RECOMBINANT ENGINEERED STRAIN FOR DE NOVO SYNTHESIS OF CDP-CHOLINE USING GLUCOSE AS SUBSTRATE AND ITS PREPARATION METHOD AND APPLICATION

Abstract

A recombinant engineered strain for de novo synthesis of CDP-choline using glucose as a substrate and its preparation method and application are provided. Using BS168N as the starting strain, firstly, the phosphatidylethanolamine N-methyltransferase gene PEM1 and phosphatidylethanolamine/phosphatidyl-N-methylethanolamine N-methyltransferase gene PEM2 from S. cerevisiae are integrated into the genome of the BS168N for induced expression, thereby opening up the synthesis pathway from phosphatidylethanolamine to phosphatidylcholine; subsequently, the CKI and CCT genes of S. cerevisiae are further integrated into the BS168N genome expressing PEM1-PEM2, opening up the synthesis pathway of choline to CDPC, thereby obtaining the recombinant engineered strain. Further, the recombinant engineered strain is subjected to shake flask fermentation to achieve de novo synthesis of CDP-choline using glucose as a substrate. The method of the present disclosure provides a fundamental research and theoretical basis for the construction of efficient cell factories for de novo synthesis of CDP-choline through synthetic biology.

Claims

1. A recombinant engineered strain for de novo synthesis of CDP-choline using glucose as a substrate, wherein the recombinant engineered strain is obtained by modifying Bacillus subtilis 168N as a chassis as follows: (1) modification of Bacillus subtilis 168N: integrating the phosphatidylethanolamine N-methyltransferase gene PEM1 and phosphatidylethanolamine/phosphatidyl-N-methylethanolamine N-methyltransferase gene PEM2 from Saccharomyces cerevisiae into an artificial operon P.sub.xyl-PEM2-PEM1, and integrating the artificial operon P.sub.xyl-PEM2-PEM1 into the lacA site of the Bacillus subtilis 168N genome for induced expression, wherein the Bacillus subtilis 168N is obtained by integrating P.sub.ara-neo into the araR site of the wild-type Bacillus subtilis 168 genome; (2) construction of recombinant engineered strain: integrating the artificial operon TP2-CCT-CKI into the genome of Bacillus subtilis expressing P.sub.xyl-PEM2-PEM2-PEM1 in step (1), thus constructing a recombinant engineered strain capable of de novo synthesis of CDP-choline using glucose as a substrate; the artificial operon TP2-CCT-CKI is composed of the choline kinase gene CKI and the phosphocholine cytidylyltransferase gene CCT as well as the constitutive strong promoter TP2.

2. The application of the recombinant engineered strain according to claim 1 in de novo synthesis of CDP-choline using glucose as the substrate.

3. The application according to claim 2, wherein the specific application comprises inoculating the single colony of the recombinant engineered strain into a test tube filled with LB liquid culture medium, conducting shake culture, then transferring the inoculum volume of 0.5% to 2% of the fermentation medium volume to a conical flask containing the fermentation medium, conducting shake culture, and then adding xylose with a final concentration of 10 g/L after 2-5 hours of fermentation.

4. The application according to claim 3, wherein the LB liquid culture medium comprises the following components: tryptone 5-15 g/L, yeast extract 1-9 g/L, NaCl 5-15 g/L, erythromycin 1-8 g/mL, neomycin 10-20 g/mL.

5. The application according to claim 3, wherein the fermentation medium comprises the following components: glucose 20-80 g/L, tryptone 5-15 g/L, yeast extract 1-9 g/L NaCl 5-15 g/L, MgSO.sub.4.Math.7H.sub.2O 0.02-3 g/L, cytidine-5-phosphate 0.01-4 g/L, and xylose 5-20 g/L; the fermentation conditions are: pH 6.0-8.0, fermentation temperature 30-45 C., rotation speed 100-250 r/min, fermentation time 20-50 h.

