阅读说明：本技术 一种安全无毒的高通量无缝构建质粒的方法 (Safe and nontoxic high-throughput seamless plasmid construction method ) 是由 刘延峰 张晓龙 董晓敏 刘龙 吕雪芹 堵国成 李江华 陈坚 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种安全无毒的高通量无缝构建质粒的方法,属于遗传工程领域。本发明通过优化组装溶液成分,排除现有方法中存在有毒试剂二硫苏糖醇,以及DNA聚合酶、DNA连接酶、NAD等较昂贵的试剂。由于不需要DNA纯化操作,使得该方法适用于基于96孔PCR板或96孔酶标板等的高通量操作平台。同时,太极组装4个片段构建质粒时,拥有95％以上的阳性率,在高通量构建质粒实验中,可以省略菌落PCR验证步骤,进一步简化高通量质粒构建流程。(The invention discloses a safe and nontoxic method for seamlessly constructing plasmids with high flux, belonging to the field of genetic engineering. The invention eliminates the toxic reagent dithiothreitol and expensive reagents such as DNA polymerase, DNA ligase, NAD and the like in the prior method by optimizing the components of the assembly solution. The method is suitable for a high-throughput operation platform based on a 96-well PCR plate or a 96-well enzyme label plate and the like because the DNA purification operation is not required. Meanwhile, when the plasmid is constructed by assembling 4 fragments by Taiji, the positive rate of more than 95 percent is obtained, and in a high-throughput plasmid construction experiment, a colony PCR verification step can be omitted, so that the high-throughput plasmid construction process is further simplified.)

1. A method for constructing plasmids is characterized in that DNA fragments are added into a reaction system containing magnesium ions, PEG8000, exonuclease and Tris-HCl to react to obtain recombinant plasmids.

2. The method according to claim 1, wherein the DNA fragment is obtained by high fidelity enzymatic amplification, and the PCR product containing the DNA fragment obtained by amplification is added to the reaction system in an amount of 5 to 50% by volume.

3. The method according to claim 2, wherein the concentration of magnesium chloride in the reaction system is 0.2-4.0 g/L.

4. The method according to claim 3, wherein the concentration of PEG8000 in the reaction system is 0-80 g/L; the exonuclease comprises but is not limited to T5 exonuclease, and the concentration of the T5 is 0.01-0.5 mu L/mL; the pH value of the Tris-HCl is 7.0-8.0, and the concentration is 40-140 mL/L.

5. The method of claim 4, wherein the reaction is carried out at 30 to 48 ℃ for 10 to 60 min.

6. The method according to claim 4, wherein the number of the DNA fragments is not less than 2, and the volume ratio of any two DNA fragments is 1 (1-10).

7. The method of claim 6, wherein the PCR products contain overlapping sequences, each overlapping sequence has a length of not less than 8 bases, and the GC content is 10-80%.

8. A kit, which is characterized by comprising PEG8000, magnesium chloride, T5 exonuclease and Tris-HCl.

9. The kit of claim 8, wherein the working concentration of PEG8000 is 0-80 g/L, the working concentration of magnesium chloride is 0.2-4.0 g/L, the working concentration of T5 exonuclease is 0.01-0.5 μ L/mL, and the working concentration of Tris-HCl is 40-140 mL/L.

10. The method of claims 1 to 7, or the use of the kit of claim 8 or 9 in high throughput screening.

Technical Field

The invention relates to a safe and nontoxic method for seamlessly constructing plasmids with high flux, belonging to the field of genetic engineering.

Background

The rapid, simple and convenient seamless construction of plasmids is the basis of molecular biology, and along with the demand of molecular biology research on a large amount of data, a high-throughput seamless construction plasmid technology platform becomes the key for promoting the field progress. Since the discovery of the first restriction enzyme and the development of a plasmid construction method based on enzyme digestion ligation, with the continuous development of technologies, three mainstream plasmid construction methods are currently formed, including Gibson assembly (Gibson assembly), gold gate assembly (Golden gate assembly) and saccharomyces cerevisiae in vivo assembly. Gold gate assembly based on type IIS restriction endonucleases can be used to assemble multiple fragments comprising repeated sequences. The endogenous recombinase-based saccharomyces cerevisiae assembly method is suitable for assembling a plurality of large fragments. However, the gold-gate assembly requires the processes of digestion, ligation, purification, etc., and is complicated and costly. Saccharomyces cerevisiae in vivo assembly is not suitable for high-throughput plasmid construction due to the need of a transformation method specific to Saccharomyces cerevisiae, and its slow growth. The Gibbson assembly method reported in 2009 is the most widely used method for conventional gene cloning due to its simplicity and high efficiency (DOI: 10.1038/nmeth.1318).

The Gibbson assembly method consists of T5 exonuclease, DNA polymerase and DNA ligase, as well as Tris-HCl, magnesium ions, PEG8000 and DTT (dithiothreitol). Magnesium ions, Tris-HCl and PEG8000 provide the enzyme reaction conditions, and DTT as a strong reducing agent provides the reducing environment. Firstly, cutting double-stranded DNA by T5 exonuclease to obtain 3 'end single-stranded end, then repairing the gap of the 3' end single-stranded end between different fragments by DNA polymerase, and finally performing ligation by DNA ligase. Among them, the T5 exonuclease is the core of the Gibbson assembly method, and the plasmid construction based on the 3' end single-stranded end annealing of the enzyme provides extremely high positive rate (more than 90%), and all methods based on the T5 exonuclease optimization have the positive rate of about 90% or more. Due to the high efficiency and simplicity of the gibson assembly method, after 2009, there have been many further studies. In 2014, researches show that after the DNA Ligase with the highest cost is removed, the Gibson assembly efficiency is not influenced, and the Method is named as Hot Fusion, so that the cost is greatly reduced by about 90 percent, but the Method needs dNTP, NAD and the like in a reaction solution, so that the configuration process is complicated, and a toxic reagent DTT (Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as introduced wastes with out library) needs to be added. In addition, the assembly method adopted in patent application of exonuclease and method of seamless cloning of CN 107760706a, and the assembly method named TEDA in patent application of CN 108841901a, a kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 and application thereof, are all assembly methods by further removing DNA polymerase on the basis of Hot Fusion. However, 2016 (research) showed that the removal of DNA polymerase resulted in a significant decrease in the Assembly Efficiency of two DNA fragments of about 70% ("Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost"). The results of this patent further show that when 4 DNA fragments are assembled, removal of the DNA polymerase results in a decrease of the assembly efficiency of about 90% (fig. 2). Thus, DNA polymerases are based on key non-removable components of the T5 exonuclease assembly method.

At present, the Gibbson assembly method and the Hot Fusion method have higher assembly efficiency due to the DNA polymerase, but have higher cost and complex configuration process; the TEDA assembly method and the assembly method mentioned in patent publication No. CN 107760706a have a low cost because they do not contain DNA polymerase, but have a low assembly efficiency, and therefore, the assembly method based on T5 exonuclease has a problem that the cost and the assembly efficiency are difficult to unify. Secondly, DTT is required to be added into the reaction solution of the 4 assembling methods, the price of DTT is high, and the irritant odor and toxicity have biological potential safety hazards. Thirdly, the above 4 assembly methods all require purification of DNA fragments to remove substances such as enzymes or primers (DNA purification is required before DNA concentration is determined), the purification process not only increases the cost, but also requires steps such as centrifugation, and the like, which is difficult to meet the requirement of high-throughput automation platform for automated plasmid construction. Fourthly, the above 4 assembly methods all need to adjust the concentration of each fragment within a proper range to obtain high assembly efficiency, additionally need a nucleic acid quantitative instrument with higher price, and need tedious concentration calculation and pipetting operation, which is not favorable for meeting the requirement of constructing plasmids with high flux. However, no method for effectively solving the four problems exists at present, and time and resources are wasted for constructing plasmids with high flux.

