaaggtcg gcttttgtac 4740
ctttgaaccg aaagaaaaac gggctccaaa agcaacttat acattccaat cgttcatcgc 4800
ctgggagcac atcaataaat tgcggctcat ctccccatca ggcgcacgag ggctcaccga 4860
tgaggaacga cgccttttgt atgaacaggc attccaaaaa aacaaaatca cctaccatga 4920
tatacggacg ttgctccatt tgcctgatga cacttacttt aaaggcattg tgtatgatcg 4980
aggcgaatca cgaaagcaaa atgaaaacat ccgattcctt gaactcgacg cctatcatca 5040
aattcggaaa gccgtcgata aagtgtatgg aaaagggaag tcaagttcat ttctcccaat 5100
cgattttgat acattcggtt acgccctgac attgtttaaa gatgatgccg acattcacag 5160
ctacttgcga aacgaatatg aacaaaacgg aaaacggatg ccaaatttag cgaacaaggt 5220
atatgacaac gagctaattg aggaactatt gaacttgtca ttcaccaaat tcggccattt 5280
gtcgctaaag gcgcttcgca gcattctccc gtacatggaa caaggagaag tctactcttc 5340
ggcttgtgag cgagcgggat acacatttac agggccaaag aagaaacaaa aaacaatgtt 5400
gctaccaaat attccgccga tcgccaatcc ggtcgtcatg cgtgcattga cacaggcgcg 5460
gaaagtggtg aatgctatta ttaaaaagta cggttcgccg gtatccattc atatcgagtt 5520
ggcccgcgat ttatcacaaa cgtttgacga acggcggaaa acaaaaaaag aacaagacga 5580
aaaccggaag aaaaacgaaa ccgccatccg ccaactcatg gagtatggct tgacgctaaa 5640
cccaaccggc catgacattg tcaagttcaa actttggagt gagcaaaatg ggaggtgcgc 5700
ctactcgctt caaccgatcg aaatcgagcg gctgcttgaa ccggggtatg tagaagtgga 5760
tcacgtcatc ccgtatagcc gaagcttgga cgacagttat accaataaag tgttggtatt 5820
gacaagagaa aaccgcgaaa aaggcaaccg cattcctgcc gaatatttag gcgtcggaac 5880
cgaacgctgg caacagtttg aaacgtttgt gttaacgaac aagcagtttt ccaaaaagaa 5940
acgggatcgg ctgctccgac tccattatga tgaaaatgaa gaaacagaat ttaaaaatcg 6000
aaatttaaat gacacccggt atatttcacg ctttttcgcc aactttattc gcgaacatct 6060
gaaattcgcc gaaagtgatg acaagcaaaa agtctatacg gtcaacggcc gcgttaccgc 6120
ccatttgcga agccgctggg agtttaacaa aaaccgtgaa gaatcggatt tgcatcatgc 6180
cgtcgatgcc gccatcgtcg cttgcacaac gccaagcgat atcgccaaag tcaccgcctt 6240
ttaccaacgg cgcgaacaaa acaaagaact ggccaaaaag acagaaccgc acttcccgca 6300
gccttggccg cacttcgccg acgaactgcg ggcgcgttta tccaaacatc caaaagagag 6360
tataaaagct ctcaatcttg gaaattatga tgatcagaaa ctcgaatcgc tacagccggt 6420
ttttgtatcc cgaatgccga agcggagcgt tacaggggcg gctcatcaag aaacattacg 6480
gcgctacgtc ggtatcgatg aacggagcgg gaaaatccaa actgttgtca aaacgaaact 6540
gtccgaaatc aagttggatg cgagcgggca ttttccgatg tacggcaaag aaagcgaccc 6600
aaggacatac gaagcgatcc gccagcgtct gcttgaacat aacaatgacc cgaaaaaggc 6660
attccaagaa cctttgtaca agccgaaaaa gaacggcgaa cccgggcctg tcattcggac 6720
cgtaaaaatc atcgacacaa aaaaccaggt catcccgctc aatgacggca aaaccgtcgc 6780
ctacaacagc aacatcgttc gggttgacgt atttgaaaaa gatgggaaat attattgtgt 6840
ccctgtctac accatggaca tcatgaaagg gattttgcca aacaaagcca tcgagccgaa 6900
caagccgtac tctgagtgga aggaaatgac ggaggactat acgttccgat ttagcctgta 6960
cccgaatgat ctcattcgca ttgagcttcc gcgggaaaaa acagtgaaaa cagccgcagg 7020
cgaagagatc aacgttaagg atgtatttgt ctactacaaa acgattgact cagccaacgg 7080
aggattagag ttgatcagcc atgaccaccg tttctcgctc cgcggcgtcg gttcaagaac 7140
cctcaaacga ttcgagaaat accaagtaga tgtgctcggc aacatctaca aagtgagagg 7200
ggaaaaaaga gttgggttgg cgtcatctgc tcattcgaaa accggggaaa ctgtccgtcc 7260
gttacaatca actcgtgatt gatctagata gtacctagat ttagatgtct aaaaagcttt 7320
ttagacatct aatcttttct gaagtacatc cgcaactgtc catactcaga acatgtgagc 7380
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 7440
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 7500
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 7560
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 7620
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 7680
