A method for bacterial gene site-directed transposition and mutant library construction and its application

Abstract

This invention discloses a method for site-directed transposition of bacterial genes and construction of mutant libraries, and its applications. The gene transposition system comprises: (1) an expression construct 1 containing genes encoding Cas12k, TnsB, TnsC, and TniQ proteins; and (2) an expression construct 2 containing transposon sequences corresponding to the Cas12k protein, a guide RNA backbone sequence, and target sequences for different target sequences. This invention enables transposition at at least one site in the bacterial genome, and enables the construction of bacterial mutant libraries, which can be used to screen for genes in bacteria related to different environmental stresses.

Description

Technical Field

[0001] This invention relates to a method for site-directed transposition of the bacterial genome in genome editing and its application in bacterial genome editing and the construction and screening of bacterial site-specific mutation libraries. Background Technology

[0002] Bacterial genomes possess rich sequence diversity, enabling bacteria to rapidly adapt to constantly changing environments. Humans have derived a wealth of useful resources from bacterial genomes, such as antibiotics, various enzymes, and bacterial fermentation agents. Simultaneously, studying the various pathogenic and drug-resistance genes in human pathogenic bacteria is crucial for developing novel therapeutic drugs and methods to combat bacterial infections. However, due to the complexity and diversity of bacterial genomes, the functions of only a small number of bacterial genes in nature have been studied to date. Genome editing and gene screening technologies are important tools for studying bacterial gene function and are significant for advancing human understanding of bacteria. To explore the vast number of untapped genomic functions within bacterial genomes, high-throughput genome engineering and screening technologies are indispensable methods for analyzing complex bacterial gene networks.

[0003] Currently, several techniques exist for high-throughput library construction and screening in bacteria, including Tn-seq, MAGE, TRMR, and CREATE. However, each of these techniques has its drawbacks. For example, while Tn-seq can construct bacterial mutant libraries by transposing genes on the bacterial genome, this transposition is random, making it impossible to construct site-specific bacterial mutant libraries, and requiring extensive screening to obtain the target mutants. MAGE, TRMR, and CREATE techniques rely on the homologous recombination efficiency of bacteria; therefore, their application is currently limited to *E. coli* and cannot be applied to other bacteria with weaker homologous recombination capabilities.

[0004] In 2019, a CRISPR-related transposition system—the CAST system—was reported. This system contains the VK-type CRISPR protein Cas12k and transposases TnsB, TnsC, and TniQ, which are similar to Tn7, enabling efficient gene transposition at specific sites in *E. coli*. This system features controllable insertion sites, ease of operation, and high efficiency, making it an ideal technology with potential for high-throughput mutant library construction, effectively addressing the shortcomings of previously developed technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a genome editing method for gene transposition in bacterial genomes and its application. This invention can (1) efficiently and rapidly transpose genes at specific genomic sites in different bacterial strains, including but not limited to *Pseudomonas aeruginosa* and *Klebsiella pneumoniae*; (2) construct bacterial transposition mutation libraries targeting specific gene sites by combining high-throughput primer pool synthesis technology and second-generation sequencing technology, and perform data analysis; (3) screen the constructed bacterial transposition mutation libraries to detect genes related to various bacterial pathways.

[0006] To achieve the above objectives, one technical solution of the present invention is a gene transposition system (or dual plasmid system) capable of transposing the bacterial genome, wherein the gene transposition system comprises:

[0007] (1) An expression construct 1 containing genes encoding Cas12k, TnsB, TnsC and TniQ proteins;

[0008] (2) An expression construct 2 comprising a transposon sequence corresponding to the Cas12k protein, a guide RNA backbone sequence, and a target sequence for different target sequences.

[0009] In short, one plasmid can express Cas12k, TnsB, TnsC and TniQ proteins, while the other plasmid can express the corresponding guide RNA (gRNA) and carry transposon sequences.

[0010] Preferably, the sequences of Cas12k, TnsB, TnsC and TniQ are as shown in SEQ ID NO:1-4 of the sequence listing.

[0011] Preferably, the corresponding gRNA backbone and transposon sequence comprise sequences as shown in SEQ ID NO:5 and SEQ ID NO:6 in the sequence listing, respectively.

[0012] Preferably, a target sequence for the target sequence is added to the 3' end of the guide RNA backbone sequence to form a complete guide RNA. The target sequence is a DNA fragment following the PAM sequence, preferably 23 bp in length; the PAM sequence is preferably GTN, where N is any base from A, T, C, and G.

[0013] In a preferred embodiment of the present invention, the expression construct 1 comprises the sequence shown in SEQ ID NO:7, and the expression construct 2 comprises the sequence shown in SEQ ID NO:14.

[0014] The second technical solution of the present invention is a method for site-directed gene transposition / mutation, which includes introducing the genome transposition system described above into bacteria containing the target sequence for genome transposition editing, and optionally performing subsequent screening and verification.

[0015] The above methods can be used, for example, for bacterial genome transposition.

[0016] The "bacteria" mentioned in this invention refer to various bacterial strains that exist in nature, including but not limited to Pseudomonas aeruginosa and Klebsiella pneumoniae mentioned in the examples.

[0017] The third technical solution of the present invention is a transposon mutant bacterium obtained by the method described in the second technical solution.

[0018] The fourth technical solution of the present invention is a method for screening homozygous transposon mutant bacteria, which includes purifying the transposon mutant bacteria by screening with antibiotics and sucrose to obtain homozygous bacteria with complete transposon mutation; the transposon mutant bacteria are preferably the transposon mutant bacteria described in the third technical solution.

[0019] The fifth technical solution of the present invention is a method for constructing a bacterial mutant library. This method allows for the autonomous selection of specific genes and sites for transposition, thereby enabling the selection of specific genes in bacteria for mutation as needed, thus constructing a specific gene mutant library. Specifically, the construction method includes:

[0020] (1) Obtain transcriptional regulatory factors from bacterial genomes and select target sequences based on them;

[0021] (2) Construct the expression construct 2 as defined in one of the different technical solutions based on different target sequences;

[0022] (3) The different expression constructs 2 obtained in step (2) and the expression construct 1 defined in one of the technical solutions are transferred into competent bacterial cells to obtain a bacterial gene mutation library.

[0023] The sixth technical solution of the present invention is a high-throughput sequencing and data analysis method for analyzing the above-mentioned bacterial mutant library.

[0024] The seventh technical solution of the present invention is a screening method for the above-mentioned bacterial mutant library, which can screen for relevant resistance genes under different screening conditions.

[0025] Compared with existing technologies, the advantages of the technology described in this invention are that it can effectively transpose specific sites in the bacterial genome, thereby inactivating specific genes or introducing specific DNA sequences, and can be used to construct specific bacterial mutant libraries. Therefore, it has broad application prospects in bacterial physiological research, drug target discovery, and the development of novel therapeutic drugs.

[0026] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0027] The reagents and raw materials used in this invention are all commercially available.

[0028] The positive and progressive effects of this invention are as follows:

[0029] The Cas12k gene transposition system was first applied to bacteria such as *Pseudomonas aeruginosa* and *Klebsiella pneumoniae*, achieving highly efficient gene transposition. A dual screening method using antibiotics and sucrose was developed to obtain homozygous transposition mutant monoclonal colonies. The Cas12k gene transposition system was also first applied to the construction of bacterial mutant libraries, enabling high-throughput mutagenesis at specific sites in the bacterial genome. Attached Figure Description

[0030] Figure 1 A schematic diagram of the plasmid structures of pCASTPA and PGTPA and the transposon editing in Pseudomonas aeruginosa mediated by the pCASTPA / PGTPA dual plasmid system.

[0031] Figure 2 This is a PCR verification diagram of the rhlR gene site transposition in PAO1 strain mediated by the pCASTPA / PGTPA dual plasmid system.

[0032] Figure 3 After screening with gentamicin and sucrose, pure transposable bacterial single clones could be obtained. The results showed that the band of the transposable Pseudomonas aeruginosa single clone on the gel image was significantly higher than that of the wild-type bacteria used as a control, indicating that the bacterial genome had successfully transposed.

[0033] Figure 4 A schematic diagram illustrating the steps involved in constructing a high-throughput genome sequencing library for a Pseudomonas aeruginosa transposon mutant library.

[0034] Figure 5 The results are from high-throughput sequencing of a Pseudomonas aeruginosa transposon mutant library. The outermost circle indicates the location of the gene, the second layer shows the GC ratio, the third layer shows the specific insertion sites of the transposon, and the fourth layer shows the off-target sites of the transposon.

[0035] Figure 6 To screen for genes associated with ceftazidime resistance in a transposon mutant library of Pseudomonas aeruginosa; the results showed that the proportion of strains with mutations in the nalC, nalD, and mexR genes increased in the total bacterial count, while the proportion of strains with mutations in the ampDh2, dksA, and algU genes decreased in the total bacterial count.

[0036] Figure 7The pCASTKP / PGTKP dual plasmid system can effectively mediate transposition reactions in Klebsiella pneumoniae; Figure (A) shows the schematic diagram of the plasmid structures of pCASTKP and PGTKP, and Figure (B) shows the PCR verification diagram of genome transposition in Klebsiella pneumoniae mediated by the pCASTPA / PGTPA dual plasmid system; the results show that gene transposition insertion was successfully achieved in all 5 bacterial clones that underwent transposition. Detailed Implementation

[0037] The embodiments of the present invention will be clearly and completely described below with reference to the examples. Obviously, the described embodiments are only used to illustrate a part of the embodiments of the present invention and should not be regarded as limiting the scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments used are not specified, they shall be regarded as conventional products that can be purchased commercially.

[0038] The sources of the biological materials used in each embodiment are as follows:

[0039] pACRISPR plasmid (Addgene, Inc., Catalog No.: 113348);

[0040] pCasPA plasmid (Addgene, Inc., Catalog No.: 113347);

[0041] pBECKP-km plasmid (Addgene, Inc., Catalog No.: 117235);

[0042] pSGKP-spe plasmid (Addgene, Inc., Catalog No.: 117234);

[0043] Pseudomonas aeruginosa PAO1 strain (ATCC, USA, catalog number: ATCC15692);

[0044] Klebsiella pneumoniae CMCC(B)46117 strain (Guangdong Huankai Microbial Technology Co., Ltd., product number: FSCC167002);

[0045] Competent Escherichia coli DH5α strain (Wuhan Miaoling Biotechnology Co., Ltd., Product No.: P1435).

[0046] The LB liquid culture medium used in each example was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A507002-0250; the LB solid culture medium was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A507003-0250; tetracycline hydrochloride was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A100422-0010; and disodium carbenicillin was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A600. 469-0005; Gentamicin sulfate was purchased from Sangon Biotech (Shanghai) Co., Ltd., item number: A506614-0005; L-arabinose was purchased from Sangon Biotech (Shanghai) Co., Ltd., item number: A610071-0100; Sucrose was purchased from Sangon Biotech (Shanghai) Co., Ltd., item number: A100335-0250; Glycerol was purchased from Sangon Biotech (Shanghai) Co., Ltd., item number: A100854-0500.

[0047] Example 1: Construction of pCASTPA plasmid

[0048] The composition of the pCASTPA plasmid is as follows: Figure 1 As shown, its sequence is SEQ ID NO:7. The specific construction method of pCASTPA plasmid is as follows:

[0049] (1) The Cas12k gene was synthesized at Sangon Biotech (Shanghai) Co., Ltd., and its sequence is SEQ ID NO:8. The TnsB-TnsC-TniQ operon sequence is SEQ ID NO:9.

[0050] (2) Using pCasPA plasmid as a template, the pCasPA plasmid backbone and sacB gene fragment were obtained by PCR amplification.

[0051] The primer sequences used for PCR amplification are as follows:

[0052] pCasPA plasmid backbone amplification 5' primer (SEQ ID NO:10):

[0053] 5'-TGAGGGTTAGTTTGACTGTATAAATAAGCTTTCCCTATAGTGAGTCGTATTAG-3'

[0054] pCasPA plasmid backbone amplification 3' primers (SEQ ID NO:11):

[0055] 5'-AATCAGGATTTTGCTGACTGTTCATTTTTTATAACCTCCTTAGAGCTCGAATTCC-3'

[0056] 5' primer for sacB gene amplification (SEQ ID NO:12):

[0057] 5'-CATGGCAAGATTATTCCTACTAGGATCTAGACGGCATCAGAGCAGATT-3'

[0058] sacB gene amplification 3' primer (SEQ ID NO:13):

[0059] 5'-GAGCCTGGATGGTGATCTGGCTCATTTTTTATAACCTCCTTAGAGCTCG AATTCC-3'

[0060] Using Novizan Max Master Mix was used to amplify the plasmid backbone and sacB gene described above. The reaction mixture consisted of 20.25 μL ddH2O and 25 μL 2x... Max Master Mix, 1.5μL 5'Primer (10μM), 1.5μL 3'Primer (10μM), 0.25μL template pCasPA DNA (100ng / μL), 1.5μL DMSO.

[0061] After the above PCR reaction system was mixed evenly, polymerase chain reaction (PCR) was performed, and the following cycles were performed: 98℃ for 2 min; then 98℃ for 20 s, 55℃ for 20 s, 72℃ for 5 min, for a total of 30 cycles; and finally 72℃ for 10 min.

[0062] The PCR products were recovered using the SanPrep column-based PCR product purification kit manufactured by Sangon Biotech (Shanghai) Co., Ltd. The specific purification steps were performed according to the kit's instruction manual.

[0063] (3) The two DNA fragments synthesized in step (1) and the two PCR products obtained in step (2) were assembled into a plasmid using Gibson assembly. The specific reaction system was as follows: 10 μL NEBuilder HiFi DNA AssemblyMaster Mix (NEB), 20 fmol TnsB-TnsC-TniQ operon DNA fragment, 20 fmol Cas12k gene, 20 fmol pCasPA plasmid backbone, 20 fmol sacB gene fragment, and an appropriate amount of ddH2O was added to a total volume of 20 μL. The reaction was carried out at 50 °C for 1 hour.

[0064] (4) Transform 20 μL of the reaction product into competent *Escherichia coli* DH5α strain and plate it on an LB agar plate containing 20 μg / mL tetracycline. After the transformation solution was absorbed by the agar plate, incubate it overnight at 37°C with the plate inverted. Transfer and preserve the transformed strain grown on the agar plate, and extract plasmids using the SanPrep column-based plasmid DNA mini-extraction kit from Sangon Biotech (Shanghai) Co., Ltd. for subsequent experiments. The specific steps for plasmid extraction were performed according to the kit's instruction manual. Simultaneously, the plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing confirmation.

[0065] Example 2: Construction of pGTPA plasmid

[0066] The composition of the pGTPA plasmid is as follows: Figure 1 As shown, its sequence is SEQ ID NO:14. The specific construction method of the pGTPA plasmid is as follows:

[0067] (1) A transposon-related DNA sequence (SEQ ID NO:15) was synthesized at Sangon Biotech (Shanghai) Co., Ltd. The sequence contains a gRNA sequence corresponding to Cas12k, two BsaI sites designed at the 3' end of the gRNA for insertion of the target sequence, and a transposon fragment containing the gentamicin gene.

[0068] (2) Using pACRISPR plasmid as a template, the pACRISPR plasmid backbone was obtained by PCR amplification.

[0069] The primer sequences used for PCR amplification are as follows:

[0070] pACRISPR plasmid backbone amplification 5' primer (SEQ ID NO:16):

[0071] 5'-ACGACAGATAATTTGTCACTGTACAAAGCTTGAGTATTCTATAGTGTCACCT-3'

[0072] pACRISPR plasmid backbone amplification 3' primers (SEQ ID NO:17):

[0073] 5'-TACCTAGGACTGAGCTAGCTGTCAAGGGCCCATGACAGCGGCCGC-3'

[0074] Using Novizan Max Master Mix was used to amplify the plasmid backbone and sacB gene described above. The reaction mixture consisted of 20.25 μL ddH2O and 25 μL 2x... Max Master Mix, 1.5μL 5'Primer (10μM), 1.5μL 3'Primer (10μM), 0.25μL template pACRISPR DNA (100ng / μL), 1.5μL DMSO.