Disclosure of Invention

In order to solve four limitations of the existing plasmid construction method, the invention aims to provide a safe and nontoxic high-flux seamless plasmid construction method (named Taiji assembly, Tai Chi assembly) based on T5 exonuclease on the basis of a Gibbson assembly method, which does not contain toxic substance DTT, does not need a DNA purification process, has low cost and high efficiency, and is suitable for high-flux seamless plasmid construction. First, existing studies have shown that DNA polymerases are key to improving assembly efficiency, but also result in higher cost and complex reaction solutions. Therefore, the invention provides a novel idea of repairing a gap generated by the T5 exonuclease by using the DNA polymerase in the PCR reaction system, which not only avoids the high cost and the complex configuration process caused by adding the DNA polymerase in the reaction solution, but also can omit the DNA purification process, further reduce the cost and simplify the operation process. Secondly, in order to remove toxic DTT in the reaction solution, by optimizing different formulas, not only the highest current multi-assembly efficiency is realized, but also the complex Gibbson reaction system is simplified to only need T5 exonuclease, magnesium chloride, PEG and Tris-HCl, 4 components, and the most simplified Gibbson optimization system TEDA still needs at least 5 components (containing toxic DTT). The Taiji assembly can realize high-efficiency assembly efficiency without optimizing the addition amount and the addition proportion of fragments, and obviously reduces the operation difficulty. Since no DNA purification operation is needed, the method is suitable for a high-throughput operation platform based on a 96-well PCR plate or a 96-well enzyme label plate and the like, and the high-throughput plasmid construction scheme suitable for Taiji assembly is optimized (figure 8).

The invention provides a method for constructing plasmids, which comprises the steps of adding a DNA fragment into a reaction system containing magnesium ions, PEG8000, exonuclease and Tris-HCl, and reacting to obtain recombinant plasmids.

In one embodiment, the DNA fragment is obtained by high fidelity enzymatic amplification, and the PCR product containing the DNA fragment obtained by amplification is added to the reaction system in an amount of 5-50% by volume.

In one embodiment, the amount of PCR product added is preferably 20%.

In one embodiment, the concentration of magnesium chloride in the reaction system is 0.2-4.0 g/L; the concentration of magnesium chloride is preferably 4.0 g/L.

In one embodiment, the concentration of PEG8000 in the reaction system is 0-80 g/L; the concentration of PEG8000 is preferably 40 g/L.

In one embodiment, the exonuclease includes but is not limited to T5 exonuclease, and the concentration of the T5 is 0.01-0.5 μ L/mL; the concentration of T5 is preferably 0.15. mu.L/L.

In one embodiment, the Tris-HCl has a pH of 7.0 to 8.0 and a concentration of 1M; the concentration is 40-140 mL/L; preferably, Tris-HCl has a pH of 7.0, 7.5 or 8.0 and a concentration of 80 mL/L.

In one embodiment, the reaction is carried out at 30-48 ℃ for 10-60 min.

In one embodiment, the number of the DNA fragments is not less than 2, and the volume ratio of any two DNA fragments is 1 (1-10).

In one embodiment, the PCR products contain overlapping sequences therebetween, each overlapping sequence having a length of not less than 8 bases and a GC content of 10% to 50%.

In one embodiment, magnesium chloride, PCEG8000, T5 exonuclease, and Tris-HCl are configured as desired into 2 ×, 3 ×, 4 ×, etc. different mother liquors.

In one embodiment, the constructed recombinant plasmid is transformed into escherichia coli, and the recombinant escherichia coli containing the recombinant plasmid is obtained.

In one embodiment, the escherichia coli includes, but is not limited to, JM109, JM110, MG1655, Top10, BL21, BL21, and DH5 α.

The invention provides a kit, which contains PEG8000, magnesium chloride, T5 exonuclease and Tris-HCl.

In one embodiment, the working concentration of PEG8000 is 0-80 g/L, the working concentration of magnesium chloride is 0.2-4.0 g/L, the working concentration of T5 exonuclease is 0.01-0.5 muL/mL, and the working concentration of Tris-HCl is 40-140 muL/L.

In one embodiment, the PEG8000 concentration is preferably 40g/L, the magnesium chloride concentration is preferably 4.0g/L, the T5 exonuclease concentration is preferably 0.15 μ L/L, and the Tris-HCl concentration is preferably 80 mL/L.

In one embodiment, the Tris-HCl has a pH of 7.0, 7.5, or 8.0.

The invention provides application of the method in high-throughput screening.

The invention provides application of the kit in high-throughput screening.

The invention has the beneficial effects that:

1. toxic substance DTT with pungent smell is not required to be added into the Tai Chi assembly reaction solution, so that potential safety hazards are avoided;

2. the Taiji assembly does not need DNA fragment purification, simplifies the operation process and reduces the cost, and is the most convenient and lowest-cost plasmid construction method at present;

3. the positive rate of Taiji assembly is consistent with the method reported at present, but the assembly efficiency is slightly higher than Gibbson assembly and is far higher than other Gibbson simplified methods;

4. the Tai Chi assembly can realize the one-time assembly of 12 segments;

5. the Taiji assembly can realize high-flux plasmid construction based on a 96-well PCR plate and a 96-shallow-well plate;

6. the Taiji assembly can obtain high assembly efficiency without adjusting the adding proportion of each fragment, and omits complicated processes such as DNA concentration determination, molar concentration calculation and the like.

Drawings

FIG. 1 is a schematic view of a Tai Chi assembly.

FIG. 2 is a graph comparing the efficiency of a Tai Chi assembly and a current similar method.

FIG. 3 is a graph of the effect of different components on the efficiency of assembly of 4 fragments.

FIG. 4 is a diagram of optimization of reaction conditions for Tai Chi assembly.

FIG. 5 is a graph of the effect of products amplified by different PCR enzymes on assembly efficiency; (A) the PCR enzyme in (A) is ApexHF HS DNA Polymerase FS Master Mix, and the PCR enzyme in (B) is 2 Xsuper Pfx Master Mix.

Figure 6 is a graph of the effect of optimization of 95 different tai chi assembly formulations on assembly efficiency.

FIG. 7 is a graph of the effect of different hosts, different numbers of fragments, different overlapping sequences on the efficiency of Tai chi assembly; .

FIG. 8 is a flow chart of the Tai Chi assembly operation for a high throughput platform.

Detailed Description

Strains E.coli JM109, E.coli JM110, E.coli MG1655, E.coli Top10, E.coli BL21 and E.coli DH5 alpha used in the present invention are all commercial strains.

1M Tris-HCl(pH7.5)、MgCl₂·6H₂O, 100mM dNTP, PEG8000, DTT, 100mM NAD were purchased from Biotechnology engineering (Shanghai) Inc.

Taq DNA ligase (40U/. mu.L), T5 exouchase (10U/. mu.L), and Phusion polymerase (2U/. mu.L) were purchased from NEB (Beijing) Ltd.

The PCR enzyme ApexHF HS DNA Polymerase FS Master Mix (Esciurel, AG12202, China); the PCR enzyme 2 Xsuper Pfx MasterMix (Kangshi, CW2965, China). PCR enzymes PrimeSTAR Max DNApolymerase (Takara, R045B, Japan), all PCR fragments were obtained using PrimeSTAR Max DNA Polymerase amplification in the examples except for the specific instructions.