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 7740
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 7800
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 7860
ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 7920
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 7980
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 8040
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 8100
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 8160
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 8220
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 8280
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 8340
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 8400
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 8460
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 8520
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 8580
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 8640
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 8700
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 8760
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 8820
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 8880
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 8940
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 9000
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 9060
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 9120
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac 9179
<210> 50
<211> 139
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
atgtggcaat cctgctactc ggtcatagtt cccctgagaa atcagggtta ctatgataag 60
ggctttctgc ctaaggcaga ctgacccgcg gcgttgggga tcgcctgtcg cccgcttttg 120
gcgggcattc cccatcctt 139
<210> 51
<211> 361
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gcgaaatttt cagctgtaga ggtagcgtat actactggta actcgattac caaatctata 60
ccggctttta atgccatttg agttcgtttc catttgtcta caatagctgg ctctcctctt 120
tgtacaaaat ttccactcat tactgctata gtataatcgc aattagttaa tttttttgaa 180
atttgcagat gataaaggtg gccattgtga aaaggattat attcgacaat aattcctaaa 240
attcccatat aacttcttac cctttcaaaa aaatttttaa gatatactta ttattttaca 300
taaaatatga taaaatgtaa aagggagatc gtgtatacaa tattatagtg ataaaattaa 360
a 361
<210> 52
<211> 6308
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg aattgacaaa gttttctatt tgtgttaaca ttgtttatat aatagtgaac 180
agtgttaaga ttaaatgtga ggtgtttgta tggatattaa tgattataaa gagaagggac 240
tttatttatt aagtagtatg gatgatttta ttaaaattaa tgatttgttt atgggtaaag 300
ttgtttctcc tggctatgtt gcttcggttt ttggtgtttc caggtctact gttacacaat 360
ggattcaaag acgtaaaatt agagctttta agtataaagg taaggaaggt gactatatgg 420
ttatacctat tgctgatatt attgattaca aaagattgag taataatgat tttatttatg 480
ataagttagt gaggtgattt attttatgtt tgacgatagc tatgttgtta atgagtgttc 540
gtctaatgtt agtgaaaatg atagagattt ttgtagtttg gttggtcgtt ttatgattat 600
taatggtata gataagttgg ttattaagat taatagaaaa tttaatagga aatctttaag 660
tttagatttt agtgttgatt tattcccttc tatcaaagtt tctgaattag ttttttttga 720
tgagtttaac aaaacgtgtg gtttttattt ttcttttaat tcttttacaa tttttaaggc 780
ttttagagat gttcataatc ataataaaat atcattttat tttgcataat ttcgggtctg 840
ggccgcagac caggcccagt gctaacaata ttaattttta atgttaggaa ttgtttaatt 900
cttaattgtg tttttaaagg tagaataatt acccattcgc cctttagcca acaaaaatta 960
aggaggtata aacatggata aaatggattt gattcttcaa gatgaaagac tgggtgagat 1020
atttaaagat atagatttaa cagataatga aaagagatat cttaaatggt tatggaaatg 1080