[0075] After the above PCR reaction system was mixed evenly, polymerase chain reaction (PCR) was performed, and the following cycles were performed: 98℃ for 2 min; then 98℃ for 20 s, 55℃ for 20 s, 72℃ for 4 min, for a total of 30 cycles; and finally 72℃ for 6 min.

[0076] The PCR products were recovered using the SanPrep column-based PCR product purification kit manufactured by Sangon Biotech (Shanghai) Co., Ltd. The specific purification steps were performed according to the kit's instruction manual.

[0077] (3) The DNA fragment synthesized in step (1) and the PCR product obtained in step (2) were assembled into a plasmid using Gibson assembly. The specific reaction system was as follows: 10 μL NEBuilder HiFi DNA AssemblyMaster Mix (NEB), 20 fmol transposon DNA fragment, 20 fmol pACRISPR plasmid backbone, and an appropriate amount of ddH2O was added to a total volume of 20 μL. The reaction was carried out at 50 °C for 1 hour.

[0078] (4) Transform 20 μL of the reaction product into competent Escherichia coli DH5α strain and plate it on an LB agar plate containing 50 μg / mL carbenicillin. After the transformation solution is absorbed by the agar plate, incubate overnight at 37°C with the plate inverted. Transfer and preserve the transformed strain grown on the agar plate, and extract plasmids using the SanPrep column-based plasmid DNA mini-extraction kit from Sangon Biotech (Shanghai) Co., Ltd. for subsequent experiments. The specific steps for plasmid extraction were performed according to the kit's instruction manual. Simultaneously, the plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing confirmation.

[0079] Example 3: Genome transposition in *Pseudomonas aeruginosa* strains using the pCASTPA / pGTPA plasmid system

[0080] The pCASTPA / pGTPA plasmid system enables efficient transposition insertion at different genomic loci in various *Pseudomonas aeruginosa* strains. In this experiment, the rhlR gene was selected as an example, and genomic transposition experiments were performed in the *Pseudomonas aeruginosa* PAO1 strain. The specific experimental mechanism is as follows: Figure 1 As shown.

[0081] (1) First, select a DNA fragment of 23 bases following a GTN (N is any base) sequence at the target genomic locus of strain PAO1 (these 23 bases are called the spacer, and the GTN is not included in it). For example, the DNA sequence (SEQ ID NO:18) of the spacer of the rhlR gene selected in the experiment is: 5'-ggacatcagcatcggatgctcca-3'. In order to insert this sequence into the BsaI site of the pGTPA plasmid and construct a guide gRNA for the rhlR gene, it is necessary to add several extra bases to the spacer according to the sequence of the pGTPA plasmid so that the spacer sequence can form sticky ends, thereby achieving successful insertion of the spacer sequence. The specific sequence design is as follows (uppercase letters are the added extra bases used to form sticky ends):

[0082] 5'-GTGGAGggacatcagcatcggatgctcca-3'(SEQ ID NO:19)

[0083] 3'-TCcctgtagtcgtagcctacgaggtCAAA-5'(SEQ ID NO:20)

[0084] The two primers were synthesized using conventional methods at Sangon Biotech (Shanghai) Co., Ltd.

[0085] (2) First, the two designed and synthesized spacer primers were annealed. The specific reaction system was as follows: 5 μL 10×T4 DNA ligase Buffer (NEB), 10 μL spacer 5' primer (10 μM), 10 μL spacer 3' primer (10 μM), and 25 μL ddH2O. The mixture was heated at 95℃ for 5 min, and then slowly cooled to room temperature in a PCR instrument to allow the two single-stranded primers to form double-stranded DNA through base pairing. The resulting product was then diluted 20-fold with ddH2O.

[0086] The double-stranded DNA obtained above was inserted into the BsaI site of the pGTPA plasmid constructed in Example 2. The specific reaction system was as follows: 1 μL 10×T4 DNA ligase Buffer (NEB), 1 μL of the above 20-fold diluted double-stranded DNA, 20 fmol of the pGTPA plasmid obtained in Example 2, 0.5 μL T4 DNA ligase (NEB), 0.5 μL BsaI-HF (NEB), and finally, an appropriate amount of ddH2O was added to a total volume of 10 μL. The reaction was carried out in a PCR instrument with the following cycles: 37℃ for 2 min; 16℃ for 5 min, for a total of 25 cycles; then 50℃ for 5 min, 80℃ for 15 min.

[0087] The 10 μL reaction product was transformed into competent Escherichia coli DH5α cells and plated on LB agar plates containing 50 μg / mL carbenicillin. After the transformation solution was absorbed by the agar plates, the plates were incubated upside down at 37°C overnight. The transformed strains that grew on the culture medium were then transferred and stored. Simultaneously, the constructed plasmid pGTPA-rhlR was extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification.

[0088] (3) The pCASTPA plasmid constructed in Example 1 was electroporated into PAO1 strain electroporation competent cells. The electroporation parameters were: 2100V, 200Ω, 25μF, and a 1mm electroporation cuvette. Immediately after electroporation, 1mL of LB culture medium was added, mixed thoroughly, and transferred to a clean EP tube. The tube was shaken at 37℃ for 1–2 hours, and then the bacterial culture was spread evenly on an LB solid culture plate containing 100μg / mL tetracycline. After the bacterial culture was absorbed by the solid culture medium, the tube was incubated upside down at 37℃ overnight. Only bacteria that were successfully transfected with the plasmid were able to grow on the culture medium.

[0089] (4) Pick a single colony that has grown on the culture plate and inoculate it into 50 mL of LB liquid medium containing 100 μg / mL tetracycline. Shake overnight at 250 rpm in a shaker at 37°C. The next day, add 0.5 mL of 20% w / v L-arabinose to induce the expression of the TnsB-TnsC-TniQ operon and Cas12k protein. Continue shaking in a shaker at 37°C for 2 hours, then cool the bacterial culture on ice for 10 minutes. Centrifuge at 6000 rpm for 5 minutes in a centrifuge at 4°C to collect the bacteria and discard the supernatant. Wash the bacterial pellet at the bottom twice with 20 mL of 10% v / v glycerol (autoclaved and pre-cooled on ice), and then resuspend the bacterial pellet at the bottom with 0.5 mL of 10% v / v glycerol. Aliquot the obtained electrocompetent bacteria into EP tubes, 50 μL per tube.

[0090] (5) Transform the pGTPA-rhlR plasmid constructed in step (2) into the PAO1 strain electrocompetent cells containing the pCASTPA plasmid prepared in step (4). The specific operation is as follows: Mix 1-2 μg of pGTPA-rhlR plasmid with 50 μL of PAO1 strain electrocompetent cells containing the pCASTPA plasmid, transfer to a 1 mm electroporation cuvette (Bio-Rad), and electroporate at room temperature using a GenePulserXcell electroporator (Bio-Rad). The electroporation parameters are: 2100V, 200Ω, 25μF. Immediately after electroporation, add 1 mL of LB culture medium, mix well, and transfer to a clean EP tube. Shake at 37℃ for 1-2 hours. Spread the bacterial culture evenly on an LB solid culture plate containing 150 μg / mL carbenicillin, 100 μg / mL tetracycline, and 0.2% w / v L-arabinose. After the bacterial culture is absorbed by the solid culture medium, it is incubated upside down in a 37°C incubator overnight. Only bacteria that have been successfully transferred with plasmids can grow on the culture medium.

[0091] (6) Pick a single colony and add it to 10 μL of ddH2O. Heat at 95°C for 10 min to lyse the bacteria. Use this lysate as a PCR template to amplify the rhlR gene. The reaction mixture is: 20 μL ddH2O, 25 μL 2x Max Master Mix, 1.5 μL 5' Primer (10 μM), 1.5 μL 3' Primer (10 μM), 0.5 μL bacterial template, 1.5 μL DMSO. After thoroughly mixing the above PCR reaction mixture, polymerase chain reaction (PCR) was performed using the following cycle: 98℃ for 2 min; then 98℃ for 20 s, 55℃ for 20 s, 72℃ for 1 min, for a total of 30 cycles; finally, 72℃ for 2 min.

[0092] The key feature of this step is that the 5' primer for PCR verification is located outside the rhlR gene in the bacterial genome, while the 3' primer is located on the transposition fragment. Therefore, only single-clone colonies that have successfully transposed at the rhlR gene site can amplify PCR bands. Thus, the success of gene transposition can be determined by the presence or absence of a single bright DNA fragment on the agarose gel.

[0093] The PCR verification primer sequences for rhlR gene site transposition are as follows:

[0094] 5' Primer: 5'-ctcaggatgatggcgatttc-3' (SEQ ID NO:21)

[0095] 3' Primer: 5'-gtgggttgaaagcaagtcct-3' (SEQ ID NO:22)

[0096] Figure 2 The results show that all single clones of *Pseudomonas aeruginosa* undergoing transposition exhibited bright bands on the gel image, indicating that transposition of the bacterial genome had occurred, while the wild-type bacteria used as controls did not show obvious bands. This demonstrates that gene transposition was successfully achieved in all five selected colonies.

[0097] Example 4: Isolation of Successfully Transposable Strains and Elimination of Plasmids

[0098] In *Pseudomonas aeruginosa* strains that undergo transposition, only a portion of the bacteria successfully transpose, while the remainder remain wild-type bacteria. Therefore, further purification and screening are necessary to obtain pure, successfully transposed bacteria. Furthermore, transposed bacteria often retain the associated pCASTPA and pGTPA plasmids, which need to be eliminated. This example describes the subsequent steps for strain purification and plasmid elimination after successful gene transposition.

[0099] (1) Pick a single clone from the P. aeruginosa PAO1 strain containing pCASTPA and pGTPA plasmids that successfully transposonized, and inoculate it into 3 mL of LB liquid medium containing 150 μg / mL carbenicillin and 100 μg / mL tetracycline, and shake overnight at 37°C.

[0100] (2) Take 3 μL of bacterial culture and dilute it at a ratio of 1:1000 into 3 mL of LB liquid medium containing 30 μg / mL gentamicin. Shake and culture at 37°C until the bacterial culture becomes turbid.

[0101] (3) Dilute the bacterial culture 10,000 times with fresh LB liquid medium. Take 100 μL of the diluted bacterial culture and spread it evenly on an LB solid culture plate containing 5% w / v sucrose and 30 μg / mL gentamicin. After the transformation solution is absorbed by the solid culture plate, incubate it upside down in an incubator at 37°C overnight.

[0102] (4) Pick single colonies from LB agar plates containing 5% w / v sucrose and inoculate them into 10 μL LB liquid medium. Streak the colonies on LB agar plates, LB agar plates containing 100 μg / mL tetracycline, and LB agar plates containing 150 μg / mL carbenicillin. Colonies that can grow on LB agar plates but not on LB agar plates containing 100 μg / mL tetracycline or LB agar plates containing 150 μg / mL carbenicillin are considered colonies with eliminated pCASTPA plasmid and pGTPA. Cultivate and preserve these colonies.

[0103] This screening method is based on the fact that both pCASTPA and pGTPA plasmids contain the sacB gene, which causes the plasmids to produce substances harmful to bacteria under sucrose conditions, leading to bacterial death. Therefore, the colonies that grow on the culture plates are plasmid-free. Furthermore, since the culture plates contain gentamicin resistance, only strains that have successfully transposed and carry the gentamicin resistance gene in their genome can survive on the plates. This method can effectively screen for pure transposable bacteria that do not contain plasmids. Experimental results showed that all 10 selected colonies were pure transposable colonies. Figure 3 ).

[0104] Example 5: Construction of transposon plasmid library

[0105] Because the pCASTPA / pGTPA dual plasmid system can efficiently and rapidly perform transposition reactions in bacteria, it can effectively inactivate specific genes on the bacterial genome. Based on this system, we further developed a high-throughput library construction technique, which allows us to construct bacterial site-specific mutant libraries for high-throughput testing and screening of the activity and function of different genes in bacteria. In our experiments, we used *Pseudomonas aeruginosa* strain PAO1 as an example, selecting transcriptional regulatory factors to construct a mutant library of transcriptional regulatory factors from *Pseudomonas aeruginosa* strain PAO1.

[0106] (1) We searched NCBI (https: / / www.ncbi.nlm.nih.gov / ) and the Pseudomonas aeruginosa website (www.pseudomonas.com) to find gene loci marked as transcriptional regulatory factors in the genome of P. aeruginosa strain PAO1. We found a total of 593 transcriptional regulatory factors.

[0107] (2) Select 15 suitable spacer sites on each transcription factor gene. This step is characterized by the spacer sites being located at the 3' end of the GTN sequence, the GC ratio of the spacer sequence generally being between 35% and 75%, and the absence of more than four consecutive T, A, G, or C sequences. For transcription factors with fewer than 15 spacer sites, all spacer sites are selected. This results in a total of 8891 spacer sites. Additionally, 100 spacer sites targeting 10 essential genes and 50 non-targeted spacer sequences are selected as positive and negative controls, respectively. The final spacer sequence library contains 9041 spacer sequences.

[0108] (3) A spacer sequence primer pool was synthesized at Genscript Biotech Inc. The synthesized spacer sequence library was inserted into the BsaI site of the pGTPA plasmid constructed in Example 2 using Golden Gate Assembly technology. The specific reaction system was as follows: 10 μL 10×T4 DNA ligase Buffer (NEB), 10 μL spacer sequence library, 200 fmol of the pGTPA plasmid obtained in Example 2, 5 μL T4 DNA ligase (NEB), 5 μL BsaI-HF (NEB), and finally, an appropriate amount of ddH2O was added to a total volume of 100 μL. The 100 μL reaction solution was divided into 10 portions, each 10 μL. The reaction was carried out in a PCR instrument with the following cycles: 37℃ for 2 min; 16℃ for 5 min, for a total of 25 cycles; then 50℃ for 5 min, 80℃ for 15 min.

[0109] (4) Ten 10 μL portions of the reaction product were transformed into competent Escherichia coli DH5α strains and spread evenly on LB agar plates containing 50 μg / mL carbenicillin. After the transformation solution was absorbed by the agar plates, the plates were incubated upside down at 37°C overnight.

[0110] (5) After scraping all bacterial colonies from 10 solid culture plates, collect them into EP tubes. Use the high-purity plasmid mini-extraction kit from Tiangen Biotech (Beijing) Co., Ltd. to extract plasmids for subsequent experiments. Follow the kit's instruction manual for specific plasmid extraction steps.

[0111] (6) The extracted plasmid was sent to GenScript Biotech Co., Ltd. for high-throughput sequencing to confirm the construction of the plasmid library.

[0112] Example 6: Construction of a bacterial transposon mutant library

[0113] (1) The plasmid library constructed in step 5 was transferred into the PAO1 strain electroporation competent cells containing the pCASTPA plasmid prepared in step (4) of step 3. The specific operation is as follows: 1-2 μg of plasmid library was mixed evenly with 50 μL of PAO1 strain electroporation competent cells containing the pCASTPA plasmid and then electroporated. The electroporation parameters were: 2100 V, 200 Ω, 25 μF.

[0114] (2) Immediately after electroporation, add 1 mL of LB culture medium containing 0.2% w / v L-arabinose, mix well, transfer to a clean EP tube, and shake in a shaker at 37°C for 1 hour.

[0115] (3) Expand 1 mL of bacterial culture to 20 mL of LB medium containing 30 μg / mL gentamicin and 0.2% w / v L-arabinose, and shake overnight in a shaker at 37°C.