TABLE 1 plasmids of the invention

TABLE 2 homologous overlap sequences for different GC content and Length validation experiments

TABLE 32-12 overlapping sequences for fragment validation experiments

TABLE 4 Tai Chi Assembly 95 different formulation profiles

Example 1 optimization of Tai Chi Assembly solution component concentrations

1. Initial taiji assembly reaction solution preparation in this example:

adding 1.0g MgCl into a 15mL test tube₂·6H₂O, diluted to 10mL to obtain MgCl₂·6H₂O solution; 1.0g of DTT was added to a 15mL tube and diluted to 10mL to obtain a DTT solution；

② adding 1.5g PEG8000 and 0.5mL MgCl into a 50mL tube₂·6H₂O solution and 2.5mL Tris-HCl (1M, pH7.5), water was added to make up to 12.5mL, dissolved at 60 ℃, cooled in an ice bath and then 0.5mL DTT solution was added. Shaking, packaging into 4.0mL/5.0mL EP tube to obtain assembly buffer solution, and storing at-20 deg.C;

③ to 4.0mL of the assembly buffer, 1.6. mu. L T5 was added, shaken well, and then dispensed into 10. mu.L/PCR tubes and stored at-20 ℃ (2 XTai Chi Mix).

2. Taiji assembling seamless cloning process:

taking sequences 1-4 (nucleotide sequences are respectively shown as SEQ ID NO. 1-4, overlapping sequences at joints are shown as table 3), assembling 4 DNA fragments into a plasmid as a research object, adding a total of 4 muL of unpurified DNA mixed fragments and 6 muL of water (the nucleotide sequences are shown as SEQ ID NO. 1-4, the volume ratio of the 4 fragments is 1:1:1:1) into a Tai Chi Mix (10 muL/PCR tube), and shaking up;

② reacting for 60 minutes at 40 ℃ in a PCR instrument;

③ after 5 minutes of ice bath, transforming the Escherichia coli DH5 alpha, coating the transformation liquid on an LB culture medium, and culturing for 12-14 hours.

3. And (3) detection of positive rate:

colony PCR is carried out by designing specific primers, and the positive rate is calculated.

4. Effect of different reaction conditions on Tai Chi Assembly efficiency

(1) According to the mode, under the condition that other components are not changed, the concentration of magnesium chloride in the 2 xTai Chi Mix is adjusted to be 0-12.0 g/L, and Mg is verified²⁺Impact on the efficiency of the tai chi assembly.

The results are shown in FIG. 3A, which shows Mg²⁺The influence on the assembly efficiency is obvious, and the optimal concentration is 8.0 g/L.

(2) According to the mode, under the condition that other components are not changed, the concentration of PEG8000 in the 2 xTai Chi Mix is set to be 0-200 g/L respectively, and the influence of the concentration of PEG8000 on the Tai Chi assembly efficiency is verified.

The results are shown in FIG. 3B, which shows that PEG8000 has a significant effect on the assembly efficiency, and the optimal concentration is 20 g/L.

(3) According to the method, under the condition that other components are not changed, the concentration of DTT in the 2 xTai Chi Mix is adjusted to be 0-16.0 g/L, and the influence of the toxic reagent DTT on the Taiji assembly efficiency is verified.

The results are shown in FIG. 3C, which shows that DTT is unfavorable for Taiji assembly, and the number of single clone colonies is 3404 without DTT, while the maximum number of single clone colonies is 2100 at a concentration of 16.0g/L after DTT is added. Therefore, the efficiency is obviously higher than that of any concentration by more than 50% when the additive is not added.

(4) According to the method, under the condition that the rest components are not changed, the concentration of the T5 exonuclease in the 2 xTai Chi Mix is replaced by 0-2.0 mu L/mL to verify the influence of the T5 exonuclease on the Taiji assembly efficiency.

As a result, as shown in FIG. 3D, the concentration was high in the range of 0.05. mu.L to 1.0. mu.L, and the optimum concentration was 0.3. mu.L/L.

(5) According to the mode, under the condition that the rest components are unchanged, Tris-HCl 7.5 is replaced by Tris-HCl 7.0, the concentration of Tris-HCl 7.0 in 2 xTai Chi Mix is set to be 0-500 mL/L respectively, and the influence of Tris-HCl 7.0 on Taiji assembly efficiency is verified.

The results are shown in FIG. 3F, which shows that the optimal concentration is 160mL/L (FIG. 3F).

(6) According to the mode, under the condition that the rest components are unchanged, the concentration of Tris-HCl 7.5 is set to be 0-500 mL/L respectively, and the influence of Tris-HCl 7.5 on the Taiji assembly efficiency is verified.

As a result, as shown in FIG. 3G, the optimum concentration was 200 mL/L.

(7) In the manner, under the condition that the rest components are unchanged, Tris-HCl 7.5 is replaced by Tris-HCl 8.0, the concentration of Tris-HCl 8.0 is respectively set to be 0-500 mL/L, the influence of Tris-HCl 8.0 on the Taiji assembly efficiency is verified,

as a result, the optimum concentration was 160mL/L as shown in FIG. 3H.

Example 2 Effect of different reaction temperatures, reaction times and DNA fragment addition amounts on Tai chi Assembly efficiency

1. Initial taiji assembly reaction solution preparation in this example:

adding 1.0g MgCl into a 15mL test tube₂·6H₂O, diluted to 10mL to obtain MgCl₂·6H₂O solution; adding 1.0g of DTT into a 15mL tube, and diluting to 10mL to obtain a DTT solution;

② adding 1.5g PEG8000 and 0.5mL MgCl into a 50mL tube₂·6H₂O solution and 2.5mL Tris-HCl (1M, pH7.5), water was added to make up to 12.5mL, dissolved at 60 ℃, cooled in an ice bath and then 0.5mL DTT solution was added. Shaking, packaging into 4.0mL/5.0mL EP tube to obtain assembly buffer solution, and storing at-20 deg.C;

③ to 4.0mL of the assembly buffer, 1.6. mu. L T5 was added, shaken well, and then dispensed into 10. mu.L/PCR tubes and stored at-20 ℃ (2 XTai Chi Mix).

2. Taiji assembling seamless cloning process:

taking 4 fragments as an example (the nucleotide sequences are respectively shown as SEQ ID NO. 1-4, and the overlapping sequences of joints are shown in Table 3), assembling 4 DNA fragments into a plasmid as a research object, adding a total of 4 mu L of unpurified DNA mixed fragments and 6 mu L of water (the volume ratio of the 4 fragments is 1:1:1) into a Tai Chi Mix (10 mu L/PCR tube), and shaking up;

② reacting for 60 minutes at 40 ℃ in a PCR instrument;

③ after 5 minutes of ice bath, transforming the Escherichia coli DH5 alpha, coating the transformation liquid on an LB culture medium, and culturing for 12-14 hours.

3. And (3) detection of positive rate:

colony PCR is carried out by designing specific primers, and the positive rate is calculated.

4. Effect of different reaction conditions on Tai Chi Assembly efficiency

(1) According to the mode, the reaction temperature is set to be 30-50 ℃, and 2 ℃ is a gradient, so that the influence of the reaction temperature on the Tai Chi assembly efficiency is verified.

The results are shown in FIG. 4A, which shows that the assembly efficiency is higher at 30-48 ℃, and the optimal temperature is 40 ℃.

(2) According to the mode, the reaction time is set to be 10-60 min, and the influence of the reaction time on the Tai Chi assembly efficiency is verified.

The results are shown in FIG. 4B, which shows that the assembly efficiency is high in all of 10 to 60 minutes, and the optimal reaction time is 60 minutes.

(3) In the above manner, the addition ratio (volume ratio) of the PCR product was set to 5% to 50% (the PCR product ratio was calculated as 1:1:1:1), and the influence of the total DNA fragment addition amount (v/v) on the Taiji assembly efficiency was verified. Wherein the PCR program is: pre-denaturation at 98 ℃ for 3 min, denaturation at 98 ℃ for 10 sec, annealing at 55 ℃ for 15 sec, extension at 72 ℃ for 1 min, extension at 72 ℃ for 5 min, and cooling at 16 ℃ for 10 min.