ggattatgaa acacgtgata cttttgtatc aatttttttg aagctaaaaa atggtggaaa 1140
atgatttttt tcttatcttg atatattaga aaaaagcgta ctcacgaagt aagaatttgt 1200
aaaaaaagaa ggggggattt ttttggatga gagtttgtac aagcagattt taagtaatat 1260
tattattact cgtgattatt gtaaaaatgt tttagataat ataaagttca atgaaaaaat 1320
aattgattat tatgttatgt tacaaaatga tgtttttatt gattttacta ataaaataaa 1380
ttcaataagg gattgtaata aatattggta tttggatgtt tataaaaagc agaaaataaa 1440
ggattttaaa aagactaatt tgtgtaaaga taagttctgt aataattgta agaaagttaa 1500
acaggcttca agaatgcaaa aatatattcc tgaattacag aaatacaaag atggcttata 1560
tcattttata tttactgttg aaaatgtgcc aggtagtgaa ttaagagata ctattgatag 1620
gttgtttaag tcttttaagt catttacaag gtatttaagt ggtaatctta aaataaaagg 1680
tgttaatttt gataaatggg gttataaagg ctgtgtaagg tctttagagg taacttatag 1740
tatgattgat aatcatatta tgtatcatcc acacttgcat gttgcgatga tattagatcc 1800
tttttacgat ggttttaatg ttgaaaggat gcatataatt aataagttta gttatagcta 1860
tggtgtttta aaaaggttgt ttactgatga tgaattatta attcaaaaaa tttggtattt 1920
attgtttaat aatattgagg ttaacatggc caatataaat aatttagagg atggttattc 1980
ttgtttagtt aataagttta gtgattatga ttatgcggag ctgtttaagt atatttgtaa 2040
aaatactgat gaacaaggtt tacttatgac ttatgatatt tttaaagatt tatattttgc 2100
attacataat gttcatcaga tacaaggcta tggttgttta tataatataa gagatgatac 2160
tcaattagat ttaaaggttg atgacattta taatgatttg attgatttat tacaagttac 2220
agaaaatcct atacagtcta tggaaactgt acaggattta ttaaaggata ctgaatatac 2280
aataataagc cgtaagcgta tatttaagta tctaacacaa ttatatcata aggattgata 2340
tttataccgt ctgtcggact catgcggagg gggacttgag ggggtctccc ctcgcattgt 2400
acgacagacg gtattattat tatacaaatt ttttttatgt aatttttttt gtgtaatttt 2460
tttatacaaa taatatttca attcgtcgac ctgcagaagc ctggctgcag gtcgataaac 2520
ccagcgaacc atttgaggtg ataggtaaga ttataccgag gtatgaaaac gagaattgga 2580
cctttacaga attactctat gaagcgccat atttaaaaag ctaccaagac gaagaggatg 2640
aagaggatga ggaggcagat tgccttgaat atattgacaa tactgataag ataatatatc 2700
ttttatatag aagatatcgc cgtatgtaag gatttcaggg ggcaaggcat aggcagcgcg 2760
cttatcaata tatctataga atgggcaaag cataaaaact tgcatggact aatgcttgaa 2820
acccaggaca ataaccttat agcttgtaaa ttctatcata attgtggttt caaaatcggc 2880
tccgtcgata ctatgttata cgccaacttt caaaacaact ttgaaaaagc tgttttctgg 2940
tatttaaggt tttagaatgc aaggaacagt gaattggagt tcgtcttgtt ataattagct 3000
tcttggggta tctttaaata ctgtagaaaa gaggaaggaa ataataaatg aaaggaccaa 3060
taataatgac tagagaagaa agaatgaaga ttgttcatga aattaaggaa cgaatattgg 3120
ataaatatgg ggatgatgtt aaggcaattg gtgtttatgg ctctcttggt cgtcagactg 3180
atgggcccta ttcggatatt gagatgatgt gtgttctgtc aacagaggga gtagagttca 3240
gctatgaatg gacaaccggt gagtggaagg cggaagtgaa tttttatagc gaagagattc 3300
tactagatta tgcatctcgg gtggaaccgg attggccgct tacacatggt cgatttttct 3360
ctattttgcc gatttatgat ccaggtggat actttgagaa agtgtaccaa actgctaaat 3420
cggtagaagc ccaaaagttc cacgatgcga tctgtgccct tatcgtagaa gagctgtttg 3480
aatatgcagg caaatggcgt aatattcgtg tgcaaggacc gacaacattt ctaccatcct 3540
tgactgtaca ggtggcaatg gcaggtgcca tgttgattgg tctgcatcat cgcatctgtt 3600
atacgacgag cgcttcggtc ttaactgaag cagttaagca accagatctt cctccaggtt 3660
atgtccaact gtgccagctc gtaatgtctg gtcaactttc cgaccctgag aaacttctgg 3720
aatcgctaga gaatttctgg aatggggttc aggagtgggc ggaacgacac ggatatatag 3780
tggatgtgtc aaaacgcata ccattttgat gacattaaga aaggtggttt ttatgctcga 3840
ggatggatcc atttataggc ctcgctccat aagcgaaatt ttcagctgta gaggtagcgt 3900
atactactgg taactcgatt accaaatcta taccggcttt taatgccatt tgagttcgtt 3960