[0116] (4) On the second day, the bacterial culture was evenly spread on 68 15cm×15cm LB agar plates containing 30μg / mL gentamicin and 0.2% w / v L-arabinose. The plates were incubated upside down overnight at 37°C.

[0117] (5) Collect all bacteria from the culture plates, mix them thoroughly, and then aliquot them. Store the aliquoted bacterial mutant libraries at -80 degrees Celsius for subsequent experiments.

[0118] Example 7: Sequencing and High-Throughput Data Analysis of Bacterial Transposon Mutant Libraries

[0119] The six successfully constructed bacterial transposon mutant libraries in Example 6 require further high-throughput sequencing and analysis to confirm that transposition did indeed occur at the selected specific sites in the experiment, and to analyze the transposon frequency at each site on the genome. Specific steps are as follows: Figure 4 As shown.

[0120] (1) Take one tube of the bacterial mutant library constructed in Example 6 and extract the genome of Pseudomonas aeruginosa PAO1 strain using the Ezup column bacterial genomic DNA extraction kit produced by Sangon Biotech (Shanghai) Co., Ltd. The specific steps are performed according to the kit user manual.

[0121] (2) After diluting the genomic DNA to 100-200 ng / μL using ddH2O, the genome was broken down by sonication into fragments of 200-1200 bp in length.

[0122] (3) Repair the ends of the DNA fragments that were broken by ultrasound, then add dA and link them to UMI_linker.

[0123] (4) Use biotin-labeled primers for the first round of PCR amplification, and then use 2ndLink_Primer and adapter_primer for the second round of PCR amplification.

[0124] (5) The high-throughput sequencing library was purified using AMPure beads (Beckman Coulter) and the concentration of the library was determined using the QubitDNA HS Assay kit (Thermo Fisher Scientific).

[0125] (6) High-throughput sequencing was performed on the Illumina HiSeq X10 PE150 platform.

[0126] (7) Perform data analysis on the high-throughput sequencing results to detect the transposition sites and transposition frequencies of different spacers in the bacterial mutant library. Figure 5 ).

[0127] Example 8: Screening for phenotype-related genes in a Pseudomonas aeruginosa transposon mutant library

[0128] The bacterial mutant library constructed in Example 6 can be used for high-throughput testing to screen the function and activity of different genes in bacteria under environmental stress, such as antibiotics, heavy metals, and oxidants. In this experiment, the transcriptional regulatory factor mutant library of *Pseudomonas aeruginosa* strain PAO1 constructed in Example 6 was used, and the antibiotic ceftazidime was selected as an environmental screening factor to screen for ceftazidime-related transcriptional regulatory factors in bacteria.

[0129] (1) Take a tube of the bacterial mutant library constructed in Example 6 and place it on ice for 10 minutes to allow the bacterial mutant library to completely dissolve.

[0130] (2) Take 100 μL of bacterial mutant library and dilute it into 100 mL of LB liquid medium without antibiotics and 100 mL of LB liquid medium containing 1 μg / mL ceftazidime, respectively. Shake overnight in a shaker at 37°C.

[0131] (3) Collect bacteria by centrifugation, and extract the bacterial genome using the Ezup column-type bacterial genomic DNA extraction kit produced by Sangon Biotech (Shanghai) Co., Ltd. The specific steps are performed according to the kit's user manual.

[0132] (4) Perform high-throughput sequencing and data analysis on the bacterial library according to the steps in Example 7. Using the bacterial mutant library without antibiotics as the control group, compare the bacterial mutant library under the condition of containing 1 μg / mL ceftazidime to see which gene mutation strains increased in proportion in the total bacterial count and which gene mutation strains decreased.

[0133] (5) Data analysis showed that the proportion of strains with mutations in the nalC, nalD, and mexR genes increased in the total bacterial load, indicating that these three gene mutations improved bacterial resistance to ceftazidime. Conversely, the proportion of strains with mutations in the ampDh2, dksA, and algU genes decreased in the total bacterial load, indicating that these three gene mutations reduced bacterial resistance to ceftazidime. Figure 6 ).

[0134] Example 9: Transposon Editing in the Klebsiella pneumoniae Genome

[0135] (1) Construct plasmids pCASTKP (SEQ ID NO:23) and pGTKP (SEQ ID NO:24) for genome transposition in Klebsiella pneumoniae. Figure 7 (A) The construction steps involve using Gibson Assembly technology to replace the backbones of pCASTPA and pGTPA plasmids with the backbones of pBECKP-Km and pSGKP-spe plasmids, thereby enabling the plasmid system to replicate and function in Klebsiella pneumoniae.

[0136] (2) Select a suitable spacer site in the genome of Klebsiella pneumoniae strain KP1.6366 and insert the spacer into the pGTKP plasmid. The specific experimental steps are the same as described in Example 3, except that the antibiotic used for screening is changed to 50 μg / mL kanamycin.

[0137] (3) Electroporate the pCASTKP plasmid constructed in step (1) into KP1.6366 strain electroporation competent cells. The electroporation parameters were: 1800V, 200Ω, 25μF, and a 1mm electroporation cuvette. After shaking 1 mL of LB culture medium in a shaker at 37℃ for 1–2 hours, spread the bacterial culture evenly on an LB solid culture plate containing 75 μg / mL spectinomycin. Incubate overnight in an inverted incubator at 37℃.

[0138] (4) Pick a single colony that has grown on the culture plate and inoculate it into 3 mL of LB liquid medium containing 75 μg / mL spectinomycin. Incubate overnight at 37°C with shaking at 250 rpm. The next day, take 0.5 mL of the bacterial culture and dilute it into 50 mL of LB liquid medium containing 75 μg / mL spectinomycin. Incubate with shaking until OD reaches 0.5. 600 ≈0.2, add 0.5 mL of 20% w / v L-arabinose, and continue shaking in a shaker at 37°C for 2 hours. Centrifuge at 6000 rpm for 5 minutes at 4°C to collect the bacteria, discarding the supernatant. Wash the bacterial pellet at the bottom twice with 20 mL of 10% v / v glycerol (autoclaved and pre-chilled on ice), then resuspend the bacterial pellet in 0.5 mL of 10% v / v glycerol. Aliquot the obtained electrocompetent bacteria into EP tubes, 50 μL per tube.

[0139] (5) The pGTKP plasmid containing the spacer constructed in step (2) was transferred into the KP1.6366 strain electrocompetent cells containing the pCASTKP plasmid prepared in step (4). The bacterial culture was spread evenly on LB agar plates containing 75 μg / mL spectinomycin, 50 μg / mL kanamycin, and 0.2% w / v L-arabinose. The plates were incubated upside down overnight at 37°C.

[0140] (6) Single colonies were selected, and PCR was used to verify whether the bacteria had successfully transposed. The results showed that all five selected colonies successfully underwent gene transposition. Figure 7 (B)