6. A method for de novo synthesis of phosphatidylcholine using glucose as a substrate, comprising the following steps: (1) modification of Bacillus subtilis 168N: combining the phosphatidylethanolamine N-methyltransferase gene PEM1 and phosphatidylethanolamine/phosphatidyl-N-methylethanolamine N-methyltransferase gene PEM2 from Saccharomyces cerevisiae into an artificial operon P.sub.xyl-PEM2-PEM1, and integrating the artificial operon P.sub.xyl-PEM2-PEM1 into the lacA site of the Bacillus subtilis 168N genome for induced expression; (2) construction of recombinant engineered strain: integrating the artificial operon TP2-CCT-CKI into the genome of Bacillus subtilis expressing P.sub.xyl-PEM2-PEM2-PEM1 in step (1), thus constructing a recombinant engineered strain capable of de novo synthesis of CDP-choline using glucose as a substrate; (3) inoculating the single colony of the recombinant engineered strain constructed in step (2) into a test tube filled with LB liquid culture medium, conducting shake culture, then transferring the inoculum volume of 0.5% to 2% of the fermentation medium volume to a conical flask containing the fermentation medium, then adding xylose with a final concentration of 10 g/L after 2-5 hours of fermentation, and then conducting shake culture in the conical flask for 20-50 hours to synthesize CDP-choline.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 is a schematic diagram of the biosynthetic pathway and engineering modification strategy of CDP-choline in Bacillus subtilis provided by the present disclosure;

[0054] Wherein the reference labels shown in FIG. 1 are as below: Glucose: glucose; Glucose-6P: glucose-6-phosphate; Frucose-6P: fructose-6-phosphate; Frucose-1,6P.sub.2: fructose-1,6-diphosphate; Glyceraldehyde-3P: glyceraldehyde 3-phosphate; Glycerone-P: dihydroxyacetone phosphate; Glycerol: glycerol; Glycerol-3P: glycerol-3-phosphate; 1-Acyl-sn-glycerol-3P: 1-acyl-sn-glycerol-3-phosphate; 1,2-Diacyl-sn-glycerol-3P: 1,2-diacyl-sn-glycerol-3-phosphate; CDP-diacylglycerol: CDP-diacylglycerol; Phosphatidyl-L-serine: phosphatidyl-L-serine; Phosphatidylethanolamine: phosphatidylethanolamine; Phosphatidyl-N-methylethanolamine: phosphatidyl-N-methylethanolamine; Phosphatidyl-N-dimethylethanolamine: phosphatidyl-N-dimethylethanolamine; Phosphatidylcholine: phosphatidylcholine; 1-Acyl-sn-glycero-3-phosphocholine: 1-Acyl-sn-glycero-3-phosphocholine; sn-Glycero-3-phosphocholine: sn-Glycero-3-phosphocholine; Choline: choline; Phosphocholine: choline phosphate; CDP-choline: cytidine diphosphate-choline;

[0055] FIG. 2 shows the growth curves of three recombinant strains BS168N/PEM21, BS168N/PEM21/ydeO, and BS168N/PEM21/yjoB in Embodiment 3 of the present disclosure;

[0056] FIG. 3 shows the high-performance liquid chromatography chromatogram of the supernatant of the fermentation broth of recombinant bacterium BS168N/PEM21 in Embodiment 3 of the present disclosure;

[0057] FIG. 4 shows the high-performance liquid chromatography chromatogram of the supernatant from the fermentation broth of recombinant strain BS168N/PEM21/ydeO in Embodiment 3 of the present disclosure;

[0058] FIG. 5 shows the high-performance liquid chromatography chromatogram of the supernatant from the fermentation broth of recombinant strain BS168N/PEM21/yjoB in Embodiment 3 of the present disclosure;

[0059] FIG. 6 shows the high-performance liquid chromatography chromatogram of the supernatant obtained from lysed cell pellets of recombinant bacterium BS168N/PEM21 in Embodiment 3 of the present disclosure;

[0060] FIG. 7 shows the high-performance liquid chromatography chromatogram of the supernatant obtained from lysed cell pellets of recombinant strain BS168N/PEM21/ydeO in Embodiment 3 of the present disclosure;

[0061] FIG. 8 shows the high-performance liquid chromatography chromatogram of the supernatant obtained from lysed cell pellets of recombinant strain BS168N/PEM21/yjoB in Embodiment 3 of the present disclosure;

[0062] FIG. 9 shows the extracellular and intracellular CDPC content, as well as the total synthesis of CDPC, of three recombinant strain BS168N/PEM21, BS168N/PEM21/ydeO, and BS168N/PEM21/yjoB in Embodiment 3 of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0063] In order to enable skilled person in the art to better understand the present disclosure, the present disclosure will be further elaborated in conjunction with specific embodiments.