tccatttgtc tacaatagct ggctctcctc tttgtacaaa atttccactc attactgcta 4020
tagtataatc gcaattagtt aatttttttg aaatttgcag atgataaagg tggccattgt 4080
gaaaaggatt atattcgaca ataattccta aaattcccat ataacttctt accctttcaa 4140
aaaaattttt aagatatact tattatttta cataaaatat gataaaatgt aaaagggaga 4200
tcgtgtatac aatattatag tgataaaatt aagatatgtg gcaatcctgc tactcggtca 4260
tagttcccct gagaaatcag ggttactatg ataagggctt tctgcctaag gcagactgac 4320
ccgcggcgtt ggggatcgcc tgtcgcccgc ttttggcggg cattccccat ccttaacgaa 4380
gaccttggcg gaaaacgtca aggtcttttt ttctagatag tacctagatt tagatgtcta 4440
aaaagctttt tagacatcta atcttttctg aagtacatcc gcaactgtcc atactcagaa 4500
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 4560
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 4620
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 4680
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 4740
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 4800
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 4860
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 4920
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 4980
taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 5040
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 5100
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 5160
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 5220
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 5280
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 5340
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 5400
gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 5460
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 5520
gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 5580
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 5640
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 5700
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 5760
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 5820
taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 5880
caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 5940
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 6000
ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 6060
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 6120
aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 6180
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 6240
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 6300
agtgccac 6308
<210> 53
<211> 9712
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg aattgacaaa gttttctatt tgtgttaaca ttgtttatat aatagtgaac 180
agtgttaaga ttaaatgtga ggtgtttgta tggatattaa tgattataaa gagaagggac 240
tttatttatt aagtagtatg gatgatttta ttaaaattaa tgatttgttt atgggtaaag 300
ttgtttctcc tggctatgtt gcttcggttt ttggtgtttc caggtctact gttacacaat 360
ggattcaaag acgtaaaatt agagctttta agtataaagg taaggaaggt gactatatgg 420
ttatacctat tgctgatatt attgattaca aaagattgag taataatgat tttatttatg 480
ataagttagt gaggtgattt attttatgtt tgacgatagc tatgttgtta atgagtgttc 540
gtctaatgtt agtgaaaatg atagagattt ttgtagtttg gttggtcgtt ttatgattat 600
taatggtata gataagttgg ttattaagat taatagaaaa tttaatagga aatctttaag 660
tttagatttt agtgttgatt tattcccttc tatcaaagtt tctgaattag ttttttttga 720
tgagtttaac aaaacgtgtg gtttttattt ttcttttaat tcttttacaa tttttaaggc 780
ttttagagat gttcataatc ataataaaat atcattttat tttgcataat ttcgggtctg 840
ggccgcagac caggcccagt gctaacaata ttaattttta atgttaggaa ttgtttaatt 900
cttaattgtg tttttaaagg tagaataatt acccattcgc cctttagcca acaaaaatta 960