[0141] The genome transposition method of this invention is universally applicable to different bacterial strains in nature. By simply changing the plasmid backbone to suit different bacteria, the system can function in various bacteria. While specific embodiments of this invention describe the application of this system in *Pseudomonas aeruginosa* and *Klebsiella pneumoniae*, those skilled in the art should understand that these are merely illustrative examples, and the system can be similarly applied to other bacteria. Various changes or modifications made to these embodiments without departing from the principles and essence of this invention should also be protected. Therefore, the scope of protection of this invention is defined by the appended claims. SEQUENCE LISTING <110> ShanghaiTech University <120> A method for bacterial gene site-directed transposition and mutant library construction and its application <130> P21015714C <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 1920 <212> DNA <213> Artificial Sequence <220> <223> Cas12k <400> 1 atgagccaga tcaccatcca ggctcgtctg atcagctttg aatctaaccg ccagcagctg 60 tggaaactga tggctgatct gaacaccccg ctgattaacg aactgctgtg tcagctgggc 120 cagcacccgg actttgaaaa atggcagcag aaaggtaaac tgccgagcac cgtggtttct 180 cagctgtgcc agccactgaa aactgacccg cgtttcgcgg gtcagccgag ccgtctgtac 240 atgtctgcaa ttcacatcgt tgattacatc tataaatcct ggctggcgat ccagaaacgc 300 ctgcaacagc agctggatgg caaaacccgt tggctggaaa tgctgaacag cgatgcagaa 360 ctggttgaac tgtctggtga tactctggaa gcgattcgcg tcaaagcggc tgaaatcttg 420 gctatcgcca tgccggcaag cgaatccgat tctgcatctc cgaaaggtaa aaaaggcaaa 480 aaagagaaaa aaccgagcag cagctccccg aaacgcagtc tttctaaaac cctgtttgac 540 gcgtaccagg aaaccgaaga tatcaaatct cgtagcgcga tctcttatct gctgaaaaac 600 ggctgtaaac tgactgataa agaagaagat agcgaaaaat ttgctaagcg ccgtcgtcag 660 gtggagattc agattcagcg tctgaccgaa aaactgatct ctcgtatgcc gaaaggtcgc 720 gacttgacca acgctaaatg gcttgaaacc ctgctgaccg cgactactac ggtggcagaa 780 gataatgcgc aggcaaaacg ttggcaggat atcctcctga cccgtagctc ttccctgccg 840 ttcccgctgg tatttgaaac caacgaagac atggtgtggt ctaaaaacca gaaaggccgt 900 ctgtgcgtc acttcaacgg cctgagcgat ctgatcttcg aagtgtactg tggcaaccgc 960 cagctccact ggttccagcg tttcctggaa gaccagcaga ccaaacgcaa aagcaaaaac 1020 cagcactcaa gcggcctgtt cactctgcgt aacggtcacc tggtgtggct ggaaggcgaa 1080 ggtaaaggtg aaccgtggaa cctgcaccac ctgaccctgt actgctgcgt ggacaaccgt 1140 ctgtggaccg aagaaggcac cgaaatcgtc cgtcaggaaa aagcagacga aatcaccaaa 1200 tttatcacca acatgaaaaa gaaatctgat ctgagcgaca cccaacaggc tctgattcag 1260 cgcaaacagt ctaccctgac ccgtattaat aactctttcg aacgcccgtc ccagccgctg 1320 taccagggcc agagccacat cctggtcggt gtttctctgg gcctggaaaa accggctacc 1380 gttgccgttg tcgatgccat tgcaaacaaa gttctggctt accgttccat caaacagctg 1440 ctgggcgata actatgaact gctgaaccgt cagcgtcgcc agcagcagta cctgagccac 1500 gaacgtcaca aagcgcagaa aaatttctct ccgaaccaat tcggcgcaag cgaactgggc 1560 cagcacatcg atcgtctgct ggccaaagcg atcgttgcgc tggcgcgtac ctataaagcc 1620 ggctctatcg tcctgccgaa actgggcgac atgcgtgaag tggttcagtc tgaaatccag 1680 gcgatcgctg aacagaaatt tccgggttat attgaaggtc agcagaaata cgcgaaacag 1740 taccgcgtga acgttcaccg ttggagctac ggtcgtctga ttcagtccat ccagtctaaa 1800 gcagcgcaga ctggcatcgt gattgaagaa ggcaaacagc cgatccgtgg ctccccgcac 1860 gataaagcga aagaactggc gctgagcgca tacaacctgc gtctgacccg cgttcctaa 1920 <210> 2 <211> 1755 <212> DNA <213> Artificial Sequence <220> <223> Facebook page TnsB <400> 2 atgaacagtc agcaaaatcc tgatttagct gttcatccct tggcaattc tatggaaggc 60 ttactaggag aaagtgctac aactcttgag aagaatgtaa ttgccacaca actctcagag 120 gaagcccaag taaagctaga ggtaatccaa agtttactgg aaccctgcga tcgcacaact 180 tatgggcaaa agttgcggga agcagcagag aaactaaatg tatcgttgcg aacggtacaa 240 aggttggtga aaaactggga acaagatggc ttagtcggac tcactcaaac aagtagggct 300 gataaaggaa aaccgcat tggtgagttt tgggaaaact tcattaccaa aacctacaag 360 gagggtaaca agggaagtaa acgtatgacc cttaaacaag ttgctctcag agtcgaggct 420 aaagcccgtg attaaaaga ctctaagccg cccaattaca aaaccgtgtt acgggtatta 480 gcacccattt tggaaaagca acaaaaagcc aagagtatcc gcagtcctgg ttggagagga 540 actacgcttt cggttaaaac ccgtgaagga aaagatttat cggttgatta cagtaaccat 600 gtttggcaat gtgaccatac ccgcgtggat gtgttgctgg tagatcaaca tggtgaaatt 660 ttaagtcgtc cctggctaac aacagtaatt gatacttact ctcgttgcat tatgggtatc 720 aacttgggct ttgatgcacc cagttctggg gtagtagcat tagcgttacg ccatgcaatt 780 ctaccaaagc gttacggttc cgagtacaaa ctgcattgtg agtggggaac ctatggaaaa 840 ccagaacatt tttatactga tggcggtaaa gactttcgct ctaaccactt gagtcagatt 900 ggggcgcaat tgggatttgt ctgtcattta cgcgatcgcc cttctgaagg tggagtagta 960 gaacgtccct tcaaaacatt aaatgaccaa ctattttcaa cgcttcctgg gtacaccgga 1020 tctaatgtgc aggaacgccc agaagatgca gagaaggacg caagacttac tttgcgagaa 1080 ctagaacagt tacttgtgcg ttacatcgta gatcgttaca accaaagtat tgatgcgcgg 1140 atgggcgacc aaacgcgctt tgagcgttgg gaagcaggat tgcctacagt gccagtacca 1200 ataccagaac gagatttgga tatttgttta atgaagcagt cacggcgcac tgtgcaaaga 1260 ggtggttgtt tgcagtttca gaatttaatg tatcgggggg aatatttggc aggttatgcc 1320 ggagaaactg tcaacttaag gtttgacccc agagacatta caacaattttt ggtttatcgc 1380 caggaaaaca atcaggaagt atttctgact cgcgctcacg ctcaaggttt ggagacagag 1440 caactggcat tagatgaggc tgaggcagca agtcgcagac tccgtaccgc agggaaaact 1500 atcagtaacc aatcattatt gcaagaagtt gttgaccgcg atgctcttgt cgctaccaag 1560 aaaagccgta aggagcgtca aaaattggaa cagactgttt tgcgatctgc tgctgttgat 1620 gaaagtaata gagaatcctt gccttctcaa atagttgaac cagatgaagt ggaatctaca 1680 gaaacggttc actctcaata cgaagacatt gaggtgtggg actatgaaca acttcgtgaa 1740 gaatatgggt tttaa 1755 <210> 3 <211> 831 <212> DNA <213> Artificial Sequence <220> <223> Read more about TnsC <400> 3 atgacagaag ctcaggcgat cgccaagcag ttgggtgggg taaaaccgga tgatgagtgg 60 ttacaagctg aaattgctcg tctcaagggt aagagcattg tgcctttaca gcaggtaaaa 120 actctccatg attggttaga tggcaagcgc aaggcaagaa aatcttgccg agtagttggg 180 gaatcgagaa ctggcaagac agttgcttgt gatgcctaca gatacaggca caaacctcag 240 caggaagctg gacgacctcc aactgtgcct gtcgtttata ttcgacctca ccaaaaatgt 300 ggccccaagg atttgtttaa aaagattact gagtacctca agtatcgggt aaaaaggg 360 actgtatctg atttcgaga taggacgata gaagtactca agggttgtgg cgtagagatg 420 ctaattattg atgaagctga ccgtctcaag cctgaaactt ttgctgatgt gcgagatatt 480 gccgaagatt taggaattgc tgtggtactg gtaggaacag accgtttgga tgcggtaatt 540 aagcgggatg agcaggttct cgaacgcttt cgggcgcatc ttcgctttgg taaattgtcg 600 ggagaggatt ttaagaacac cgtagaaatg tgggaacaaa tggttttgaa actgccagta 660 tcttctaatc taaagagcaa ggagatgcta cggattctca cgtcagcaac tgaaggctac 720 attggtcgcc ttgatgagat tcttagggaa gctgcaattc gttccttatc aagaggattg 780 aagaagattg acaaggctgt tttacaggaa gtagctaagg agtacaaatg a 831 <210> 4 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding TniQ <400> 4 atgatagaag caccagatgt taaaccttgg ctattcttga ttaaacccta tgaaggggaa 60 agcctgagcc actttcttgg caggttcaga cgtgccaacc atttatccgc aagtggattg 120 ggtactttgg caggaattgg tgctatagtg gcacgttggg aaagatttca ttttaatcct 180 cgccctagtc agcaagaatt ggaagcgatc gcatctgtag tagaagtgga tgctcaaagg 240 ttagcccaga tgttaccgcc tgctggagtg ggaatgcagc atgagccaat tcgcttgtgt 300 ggggcttgtt atgccgagtc gccttgtcac cgaattgaat ggcagtacaa gtcggtgtgg 360 aagtgcgatc gccatcaact caagatttta gcaaagtgtc caaactgtca agcacctttt 420 aaaatgcctg cgctgtggga ggatgggtgc tgtcacagat gtaggatgcc gtttgcagaa 480 atggcaaagc tacagaaggt ttga 504 <210> 5 <211> 235 <212> DNA <213> Artificial Sequence <220> <223> gRNA <400> 5 atattaatag cgccgcaatt catgctgctt gcagcctctg aattttgtta aatgagggtt 60 agtttgactg tataaataca gtcttgcttt ctgaccctgg tagctgctca ccctgatgct 120 gctgtcaata gacaggatag gtgcgctccc agcaataagg gcgcggatgt actgctgtag 180 tggctactga atcacccccg atcaaggggg aaccctccaa aaggtgggtt gaaag 235 <210> 6 <211> 1329 <212> DNA <213> Artificial Sequence <220> <223> transposon <400> 6 tgtacagtga ctaattatat gtcgttgtga caaattattg tcatcagtaa aatccttata 60 cagtatagat tatagcgctt tggcagtttt agcataacct ctttgcagtg acaaaataga 120 tgtcgttgtc cgtgattgtg acaaattagc tgtcgctttg caagatagga aaaagctttt 180 gtgtattttc ataatgacaa attgactgtc gcctcgagga ataggaactt cggaatagga 240 acttcaagat cccctgattc cctttgtcaa cagcaatgga tcgaattgac ataagcctgt 300 tcggttcgta aactgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg 360 accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt 420 tttgtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt 480 ttgatgttat ggagcagcaa cgatgttacg cagcagcaac gatgttacgc agcagggcag 540 tcgccctaaa acaaagttag gtggctcaag tatgggcatc attcgcacat gtaggctcgg 600 ccctgaccaa gtcaaatcca tgcgggctgc tcttgatctt ttcggtcgtg agttcggaga 660 cgtagccacc tactcccaac atcagccgga ctccgattac ctcgggaact tgctccgtag 720 taagacattc atcgcgcttg ctgccttcga ccaagaagcg gttgttggcg ctctcgcggc 780 ttacgttctg cccaagttg agcagccgcg tagtgagatc tatatctatg atctcgcagt 840 ctccggcgag caccggaggc agggcattgc caccgcgctc atcaatctcc tcaagcatga 900 ggccaacgcg cttggtgctt atgtgatcta cgtgcaagca gattacggtg acgatcccgc 960 agtggctctc tatacaagt tgggcatacg ggaagaagtg atgcactttg atatcgaccc 1020 aagtaccgcc acctacaat tcgttcaagc cgagatcggc ttcccggccg cggagttgtt 1080 cggtaaattg tcacaacgcc gcggccaatt cgatctagat cagtaactat taaacttagg 1140 ggtgggttga aagcaagtcc ttttaccgc ttgttttaat tgcttgtat aaattgca 1200 gagcatatta tattgatgac atttaatttg tcatcaatta attaagcaac gctgatgggt 1260 cacgacgaca attaatagt cacaatgaca ttaatctgtc accgacgaca gataatttgt 1320 cactgtaca 1329 <210> 7 <211> 16547 <212> DNA <213> Artificial Sequence <220> <223> pCASTPA <400> 7 aagctttccc tatagtgagt cgtattagag cttggcgtaa tcatggtcat agctgtttcc 60 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 120 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 180 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 240 gagaggcggt ttgcgtattg ggcgccggct gctgaacccc caaccgttcc gccagtttgc 300 gtgtcgtcag accgtctacg ccgacctcgt tcaacaggtc cagggcggca cggatcactg 360 tattcggctg caactttgtc atgattgaca ctttatcact gataaacata atatgtccac 420 caacttatca gtgataaaga atccgcgcgt tcaatcggac cagcggaggc tggtccggag 480 gccagacgtg aaacccaaca tacccctgat cgtaattctg agcactgtcg cgctcgacgc 540 tgtcggcatc ggcctgatta tgccggtgct gccgggcctc ctgcgcgatc tggttcactc 600 gaacgacgtc accgcccact atggcattct gctggcgctg tatgcgttgg tgcaatttgc 660 ctgcgcacct gtgctgggcg cgctgtcgga tcgtttcggg cggcggccaa tcttgctcgt 720 ctcgctggcc ggcgccactg tcgactacgc catcatggcg acagcgcctt tcctttgggt 780 tctctatatc gggcggatcg tggccggcat caccggggcg actggggcgg tagccggcgc 840 ttatattgcc gatatcactg atggcgatga gcgcgcgcgg cacttcggct tcatgagcgc 900 ctgtttcggg ttcgggatgg tcgcgggacc tgtgctcggt gggctgatgg gcggtttctc 960 cccccacgct ccgttcttcg ccgcggcagc cttgaacggc ctcaatttcc tgacgggctg 1020 tttccttttg ccggagtcgc acaaaggcga acgccggccg ttacgccggg aggctctcaa 1080 cccgctcgct tcgttccggt gggcccgggg catgaccgtc gtcgccgccc tgatggcggt 1140 cttcttcatc atgcaacttg tcggacaggt gccggccgcg ctttgggtca ttttcggcga 1200 ggatcgcttt cactgggacg cgaccacgat cggcatttcg cttgccgcat ttggcattct 1260 gcattcactc gcccaggcaa tgatcaccgg ccctgtagcc gcccggctcg gcgaaaggcg 1320 ggcactcatg ctcggaatga ttgccgacgg cacaggctac atcctgcttg ccttcgcgac 1380 acggggatgg atggcgttcc cgatcatggt cctgcttgct tcgggtggca tcggaatgcc 1440 ggcgctgcaa gcaatgttgt ccaggcaggt ggatgaggaa cgtcaggggc agctgcaagg 1500 ctcactggcg gcgctcacca gcctgacctc gatcgtcgga cccctcctct tcacggcgat 1560 ctatgcggct tctataacaa cgtggaacgg gtgggcatgg attgcaggcg ctgccctcta 1620 cttgctctgc ctgccggcgc tgcgtcgcgg gctttggagc ggcgcagggc aacgagccga 1680 tcgctgatcg tggaaacgat aggcctatgc catgcgggtc aaggcgactt ccggcaagct 1740 atacgcgccc taggagtgcg gttggaacgt tggcccagcc agatactccc gatcacgagc 1800 aggacgccga tgatttgaag cgcactcagc gtctgatcca agaacaacca tcctagcaac 1860 acggcggtcc ccgggctgag aaagcccagt aaggaaaacaa ctgtaggttc gagtcgcgag 1920 atccccccgga accaaaggaa gtaggttaaa cccgctccga tcaggccgag ccacgccagg 1980 ccgagaacat tggttcctgt aggcatcggg attggcggat caaacactaa agctactgga 2040 aggagcagaa gtcctccggc cgccagttgc caggccgtaa aggtgagcag aggcacggga 2100 ggttgccact tgcgggtcag cacggttccg aacgccatgg aaaccgcccc cgccaggccc 2160 gctgcgacgc cgacaggatc tagcgctgcg tttggtgtca acaccaacag cgccacgccc gcagttccgc aaatagcccc caggaccgcc atcaatcgta tcgggctacc tagcagagcg 2280. gcagagatga acacgaccat cagcggctgc acagcgccta ccgtcgccgc gaccccgccc ggcaggcggt agaccgaaat aaacaacaag ctccagaata gcgaaatatt aagtgcgccg aggatgaaga tgcgcatcca ccagattccc gttggaatct gtcggacgat catcacgagc aataacccg ccggcaacgc ccgcagcagc ataccggcga cccctcggcc tcgctgttcg ggctccacga aaacgccgga cagatgcgcc ttgtgagcgt ccttggggcc gtcctcctgt 2580 ttgaagaccg acagcccaat gatctcgccg tcgatgtagg cgccgaatgc cacggcatct cgcaaccgtt cagcgaacgc ctccatgggc tttttctcct cgtgctcgta aacggacccg 2700. aacatctctg gagctttctt cagggccgac aatcggatct cgcggaatc ctgcacgtcg gccgctccaa gccgtcgaat ctgagcctta atcacaattg tcaattttaa tcctctgttt atcggcagtt cgtagcgc gccgtgcgtc ccgagcgata ctgagcgaag caagtgcgtc 2880. gagcagtgcc cgcttgttcc tgaaatgcca gtaaagcgct ggctgctgaa cccccagccg 2940 gaactgaccc cacaaggccc tagcgtttgc aatgcaccag gtcatcattg acccaggcgt 3000 gttccaccag gccgctgcct cgcaactctt cgcaggcttc gccgacctgc tcgcgccact 3060 tcttcacgcg ggtggaatcc gatccgcaca tgaggcggaa ggtttccagc ttgagcgggt 3120 acggctcccg gtgcgagctg aaatagtcga acatccgtcg ggccgtcggc gacagcttgc 3180 ggtacttctc ccatatgaat ttcgtgtagt ggtcgccagc aaacagcacg acgatttcct 3240 cgtcgatcag gacctggcaa cgggacgttt tcttgccacg gtccaggacg cggaagcggt 3300 gcagcagcga caccgattcc aggtgcccaa cgcggtcgga cgtgaagccc atcgccgtcg 3360 cctgtaggcg cgacaggcat tcctcggcct tcgtgtaata ccggccattg atcgaccagc 3420 ccaggtcctg gcaaagctcg tagaacgtga aggtgatcgg ctcgccgata ggggtgcgct 3480 tcgcgtactc caacacctgc tgccacacca gttcgtcatc gtcggcccgc agctcgacgc 3540 cggtgtaggt gatcttcacg tccttgttga cgtggaaaat gaccttgttt tgcagcgcct 3600 cgcgcgggat tttcttgttg cgcgtggtga acagggcaga gcgggccgtg tcgtttggca 3660 tcgctcgcat cgtgtccggc cacggcgcaa tatcgaacaa ggaaagctgc atttccttga 3720 tctgctgctt cgtgtgtttc agcaacgcgg cctgcttggc ctcgctgacc tgttttgcca 3780 ggtcctcgcc ggcggttttt cgcttcttgg tcgtcatagt tcctcgcgtg tcgatggtca 3840 tcgacttcgc caaacctgcc gcctcctgtt cgagacgacg cgaacgctcc acggcggccg 3900 atggcgcggg cagggcaggg ggagccagtt gcacgctgtc gcgctcgatc ttggccgtag 3960 cttgctggac catcgagccg acggactgga aggtttcgcg gggcgcacgc atgacggtgc 4020 ggcttgcgat ggtttcggca tcctcggcgg aaaaccccgc gtcgatcagt tcttgcctgt 4080 atgccttccg gtcaaacgtc cgattcattc accctccttg cgggattgcc ccgactcacg 4140 ccggggcaat gtgcccttat tcctgatttg acccgcctgg tgccttggtg tccagataat 4200 ccaccttatc ggcaatgaag tcggtcccgt agaccgtctg gccgtccttc tcgtacttgg 4260 tattccgaat cttgccctgc acgaatacca gctccgcgaa gtcgctcttc ttgatggagc 4320 gcatggggac gtgcttggca atcacgcgca ccccccggcc gttttagcgg ctaaaaaagt 4380 catggctctg ccctcgggcg gaccacgccc atcatgacct tgccaagctc gtcctgcttc 4440 tcttcgatct tcgccagcag ggcgaggatc gtggcatcac cgaaccgcgc cgtgcgcggg 4500 tcgtcggtga gccagagttt cagcaggccg cccaggcggc ccaggtcgcc attgatgcgg 4560 gccagctcgc ggacgtgctc atagtccacg acgcccgtga ttttgtagcc ctggccgacg 4620 gccagcaggt aggccgacag gctcatgccg gccgccgccg ccttttcctc aatcgctctt 4680 cgttcgtctg gaaggcagta caccttgata ggtgggctgc ccttcctggt tggcttggtt 4740 tcatcagcca tccgcttgcc ctcatctgtt acgccggcgg tagccggcca gcctcgcaga 4800 gcaggattcc cgttgagcac cgccaggtgc gaataaggga cagtgaagaa ggaacacccg 4860 ctcgcgggtg ggcctacttc acctatcctg cccggctgac gccgttggat acaccaagga 4920 aagtctacac gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc 4980 gaaaaaatcg ctataatgac cccgaagcag ggttatgcag cggaaaagat ccgtcgaccc 5040 tttccgacgc tcaccgggct ggttgccctc gccgctgggc tggcggccgt ctatggccct 5100 gcaaacgcgc cagaaacgcc gtcgaagccg tgtgcgagac accgcggccg ccggcgttgt 5160 ggatacctcg cggaaaactt ggccctcact gacagatgag gggcggacgt tgacacttga 5220 ggggccgact cacccggcgc ggcgttgaca gatgaggggc aggctcgatt tcggccggcg 5280 acgtggagct ggccagcctc gcaaatcggc gaaaacgcct gattttacgc gagtttccca 5340 cagatgatgt ggacaagcct ggggataagt gccctgcggt attgacactt gaggggcgcg 5400 actactgaca gatgaggggc gcgatccttg acacttgagg ggcagagtgc tgacagatga 5460 ggggcgcacc tattgacatt tgaggggctg tccacaggca gaaaatccag catttgcaag 5520 ggtttccgcc cgtttttcgg ccaccgctaa cctgtctttt aacctgcttt taaaccaata 5580 tttataaacc ttgtttttaa ccagggctgc gccctgtgcg cgtgaccgcg cacgccgaag 5640 gggggtgccc ccccttctcg aaccctcccg gcccgctaac gcgggcctcc catcccccca 5700 ggggctgcgc ccctcggccg cgaacggcct caccccaaaa atggcaggac aactggtgag 5760 tctggatctg aagttcattg tgtcatga tgatctgac cgactgagc catcccaggt 5820 ggcggaagtg ttcaggcttg tgagtgcagt agccgatctg ccccgcttta cccatattct 5880 ctgttatgac agggatta tcactcatgc cgttgaacat gcgctgaata tcgagatgg 5940 cagccgttat ctccagaaaa tcattcagct tagttttaaa ttaccccgac ctgaagcctt 6000 tgatttacgt atgaatttc gccagcgggc tgaggctta tatcagcaa ttataatca 6060 accgccagac tctggaatgg tagggatt catcgcggtg actgatacct atggtgccgc 6120 actttcgacg ccacgggaaa tccatcaggc cattaatttcc ctgatttttc ttatccggg 6180 gatgcgggat tttgtttt tccctgatt gtgcctgctt cagcttatac gggtgacaaa 6240 cccggctctg tatgactgga cagagcatta cctgacagaa cggtccgtga ttgaaccgg 6300 tcagggtag ctttctgacg gagagaaagc agactccgg gaggggctta tcagatgtat 6360 gaagacgttc agggcatcaa atgcagactc gtttctgaca cttgcagact ggatctatct 6420 catctgcgca