1. Preparation and Sources of Strains, Plasmids, and Culture Media.

[0064] All strains and plasmid information involved in the present disclosure are detailed in Table 1, and the primers were synthesized by GenScript company.

[0065] LB medium: tryptone 10 g/L, yeast extract 5 g/L NaCl 10 g/L, add 15 g/L agar powder to solid culture medium, and add 2 g/mL erythromycin (E2), 16 g/mL neomycin (N16), and 8 g/mL chloramphenicol (C8) according to actual need for screening Bacillus subtilis transformants and seed cultivation.

[0066] Fermentation medium: glucose 40 g/L, tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, MgSO.sub.4.Math.7H.sub.2O 1 g/L, CMP 0.02 g/L, xylose 10 g/L, and pH 6.0-8.0.

TABLE-US-00001 TABLE 1 Strains and plasmids involved in the experiment Name Genetic background Source Strains S. cerevisiae Saccharomyces cerevisiae Laboratory preservation BS168N trpC 2, araR::P.sub.ara-neo Laboratory preservation BS168NCm trpC 2, araR::neo, yrpC::cat-araR Laboratory preservation BSC1-2 BS168N, yrpC::TP2-CCT-CKI.sup.E216G Laboratory preservation BS168N/PEM21 BS168N, lacA::P.sub.xyl-PEM2-PEM1 Constructed in this present disclosure BS168N/PEM21/ydeO BS168N/PEM21, Constructed ydeO::TP2-CCT-CKI.sup.E216G in this present disclosure BS168N/PEM21/yjoB BS168N/PEM21, Constructed yjoB::TP2-CCT-CKI.sup.E216G in this present disclosure Plasmid pJMP1 Bacillus subtilis dCas9 expression vector; Laboratory integrates into lacA preservation

2. Reagents and Instruments.

[0067] High fidelity DNA polymerase 2Phanta Flash Master Mix (Dye Plus) and high-purity heat-resistant DNA polymerase 2Taq Master Mix (Dye Plus) were both purchased from Nanjing Vazyme Biotechnology Co., Ltd; the plasmid extraction kit, yeast genomic DNA extraction kit, and bacterial genomic DNA extraction kit were all purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd; standard CDP-choline sodium was purchased from Sigma Aldrich (USA); the remaining biochemical reagents are domestic analytical grade reagents.

[0068] Instruments used: PCR instrument (LifeECO), microplate reader (Thermo), ultrasonic cell disruptor (Ningbo Xinzhi), high-performance liquid chromatography chromatogram (Agilent).

3. Primers.

[0069] The primers used for PCR are shown in Table 2.