As shown in FIG. 4C, the results showed that the assembly efficiency was high in all of the cases where the amount of addition was 5% to 50%, and the optimum amount of addition was 20%.

(4) According to the mode, 4 fragments with nucleotide sequences shown as SEQ ID No. 1-4 are added according to different volume ratios, the influence of different fragment concentrations on the Taiji assembly efficiency is verified, and the results show that the assembly efficiency is higher when the fragment ratio is 1-10 times (FIGS. 4D, 4E and 4F).

Example 3 Effect of different commercial PCR premix enzymes on Taiji Assembly efficiency

1. Initial taiji assembly reaction solution preparation in this example:

adding 1.0g MgCl into a 15mL test tube₂·6H₂O, diluted to 10mL to obtain MgCl₂·6H₂And (4) O solution. Adding 1.0g of DTT into a 15mL tube, and diluting to 10mL to obtain a DTT solution;

② adding 1.5g PEG8000 and 0.5mL MgCl into a 50mL tube₂·6H₂O solution and 2.5mL Tris-HCl (1M, pH7.5), water was added to make up to 12.5mL, dissolved at 60 ℃, cooled in an ice bath and then 0.5mL DTT solution was added. Shaking, packaging into 4.0mL/5.0mL EP tube to obtain assembly buffer solution, and storing at-20 deg.C;

③ to 4.0mL of the assembly buffer, 1.6. mu. L T5 was added, shaken well, and then dispensed into 10. mu.L/PCR tubes and stored at-20 ℃ (2 XTai Chi Mix).

2. Taiji assembling seamless cloning process:

taking a sequence 1-a sequence 4 (overlapping sequences at joints are shown in an attached table 3), assembling 4 DNA fragments into a plasmid as a research object, adding a total of 4 mu L of unpurified DNA mixed fragments and 6 mu L of water (the nucleotide sequence is shown as SEQ ID NO. 1-4, the volume ratio of the 4 fragments is 1:1:1:1) into a Tai Chi Mix (10 mu L/PCR tube), and shaking up;

② reacting for 60 minutes at 40 ℃ in a PCR instrument;

③ after 5 minutes of ice bath, transforming the Escherichia coli DH5 alpha, coating the transformation liquid on an LB culture medium, and culturing for 12-14 hours.

3. And (3) detection of positive rate:

colony PCR is carried out by designing specific primers, and the positive rate is calculated.

4. Fragments amplified using different PCR premixed enzymes and used in Taiji Assembly

(1) According to the above manner, 4 fragments were assembled as a test pair object (SEQ ID No. 1-4 is adopted for 4 fragments, and the overlapping sequence is shown in table 3), and the influence of the PCR product obtained by using the PCR enzyme ApexHF HS DNA Polymerase FS Master Mix (icovery, AG12202, china) on the assembly efficiency of tai chi was verified, wherein the PCR procedure was: pre-denaturation at 94 ℃ for 1 min, denaturation at 98 ℃ for 10 sec, annealing at 55 ℃ for 15 sec, extension at 72 ℃ for 1 min, extension at 72 ℃ for 5 min, and cooling at 16 ℃ for 10 min.

The results show that the PCR products have higher assembly efficiency when the addition amount of the PCR products in the assembly reaction solution is 5-50%, and the optimal addition amount is 20% (FIG. 5A).

(2) The influence of PCR products obtained by using 2 XSuperPfx MasterMix (Kangshi, CW2965, China) on Taiji assembly efficiency is verified by using 4 fragments as an experimental pair object (detailed sequence information is shown in SEQ ID NO. 1-4), wherein the PCR program is as follows: pre-denaturation at 98 ℃ for 3 min, denaturation at 98 ℃ for 10 sec, annealing at 55 ℃ for 15 sec, extension at 72 ℃ for 1 min, extension at 72 ℃ for 5 min, and cooling at 16 ℃ for 10 min.

The results show that the PCR products have higher assembly efficiency when the addition amount of the PCR products in the assembly reaction solution is 5-50%, and the optimal addition amount is 15% (FIG. 5B).

Example 4 influence of different reagents in reaction solution for Tai Chi Assembly on Assembly efficiency

The method comprises the steps of taking 4 fragments as a test pair object (the 4 fragments adopt SEQ ID NO. 1-4, and the overlapping sequence is shown in a table 3), verifying the different concentration compounding of reagents such as magnesium chloride, PEG8000, Tris-HCl and exonuclease in Taiji reaction liquid, and totally determining the influence of 95 formulas on Taiji assembly efficiency (different formulas and assembly processes of Taiji assembly are shown in a table 5), wherein the results show that when the 4 fragments are assembled, more than 3000 positive clone colonies can be obtained by formulas of No. 100, No. 93, No. 92, No. 12, No. 95 and No. 85, and high-efficiency assembly efficiency is realized (figure 6).

The 6 fragments are assembled into a test pair object (the detailed sequence information is shown in appendix sequences 1-6, the overlapping sequences are shown in A1, A2, A5, A7 and A8 of Table 3), the formulas No. 12, 36, 50, 60, 87, 85, 86, 92, 93, 95 and 100 with higher efficiency in different Taiji assembly formulas are further selected, the influence on the multi-fragment assembly efficiency is verified, and the result shows that when 6 fragments are assembled, No. 12 and No. 85 have a positive rate higher than 20%, and effective assembly can be realized (FIG. 4H).

The 7 fragments are assembled into a test pair object (the detailed sequence information is shown in appendix sequences 1-7, the overlapping sequences are shown in A1, A2, A5, A7, A9 and A10 of Table 3), the efficiency of different Taiji assembly formulas is further selected to be higher, and the formulas 12, 36, 50, 60, 87, 85, 86, 92, 93, 95 and 100 are selected to verify the influence on the multi-fragment assembly efficiency, and the result shows that when 7 fragments are assembled, the numbers 36, 85 and 12 have the positive rate higher than 20%, and the effective assembly can be realized (FIG. 4I). Thus, when assembling multiple fragments, formulation No. 85, which does not contain the toxic agent DTT, is the optimal tai chi assembly formulation.

Example 5 Effect of homologous overlap sequence Length and GC content on Taiji Assembly efficiency

1. Initial taiji assembly reaction solution preparation in this example:

adding 1.0g MgCl into a 15mL test tube₂·6H₂O, diluted to 10mL to obtain MgCl₂·6H₂And (4) O solution.

② 0.5g PEG8000 and 1.0mL MgCl were added into a 50mL tube₂·6H₂O solution and 2.5mL Tris-HCl (1M, pH7.5), make up to 12.5mL with water, dissolve at 60 ℃. Shaking, packaging into 4.0mL/5.0mL EP tube to obtain assembly buffer solution, and storing at-20 deg.C;

③ to 4.0mL of the assembly buffer, 1.2. mu. L T5 was added, shaken, and then dispensed into 10. mu.L/PCR tubes and stored at-20 ℃.

2. Taiji assembling seamless cloning process:

adding a total of 4 mu L of unpurified DNA mixed fragment and 6 mu L of water into a Tai Chi Mix (10 mu L/PCR tube), and shaking up;

② reacting for 60 minutes at 40 ℃ in a PCR instrument;

③ after 5 minutes of ice bath, transforming the Escherichia coli DH5 alpha, coating the transformation liquid on an LB culture medium, and culturing for 12-14 hours.

3. And (3) detection of positive rate:

colony PCR is carried out by designing specific primers, and the positive rate is calculated.