aggaggtata aacatggata aaatggattt gattcttcaa gatgaaagac tgggtgagat 1020
atttaaagat atagatttaa cagataatga aaagagatat cttaaatggt tatggaaatg 1080
ggattatgaa acacgtgata cttttgtatc aatttttttg aagctaaaaa atggtggaaa 1140
atgatttttt tcttatcttg atatattaga aaaaagcgta ctcacgaagt aagaatttgt 1200
aaaaaaagaa ggggggattt ttttggatga gagtttgtac aagcagattt taagtaatat 1260
tattattact cgtgattatt gtaaaaatgt tttagataat ataaagttca atgaaaaaat 1320
aattgattat tatgttatgt tacaaaatga tgtttttatt gattttacta ataaaataaa 1380
ttcaataagg gattgtaata aatattggta tttggatgtt tataaaaagc agaaaataaa 1440
ggattttaaa aagactaatt tgtgtaaaga taagttctgt aataattgta agaaagttaa 1500
acaggcttca agaatgcaaa aatatattcc tgaattacag aaatacaaag atggcttata 1560
tcattttata tttactgttg aaaatgtgcc aggtagtgaa ttaagagata ctattgatag 1620
gttgtttaag tcttttaagt catttacaag gtatttaagt ggtaatctta aaataaaagg 1680
tgttaatttt gataaatggg gttataaagg ctgtgtaagg tctttagagg taacttatag 1740
tatgattgat aatcatatta tgtatcatcc acacttgcat gttgcgatga tattagatcc 1800
tttttacgat ggttttaatg ttgaaaggat gcatataatt aataagttta gttatagcta 1860
tggtgtttta aaaaggttgt ttactgatga tgaattatta attcaaaaaa tttggtattt 1920
attgtttaat aatattgagg ttaacatggc caatataaat aatttagagg atggttattc 1980
ttgtttagtt aataagttta gtgattatga ttatgcggag ctgtttaagt atatttgtaa 2040
aaatactgat gaacaaggtt tacttatgac ttatgatatt tttaaagatt tatattttgc 2100
attacataat gttcatcaga tacaaggcta tggttgttta tataatataa gagatgatac 2160
tcaattagat ttaaaggttg atgacattta taatgatttg attgatttat tacaagttac 2220
agaaaatcct atacagtcta tggaaactgt acaggattta ttaaaggata ctgaatatac 2280
aataataagc cgtaagcgta tatttaagta tctaacacaa ttatatcata aggattgata 2340
tttataccgt ctgtcggact catgcggagg gggacttgag ggggtctccc ctcgcattgt 2400
acgacagacg gtattattat tatacaaatt ttttttatgt aatttttttt gtgtaatttt 2460
tttatacaaa taatatttca attcgtcgac ctgcagaagc ctggctgcag gtcgataaac 2520
ccagcgaacc atttgaggtg ataggtaaga ttataccgag gtatgaaaac gagaattgga 2580
cctttacaga attactctat gaagcgccat atttaaaaag ctaccaagac gaagaggatg 2640
aagaggatga ggaggcagat tgccttgaat atattgacaa tactgataag ataatatatc 2700
ttttatatag aagatatcgc cgtatgtaag gatttcaggg ggcaaggcat aggcagcgcg 2760
cttatcaata tatctataga atgggcaaag cataaaaact tgcatggact aatgcttgaa 2820
acccaggaca ataaccttat agcttgtaaa ttctatcata attgtggttt caaaatcggc 2880
tccgtcgata ctatgttata cgccaacttt caaaacaact ttgaaaaagc tgttttctgg 2940
tatttaaggt tttagaatgc aaggaacagt gaattggagt tcgtcttgtt ataattagct 3000
tcttggggta tctttaaata ctgtagaaaa gaggaaggaa ataataaatg aactttaata 3060
aaattgattt agacaattgg aagagaaaag agatatttaa tcattatttg aaccaacaaa 3120
cgacttttag tataaccaca gaaattgata ttagtgtttt ataccgaaac ataaaacaag 3180
aaggatataa attttaccct gcatttattt tcttagtgac aagggtgata aactcaaata 3240
cagcttttag aactggttac aatagcgacg gagagttagg ttattgggat aagttagagc 3300
cactttatac aatttttgat ggtgtatcta aaacattctc tggtatttgg actcctgtaa 3360
agaatgactt caaagagttt tatgatttat acctttctga tgtagagaaa tataatggtt 3420
cggggaaatt gtttcccaaa acacctatac ctgaaaatgc tttttctctt tctattattc 3480
catggacttc atttactggg tttaacttaa atatcaataa taatagtaat taccttctac 3540
ccattattac agcaggaaaa ttcattaata aaggtaattc aatatattta ccgctatctt 3600
tacaggtaca tcattctgtt tgtgatggtt atcatgcagg attgtttatg aactctattc 3660
aggaattgtc agataggcct aatgactggc ttttataatg acattaagaa aggtggtttt 3720
tatgctcgag gtgagatacc cttggaacag aatgtttgaa atagaagttt tacctaaaat 3780
aatattacaa cagggttcgt aatgttaaat taatattaca gaaatattac aaattggtaa 3840