TABLE-US-00002 TABLE2 PCRPrimerSequences Sequence Primers name Sequence(5.fwdarw.3) lacA-U1 SEQID CAATAACGCTGGATACATCTG No.1 lacA-U2 SEQID TGAATCTGCTGTCATCCAT No.2 lacA-P1q SEQID ATGGATGACAGCAGATTCACAGTGTATTTAC No.3 TTGAGAGGAG lacA-P2 SEQID CATTTCCCCCTTTGATTITTAG No.4 PEM2-1q SEQID TCTAAAAATCAAAGGGGGAAATGAAGGAGT No.5 CAGTCCAAGAGAT PEM2-2 SEQID AGAAATAGAGAACTTACATATTC No.6 PEM1-1q SEQID GAATATGTAAGTTCTCTATTTCTAAAGGAGG No.7 AATAAAAAATGTCCAGITGTAAAACCACT PEM1-2 SEQID TCAAGCAAGACTATCAAGC No.8 lacA-ED1q SEQID CGCTTGATAGTCTTGCTTGAGAATTGAACCT No.9 CTACCACAA lacA-ED2 SEQID GGGTGATGTCTAAGGTTGA No.10 CX-PP1 SEQID TTGAGAGGAGACAGTAGACTT No.11 CX-PP2 SEQID GCATCTAACTTGACCCAGA No.12 CX-PP3 SEQID TCCTATGAGCAATGGCAAT No.13 CX-PP4 SEQID ATTGCTAGTCTCGTTTCCTT No.14 CX-PP5 SEQID CGTTTCCGCCATTCTTTG No.15 ydeO-U1 SEQID CTTCATATAACGCAGCATCA No.16 ydeO-U2q SEQID ACCATCAACGCAACCATAAACTGAACCAAC No.17 GACGCTAAGA ydeO-PCC2 SEQID AGATGTTAGCGACAGTAATAG No.18 ydeO-D1q SEQID TCTATTACTGTCGCTAACATCTCGGTGAAGG No.19 TCTGAATCA ydeO-D2 SEQID AGGAAGGCTGATAGGTCAT No.20 ydeO-CR1q SEQID CATGACCTATCAGCCTTCCTTCTTCAACTAA No.21 AGCACCCAT ydeO-CR2 SEQID TTATTCATTCAGTTTTCGTG No.22 ydeO-G1q SEQID GCACGAAAACTGAATGAATAACGGCTAACAT No.23 AGAAGGAGAT ydeO-G2 SEQID GCTACATCATACACGGTAAC No.24 CX-CC5 SEQID ATCTCTGGTGCGATGACA No.25 yjoB-U1 SEQID GACGATAAACCCAAACACAA No.26 yjoB-U2q SEQID ACCATCAACGCAACCATAAACCGCAACTCTT No.27 TCATTCTCTT yjoB-PCC2 SEQID TGGAGTTAGCCACAGTAATAG No.28 yjoB-D1q SEQID TCTATTACTGTGGCTAACTCCATCATTGGCAC No.29 GAAGAAGG yjoB-D2 SEQID GATTCTGCGGACGGTGTA No.30 yjoB-CR1q SEQID GTACACCGTCCGCAGAATCTCTTCAACTAAA No.31 GCACCCAT yjoB-G1q SEQID GCACGAAAACTGAATGAATAAGGTTGATTG No.32 GAGCCTGAG yjoB-G2 SEQID CGTCCTGCGTATTGAGAA No.33 2-yjoB-U1 SEQID TCTTCCTGATGCTGGTGAA No.34

[0070] The present disclosure provides a recombinant engineered strain, preparation method, and application for de novo synthesis of CDP-choline using glucose as a substrate, as follows:

[0071] In the present disclosure, first the phosphatidylethanolamine N-methyltransferase gene PEM1 and phosphatidylethanolamine/phosphatidyl-N-methylethanolamine N-methyltransferase gene PEM2 of S. cerevisiae were integrated with the xylose induced promoter P.sub.xyl to form an artificial operon P.sub.xyl-PEM2-PEM1, which was then integrated into the genome of Bacillus subtilis 168N, aiming to open up the synthesis pathway from glucose to choline. Then, the artificial operon TP2-CCT-CKI constructed in patent CN116790466B was integrated into the genome of Bacillus subtilis expressing P.sub.xyl-PEM2-PEM1, opening up the synthetic pathway from choline to CDPC, and then shake flask fermentation culture was performed on the constructed recombinant engineered strain, measuring the growth of the strain and the synthesis of CDPC separately.

Embodiment 1

[0072] Construction of recombinant strain BS168N/PEM21: co-expressing the PEM2 and PEM1 genes of S. cerevisiae at the lacA site on the BS168N genome.

[0073] Firstly, taking the genome of BS168N as a template, primers lacA-U1/lacA-U2 were used to amplify the fragment U containing the upstream homologous arm (as shown in SEQ ID No. 35, 1309 bp).