Assembling 2 fragments as an experiment on a subject (nucleotide sequences are shown as SEQ ID NO.1 and 2, adding an overlapping sequence 1 and an overlapping sequence 2 with the same base number at two ends of the SEQ ID NO.1 respectively, such as adding the overlapping sequence 1 and the overlapping sequence 2 of L6, correspondingly, adding the overlapping sequence 1 and the overlapping sequence 2 of L6 at two ends of the SEQ ID NO.2 respectively, designing a total of 11 homologous overlapping sequence experimental groups with low GC content (10%) of L6, M6, an overlapping sequence experimental group with medium GC content (50%) of M6, and H3680), and carrying out a detailed overlapping sequence experimental group with high GC content (see the above-in-for a detailed table), the influence of different lengths and GC contents of homologous overlapping sequences on the Taiji assembly efficiency is verified.

The results show that the homologous overlapping sequence of more than 6bp can realize fragment assembly, the high GC content (80%) is not beneficial to Taiji assembly efficiency, and the homologous sequence of more than 12bp can realize high-efficiency assembly under the conditions of low GC content (10%) and medium GC content (50%) (FIGS. 7A and 7B).

Example 6 testing the effect of different hosts on the efficiency of Taiji Assembly

The procedure of example 4 was followed, and 4 fragments were assembled as test pair subjects (4 fragments were SEQ ID NO. 1-4, and overlapping sequences are shown in Table 3 as A1, A2, A3 and A4), and the effects of different E.coli hosts JM109, JM110, MG1655, Top10, BL21 and DH5 alpha on Taiji assembly efficiency were verified, which indicated that E.coli Top10 had the highest assembly efficiency (FIG. 7C).

Example 7 testing of Taiji Assembly for Multi-segment Assembly capability

Assembling 4-12 fragments as test pair objects, wherein the sequences are respectively shown in SEQ ID No. 1-12, and verifying the assembling capability of Taiji assembly on the multiple fragments.

(1) Preparation of a tai chi assembly reaction solution in this example:

adding 1.0g MgCl into a 15mL test tube₂·6H₂O, diluted to 10mL to obtain MgCl₂·6H₂And (4) O solution.

② 0.5g PEG8000 and 1.0mL MgCl were added into a 50mL tube₂·6H₂O solution and 2.5mL Tris-HCl (1M, pH7.5), make up to 12.5mL with water, dissolve at 60 ℃. Shaking, packaging into 4.0mL/5.0mL EP tube to obtain assembly buffer solution, and storing at-20 deg.C;

③ to 4.0mL of the assembly buffer, 1.2. mu. L T5 was added, shaken, and then dispensed into 10. mu.L/PCR tubes and stored at-20 ℃.

(2) Taiji assembling seamless cloning process:

1. to 10. mu.L of the reaction mixture, a total of 4. mu.L of the fragment and 6. mu.L of water were added and mixed well.

In a PCR instrument, 60 minutes at 40 ℃.

3. After 5 minutes in ice bath, E.coli DH 5. alpha. was transformed.

When assembling 4 fragments, adopting SEQ ID NO. 1-4 (the overlapping sequences are shown in Table 3, the overlapping sequences of the SEQ ID NO.1 and the SEQ ID NO.2 fragments are respectively added with an overlapping sequence A1 for Taiji assembly at the joint in a mode of adding additional sequences through nonspecific primers, and similarly, the overlapping sequence of the SEQ ID NO.2 and the SEQ ID NO.3 is A2, the overlapping sequence of the SEQ ID NO.3 and the SEQ ID NO.4 is A3, and the overlapping sequence of the SEQ ID NO.4 and the SEQ ID NO.1 is A4);

when 5 fragments are assembled, 1-5 of SEQ ID NO.1, 6 fragments are assembled, 1-6 of SEQ ID NO.1, 7 fragments are assembled, 1-7 of SEQ ID NO.1, 8 fragments are assembled, 1-8 of SEQ ID NO.1, 9 fragments are assembled, 1-9 of SEQ ID NO.1, 10 fragments are assembled, 1-10 of SEQ ID NO.1, 11 fragments are assembled, 1-11 of SEQ ID NO.1, 12 fragments are assembled, and the overlapping sequences are shown in table 3, and the overlapping sequences are similar to those used for assembling 4 fragments, and corresponding overlapping sequences are selected among the fragments.

The results show that taiji assembly can achieve high efficiency assembly of 4-7 segments, and can achieve efficient assembly of up to 12 segments (fig. 7D).

Example 8 testing of Taiji Assembly for high throughput construction of plasmids

(1) Preparation of a tai chi assembly reaction solution in this example:

adding 1.0g MgCl into a 15mL test tube₂·6H₂O, diluted to 10mL to obtain MgCl₂·6H₂And (4) O solution.

② 0.5g PEG8000 and 1.0mL MgCl were added into a 50mL tube₂·6H₂O solution and 2.5mL Tris-HCl (1M, pH7.5), make up to 12.5mL with water, dissolve at 60 ℃. Shaking, packaging into 4.0mL/5.0mL EP tube to obtain assembly buffer solution, and storing at-20 deg.C;

③ to 4.0mL of the assembly buffer, 1.2. mu. L T5 was added, shaken, and then dispensed into 10. mu.L/PCR tubes and stored at-20 ℃.

(2) Taiji assembling seamless cloning process:

1. to 10. mu.L of the reaction mixture, a total of 4. mu.L of the fragment and 6. mu.L of water were added and mixed well.

In a PCR instrument, 60 minutes at 40 ℃.

3. After 5 minutes in ice bath, E.coli DH 5. alpha. was transformed.

Transformation protocol for 96-well PCR plate:

(1) adding 8 mu L of Tai Chi reaction solution, adding 2 mu L of DNA fragment, preserving the temperature for 60 minutes at 40 ℃, and then carrying out ice bath for 5 minutes;

(2) adding 50 mu L of escherichia coli competent cells, and carrying out ice bath for 30 minutes;

(3) the heat shock is carried out for 90 seconds, and the ice bath is carried out for 5 minutes;

(4) 140 μ L of LB was added, incubated at 37 ℃ for 1 hour, and the resistant plates were spread.

A 96-well enzyme label plate conversion process:

(1) adding 32 mu L of Tai Chi reaction solution, adding 8 mu L of DNA fragment, preserving the temperature for 60 minutes at 40 ℃, and then carrying out ice bath for 5 minutes;

(2) adding 60 mu L of escherichia coli competent cells, and carrying out ice bath for 30 minutes;

(3) the heat shock is carried out for 90 seconds, and the ice bath is carried out for 5 minutes;

(4) 100 μ L of LB was added, incubated at 37 ℃ for 1 hour, and the resistant plates were spread.

The results show that the 96-well PCR plate or 96-well enzyme label plate high-throughput transformation process can obtain more than 100 positive clone colonies when used for assembling 4 fragments, and can obtain more than 50 positive clone colonies when assembling 5 fragments, thereby realizing high-throughput and high-efficiency plasmid construction (FIGS. 8A, 8B, 8C and 8D).

Comparative example 1: gibbson Assembly, Hot Fusion, TEDA and Taiji Assembly cost comparison

The T5 exonuclease based plasmid construction method has the main cost of T5 exonuclease, DNA polymerase and DNA ligase, and the secondary cost of the purification process of DNA fragments and NAD, dNTP and DTT in the reaction liquid. Without considering the two inevitable costs of primer synthesis and PCR enzymes, 5 fragments were assembled as an example:

the Gibbson assembly consists of T5 exonuclease, DNA polymerase and DNA ligase, as well as Tris-HCl, magnesium ions, PEG8000 and DTT, reagents lead to the highest preparation cost at most, and the reagent configuration cost of laboratory self-purchase is about 50 yuan/reaction (wherein the cost of 5 fragment DNA purification is 10 yuan, and the Samerfei DNA purification kit is used). Hot Fusion reduces the cost to about 15M/reaction (10M for 5 fragment DNA purification) without reducing assembly efficiency due to the elimination of the most expensive component DNA ligase in Gibbson reaction. The TEDA assembly further removes the higher cost of DNA polymerase and NAD and dNTP components in the reaction solution on the basis of Hot Fusion, so that the cost is reduced to about 10.06 yuan/reaction (wherein the cost of DNA purification of 5 fragments is 10 yuan). All three methods need to purify DNA fragments, which results in higher construction cost, and the Taiji assembly needs DNA polymerase in a PCR reaction system, so that the purification process of the DNA fragments is eliminated, and the cost is reduced to 0.06 yuan/reaction.