catttattga ctcattaaat acctgatgct ataatataat caggttgttt atccaaaaca 3900
tactacatgc tctaaaatgt acttacatag gtaaaaaatt tttgaaaggc attatgcctt 3960
tcaaaacaaa aaatataaaa attaaaatac aaatacaagg aggaggattg actgtagata 4020
caaaatcggc cttgatatcg gcattacctc tgtaggttgg gcagtcatga acttggatat 4080
tccccgcatc gaagatttgg gcgtccgcat ttttgacaga gccgaaaatc cgcagacggg 4140
agaatcccta gctcttcccc gacgcctcgc ccgctccgcc cggcgtcgat tgcgccgccg 4200
caagcatcgg ctggagcgca ttcgccgtct ggtcattcgc gaaggaattt taacgaaaga 4260
ggaactggac aaactattcg aggaaaaaca cgaaatcgac gtctggcagc tgcgtgttga 4320
ggcattggat cgaaaattaa acaacgacga gctggctcgc gtcctccttc atctagccaa 4380
gcggcgcggc ttcaaatcca accgcaaaag cgagcgcagc aacaaagaaa acagcacgat 4440
gctcaaacat atcgaagaaa accgggccat tctctcaagc taccggaccg tcggcgaaat 4500
gattgtgaaa gatccaaagt ttgcgctcca taagcgcaat aaaggagaaa attatacaaa 4560
cacaatcgcc cgcgatgact tagaacgcga gatccgactc attttctcca aacagcgtga 4620
gtttggaaac atgagttgca cggaagagtt tgagaatgaa tatatcacca tttgggcttc 4680
ccagcgtcct gtcgcttcca aagatgacat cgagaaaaag gtcggctttt gtacctttga 4740
accgaaagaa aaacgggctc caaaagcaac ttatacattc caatcgttca tcgcctggga 4800
gcacatcaat aaattgcggc tcatctcccc atcaggcgca cgagggctca ccgatgagga 4860
acgacgcctt ttgtatgaac aggcattcca aaaaaacaaa atcacctacc atgatatacg 4920
gacgttgctc catttgcctg atgacactta ctttaaaggc attgtgtatg atcgaggcga 4980
atcacgaaag caaaatgaaa acatccgatt ccttgaactc gacgcctatc atcaaattcg 5040
gaaagccgtc gataaagtgt atggaaaagg gaagtcaagt tcatttctcc caatcgattt 5100
tgatacattc ggttacgccc tgacattgtt taaagatgat gccgacattc acagctactt 5160
gcgaaacgaa tatgaacaaa acggaaaacg gatgccaaat ttagcgaaca aggtatatga 5220
caacgagcta attgaggaac tattgaactt gtcattcacc aaattcggcc atttgtcgct 5280
aaaggcgctt cgcagcattc tcccgtacat ggaacaagga gaagtctact cttcggcttg 5340
tgagcgagcg ggatacacat ttacagggcc aaagaagaaa caaaaaacaa tgttgctacc 5400
aaatattccg ccgatcgcca atccggtcgt catgcgtgca ttgacacagg cgcggaaagt 5460
ggtgaatgct attattaaaa agtacggttc gccggtatcc attcatatcg agttggcccg 5520
cgatttatca caaacgtttg acgaacggcg gaaaacaaaa aaagaacaag acgaaaaccg 5580
gaagaaaaac gaaaccgcca tccgccaact catggagtat ggcttgacgc taaacccaac 5640
cggccatgac attgtcaagt tcaaactttg gagtgagcaa aatgggaggt gcgcctactc 5700
gcttcaaccg atcgaaatcg agcggctgct tgaaccgggg tatgtagaag tggatcacgt 5760
catcccgtat agccgaagct tggacgacag ttataccaat aaagtgttgg tattgacaag 5820
agaaaaccgc gaaaaaggca accgcattcc tgccgaatat ttaggcgtcg gaaccgaacg 5880
ctggcaacag tttgaaacgt ttgtgttaac gaacaagcag ttttccaaaa agaaacggga 5940
tcggctgctc cgactccatt atgatgaaaa tgaagaaaca gaatttaaaa atcgaaattt 6000
aaatgacacc cggtatattt cacgcttttt cgccaacttt attcgcgaac atctgaaatt 6060
cgccgaaagt gatgacaagc aaaaagtcta tacggtcaac ggccgcgtta ccgcccattt 6120
gcgaagccgc tgggagttta acaaaaaccg tgaagaatcg gatttgcatc atgccgtcga 6180
tgccgccatc gtcgcttgca caacgccaag cgatatcgcc aaagtcaccg ccttttacca 6240
acggcgcgaa caaaacaaag aactggccaa aaagacagaa ccgcacttcc cgcagccttg 6300
gccgcacttc gccgacgaac tgcgggcgcg tttatccaaa catccaaaag agagtataaa 6360
agctctcaat cttggaaatt atgatgatca gaaactcgaa tcgctacagc cggtttttgt 6420
atcccgaatg ccgaagcgga gcgttacagg ggcggctcat caagaaacat tacggcgcta 6480
cgtcggtatc gatgaacgga gcgggaaaat ccaaactgtt gtcaaaacga aactgtccga 6540
aatcaagttg gatgcgagcg ggcattttcc gatgtacggc aaagaaagcg acccaaggac 6600
atacgaagcg atccgccagc gtctgcttga acataacaat gacccgaaaa aggcattcca 6660
agaacctttg tacaagccga aaaagaacgg cgaacccggg cctgtcattc ggaccgtaaa 6720