[0074] Taking the plasmid pJMP1 as a template, primers lacA-P1q/lacA-P2 were used to amplify the fragment P containing xylose inducible promoter P.sub.xyl (as shown in SEQ ID No. 36, 426 bp).

[0075] Primers lacA-ED1q/lacA-ED2 were used to amplify the fragment ED containing the erythromycin resistance gene and downstream homologous arm (as shown in SEQ ID No. 37, 1976 bp).

[0076] Taking the genome of S. cerevisiae as a template, primers PEM2-1q/PEM2-2 and PEM1-1q/PEM1-2 were used to amplify the fragment M2 containing the PEM2 gene sequence (as shown in SEQ ID No. 38, 631 bp) and the fragment M1 containing the PEM1 gene sequence (as shown in SEQ ID No. 39, 2626 bp), respectively.

[0077] Secondly, primers lacA-P1q/PEM1-2 were used to splice the fragment P, the fragment M2, and the fragment M1 together to form the fragment PM2M1 (3683 bp) though overlap-PCR method; then, primers lacA-U1/lacA-ED2 were used to splice the fragment U, the fragment PM2M1, and the fragment ED into the fragment UPM2M1ED (as shown in SEQ ID No.40, 6968 bp).

[0078] Finally, the fragment UPM2M1ED was transformed into competent cells of the receptor bacterium BS168N and coated onto LBE2 solid plates. The grown transformants were selected and transferred to LBE2 liquid medium for shaking culture for 2 hours. Primers CX-PP1/CX-PP5 were used for bacterial liquid PCR verification. The correct transformant was one with a band size of 3814 bp.

[0079] A correct transformant is randomly selected and transferred into a test tube containing 5 mL of LBE2 liquid medium for overnight culture. Extracting its genome as a template, and primers CX-PP1/CX-PP5 were used for amplification. Then, the PCR product was sent for testing, with sequencing primers as follows: CX-PP1/CX-PP2/CX-PP3/CX-PP4/CX-PP5.

[0080] CX-PP1/CX-PP2/CX-PP3/CX-PP4/CX-PP5 were sequenced, and a recombinant strain without additional point mutations was obtained. The strain was named BS168N/PEM21 and deposited.

Embodiment 2

[0081] Construction of recombinant engineered strain BS168N/PEM21/ydeO and BS168N/PEM21/yjoB: co-expressing The CCT and CKI genes of S. cerevisiae at ydeO and yjoB sites on the BS168N/PEM2 genome, respectively.

(1) Construction of Recombinant Strain BS168N/PEM21/ydeO

[0082] Firstly, taking the genome of BS168NCm as a template, primers ydeO-U1/ydeO-U2q, ydeO-D1q/ydeO-D2, ydeO-CR1q/ydeO-CR2, and ydeO-D1q/ydeO-G2 were used to amplify the fragment U containing upstream homologous arms (as shown in SEQ ID No. 41, 1302 bp), the fragment D containing homologous recombination (as shown in SEQ ID No. 42, 1005 bp), the fragment CR containing chloramphenicol resistance gene and arabinose operon repressor gene (as shown in SEQ ID No. 43, 2069 bp), and the fragment G containing downstream homologous arms (as shown in SEQ ID No. 44, 514 bp).

[0083] Taking the genome of BSC1-22 as a template, primers TP2-1/ydeO-PCC2 were used to amplify the fragment PCC (as shown in SEQ ID No. 45, 3329 bp) containing the constitutive strong promoter TP2, CCT gene and CKI gene derived from S. cerevisiae.

[0084] Secondly, primers ydeO-U1/ydeO-PCC2 were used to splice the fragment U, the fragment PCC, and the fragment D into the fragment UPCCD (5636 bp) through the overlap-PCR method; then, primers ydeO-U1/ydeO-G2 were used to splice the fragment UPCCD, the fragment CR, and the fragment G into the fragment UPCCDCRG (as shown in SEQ ID No. 46, 8219 bp).