Comparative example 2: gibbson Assembly, Hot Fusion, TEDA and Taiji Assembly efficiency comparison

The 4 assembly methods used the reaction solutions and the assembly steps are shown in Table 5. Taking 4 fragments as an example, the assembly methods of Gibbson assembly (T5+ Phusion + Taq) and Hot Fusion (T5+ Phusion) have high assembly efficiency due to the DNA polymerase contained in the system, and can obtain more than 3000 clone colonies. To identify the effect of DNA polymerase on assembly efficiency, the amount of DNA polymerase added in Hot Fusion was reduced to one third, and not added. The results show that the assembly efficiency decreases significantly by about 90% when the DNA polymerase is reduced to one-third, and results in about 95% reduction in assembly efficiency when no DNA polymerase is added, thus indicating that DNA polymerase is critical for assembly efficiency in gibson assembly (fig. 2). TEDA assembly, because it does not contain DNA polymerase, is about 99% less efficient than gibson assembly. Taiji assembly was performed by using a DNA fragment that was not purified to borrow DNA polymerase in a PCR system, so that the assembly efficiency was not decreased by deletion of DNA polymerase, but was about 1.5 times higher than gibson (fig. 2).

TABLE 5 different Assembly method solution formulations and procedures

Comparative example 3: comparison of operation flows of different assembly methods in construction of multiple plasmids

With the continuous development of molecular biology, the simultaneous construction of multiple plasmids is becoming increasingly required. Taking the simultaneous construction of 10 plasmids respectively consisting of 5 fragments as an example, the basic operation flow required by the Gibbson assembly, Hot Fusion and TEDA assembly methods is as follows: DNA fragment purification (1 hour), DNA fragment concentration determination and molar mass calculation (0.5 hour), multi-fragment assembly construction (1.5 hours including sample addition 0.5 hour and incubation reaction 1 hour), and transformation of Escherichia coli (2 hours), totaling about 5 hours. The basic flow required for Taiji assembly is as follows: assembling the fragments (1.25 hours including sample adding 0.25 hour and heat preservation reaction for 1 hour) and transforming the escherichia coli (2 hours), wherein the total time is 3.25 hours, and the time can be saved by 35%. Particularly, the procedures of purifying fragments, determining concentration, calculating molar weight, adjusting the volume of added fragments and the like are complicated to operate and account for more than 50 percent of the workload of the whole constructed plasmid. If the method is applied to the construction of plasmids by a high-flux automatic operation platform, the redundant operation process can greatly improve the design difficulty and the construction cost.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> safe and nontoxic method for seamlessly constructing plasmid with high flux