aatcatcgac acaaaaaacc aggtcatccc gctcaatgac ggcaaaaccg tcgcctacaa 6780
cagcaacatc gttcgggttg acgtatttga aaaagatggg aaatattatt gtgtccctgt 6840
ctacaccatg gacatcatga aagggatttt gccaaacaaa gccatcgagc cgaacaagcc 6900
gtactctgag tggaaggaaa tgacggagga ctatacgttc cgatttagcc tgtacccgaa 6960
tgatctcatt cgcattgagc ttccgcggga aaaaacagtg aaaacagccg caggcgaaga 7020
gatcaacgtt aaggatgtat ttgtctacta caaaacgatt gactcagcca acggaggatt 7080
agagttgatc agccatgacc accgtttctc gctccgcggc gtcggttcaa gaaccctcaa 7140
acgattcgag aaataccaag tagatgtgct cggcaacatc tacaaagtga gaggggaaaa 7200
aagagttggg ttggcgtcat ctgctcattc gaaaaccggg gaaactgtcc gtccgttaca 7260
atcaactcgt gattgagcga aattttcagc tgtagaggta gcgtatacta ctggtaactc 7320
gattaccaaa tctataccgg cttttaatgc catttgagtt cgtttccatt tgtctacaat 7380
agctggctct cctctttgta caaaatttcc actcattact gctatagtat aatcgcaatt 7440
agttaatttt tttgaaattt gcagatgata aaggtggcca ttgtgaaaag gattatattc 7500
gacaataatt cctaaaattc ccatataact tcttaccctt tcaaaaaaat ttttaagata 7560
tacttattat tttacataaa atatgataaa atgtaaaagg gagatcgtgt atacaatatt 7620
atagtgataa aattaagata tgtggcaatc ctgctactcg gtcatagttc ccctgagaaa 7680
tcagggttac tatgataagg gctttctgcc taaggcagac tgacccgcgg cgttggggat 7740
cgcctgtcgc ccgcttttgg cgggcattcc ccatccttaa cgaagacctt ggcggaaaac 7800
gtcaaggtct ttttttctag atagtaccta gatttagatg tctaaaaagc tttttagaca 7860
tctaatcttt tctgaagtac atccgcaact gtccatactc agaacatgtg agcaaaaggc 7920
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 7980
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 8040
ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 8100
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 8160
agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 8220
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 8280
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 8340
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 8400
agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 8460
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 8520
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 8580
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 8640
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 8700
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 8760
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 8820
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 8880
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 8940
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 9000
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 9060
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 9120
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 9180
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 9240
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 9300
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 9360
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 9420
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 9480
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 9540
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 9600
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 9660
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac 9712
Claims (3)