agcagaacg tgagacggc cgccctggac ctcgcccgcg agcgccaggc 6480 gcacgaggcc ggcgcgcgga cccgcgccac ggcccacgag cggacgccgc agcaggagcg 6540 ccagaaggcc gccagagagg ccgagcgcgg ccgtgaggct tggacgctag ggcagggcat 6600 gaaaaagccc gtagcgggct gctacgggcg tctgacgcgg tggaaagggg gaggggatgt 6660 tgtctacatg gctctgctgt agtgagtggg ttgcgctccg gcagcggtcc tgatcaatcg 6720 tcaccctttc tcggtccttc aacgttcctg acaacgagcc tccttttcgc caatccatcg 6780 acaatcaccg cgagtccctg ctcgaacgct gcgtccggac cggcttcgtc gaaggcgtct 6840 atcgcggccc gcaacagcgg cgagagcgga gcctgttcaa cggtgccgcc gcgctcgccg 6900 gcatcgctgt cgccggcctg ctcctcaagc acggccccaa cagtgaagta gctgattgtc 6960 atcagcgcat tgacggcgtc cccggccgaa aaacccgcct cgcagaggaa gcgaagctgc 7020 gcgtcggccg tttccatctg cggtgcgccc ggtcgcgtgc cggcatggat gcgcgcgcca 7080 tcgcggtagg cgagcagcgc ctgcctgaag ctgcgggcat tcccgatcag aaatgagcgc 7140 cagtcgtcgt cggctctcgg caccgaatgc gtatgattct ccgccagcat ggcttcggcc 7200 agtgcgtcga gcagcgcccg cttgttcctg aagtgccagt aaagcgcccg ctcctttcgc 7260 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 7320 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 7380 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 7440 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 7500 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 7560 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc 7620 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 7680 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 7740 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcgagctcgg taccctttcc 7800 tgcgttgtcg actgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 7860 aaagtgccac ctgcatcgat ttattatgac aacttgacgg ctacatcatt cactttttct 7920 tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag 7980 aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg 8040 gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta 8100 atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt 8160 gcgacgctgg cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc 8220 tcgcgtaccc gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc 8280 agtaacaatt gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc 8340 ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg 8400 cgaaagaacc ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg 8460 cgcggacgaa agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag 8520 tgatgaatct ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt 8580 ccctgatttt tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt 8640 cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc 8700 gccaccagat gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc 8760 atacttttca tactcccgcc attcagagaa gaaaccaatt gtccatattg catcagacat 8820 tgccgtcact gcgtctttta ctggctcttc tcgctaacca aaccggtaac cccgcttatt 8880 aaaagcattc tgtaacaaag cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc 8940 tataatcacg gcagaaaagt ccacattgat tatttgcacg gcgtcacact ttgctatgcc 9000 atagcatttt tatccataag attagcggat cctacctgac gctttttatc gcaactctct 9060 actgtttctc catacccgtt tttttgggaa ttcgagctct areaggtta taaaaaatga 9120 acagtcagca aaatcctgat ttagctgttc atcccttggc aattcctatg gaaggcttac 9180 9240 cccaagtaaa gctagaggta atccaaagtt tactggaacc ctgcgatcgc acaacttatg 9300 ggcaaaagtt gcgggaagca gcagagaaac taaatgtatc gttgcgaacg gtacaaaggt 9360 tggtgaaaaa ctgggaacaa gatggcttag tcggactcac tcaacaagt agggctgata 9420 aaggaaaaca ccgcattggt gagttttggg aaaacttcat taccaaacc tacaaggagg 9480 gtaacaaggg aagtaaacgt atgaccccta aacaagttgc tctcagagtc gaggctaaag 9540 cccgtgaatt aaagactct aagccgccca attacaaac cgtgttacgg gtattagcac 9600 ccattttgga aaagcacaa aaagccaaga gtatccgcag tcctggttgg agaggaacta 9660 cgctttcggt taaaacccgt gaaggaaaag atttatcggt tgattacagt aaccatgttt 9720 ggcaatgtga ccataccgc gtggatgtgt tgctggtaga tcacatggt gaatttta 9780 gtcgtccctg gctaacaaca gtaattgata cttactctcg ttgcattatg ggtatcaact 9840 tgggctttga tgcatcagt tctggggtag tgtcgccat gcatchtac 9900 caaagcgtta cggttccgag tacaactgc attgtgagtg gggaacctat ggaaaaccag 9960 aacatttta tactgatggc ggtaagact ttcgctctaa ccacttgagt cagattgggg 10020 cgcaattggg atttgtctgt catttacgcg atcgccctc tgaaggtgga gtagtagaac 10080 gtcccttcaa aacattaaat gaccaactat tttcaacgct tcctgggtac accggatcta 10140 atgtgcagga acgcccagaa gatgcagaga aggacgcaag acttactttg cgagaactag 10200 aacagttact tgtgcgttac atcgtagatc gttacaacca aagtattgat gcgcggatgg 10260 gcgaccaaac gcgctttgag cgttgggaag caggattgcc tacagtgcca gtaccaatac 10320 cagaacgaga tttggatatt tgtttaatga agcagtcacg gcgcactgtg caaagaggtg 10380 gttgtttgca gtttcagaat ttaatgtatc ggggggaata tttggcaggt tatgccggag 10440 aaactgtcaa cttaaggttt gaccccagag acattacaac aattttggtt tatcgccagg 10500 aaaacaatca ggaagtattt ctgactcgcg ctcacgctca aggtttggag acagagcaac 10560 tggcattaga tgaggctgag gcagcaagtc gcagactccg taccgcaggg aaaactatca 10620 gtaaccaatc attattgcaa gaagttgttg accgcgatgc tcttgtcgct accaagaaaa 10680 gccgtaagga gcgtcaaaaa ttggaacaga ctgttttgcg atctgctgct gttgatgaaa 10740 gtaatagaga atccttgcct tctcaaatag ttgaaccaga tgaagtggaa tctacagaaa 10800 cggttcactc tcaatacgaa gacattgagg tgtgggacta tgaacaactt cgtgaagaat 10860 atgggtttta aacaatgaca gaagctcagg cgatcgccaa gcagttgggt ggggtaaaac 10920 cggatgatga gtggttacaa gctgaaattg ctcgtctcaa gggtaagagc attgtgcctt 10980 tacagcaggt aaaaactctc catgattggt tagatggcaa gcgcaaggca agaaaatctt 11040 gccgagtagt tggggaatcg agaactggca agacagttgc ttgtgatgcc tacagataca 11100 ggcacaaacc tcagcaggaa gctggacgac ctccaactgt gcctgtcgtt tatattcgac 11160 ctcaccaaaa atgtggcccc aaggatttgt ttaaaaagat tactgagtac ctcaagtatc 11220 gggtaacaaa agggactgta tctgattttc gagataggac gatagaagta ctcaagggtt 11280 gtggcgtaga gatgctaatt attgatgaag ctgaccgtct caagcctgaa acttttgctg 11340 atgtgcgaga tattgccgaa gatttaggaa ttgctgtggt actggtagga acagaccgtt 11400 tggatgcggt aattaagcgg gatgagcagg ttctcgaacg cttcgggcg catcttcgct 11460 ttggtaaatt gtcgggagag gattttaaga acaccgtaga aatgtgggaa caaatggttt 11520 tgaaactgcc agtatcttct aatctaaaga gcaaggagat gctacggatt ctcacgtcag 11580 caactgaagg ctacattggt cgccttgatg agattcttag ggaagctgca attcgttcct 11640 tatcaagagg attgaagaag attgacaagg ctgttttaca ggaagtagct aaggagtaca 11700 aatgatagaa gcaccagatg ttaaaccttg gctattcttg attaaaccct atgaagggga 11760 aagcctgagc cactttcttg gcaggttcag acgtgccaac catttatccg caagtggatt 11820 gggtactttg gcaggaattg gtgctagat ggcacgttgg gaaagatttc attttaatcc 11880 tcgccctagt cagcaagaat tggaagcgat cgcatctgta gtagaagtgg atgctcaaag 11940 gttagcccag atgttaccgc ctgctggagt gggaatgcag catgagccaa ttcgcttgtg 12000 tggggcttgt tatgccgagt cgccttgtca ccgaattgaa tggcagtaca agtcggtgtg 12060 gaagtgcgat cgccatcaac tcaagatttt agcaaagtgt ccaaactgtc aagcaccttt 12120 taaaatgcct gcgctgtggg aggatgggtg ctgtcacaga tgtaggatgc cgtttgcaga 12180 aatggcaaag ctacagaagg tttgatgata aaaccagaaa aaggtgtgaa attaactaag 12240 tccctgaatt gatctggttg tccaaaaaat ttgtgcgatc gcatggcaag attattccta 12300 ctaggatcta gacggcatca gagcagattg tactgagagt gcaccataat cggcattttc 12360 ttttgcgttt ttattgtta actgttaatt gtccttgttc areatgctg tctttgacaa 12420 cagatgtttt cttgcctttg atgttcagca ggaagctagg cgcaaacgtt gattgtttgt 12480 ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac gacattgttt cctttcgctt 12540 gaggtacagc gaagtgtgag tagtaagg ttacatcgtt aggatcaaga tccattttta 12600 acacaaggcc agttttgttc agcggcttgt atgggccagt taaagaatta gaaacataac 12660 caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt catttttgat ccgcgggagt 12720 cagtgaacag ataccatttg ccgttcattt taagacgtt cgcgcgttca atttcatctg 12780 ttactgtgtt agatgcaatc agcggtttca tcactttttt cagtgtgtaa tcatcgttta 12840 gctcaatcat accgagagcg ccgtttgcta actcagccgt gcgtttttta tcgctttgca 12900 gaagtttttg actttcttga cggaagaatg atgtgctttt gccatagtat gctttgttaa 12960 ataaagattc ttcgccttgg tagccatctt cagttccagt gtttgcttca aatactaagt 13020 atttgtggcc tttatcttct acgtagtgag gatctctcag cgtatggttg tcgcctgagc 13080 tgtagttgcc ttcatcgatg aactgctgta cattttgata cgtttttccg tcaccgtcaa 13140 agattgattt ataatcctct acaccgttga tgttcaaaga gctgtctgat gctgatacgt 13200 taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt accggagaaa tcagtgtaga 13260 ataaacggat ttttccgtca gatgtaaatg tggctgaacc tgaccattct tgtgtttggt 13320 cttttaggat agaatcattt gcatcgaatt tgtcgctgtc tttaaagacg cggccagcgt 13380 ttttccagct gtcaatagaa gtttcgccga ctttttgata gaacatgtaa atcgatgtgt 13440 catccgcatt tttaggatct ccggctaatg caaagacgat gtggtagccg tgatagtttg 13500 cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca aacgtccagg ccttttgcag 13560 aagagatatt tttaattgtg gacgaatcga actcaggaac ttgatatttt tcattttttt 13620 gctgttcagg gatttgcagc atatcatggc gtgtaatatg ggaaatgccg tatgtttcct 13680 tatatggctt ttggttcgtt tctttcgcaa acgcttgagt tgcgcctcct gccagcagtg 13740 cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt tttgatgttc atcgttcatg 13800 tctcctttt tatgtactgt gttagcggtc tgcttcttc agccctcctg tttgaagatg 13860 gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg taaggggtga cgccaaagta 13920 tacactttgc cctttacaca ttttaggtct tgcctgcttt atcagtaaca aacccgcgcg 13980 attacttt cgacctcatt ctattagact ctcgtttgga ttgcaactgg tctattttcc 14040 tcttttgttt gatagaaaat cataaaagga tttgcagact acgggcctaa agaactaaaa 14100 aatctatctg tttctttca ttctctgtat ttttatag ttctgttgca tgggcataaa 14160 gttgcctcga gacttttcat actcccgcca ttcagagaag aaaccaattg tccatattgc 14220 atcagacatt gccgtcactg cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct fathercacgg cagaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta ctgtttctcc atacccgttt ttttgggaat tcgagctcta aggaggttat 14580. aaaaaatgag ccagatcacc atccaggctc gtctgatcag ctttgaatct aaccgccagc agctgtggaa actgatggct gatctgaaca ccccgctgat taacgaactg ctgtgtcagc tgggccagca cccggacttt gaaaaatggc agcagaaagg taaactgccg agcaccgtgg tttctcagct gtgccagcca ctgaaaactg acccgcgttt cgcgggtcag ccgagccgtc tgtacatgtc tgcaattcac atcgttgatt acatctaa atcctggctg gcgatccaga aacgcctgca acagcagctg gatggcaaaa cccgttggct ggaaatgctg aacagcgatg cagaactggt tgaactgtct ggtgatactc tggaagcgat tcgcgtcaaa gcggctgaaa tcttggctat cgccatgccg gcaagcgaat ccgattctgc atctccgaaa ggtaaaaaag 15000 gcaaaaaaga gaaaaaaccg agcagcagct ccccgaaacg cagtctttct aaaacccctgt 15060 ttgacgcgta ccaggaaacc gaagaatatca aatctcgtag cgcgatctct tatctgctga 15120 aaaacggctg taaactgact gataaagaag aagatagcga aaaatttgct aagcgccgtc 15180 gtcaggtgga gattcagatt cagcgtctga ccgaaaaact gatctctcgt atgccgaaag 15240 gtcgcgactt gaccaacgct aaatggcttg aaaccctgct gaccgcgact actacggtgg 15300 cagaagaataa tgcgcaggca aaacgttggc aggatatcct cctgacccgt agctcttccc 15360 tgccgttccc gctggtattt gaaaccaacg aagacatggt gtggtctaaa aaccagaaag 15420 gccgtctgtg cgttcacttc aacggcctga gcgatctgat cttcgaagtg tactgtggca 15480 accgccagct ccactggttc cagcgtttcc tggaagacca gcagaccaaa cgcaaaagca 15540 aaaaccagca ctcaagcggc ctgttcactc tgcgtaacgg tcacctggtg tggctgggaag 15600 gcgaaggtaa aggtgaaccg tggaacctgc accacctgac cctgtactgc tgcgtggaca 15660 accgtctgtg gaccgaagaa ggcaccgaaa tcgtccgtca ggaaaaagca gacgaaatca 15720 ccaaatttat caccaacatg aaaaagaaat ctgatctgag cgacacccaa caggctctga 15780 ttcagcgcaa acagtctacc ctgacccgta ttaataactc tttcgaacgc ccgtcccagc 15840 cgctgtacca gggccagagc cacatcctgg tcggtgtttc tctgggcctg gaaaaaccgg 15900 ctaccgttgc cgttgtcgat gccattgcaa acaaagttct ggcttaccgt tccatcaaac 15960 agctgctggg cgataactat gaactgctga accgtcagcg tcgccagcag cagtacctga 16020 gccacgaacg tcacaaagcg cagaaaaatt tctctccgaa ccaattcggc gcaagcgaac 16080 tgggccagca catcgatcgt ctgctggcca aagcgatcgt tgcgctggcg cgtacctata 16140 aagccggctc tatcgtcctg ccgaaactgg gcgacatgcg tgaagtggtt cagtctgaaa 16200 tccaggcgat cgctgaacag aaatttccgg gttatattga aggtcagcag aaatacgcga 16260 aacagtaccg cgtgaacgtt caccgttgga gctacggtcg tctgattcag tccatccagt 16320 ctaaagcagc gcagactggc atcgtgattg aagaaggcaa acagccgatc cgtggctccc 16380 cgcacgataa agcgaaagaa ctggcgctga gcgcatacaa cctgcgtctg acccgccgtt 16440 cctaacaaat atctgaacct tgataataga atattaatag cgccgcaatt catgctgctt 16500 gcagcctctg aattttgtta aatgagggtt agtttgactg tataaat 16547 <210> 8 <211> 2022 <212> DNA <213> Artificial Sequence <220> <223> Cas12k‐synthesis <400> 8 atgagccaga tcaccatcca ggctcgtctg atcagctttg aatctaaccg ccagcagctg 60 tggaaactga tggctgatct gaacaccccg ctgattaacg aactgctgtg tcagctgggc 120 cagcacccgg actttgaaaa atggcagcag aaaggtaaac tgccgagcac cgtggttttct 180 cagctgtgcc agccactgaa aactgacccg cgtttcgcgg gtcagccgag ccgtctgtac 240 atgtctgcaa ttcacatcgt tgattacatc tataaatcct ggctggcgat ccagaaacgc 300 ctgcaacagc agctggatgg caaaacccgt tggctggaaa tgctgaacag cgatgcagaa 360 ctggttgaac tgtctggtga tactctggaa gcgattcgcg tcaaagcggc tgaaatcttg 420 gctatcgcca tgccggcaag cgaatccgat tctgcatctc cgaaaggtaa aaaaggcaaa 480 aaagagaaaa aaccgagcag cagctccccg aaacgcagtc tttctaaaac cctgtttgac 540 gcgtaccagg aaaccgaaga tatcaaatct cgtagcgcga tctcttatct gctgaaaaac 600 ggctgtaaac tgactgataa agaagaagat agcgaaaaat ttgctaagcg ccgtcgtcag 660 gtggagattc agattcagcg tctgaccgaa aaactgatct ctcgtatgcc gaaaggtcgc 720 gacttgacca acgctaaatg gcttgaaacc ctgctgaccg cgactactac ggtggcagaa 780 gataatgcgc aggcaaaacg ttggcaggat atcctcctga cccgtagctc ttccctgccg 840 ttcccgctgg tatttgaaac caacgaagac atggtgtggt ctaaaaacca gaaaggccgt 900 ctgtgcgtc acttcaacgg cctgagcgat ctgatcttcg aagtgtactg tggcaaccgc 960 cagctccact ggttccagcg tttcctggaa gaccagcaga ccaaacgcaa aagcaaaaac 1020 cagcactcaa gcggcctgtt cactctgcgt aacggtcacc tggtgtggct ggaaggcgaa 1080 ggtaaaggtg aaccgtggaa cctgcaccac ctgaccctgt actgctgcgt ggacaaccgt 1140 ctgtggaccg aagaaggcac cgaaatcgtc cgtcaggaaa aagcagacga aatcaccaaa 1200 tttatcacca acatgaaaaa gaaatctgat ctgagcgaca cccaacaggc tctgattcag 1260 cgcaaacagt ctaccctgac ccgtattaat aactctttcg aacgcccgtc ccagccgctg 1320 taccagggcc agagccacat cctggtcggt gtttctctgg gcctggaaaa accggctacc 1380 gttgccgttg tcgatgccat tgcaaacaaa gttctggctt accgttccat caaacagctg 1440 ctgggcgata actatgaact gctgaaccgt cagcgtcgcc agcagcagta cctgagccac 1500 gaacgtcaca aagcgcagaa aaatttctct ccgaaccaat tcggcgcaag cgaactgggc 1560 cagcacatcg atcgtctgct ggccaaagcg atcgttgcgc tggcgcgtac ctataaagcc 1620 ggctctatcg tcctgccgaa actgggcgac atgcgtgaag tggttcagtc tgaaatccag 1680 gcgatcgctg aacagaaatt tccgggttat attgaaggtc agcagaaata cgcgaaacag 1740 taccgcgtga acgttcaccg ttggagctac ggtcgtctga ttcagtccat ccagtctaaa 1800 gcagcgcaga ctggcatcgt gattgaagaa ggcaaacagc cgatccgtgg ctccccgcac 1860 gataaagcga aagaactggc gctgagcgca tacaacctgc gtctgacccg ccgttcctaa 1920 caaatatctg aaccttgata atagaatatt aatagcgccg caattcatgc tgcttgcagc 1980 ctctgaattt tgttaaatga gggttagttt gactgtataa at 2022 <210> 9 <211> 3187 <212> DNA <213> Artificial Sequence <220> <223> TnsB‑TnsC‑TniQ <400> 9 atgaacagtc agcaaaatcc tgatttagct gttcatccct tggcaattcc tatggaaggc 60 ttactaggag aaagtgctac aactcttgag aagaatgtaa ttgccacaca actctcagag 120 gaagcccaag taaagctaga ggtaatccaa agtttactgg aaccctgcga tcgcacaact 180 tatgggcaaa agttgcggga agcagcagag aaactaaatg tatcgttgcg aacggtacaa 240 aggttggtga aaaactggga acaagatggc ttagtcggac tcactcaaac aagtagggct 300 gataaaggaa aacaccgcat tggtgagttt tgggaaaact tcattaccaa aacctacaag 360 gagggtaaca agggaagtaa acgtatgacc cttaaacaag ttgctctcag agtcgaggct 420 aaagcccgtg attaaaaga ctctaagccg cccaattaca aaaccgtgtt acgggtatta 480 gcacccattt tggaaaagca acaaaaagcc aagagtatcc gcagtcctgg ttggagagga 540 actacgcttt cggttaaaac ccgtgaagga aaagatttat cggttgatta cagtaaccat 600 gtttggcaat gtgaccatac ccgcgtggat gtgttgctgg tagatcaaca tggtgaaatt 660 ttaagtcgtc cctggctaac aacagtaatt gatacttact ctcgttgcat tatgggtatc 720 aacttgggct ttgatgcacc cagttctggg gtagtagcat tagcgttacg ccatgcaatt 780 ctaccaaagc gttacggttc cgagtacaaa ctgcattgtg agtggggaac ctatggaaaa 840 ccagaacatt tttatactga tggcggtaaa gactttcgct ctaaccactt gagtcagatt 900 ggggcgcaat tgggatttgt ctgtcattta cgcgatcgcc cttctgaagg tggagtagta 960 gaacgtccct tcaaaacatt aaatgaccaa ctattttcaa cgcttcctgg gtacaccgga 1020 tctaatgtgc aggaacgccc agaagatgca gagaaggacg caagacttac tttgcgagaa 1080 ctagaacagt tacttgtgcg ttacatcgta gatcgttaca accaaagtat tgatgcgcgg 1140 atgggcgacc aaacgcgctt tgagcgttgg gaagcaggat tgcctacagt gccagtacca 1200 ataccagaac gagatttgga tatttgttta atgaagcagt cacggcgcac tgtgcaaaga 1260 ggtggttgtt tgcagtttca gaatttaatg tatcgggggg aatatttggc aggttatgcc 1320 ggagaaactg tcaacttaag gtttgacccc agagacatta caacaatttt ggtttatcgc 1380 caggaaaaca atcaggaagt atttctgact cgcgctcacg ctcaaggttt ggagacagag 1440 caactggcat tagatgaggc tgaggcagca agtcgcagac tccgtaccgc agggaaaact 1500 atcagtaacc aatcattatt gcaagaagtt gttgaccgcg atgctcttgt cgctaccaag 1560 aaaagccgta aggagcgtca aaaattggaa cagactgttt tgcgatctgc tgctgttgat 1620 gaaagtaata gagaatcctt gccttctcaa atagttgaac cagatgaagt ggaatctaca 1680 gaaacggttc actctcaata cgaagacatt gaggtgtggg actatgaaca acttcgtgaa 1740 gaatatgggt tttaaacaat gacagaagct caggcgatcg ccaagcagtt gggtggggta 1800 aaaccggatg atgagtggtt acagctgaa attgctcgtc tcaagggtaa gagcattgtg 1860 cctttacagc aggtaaaac tctcatgat tggttagatg gcaagcgcaa ggcaagaaaaa 1920 tcttgccgag tagttgggga atcgagaact ggcagacag tgcttgtga tgcctacaga 1980 tacaggcaca aacctcagca ggaagctgga cgacctccaa ctgtgcctgt cgtttatatt 2040 cgacctcacc aaaaatgtgg ccccaaggat ttgtttaaaa agattactga gtacctcaag 2100 