<130> BAA210931A

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 4221

<212> DNA

<213> Artificial sequence

<400> 1

tgtacctcag aacgtaagag tgaccatggt gactaagtgt ctattgcaga ataacttgtc 60

agactgccgg gaaatcccgg cagtcttttt tccattttca cgtcacgcgt ccatggagat 120

ctttgtctgc aactgaaaag tttatacctt acctggaaca aatggttgaa acatacgagg 180

ctaatatcgg cttattagga atagtccctg tactaataaa atcaggtgga tcagttgatc 240

agtatatttt ggacgaagct cggaaagaat ttggagatga cttgcttaat tccacaatta 300

aattaaggga aagaataaag cgatttgatg ttcaaggaat cacggaagaa gatactcatg 360

ataaagaagc tctaaaacta ttcaataacc ttacaatgga attgatcgaa agggtggaag 420

gttaatggta cgaaaattag gggatctacc tagaaagcca caaggcgata ggtcaagctt 480

aaagaaccct tacatggatc ttacagattc tgaaagtaaa gaaacaacag aggttaaaca 540

aacagaacca aaaagaaaaa aagcattgtt gaaaacaatg aaagttgatg tttcaatcca 600

taataagatt aaatcgctgc acgaaattct ggcagcatcc gaagggaatt catattactt 660

agaggatact attgagagag ctattgataa gatggttgag acattacctg agagccaaaa 720

aactttttat gaatatgaat taaaaaaaag aaccaacaaa ggctgagaca gactccaaac 780

gagtctgttt ttttaaaaaa aatattagga gcattgaata tatattagag aattaagaaa 840

gacatgggaa taaaaatatt ttaaatccag taaaaatatg ataagattat ttcagaatat 900

gaagaactct gtttgttttt gatgaaaaaa caaacaaaaa aaatccacct aacggaatct 960

caatttaact aacagcggcc aaactgagaa gttaaatttg agaaggggaa aaggcggatt 1020

tatacttgta tttaactatc tccattttaa cattttatta aaccccatac aagtgaaaat 1080

cctcttttac actgttcctt taggtgatcg cggagggaca ttatgagtga agtaaaccta 1140

aaaggaaata cagatgaatt agtgtattat cgacagcaaa ccactggaaa taaaatcgcc 1200

aggaagagaa tcaaaaaagg gaaagaagaa gtttattatg ttgctgaaac ggaagagaag 1260

atatggacag aagagcaaat aaaaaacttt tctttagaca aatttggtac gcatatacct 1320

tacatagaag gtcattatac aatcttaaat aattacttct ttgatttttg gggctatttt 1380

ttaggtgctg aaggaattgc gctctatgct cacctaactc gttatgcata cggcagcaaa 1440

gacttttgct ttcctagtct acaaacaatc gctaaaaaaa tggacaagac tcctgttaca 1500

gttagaggct acttgaaact gcttgaaagg tacggtttta tttggaaggt aaacgtccgt 1560

aataaaacca aggataacac agaggaatcc ccgattttta agattagacg taaggttcct 1620

ttgctttcag aagaactttt aaatggaaac cctaatattg aaattccaga tgacgaggaa 1680

gcacatgtaa agaaggcttt aaaaaaggaa aaagagggtc ttccaaaggt tttgaaaaaa 1740

gagcacgatg aatttgttaa aaaaatgatg gatgagtcag aaacaattaa tattccagag 1800

gccttacaat atgacacaat gtatgaagat atactcagta aaggagaaat tcgaaaagaa 1860

atcaaaaaac aaatacctaa tcctacaaca tcttttgaga gtatatcaat gacaactgaa 1920

gaggaaaaag tcgacagtac tttaaaaagc gaaatgcaaa atcgtgtctc taagccttct 1980

tttgatacct ggtttaaaaa cactaagatc aaaattgaaa ataaaaattg tttattactt 2040

gtaccgagtg aatttgcatt tgaatggatt aagaaaagat atttagaaac aattaaaaca 2100

gtccttgaag aagctggata tgttttcgaa aaaatcgaac taagaaaagt gcaataaact 2160

gctgaagtat ttcagcagtt ttttttattt agaaatagtg aaaaaaatat aatcagggag 2220

gtatcaatat ttaatgagta ctgatttaaa tttatttaga ctggaattaa taattaacac 2280

gtagactaat taaaatttaa tgagggataa agaggataca aaaatattaa tttcaatccc 2340

tattaaattt taacaagggg gggattaaaa tttaattaga ggtttatcca caagaaaaga 2400

ccctaataaa atttttacta gggttataac actgattaat ttcttaatgg gggagggatt 2460

aaaatttaat gacaaagaaa acaatctttt aagaaaagct tttaaaagat aataataaaa 2520

agagctttgc gattaagcaa aactctttac tttttcattg acattatcaa attcatcgat 2580

ttcaaattgt tgttgtatca taaagttaat tctgttttgc acaacctttt caggaatata 2640

aaacacatct gaggcttgtt ttataaactc agggtcgcta aagtcaatgt aacgtagcat 2700

atgatatggt atagcttcca cccaagttag cctttctgct tcttctgaat gtttttcata 2760

tacttccatg ggtatctcta aatgattttc ctcatgtagc aaggtatgag caaaaagttt 2820

atggaattga tagttcctct ctttttcttc aactttttta tctaaaacaa acactttaac 2880

atctgagtca atgtaagcat aagatgtttt tccagtcata atttcaatcc caaatctttt 2940

agacagaaat tctggacgta aatcttttgg tgaaagaatt tttttatgta gcaatatatc 3000

cgatacagca ccttctaaaa gcgttggtga atagggcatt ttacctatct cctctcattt 3060

tgtggaataa aaatagtcat attcgtccat ctacctatcc tattatcgaa cagttgaact 3120

ttttaatcaa ggatcagtcc tttttttcat tattcttaaa ctgtgctctt aactttaaca 3180

actcgatttg tttttccaga tctcgagggt aactagcctc gccgatcccg caagaggccc 3240

ggcagtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 3300

aaatacagag cgcctccttg acactgaatt tagcatgtga tataattaac ttacccaatt 3360

aaaggaggaa ggatccaatg agccatattc aacgggaaac gtcttgctct aggccgcgat 3420

taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc 3480

aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga 3540

aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc 3600

tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat 3660

ggttactcac cactgcgatc cctgggaaaa cagcattcca ggtattagaa gaatatcctg 3720

attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc 3780

ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac 3840

gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg 3900

ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca 3960

ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta 4020

ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact 4080

gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata 4140

atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taactgtcag 4200

accaagttta ctcatatata c 4221

<210> 2

<211> 860

<212> DNA

<213> Artificial sequence

<400> 2

gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct 60

tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact aacatgagaa 120

ttacaactta tatcgtatgg ggctgacttc aggtgctaca tttgaagaga taaattgcac 180

tgaaatctag aaatatttta tctgattaat aagatgatct tcttgagatc gttttggtct 240

gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt tttcgaaggt 300

tctctgagct accaactctt tgaaccgagg taactggctt ggaggagcgc agtcaccaaa 360

acttgtcctt tcagtttagc cttaaccggc gcatgacttc aagactaact cctctaaatc 420

aattaccagt ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg gactcaagac 480

gatagttacc ggataaggcg cagcggtcgg actgaacggg gggttcgtgc atacagtcca 540

gcttggagcg aactgcctac ccggaactga gtgtcaggcg tggaatgaga caaacgcggc 600

cataacagcg gaatgacacc ggtaaaccga aaggcaggaa caggagagcg cacgagggag 660

ccgccagggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg 720

agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg 780

cggccctctc acttccctgt taagtatctt cctggcatct tccaggaaat ctccgccccg 840

ttcgtaagcc atttccgctc 860

<210> 3

<211> 956

<212> DNA

<213> Artificial sequence

<400> 3

gaaaaccgac tgtaaaaagt acagtcggca ttatctcata tttattacaa aacgcctgcg 60

caataacgca ggcgttctgt gacattaact tatttcttag tcatgactag actagatcgt 120

gtctcgacag gtcttgagga atcatagaat ttttaattta aattttattt gacaaaaatg 180

ggctcgtgtt gtataatcta agctagtgta tttaaaggag gtgataaaaa tggtttctga 240

actgatcaaa gaaaacatgc acatgaaact gtacatggaa ggtaccgtta acaaccacca 300

cttcaaatgc acctctgaag gtgaaggtaa accgtacgaa ggtacccaga ccatgcgtat 360

caaagctgtt gaaggtggtc cgctgccgtt cgcttttgac atcctggcta cctctttcat 420

gtacggttct aaaaccttca tcaaccacac ccagggtatc ccggactttt tcaaacagtc 480

tttcccggaa ggtttcacct gggaacgtgt taccacctac gaagacggtg gtgttctgac 540

cgctacccag gacacctctc tgcaagacgg ttgcctgatc tacaacgtta aaatccgtgg 600

tgttaacttc ccgtctaacg gtccggttat gcagaaaaaa accctgggtt gggaagcttc 660

taccgaaacc ctgtacccgg ctgacggtgg tctggaaggt cgtgctgaca tggctctgaa 720

actggttggt ggtggtcacc tgatctgcaa cctgaaaacc acctaccgtt ctaaaaaacc 780