1. The genome editing vector of the thermophilic anaerobic ethanol bacillus is characterized in that the editing vector is an expression vector constructed by constructing an expression element into an escherichia coli-thermophilic anaerobic ethanol bacillus shuttle expression vector; the expression element comprises a thermostable Cas9 expression element and/or a sgRNA expression element; the escherichia coli-thermophilic anaerobic ethanol bacillus shuttle expression vector is a karamycin resistant shuttle expression vector pBlu-Htk or a chloramphenicol resistant shuttle expression vector pBlu-Cm, the editing vector is one of a Cas9 expression frame plasmid pBlu-Cm-ThermoCas9 expressed by a Psly promoter and a sgRNA expression frame plasmid pBlu-Pat-sgRNA expressed by a Pcat promoter or an editing vector pBlu-Cas9-Pat-sgRNA containing a Cas9 expression frame and a sgRNA expression frame plasmid; the nucleotide sequences of the editing carrier are respectively shown as SEQ ID NO 49, SEQ ID NO 52 and SEQ ID NO 53.
2. A method for gene editing of the genome of EtOH thermophilus, characterized in that the gene editing is carried out in the EtOH thermophilus by using the editing vector of claim 1, which comprises the following steps:
(1) Designing upstream and downstream homology arm repair sequences aiming at a target gene by taking a thermophilic anaerobic ethanol bacillus genome as a template to obtain a target gene donor DNA sequence;
(2) Constructing a plasmid containing a donor DNA sequence and sgRNA expression; (3) Transforming Cas9 expression frame plasmids into thermophilic anaerobic ethanol bacillus, transforming the plasmids constructed in the step (2) into thermophilic anaerobic ethanol bacillus, screening markers and selecting transformants to obtain gene editing mutant strains;
or connecting the Cas9 expression frame plasmid with the plasmid containing the donor DNA sequence and the sgRNA expression frame constructed in the step (2) into a simple plasmid editing vector, and directly converting the simple plasmid editing vector into the thermophilic anaerobic ethanol bacillus to obtain a gene editing mutant strain; the genome of the thermophilic anaerobic ethanol bacillus is derived from thermophilic anaerobic ethanol bacillus and is DSM 2246; the objective gene described in step (1) isTdkGene, gene,adhEGenes andRSPa gene; the above-mentionedTdkThe homologous arm amplification primers of the gene are Tdk-N and Tdk-C, and the nucleotide sequence of the primers is shown as SEQ ID NO:23 to 24; the describedadhEThe homologous arm amplification primers of the gene are adhE-N and a dhE-C, and the nucleotide sequence of the primer is shown as SEQ ID NO:21 to 22; the above-mentionedRSPThe homologous arm amplification primers of the gene are RSP-N and RSP-C, and the nucleotide sequences of the primers are shown in SEQ ID NO:37 to 38; the step (2) is as describedThe nucleotide sequence of sgRNA is shown in SEQ ID NO: 50.
3. Use of the editing vector of claim 1 for thermostable Cas 9-based gene editing in the genome of anoxella thermophila.
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| Title |
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| Characterizing a thermostable Cas9 for bacterial genome editing and silencing;Ioannis Mougiakos et al;《nature communications》;20171121;参见摘要 * |
| Genome Editing of the Anaerobic ThermophileThermoanaerobacter ethanolicus Using Thermostable Cas9;Yilin Le etal;《Applied and Environmental Microbiology》;20210131;全文 * |
| 嗜热厌氧乙醇杆菌-大肠杆菌穿梭载体的构建;傅百灵;《南京师范大学硕士论文》;20170412;参见摘要、第1.7.2节 * |
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