tatcggtaa caaagggac tgtatctgat tttcgagata ggacgataga agtactcaag 2160 ggttgtggcg tagagatgct aattattgat gaagctgacc gtctcaagcc tgaaactttt 2220 gctgatgtgc gagatattgc cgagattta ggaattgctg tggtactggt aggaacagac 2280 cgtttggatg cggtaattaa gcgggatgag caggttctcg aacgctttcg ggcgcatctt 2340 cgctttgta aattgtcgggg agaggatttt aagaacaccg tagaatgtg ggaacaatg 2400 gttttgaac tgccagtatc ttctaatcta agagcaagg agatgctacg gattctcacg 2460 tcagcactg aaggctacat tggtcgcctt gatgagattc tcagggagc tgcaatcgt 2520 tccttatcaa gaggattgaa gaagattgac aaggctgttt tacaggaagt agctaaggag 2580 tacaaatgat agaagcacca gatgttaaac cttggctatt cttgattaaa ccctatgaag 2640 gggaaagcct gagccacttt cttggcaggt tcagacgtgc caaccattta tccgcaagtg 2700 gattgggtac tttggcagga attggtgcta tagtggcacg ttgggaaaga tttcatttta 2760 atcctcgccc tagtcagcaa gaattggaag cgatcgcatc tgtagtagaa gtggatgctc 2820 aaaggttagc ccagatgtta ccgcctgctg gagtgggaat gcagcatgag ccaattcgct 2880 tgtgtggggc ttgttatgcc gagtcgcctt gtcaccgaat tgaatggcag tacaagtcgg 2940 tgtggaagtg cgatcgccat caactcaaga ttttagcaaa gtgtccaaac tgtcaagcac 3000 cttttaaaat gcctgcgctg tgggaggatg ggtgctgtca cagatgtagg atgccgtttg 3060 cagaaatggc aaagctacag aaggtttgat gataaaacca gaaaaaggtg tgaaattaac 3120 taagtccctg aattgatctg gttgtccaaa aaatttgtgc gatcgcatgg caagattatt 3180 cctacta 3187 <210> 10 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> pCasPA‐5P <400> 10 tgagggttag tttgactgta taaataagct ttccctatag tgagtcgtat tag 53 <210> 11 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> pCasPA‐3P <400> 11 aatcaggatt ttgctgactg ttcatttttt ataacctcct tagagctcga attcc 55 <210> 12 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> sacB‐5P <400> 12 catggcaaga ttattcctac taggatctag acggcatcag agcagatt 48 <210> 13 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> SacB‐3P <400> 13 gagcctggat ggtgatctgg ctcattttt ataacctcct tagagctcga attcc 55 <210> 14 <211> 8418 <212> DNA <213> Artificial Sequence <220> <223> pGTPA <400> 14 ttgacagcta gctcagtcct aggtataata ctagtatatt atagcgccg caattcatgc 60 tgcttgcagc ctctgaattt tgttaaatt gggttagttt gactgtataa atacagtctt 120 gctttctgac cctggtagct gctcaccctg atgctgctgt caatagacag gataggtgcg 180 ctcccagcaa taagggcgcg gatgtactgc tgtagtggct actgaatcac ccccgatcaa 240 gggggaaccc tccaaaggt gggttgaaag agaccattgg tctcattttt ttagccgtga 300 tagtttgcga cagatatcga attcctgcag cccgggggat ccactagttg tacagtgact 360 aattatatgt cgttgtgaca aattattgtc atcagtaaaa tccttataca gtatagatta 420 tagcgctttg gcagttttag cataacctct ttgcagtgac aaatagatg tcgttgtccg 480 tgattgtgac aattagctg tcgcttgca agataggaaa aagctttgt gtattttcat 540 aatgacaaat tgactgtcgc ctcgaggaat aggaacttcg gataggaac ttcagatcc 600 cctgattccc ttgtcaca gcaatggatc gattgacat aagcctgttc ggttcgtaaa 660 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc 720 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tgtacagtct 780 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg 840 agcagcaacg atgttacgca gcagcaacga tgttacgcag cagggcagtc gccctaaaac 900 aaagttaggt ggctcaagta tgggcatcat tcgcacatgt aggctcggcc ctgaccaagt 960 caaatccatg cgggctgctc ttgatctttt cggtcgtgag ttcggagacg tagccaccta 1020 ctcccaacat cagccggact ccgattacct cgggaacttg ctccgtagta agacattcat 1080 cgcgcttgct gccttcgacc aagaagcggt tgttggcgct ctcgcggctt acgttctgcc 1140 caagtttgag cagccgcgta gtgagatcta tatctatgat ctcgcagtct ccggcgagca 1200 ccggaggcag ggcattgcca ccgcgctcat caatctcctc aagcatgagg ccaacgcgct 1260 tggtgcttat gtgatctacg tgcaagcaga ttacggtgac gatcccgcag tggctctcta 1320 tacaaagttg ggcatacggg aagaagtgat gcactttgat atcgacccaa gtaccgccac 1380 ctaacaattc gttcaagccg agatcggctt cccggccgcg gagttgttcg gtaaattgtc 1440 acaacgccgc ggccaattcg atctagatca gtaactatta aacttagggg tgggttgaaa 1500 gcaagtcctt ttatccgctt gttttaattg ctttgtataa taattgcaga gcatattata 1560 ttgatgacat ttaatttgtc atcaattaat taagcaacgc tgatgggtca cgacgacaat 1620 taaatagtca caatgacatt aatctgtcac cgacgacaga taatttgtca ctgtacaaag 1680 cttgagtatt ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc 1740 tgtgtgaaat tgtgtatccg ctcacaattc cacacaacat acgagccgga agcataaagt 1800 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 1860 ccgctttcca gtcgggaaac ctgtcgtgcc agaattcttg aagacgaaag ggcctcgtga 1920 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 1980 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 2040 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 2100 gtatgagtat tcaacatttc cgtgtcgccc ttatccctt ttttgcggca ttttgccttc 2160 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 2220 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 2280 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 2340 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 2400 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 2460 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 2520 tcggaggacc gaaggagcta accgcttttt tgcacaacat ggggatcat gtaactcgcc 2580 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 2640 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 2700 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 2760 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggat 2820 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 2880 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 2940 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 3000 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 3060 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc aattacacgc 3120 cactggctgt gcttgctggg gtgacggtgg caacggtggc ggccttgctg ggctatcgcg 3180 ttggaaagaa acgagggaaa ggggactgat aaaccggtct tagcccctcc ccttggtgtc 3240 caaccgctct gtaggcctct caggcgccgc tggtgccgct ggttggacgc caagggtgaa 3300 tccgcctcga taccctgatt actcgcttcc tgcgccctct caggcggcga taggggactg 3360 gtaaaacggg gattgcccag acgcctcccc cgccccttca ggggcacaaa tgcggcccca 3420 acggggccac gtagtggtgc gttttttgcg tttccaccct tttcttcctt ttccctttta 3480 aaccttttag gacgtctaca ggccacgtaa tccgtggcct gtagagttta aaaagggacg 3540 gatttgttgc cattaaggga cggatttgtt gttaagaagg gacggatttg ttgttgtaaa 3600 gggacggatt tgttgtattg tgggacgcag atacagtgtc cccttataca caaggaatgt 3660 cgaacgtggc ctcaccccca atggtttaca aaagcaatgc cctggtcgag gccgcgtatc 3720 gcctcagtgt tcaggaacag cggatcgttc tggcctgtat tagccaggtg aagaggacg 3780 agcctgtcac cgatgaagtg atgtattcag tgacggcgga ggacatagcg acgatggcgg 3840 gtgtccctat cgaatcttcc tacaaccagc tcaaagaagc ggccctgcgc ctgaaacggc 3900 gggaagtccg gttaacccaa gagcccaatg gcaaggggaa aagaccgagt gtgatgatta 3960 ccggctgggt gcaaacaatc atctaccggg agggtgaggg ccgtgtagaa ctcaggttca 4020 ccaaagacat gctgccgtac ctgacggaac tcaccaaaca gttcaccaaa tacgccttgg 4080 ctgacgtggc caagatggac agcaccacg cgatcaggct ttacgagctg ctcatgcaat 4140 gggacagcat cggccagcgc gaagaaa ttgaccagct gcgaaagtgg tttcaactgg 4200 aaggccggta tccctcgatc aaggacttca agttgcgagt gcttgatcca gccgtgacgc 4260 agatcaacga gcacagcccg ctacaggtgg agtgggcgca gcgaaagacc gggcgcaagg 4320 tcacacatct gttgttcagt tttggaccga agaagcccgc caaggcggtg ggtaaggccc 4380 cagcgaagcg caaggccggg aagatttcag atgctgagat cgcgaaacag gctcgccctg 4440 gtgagacatg ggaagcggcc cgcgctcgac taacccagat gccgcttgcg caaactatta 4500 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 4560 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 4620 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 4680 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 4740 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 4800 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 4860 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 4920 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 4980 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 5040 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 5100 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 5160 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 5220 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 5280 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 5340 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 5400 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 5460 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 5520 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 5580 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 5640 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 5700 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg 5760 tgcggtattt cacaccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag 5820 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 5880 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 5940 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 6000 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 6060 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 6120 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 6180 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 6240 gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa 6300 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca 6360 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc 6420 agtgaattgt aatacgactc actatagggc gaattcgagc tcggtacccg gggatcccgg 6480 catcagagca gattgtactg agagtgcacc ataatcggca ttttcttttg cgtttttatt 6540 tgttaactgt taattgtcct tgttcaagga tgctgtcttt gacaacagat gttttcttgc 6600 ctttgatgtt cagcaggaag ctaggcgcaa acgttgattg tttgtctgcg tagaatcctc 6660 tgtttgtcat atagcttgta atcacgacat tgtttccttt cgcttgaggt acagcgaagt 6720 gtgagtaagt aaaggttaca tcgttaggat caagatccat ttttaacaca aggccagttt 6780 tgttcagcgg cttgtatggg ccagttaaag aattagaaac ataaccaagc atgtaaatat 6840 cgttagacgt aatgccgtca atcgtcattt ttgatccgcg ggagtcagtg aacagatacc 6900 atttgccgtt cattttaaag acgttcgcgc gttcaatttc atctgttact gtgttagatg 6960 caatcagcgg tttcatcact tttttcagtg tgtaatcatc gtttagctca atcataccga 7020 gagcgccgtt tgctaactca gccgtgcgtt ttttatcgct ttgcagaagt ttttgacttt 7080 cttgacggaa gaatgatgtg cttttgccat agtatgcttt gttaaataaa gattcttcgc 7140 cttggtagcc atcttcagtt ccagtgtttg cttcaaatac taagtatttg tggcctttat 7200 cttctacgta gtgaggatct ctcagcgtat ggttgtcgcc tgagctgtag ttgccttcat 7260 cgatgaactg ctgtacattt tgatacgttt ttccgtcacc gtcaaagatt gatttataat 7320 cctctacacc gttgatgttc aaagagctgt ctgatgctga tacgttaact tgtgcagttg 7380 tcagtgtttg tttgccgtaa tgtttaccgg agaaatcagt gtagaaataaa cggatttttc 7440 cgtcagatgt aaatgtggct gaacctgacc attcttgtgt ttggtctttt aggatagaat 7500 catttgcatc gaatttgtcg ctgtctttaa agacgcggcc agcgtttttc cagctgtcaa 7560 tagaagtttc gccgactttt tgatagaaca tgtaaatcga tgtgtcatcc gcatttttag 7620 gatctccggc taatgcaaag acgatgtggt agccgtgata gtttgcgaca gtgccgtcag 7680 cgttttgtaa tggccagctg tcccaaacgt ccaggccttt tgcagaagag atatttttaa 7740 ttgtggacga atcgaactca ggaacttgat attttcatt tttttgctgt tcagggattt 7800 gcagcatatc atggcgtgta atatgggaaa tgccgtatgt ttccttatat ggcttttggt 7860 tcgtttcttt cgcaaacgct tgagttgcgc ctcctgccag cagtgcggta gtaaaggtta 7920 atactgttgc ttgttttgca aactttttga tgttcatcgt tcatgtctcc ttttttatgt 7980 actgtgttag cggtctgctt cttccagccc tcctgtttga agatggcaag ttagttacgc 8040 acaataaaaa aagacctaaa atatgtaagg ggtgacgcca aagtatacac tttgcccttt 8100 acacatttta ggtcttgcct gctttatcag taacaaaccc gcgcgattta cttttcgacc 8160 tcattctatt agactctcgt ttggattgca actggtctat tttcctcttt tgtttgatag 8220 aaaatcataa aaggatttgc agactacggg cctaaagaac taaaaaatct atctgtttct 8280 tttcattctc tgtatttttt atagtttctg ttgcatgggc ataaagttgc gtcgacgcct 8340 tgctcaaatt ggaatcaggt ttgtgccaat accagtagaa acagacgaag aatccatggc 8400 ggccgctgtc atgggccc 8418 <210> 15 <211> 1677 <212> DNA <213> Artificial Sequence <220> <223> gRNA‑transposon <400> 15 ttgacagcta gctcagtcct aggtataata ctagtatatt atagcgccg caattcatgc 60 tgcttgcagc ctctgaattt tgttaaatt gggttagttt gactgtataa atacagtctt 120 gctttctgac cctggtagct gctcaccctg atgctgctgt caatagacag gataggtgcg 180 ctcccagcaa taagggcgcg gatgtactgc tgtagtggct actgaatcac ccccgatcaa 240 gggggaaccc tccaaaggt gggttgaaag agaccattgg tctcattttt ttagccgtga 300 tagtttgcga cagatatcga attcctgcag cccgggggat ccactagttg tacagtgact 360 aattatatgt cgttgtgaca aattattgtc atcagtaaaa tccttataca gtatagatta 420 tagcgctttg gcagttttag cataacctct ttgcagtgac aaatagatg tcgttgtccg 480 tgattgtgac aattagctg tcgcttgca agataggaaa aagctttgt gtattttcat 540 aatgacaaat tgactgtcgc ctcgaggaat aggaacttcg gataggaac ttcagatcc 600 cctgattccc ttgtcaca gcaatggatc gattgacat aagcctgttc ggttcgtaaa 660 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc 720 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tgtacagtct 780 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg 840 agcagcaacg atgttacgca gcagcaacga tgttacgcag cagggcagtc gccctaaaac 900 aaagttaggt ggctcaagta tgggcatcat tcgcacatgt aggctcggcc ctgaccaagt 960 caaatccatg cgggctgctc ttgatctttt cggtcgtgag ttcggagacg tagccaccta 1020 ctcccaacat cagccggact ccgattacct cgggaacttg ctccgtagta agacattcat 1080 cgcgcttgct gccttcgacc aagaagcggt tgttggcgct ctcgcggctt acgttctgcc 1140 caagtttgag cagccgcgta gtgagatcta tatctatgat ctcgcagtct ccggcgagca 1200 ccggaggcag ggcattgcca ccgcgctcat caatctcctc aagcatgagg ccaacgcgct 1260 tggtgcttat gtgatctacg tgcaagcaga ttacggtgac gatcccgcag tggctctcta 1320 tacaaagttg ggcatacggg aagaagtgat gcactttgat atcgacccaa gtaccgccac 1380 ctaacaattc gttcaagccg agatcggctt cccggccgcg gagttgttcg gtaaattgtc 1440 acaacgccgc ggccaattcg atctagatca gtaactatta aacttagggg tgggttgaaa gcaagtcctt ttatccgctt gttttaattg ctttgtata taattgcaga gcatatta ttgatgacat ttaatttgtc atcaattaat tagcaacgc tgatgggtca cgacgacaat 1677. 1677. 1677. 1677. 1677. 1677. 1677. 1677. 1677. 1677 <210> 16 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> pACRISPR‐5P <400> 16 52. acgacagata atttgtcact gtacaaagct tgagtattct atagtgtcac ct <210> 17 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> pACRISPR‐3P <400> 17 45. tacctaggac tgagctagct gtcaagggcc catgacagcg gccgc <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rhlR‐spacer <400> 18 ggacatcagc atcggatgct cca 23 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> rhlR‑spacer‑5P <400> 19 gtggagggac atcagcatcg gatgctcca 29 <210> 20 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> rhlR‑spacer‑3P <400> 20 aaactggagc atccgatgct gatgtccct 29 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rhlR‑verify‑5P <400> 21 ctcaggatga tggcgatttc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rhlR‑verify‑3P <400> 22 gtgggttgaa agcaagtcct 20 <210> 23 <211> 11911 <212> DNA <213> Artificial Sequence <220> <223> pCASTKP <400> 23 tgtaaaacga cggccagtga attgacgcgt attgggatgg taccgggccc cccctcgagg 60 tcgacggtat cgatacggca tcagagcaga ttgtactgag agtgcaccat aatcggcatt 120 ttcttttgcg tttttatttg ttaactgtta attgtccttg ttcaaggatg ctgtctttga 180 caacagatgt tttcttgcct ttgatgttca gcaggaagct aggcgcaaac gttgattgtt 240 tgtctgcgta gaatcctctg tttgtcatat agcttgtaat cacgacattg tttcctttcg 300 cttgaggtac agcgaagtgt gagtaagtaa aggttacatc gttaggatca agatccattt 360 ttaacacaag gccagttttg ttcagcggct tgtatgggcc agttaaagaa ttagaaacat 420 aaccaagcat gtaaatatcg ttagacgtaa tgccgtcaat cgtcattttt gatccgcggg 480 agtcagtgaa cagataccat ttgccgttca ttttaaagac gttcgcgcgt tcaatttcat 540 ctgttactgt gttagatgca atcagcggtt tcatcacttt tttcagtgtg taatcatcgt 600 ttagctcaat cataccgaga gcgccgtttg ctaactcagc cgtgcgtttt ttatcgcttt 660 gcagaagttt ttgactttct tgacggaaga atgatgtgct tttgccatag tatgctttgt 720 taaataaaga ttcttcgcct tggtagccat cttcagttcc agtgtttgct tcaaatacta 780 agtatttgtg gcctttatct tctacgtagt gaggatctct cagcgtatgg ttgtcgcctg 840 agctgtagtt gccttcatcg atgaactgct gtacattttg atacgttttt ccgtcaccgt 900 caaagattga tttataatcc tctacaccgt tgatgttcaa agagctgtct gatgctgata 960 cgttaacttg tgcagttgtc agtgtttgtt tgccgtaatg tttaccggag aaatcagtgt 1020 agaataaacg gatttttccg tcagatgtaa atgtggctga acctgaccat tcttgtgttt 1080 ggtcttttag gatagaatca tttgcatcga atttgtcgct gtctttaaag acgcggccag 1140 cgtttttcca gctgtcaata gaagtttcgc cgactttttg atagaacatg taaatcgatg 1200 tgtcatccgc atttttagga tctccggcta atgcaaagac gatgtggtag ccgtgatagt 1260 ttgcgacagt gccgtcagcg ttttgtaatg gccagctgtc ccaaacgtcc aggccttttg 1320 cagaagagat atttttaatt gtggacgaat cgaactcagg aacttgatat ttttcatttt 1380 tttgctgttc agggatttgc agcatatcat ggcgtgtaat atgggaaatg ccgtatgttt 1440 ccttatatgg cttttggttc gttctttcg caaacgcttg agttgcgcct cctgccagca 1500 gtgcggtagt aaaggttaat actgttgctt gttttgcaaa ctttttgatg ttcatcgttc 1560 atgtctcctt tttatgtac tgtgttagcg gtctgcttct tccagccctc ctgtttgaag 1620 atggcaagtt agttacgcac aataaaaaaa gacctaaaat atgtaagggg tgacgccaaa 1680 gtatacactt tgccctttac acattttagg tcttgcctgc tttatcagta acaaacccgc 1740 gcgatttact ttcgacctc attctattag actctcgttt ggattgcaac tggtctattt 1800 tcctctttg tttgatagaa aatcataaaa ggatttgcag actacgggcc taaagaacta 1860 aaaaatctat ctgtttcttt tcattctctg tattttttat agtttctgtt gcatgggcat 1920 aaagttgcaa gcttgcaaac atgctttggg taaacatgta ggtattaacg tcaatgcgac 1980 atagtagca ttgattaaat gagaattagt aagtgtttta cttactaaat tttatttaac 2040 ctaaaaatga accacctgga tgtgtgggat taaaaagtga agagaggagg acatatcaca 2100 tgaacagtca gcaaaatcct gatttagctg ttcatccctt ggcaattcct atggaaggct 2160 tactaggaga aagtgctaca actcttgaga agaatgtaat tgccacacaa