ggctaaaaac ctgaaaatgc cgggtgttta ctacgttgac cgtcgtctgg aacgtatcaa 840

agaagctgac aaagaaacct acgttgaaca gcacgaagtt gctgttgctc gttactgcga 900

cctgccgtct aaactgggtc accgttaaga ctgagtcatg ctcagagtga cgtgct 956

<210> 4

<211> 743

<212> DNA

<213> Artificial sequence

<400> 4

aaaggaggtg aaatgtacac atgggccgta aaggtgaaga actgttcacc ggcgttgttc 60

cgatcctgat cgaactggat ggtgatgtta acggtcataa attcttcgtt cgtggtgaag 120

gcgaaggtga tgcgaccatc ggtaaactga gcctgaaatt catctgcacc accggcaaac 180

tgccggttcc gtggccgacc ctggttacca ccctgaccta cggcgttcag tgcttctctc 240

gttacccgga tcacatgaaa cgtcacgatt tcttcaaaag cgcgatgccg gaaggttatg 300

ttcaggaacg taccatctac ttcaaagatg atggcaccta caaaacccgt gcggaagtta 360

aattcgaagg tgataccctg gttaaccgta tcgaactgaa aggcatcgat ttcaaagaag 420

atggtaacat cctgggccac aaactggaat acaacttcaa ctcgcacaag gtttacatca 480

ctgcggataa gcagaataac ggtattaaag ccaactttac tattcgtcat aatgttgagg 540

acggtagcgt tcaactggct gaccactacc agcagaacac tcccattggt gacggcccgg 600

tcgatctgcc ggatgatcac tatctgtcta cccagaccat cctgtctaaa gatctgaacg 660

gtaccgatgt tacttctgaa aaacgtgatc acatggttct gctggaatat gttaccgctg 720

cgggtattac cgatgcttct taa 743

<210> 5

<211> 498

<212> DNA

<213> Artificial sequence

<400> 5

atgagccata tcttcgacgc atctgtactg gctccacata ttcctagtaa ccttcctgat 60

aatttcaagg tgagaccact ggcaaaggat gatttttcga agggatatgt cgacctgctg 120

tcacaattga cgtcagttgg aaaccttgac caagaagcat ttgagaaacg atttgaggcg 180

atgagaacaa gcgtaccgaa ttatcacatc gtagtaattg aggattccaa cagccagaaa 240

gtggtggcgt ctgctagttt ggttgttgaa atgaaattca ttcatggggc cggatcaagg 300

ggtcgtgttg aagatgttgt cgtcgataca gaaatgcgcc ggcaaaaatt aggtgccgtg 360

cttttaaaaa ctttggtgtc acttggcaaa tctttaggcg tctacaaaat aagcctcgaa 420

tgcgtcccgg aattactccc gttctattcc caatttggct ttcaggatga ctgtaatttt 480

atgacccagc gcttttaa 498

<210> 6

<211> 547

<212> DNA

<213> Artificial sequence

<400> 6

atgggcaaaa acttacaagc tctggcccag ctttataaaa atgccctgct taacgatgtg 60

cttccgtttt gggaaaatca ttcattagat agcgaaggcg gatattttac atgcctggat 120

agacagggca aagtctacga tacagataaa tttatctggc ttcaaaaccg ccaggtttgg 180

acattttcta tgctttgtaa ccagctggaa aaaagagaaa actggctgaa aatcgctcgc 240

aatggagcca aatttctggc acaacatggc agagatgatg aaggaaactg gtattttgct 300

ttaacacgcg gcggagaacc gctggttcaa ccgtataata tttttagcga ttgctttgca 360

gcgatggcct tttctcagta tgcattagcg tcaggagaag aatgggcaaa agatgttgct 420

atgcaagcct ataataacgt gctgagacgc aaagataacc cgaaaggcaa atacacaaaa 480

acatatccgg gaacaagacc gatgaaagct ttagccgttc cgatgattct ggcgaacctg 540

acacttg 547

<210> 7

<211> 617

<212> DNA

<213> Artificial sequence

<400> 7

aaatggaatg gttactgccg caagaaacac tggaaaatgt gcttgctgcc acagtccagg 60

aagttatggg cgattttctt gatcaagaac agggattaat gtatgaaaac gtcgctccgg 120

atggctcaca tatcgattgc tttgaaggac gcctgattaa tccgggccat ggaatcgaag 180

cgatgtggtt tattatggat atcgctagac gcaaaaacga tagcaaaaca atcaaccagg 240

cggttgatgt tgtgttaaat atcctgaact ttgcttggga taacgaatac ggcggacttt 300

actactttat ggatgcagcg ggccatccgc cgcaacagct ggaatgggat caaaaacttt 360

ggtgggtgca tcttgaaagc ttagtcgcac tggcgatggg ctatagatta acaggacgcg 420

atgcatgttg ggcgtggtat caaaaaatgc atgattattc ttggcagcat tttgcagatc 480

cggaatatgg cgaatggttt ggatatctta acagacgcgg cgaagtgctt ctgaacctga 540

aaggcggaaa atggaaagga tgctttcatg tcccgagagc catgtatctg tgttggcaac 600

agtttgaagc actttca 617

<210> 8

<211> 541

<212> DNA

<213> Artificial sequence

<400> 8

atgcaaaaca acaacgaatt taaaatcggc aacagatcag tcggatataa tcatgaaccg 60

cttattatct gcgaaattgg catcaaccat gaaggaagct taaaaacagc ctttgaaatg 120

gtcgatgcag cgtataatgc cggagcagaa gttgtgaaac atcaaacaca tatcgttgaa 180

gatgaaatgt ctgatgaagc caaacaggtg atcccgggca acgcagatgt ctcaatctac 240

gaaatcatgg aaagatgtgc gctgaacgaa gaagatgaaa tcaaactgaa agaatacgtt 300

gaaagcaaag gaatgatctt tatctctaca ccgttttcac gcgctgccgc acttagatta 360

cagcgcatgg atattccggc ctataaaatc ggctctggag aatgcaacaa ctacccgctg 420

atcaaactgg tggcaagctt tggcaaaccg atcatcctgt ctacaggaat gaactcaatc 480

gaaagcatca aaaaatcagt tgaaatcatc agagaagcgg gcgtgccgta tgctctgctt 540

c 541

<210> 9

<211> 506

<212> DNA

<213> Artificial sequence

<400> 9

attgtacaaa catttatccg acaccgtatg aagatgttcg cctgggcgga atgaatgatc 60

tttcagaagc ctttccggat gcaattatcg gccttagcga tcatacatta gataactatg 120

catgcctggg agcggtggct cttggcggat ctatcctgga aagacatttt acagatagaa 180

tggatcgccc gggcccggat atcgtctgtt caatgaatcc ggatacattt aaagaactga 240

aacaaggagc ccatgcactg aaacttgcga gaggcggcaa gaaagataca attatcgctg 300

gcgaaaaacc gacaaaagat tttgcgtttg ctagcgtcgt tgcggataaa gatattaaga 360

aaggcgaact gctgtctgga gataacctgt gggtcaaaag accgggcaac ggagatttta 420

gcgttaacga atacgaaaca ctttttggca aagtggcggc ttgcaatatc cgcaaaggag 480

ctcagattaa gaaaacagat atcgaa 506

<210> 10

<211> 687

<212> DNA

<213> Artificial sequence

<400> 10

atgtctctgc ttgcgcaact tgatcagaaa attgcagcga atggcggatt aatcgtttca 60

tgccaaccgg tgccggatag cccgctggat aaaccggaaa ttgtggctgc catggcgctt 120

gcagcggaac aagcaggagc ggtggctatt agaatcgaag gcgtcgctaa cttacaggcg 180

acacgcgctg ttgtgtctgt cccgattatc ggaatcgtta aaagagatct tgaagattca 240

ccggttcgca ttacagccta tatcgaagat gtggatgcct tagcacaagc gggagctgat 300

attatcgcta ttgatggcac agatagaccg cgcccggtcc cggttgaaac attactggcc 360

agaatccatc atcatggact tttagcaatg acagattgct caacaccgga agatggctta 420

gcctgtcaga aactgggcgc agaaattatc ggaacaacac tgagcggcta tacaacaccg 480

gaaacaccgg aagaaccgga tctggcgctt gtcaaaacac tttctgatgc gggatgcaga 540

gttattgctg aaggccgcta taatacaccg gcgcaagctg ccgatgctat gagacatgga 600

gcctgggcag tgacagtcgg cagcgcaatt acacgcttag aacatatctg tcagtggtat 660

aacacagcca tgaagaaagc agttctg 687

<210> 11

<211> 550

<212> DNA

<213> Artificial sequence

<400> 11

atgaaaagaa ttttatgcat cacaggaaca cgcgcagatt ttggcaaact gaaaccgctg 60

cttgcgtata ttgaaaatca tccggatctg gaacttcatt taatcgttac aggaatgcat 120

atgatgaaaa catacggcag aacatacaaa gaagtgacac gcgaaaacta ccaacataca 180

tacctgtttt caaaccaaat tcagggcgaa ccgatgggag cagtgctggg caacacaatc 240

acatttatct ctagactttc agatgaaatc gaaccggata tggtcatgat ccatggagat 300

agacttgaag cattagcggg agcagcggtg ggcgcgttat caagccgcct ggtctgtcat 360

attgaaggcg gagaattaag cggcacagtc gatgattcta ttcgccattc aatcagcaaa 420

cttagccata tccatctggt tgctaacgaa caagccgtta caagacttgt gcagatggga 480

gaaaaacgca aacatatcca tattatcggc tcaccggatt tagatgtgat ggcttcttca 540

acactgccga 550

<210> 12

<211> 581

<212> DNA

<213> Artificial sequence

<400> 12

gccttgaaga agtcaaagaa tattatggac tgccgtacga aaactacggc atctcaatgt 60

ttcatccggt tacaacagaa gctcatctta tgccgcaata tgctgcccag tattttaaag 120

ccctggaact ttcaggacag aacattatca gcatttatcc gaataacgat acaggcacag 180

aaagcatcct tcaagaactg ctgaaatacc agagcgataa atttatcgct tttccgtcta 240

tcagatttga atattttctg gttcttctga aacatgccaa atttatggtg ggaaatagct 300

ctgctggcat tcgcgaagcc ccgctgtatg gagtcccgag catcgatgtt ggcacaagac 360

aatctaatcg ccatatggga aaatcaatca tccatacaga ttacgaaaca aaaaacattt 420

ttgatgcaat ccaacaggcg tgctctctgg gcaaatttga agcagatgat acatttaacg 480

gcggagatac aagaacatct acagaacgct ttgcagaagt cattaataac ccggaaacat 540

ggaatgtttc agcgcagaaa agatttatcg atttaaacct g 581