ctctcagagg 2220 aagcccaagt aaagctagag gtaatccaaa gtttactgga accctgcgat cgcacaactt 2280 atgggcaaaa gttgcgggaa gcagcagaga aactaaatgt atcgttgcga acggtacaaa 2340 ggttggtgaa aaactgggaa caagatggct tagtcggact cactcaaaca agtagggctg 2400 ataaaggaaa acaccgcatt ggtgagtttt gggaaaactt cattaccaaa acctacaagg 2460 agggtaacaa gggaagtaaa cgtatgaccc ctaaacaagt tgctctcaga gtcgaggcta 2520 aagcccgtga attaaaagac tctaagccgc ccaattacaa aaccgtgtta cgggtattag 2580 cacccatttt ggaaaagcaa caaaaagcca agagtatccg cagtcctggt tggagaggaa 2640 ctacgctttc ggttaaaacc cgtgaaggaa aagatttatc ggttgattac agtaaccatg 2700 tttggcaatg tgaccatacc cgcgtggatg tgttgctggt agatcaacat ggtgaaattt 2760 taagtcgtcc ctggctaaca acagtaattg atacttactc tcgttgcatt atgggtatca 2820 acttgggctt tgatgcaccc agttctgggg tagtagcatt agcgttacgc catgcaattc 2880 taccaaagcg ttacggttcc gagtacaaac tgcattgtga gtggggaacc tatggaaaac 2940 cagaacattt ttatactgat ggcggtaaag actttcgctc taaccacttg agtcagattg 3000 gggcgcaatt gggatttgtc tgtcatttac gcgatcgccc ttctgaaggt ggagtagtag 3060 aacgtccctt caaaacatta aatgaccaac tattttcaac gcttcctggg tacaccggat 3120 ctaatgtgca ggaacgccca gaagatgcag agaaggacgc aagacttact ttgcgagaac 3180 tagaacagtt acttgtgcgt tacatcgtag atcgttacaa ccaaagtatt gatgcgcgga 3240 tgggcgacca aacgcgcttt gagcgttggg aagcaggatt gcctacagtg ccagtaccaa 3300 taccagaacg agatttggat atttgtttaa tgaagcagtc acggcgcact gtgcaaagag 3360 gtggttgttt gcagtttcag aatttaatgt atcgggggga atatttggca ggttatgccg 3420 gagaaactgt caacttaagg tttgacccca gagacattac aacaattttg gtttatcgcc 3480 aggaaaacaa tcaggaagta tttctgactc gcgctcacgc tcaaggtttg gagacagagc 3540 aactggcatt agatgaggct gaggcagcaa gtcgcagact ccgtaccgca gggaaaacta 3600 tcagtaacca atcattattg caagaagttg ttgaccgcga tgctcttgtc gctaccaaga 3660 aaagccgtaa ggagcgtcaa aaattggaac agactgtttt gcgatctgct gctgttgatg 3720 aaagtaatag agaatccttg ccttctcaaa tagttgaacc agatgaagtg gaatctacag 3780 aaacggttca ctctcaatac gaagacattg aggtgtggga ctatgaacaa cttcgtgaag 3840 aatatgggtt ttaaacaatg acagaagctc aggcgatcgc caagcagttg ggtggggtaa 3900 aaccggatga tgagtggtta caagctgaaa ttgctcgtct caagggtaag agcattgtgc 3960 ctttacagca ggtaaaaact ctccatgatt ggttagatgg caagcgcaag gcaagaaaat 4020 cttgccgagt agttggggaa tcgagaactg gcaagacagt tgcttgtgat gcctacagat 4080 acaggcacaa acctcagcag gaagctggac gacctccaac tgtgcctgtc gtttatattc 4140 gacctcacca aaaatgtggc cccaaggatt tgtttaaaaa gattactgag tacctcaagt 4200 atcgggtaac aaaagggact gtatctgatt ttcgagatag gacgatagaa gtactcaagg 4260 gttgtggcgt agagatgcta attattgatg aagctgaccg tctcaagcct gaaacttttg 4320 ctgatgtgcg agatattgcc gaagatttag gaattgctgt ggtactggta ggaacagacc 4380 gttggatgc ggtaattaag cgggatgagc aggttctcga acgctttcgg gcgcatcttc 4440 gctttggtaa attgtcggga gaggatttta agaacaccgt agaaatgtgg gaacaaatgg 4500 ttttgaaact gccagtatct tctaatctaa agagcaagga gatgctacgg attctcacgt 4560 cagcaactga aggctacatt ggtcgccttg atgagattct tagggaagct gcaattcgtt 4620 ccttatcaag aggattgaag aagattgaca aggctgtttt acaggaagta gctaaggagt 4680 acaaatgata gaagcaccag atgttaaacc ttggctattc ttgattaaac cctatgaagg 4740 ggaaagcctg agccactttc ttggcaggtt cagacgtgcc aaccatttat ccgcaagtgg 4800 attgggtact ttggcaggaa ttggtgctat agtggcacgt tgggaaagat ttcattttaa 4860 tcctcgccct agtcagcaag aattggaagc gatcgcatct gtagtagaag tggatgctca 4920 aaggttagcc cagatgttac cgcctgctgg agtgggaatg cagcatgagc caattcgctt 4980 gtgtggggct tgttatgccg agtcgccttg tcaccgaatt gaatggcagt acaagtcggt 5040 gtggaagtgc gatcgccatc aactcaagat tttagcaaag tgtccaaact gtcaagcacc 5100 ttttaaaatg cctgcgctgt gggaggatgg gtgctgtcac agatgtagga tgccgtttgc 5160 agaaatggca aagctacaga aggtttgatg ataaaaccag aaaaaggtgt gaaattaact 5220 aagtccctga attgatctgg ttgtccaaaa aatttgtgcg atcgcatggc aagattattc 5280 ctactaggag aacgagatga cgttggaggg gcaaggtcgc gctgattgct ggggcaacac 5340 gtgaaaggcg agatcaccaa ggtagtcggc aaataatgtc taacggtacc cggggatcct 5400 ctagagtcga cctgcataat gtgcctgtca aatggacgaa gcagggattc tgcaaaccct 5460 atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca acttgacggc 5520 tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg ccccggtgca 5580 ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc gaccgacggt 5640 ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac gttggtcctc 5700 gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca gacgcgacgg 5760 cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc 5820 gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga gcgactcgtt 5880 aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca gcagctccga 5940 atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg 6000 ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg gccagttaag 6060 ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc attcgcgagc 6120 ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa tatcacccgg 6180 tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa tggtgagatt 6240 gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc 6300 ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg gcagcagggg 6360 atcattttgc gcttcagcca tacttttcat actcccgcca ttcagagaag aaaccaattg 6420 tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct cgctaaccaa 6480 accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag ccatgacaaa 6540 aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt atttgcacgg 6600 cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc ctacctgacg 6660 ctttttatcg caactctcta ctgtttctcc atacccgttt ttttgggcta gcgaaaagat 6720 gtttcgtgaa gccgtcgacg cttataaaaa atgagccaga tcaccatcca ggctcgtctg 6780 atcagctttg aatctaaccg ccagcagctg tggaaactga tggctgatct gaacaccccg 6840 ctgattaacg aactgctgtg tcagctgggc cagcacccgg actttgaaaa atggcagcag 6900 aaaggtaaac tgccgagcac cgtggtttct cagctgtgcc agccactgaa aactgacccg 6960 cgtttcgcgg gtcagccgag ccgtctgtac atgtctgcaa ttcacatcgt tgattacatc 7020 tataaatcct ggctggcgat ccagaaacgc ctgcaacagc agctggatgg caaaacccgt 7080 tggctggaaa tgctgaacag cgatgcagaa ctggttgaac tgtctggtga tactctggaa 7140 gcgattcgcg tcaaagcggc tgaaatcttg gctatcgcca tgccggcaag cgaatccgat 7200 tctgcatctc cgaaaggtaa aaaggcaaa aaagagaaaa aaccgagcag cagctccccg 7260 aaacgcagtc tttctaaaac cctgtttgac gcgtaccagg aaaccgaaga tatcaaatct 7320 cgtagcgcga tctcttatct gctgaaaaac ggctgtaaac tgactgataa agaagaagat 7380 agcgaaaaat ttgctaagcg ccgtcgtcag gtggagattc agattcagcg tctgaccgaa 7440 aaactgatct ctcgtatgcc gaaaggtcgc gacttgacca acgctaaatg gcttgaaacc 7500 ctgctgaccg cgactactac ggtggcagaa gataatgcgc aggcaaaacg ttggcaggat 7560 atcctcctga cccgtagctc ttccctgccg ttcccgctgg tatttgaaac caacgaagac 7620 atggtgtggt ctaaaaacca gaaaggccgt ctgtgcgttc acttcaacgg cctgagcgat 7680 ctgatcttcg aagtgtactg tggcaaccgc cagctccact ggttccagcg tttcctggaa 7740 gaccagcaga ccaaacgcaa aagcaaaaac cagcactcaa gcggcctgtt cactctgcgt 7800 aacggtcacc tggtgtggct ggaaggcgaa ggtaaaggtg aaccgtggaa cctgcaccac 7860 ctgaccctgt actgctgcgt ggacaaccgt ctgtggaccg aagaaggcac cgaaatcgtc 7920 cgtcaggaaa aagcagacga aatcaccaaa tttatcacca acatgaaaaa gaaatctgat 7980 ctgagcgaca cccaacaggc tctgattcag cgcaaacagt ctaccctgac ccgtattaat 8040 aactctttcg aacgccccgtc ccagccgctg taccagggcc agagccacat cctggtcggt 8100 gtttctctgg gcctggaaaa accggctacc gttgccgttg tcgatgccat tgcaaacaaa 8160 gttctggctt accgttccat caaacagctg ctgggcgata actatgaact gctgaaccgt 8220 cagcgtcgcc agcagcagta cctgagccac gaacgtcaca aagcgcagaa aaatttctct 8280 ccgaaccaat tcggcgcaag cgaactgggc cagcacatcg atcgtctgct ggccaaagcg 8340 atcgttgcgc tggcgcgtac ctataaagcc ggctctatcg tcctgccgaa actgggcgac 8400 atgcgtgaag tggttcagtc tgaaatccag gcgatcgctg aacagaaatt tccgggttat 8460 attgaaggtc agcagaaata cgcgaaacag taccgcgtga acgttcaccg ttggagctac 8520 ggtcgtctga ttcagtccat ccagtctaaa gcagcgcaga ctggcatcgt gattgaagaa 8580 ggcaaacagc cgatccgtgg ctccccgcac gataaagcga aagaactggc gctgagcgca 8640 tacaacctgc gtctgacccg ccgttcctaa caaatatctg aaccttgata atagaatatt 8700 aatagcgccg caattcatgc tgcttgcagc ctctgaattt tgttaaatga gggttagttt 8760 gactgtataa atgatatcga attcctgcag cccgggggat ccactagttc tagagcggcc 8820 gccaccgcgg tggagctcat cccaatggcg cgccgagctt ggctcgagca tggtcatagc 8880 tgtttcctgc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac 8940 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc 9000 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag 9060 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg 9120 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag 9180 gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg 9240 acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag 9300 cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc 9360 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg 9420 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta 9480 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc 9540 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc 9600 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac 9660 acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga 9720 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac 9780 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc 9840 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 9900 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 9960 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 10020 cggcatcaga gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag 10080 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct 10140 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 10200 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 10260 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 10320 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 10380 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 10440 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 10500 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 10560 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 10620 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 10680 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 10740 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 10800 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 10860 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 10920 gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca 10980 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc 11040 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg 11100 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt 11160 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa 11220 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg 11280 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat 11340 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt 11400 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc 11460 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct 11520 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag 11580 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag 11640 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat 11700 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta 11760 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag 11820 attaattca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 11880 ccgcgcacat ttccccgaaa agtgccacct g 11911 <210> 24 <211> 5304 <212> DNA <213> Artificial Sequence <220> <223> pGTKP <400> 24 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgcatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatggtac 420 cgggcccccc ctcgaggtcg acggtatcga tacggcatca gagcagattg tactgagagtg 480 gcaccataat cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc 540 areatgctg tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctagg 600 cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac 660 gacattgttt cctttcgctt gaggtacagc gaagtgtgag tagtaagg ttacatcgtt 720 aggatcaaga tccatttta acacaaggcc agttttgttc agcggcttgt atgggccagt 780 taaagaatta gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt 840 catttttgat ccgcgggagt cagtgaacag ataccatttg ccgttcattt taaagacgtt 900 cgcgcgttca atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt 960 cagtgtgtaa tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt 1020 gcgtttttta tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt 1080 gccatagtat gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt 1140 gtttgcttca aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag 1200 cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata 1260 cgtttttccg tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga 1320 gctgtctgat gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt 1380 accggagaaa tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc 1440 tgaccattct tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc 1500 tttaaagacg cggccagcgt ttttccagct gtcaatagaa gtttcgccga cttttgata 1560 gaacatgtaa atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat 1620 gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca 1680 aacgtccagg ccttttgcag aagagatatt tttaattgtg gacgaatcga actcaggaac 1740 ttgatatttt tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg 1800 ggaaatgccg tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt 1860 tgcgcctcct gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt 1920 tttgatgttc atcgttcatg tctcctttt tatgtactgt gttagcggtc tgcttcttc 1980 agccctcctg tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg 2040 taaggggtga cgccaaagta tacactttgc cctttacaca ttttaggtct tgcctgcttt 2100 atcagtaaca aacccgcgcg attactttt cgacctcatt ctattagact ctcgtttgga 2160 ttgcaactgg tctattttcc tcttttgttt gatagaaaat cataaaagga tttgcagact 2220 acgggcctaa agaactaaaa aatctatctg tttctttca ttctctgtat tttttatagt 2280 ttctgttgca tgggcataaa gttgcaagct tgatatcttg acagctagct cagtcctagg 2340 tataatacta gtatattaat agcgccgcaa ttcatgctgc ttgcagcctc tgaatttgtgt 2400 taaatgaggg ttagtttgac tgtataaata cagtcttgct ttctgaccct ggtagctgct 2460 caccctgatg ctgctgtcaa tagacaggat aggtgcgctc ccagcaataa gggcgcggat 2520 gtactgctgt agtggctact gaatcacccc cgatcaaggg ggaaccctcc aaaaggtggg 2580 ttgaaagaga ccattggtct cattttttta gccgtgatag tttgcgacag atatcgaatt 2640 cctgcagccc gggggatcca ctagttgtac agtgactaat tatatgtcgt tgtgacaaat 2700 tattgtcatc agtaaaatcc ttatacagta tagattatag cgctttggca gttttagcat 2760 aacctcttg cagtgacaaa atagatgtcg ttgtccgtga ttgtgacaaa ttagctgtcg 2820 ctttgcaaga taggaaaaag cttttgtgta ttttcataat gacaaattga ctgtcgcctc 2880 gagccaagct cggcgcgcca ttgggatgag ctccaccgcg gtggcggccg ctctagatca 2940 gtaactatta aacttagggg tgggttgaaa gcaagtcctt ttatccgctt gttttaattg 3000 ctttgtataa taattgcaga gcatattata ttgatgacat ttaatttgtc atcaattaat 3060 taagcaacgc tgatgggtca cgacgacaat taaatagtca caatgacatt aatctgtcac 3120 cgacgacaga taatttgtca ctgtacacat ggtcatagct gtttcctgtg tgaaattgtt 3180 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 3240 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 3300 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 3360 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3420 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3480 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3540 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3600 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3660 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgccttc 3720 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 3780 aggtcgtcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3840 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3900 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3960 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 4020 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 4080 ctggtagcgg tggtttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 4140 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 4200 aagggatttt ggtcatgaga tttcaaaaa ggatcttcac ctagatcctt ttaaattaaa 4260 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttattgc 4320 cgactacctt ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc 4380 gcgaggccaa gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg 4440 gctgatactg ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga 4500 tttgccggt tactgcgctg taccaaatgc gggacaacgt aagcactaca ttcgctcat 4560 cgccagccca gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata 4620 gatcctgttc aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc 4680 tatgttctct tgctttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga 4740 agatacctgc aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg 4800 gataacgcca cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa 4860 tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg 4920 ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca 4980 ggccgccatc cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc 5040 gctcgatgac gccaactacc tctgatagtt gagcgatac ttcggcgatc accgcttccc 5100 ccatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 5160 gatacatatt tgaatgtatt tagaaaata aaaatagg ggttccgcgc acatttcccc 5220 gaaaagtgcc acctgacgtc windowaacca ttatcat gatacacc tataaaaata 5280 ggcgtatcac gaggcccttt cgtc 5304

Claims

1. A gene transposition system for transposing the bacterial genome, comprising: (1) An expression construct 1 containing genes encoding Cas12k, TnsB, TnsC and TniQ proteins; (2) An expression construct 2 comprising a transposon sequence corresponding to the Cas12k protein, a guide RNA backbone sequence, and a targeting sequence for different target sequences; The expression construct 1 comprises the sequence shown in SEQ ID NO: 7, and the expression construct 2 comprises a backbone sequence shown in SEQ ID NO: 14, and a targeting sequence added to the 3' end of the guide RNA backbone sequence in the backbone sequence shown in SEQ ID NO: 14, wherein the guide RNA backbone sequence is shown in SEQ ID NO: 5; the bacteria is Pseudomonas aeruginosa.

2. A method for site-directed gene transposition / mutation, characterized in that, The method includes introducing the gene transposon system as described in claim 1 into bacteria containing the target sequence for genome editing; the bacteria is Pseudomonas aeruginosa.

3. The method as described in claim 2, characterized in that, The method also includes purifying the genome-edited bacteria by screening with antibiotics and sucrose to obtain homozygous bacteria with complete transposon mutations.

4. A method for constructing a bacterial gene mutation library, characterized in that, The method includes: (1) Obtain transcriptional regulatory factors from bacterial genomes and select target sequences based on them; (2) Construct different expression constructs 2 as described in claim 1 based on different target sequences; (3) The different expression constructs 2 obtained in step (2) and the expression construct 1 described in claim 1 are transferred into competent bacterial cells to obtain a bacterial gene mutation library; The bacteria in question is Pseudomonas aeruginosa.

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