[0085] Finally, the fragment UPCCDCRG was transformed into competent cells of the receptor bacterium BS168N/PEM21 and coated onto LBC8 solid plates. The grown transformants were selected and transferred to LBC8 liquid medium for shaking culture for 2 hours. Primers CX-CC3/ydeO-D2 were used for bacterial liquid PCR verification. The correct transformant was the one with a band size of 2395 bp.

[0086] A correct transformant was randomly selected and transferred to the antibiotic-free LB liquid medium for shaking cultivation for 4 hours, and then 1 L of bacterial solution was taken to coat onto a LBN16 solid plate. The grown transformants were selected and transfer to the LBN16 liquid medium for shaking cultivation for 2 hours, followed by another round of bacterial liquid PCR validation. Finally, the two rounds of validated single colonies were selected to incubate into in a test tube containing 5 mL of LBN16 liquid medium for overnight culture, extracting its genome as a template, and primers TP2-1/ydeO-PCC2 were used for amplification, with a band size of 3329 bp. Sending the PCR product for testing, and the primers were TP2-1/CCT-2/CX-CC3/CX-CC5/ydeO-PCC2 for sequencing. As a result, a recombinant strain with the same point mutation as the CKI gene mutation of strain BSC1-22 in patent CN202310882430. X was obtained. The strain was named BS168N/PEM21/ydeO and deposited, and the sequence of primers TP2-1/CCT-2/CX-CC3 can be found in patent CN116790466B.

(2) Construction of Recombinant Strain BS168N/PEM21/yjoB

[0087] Firstly, taking the genome of BS168NCm as the template, yjoB-U1/yjoB-U2q, yjoB-D1q/yjoB-D2, yjoB-CR1q/ydeO-CR2, yjoB D1q/yjoB-G2 were used to amplify the fragment U (as shown in SEQ ID No. 47, 1362 bp), the fragment D (as shown in SEQ ID No. 48, 808 bp), the fragment CR (as shown in SEQ ID No. 43, 2069 bp), and the fragment G (as shown in SEQ ID: 49, 697 bp); taking the genome of BSC1-22 as the template, TP2-1/yjoB-PCC2 was used to amplify the fragment PCC (as shown in SEQ ID No. 50, 3329 bp).

[0088] Secondly, primers yjoB-U1/yjoB-PCC2 were used to splice the fragment U, the fragment PCC, and the fragment D into the fragment UPCCD (5333 bp) through the overlap-PCR method; then, primers 2-yjoB-U1/yjoB-G2 were used to splice the fragment UPCCD, the fragment CR, and the fragment G into the fragment UPCCDCRG (as shown in SEQ ID No. 51, 8099 bp).

[0089] Finally, the fragment UPCCDCRG was transformed into competent cells of the receptor bacterium BS168N/PEM21 and coated onto LBC8 solid plates. After two rounds of screening, the correct transformant was obtained, extracting its genome as a template, and primers TP2-1/yjoB-PCC2 were used for amplification, with a band size of 3329 bp. Sending the PCR product for testing, and the sequencing primers were TP2-1/CCT-2/CX-CC3/CX-CC5/yjoB-PCC2. As a result, a recombinant strain with the same point mutation as the CKI gene mutation of strain BSC1-22 in patent CN202310882430. X was obtained, and the strain was named BS168N/PEM21/yjoB and deposited.

Embodiment 3

[0090] Two artificial operons, P.sub.xyl-PEM2-PEM1 and TP2-CCT-CKI, were integrated into the genome of Bacillus subtilis for expression and detect the synthesis of CDPC.

(1) Shake Flask Fermentation Culture and Biomass Determination

[0091] Newly activated single colonies of BS168N/PEM21, BS168N/PEM21/ydeO, and BS168N/PEM21/yjoB were selected from the solid plate of LBN16+E2 and transferred to a test tube containing 5 mL of LBN16+E2 liquid medium, shaking at 37 C. and 200 r/min for 12 hours. The culture was transferred to a 250 mL conical flask containing 30 mL of fermentation medium at 1% inoculated volume, then conducting shaking culture at 37 C. and 220 r/min for 26 hours. After 3 hours of fermentation, xylose with a final concentration of 10 g/L was added for induction.

[0092] 1 mL of fermentation broths that has been fermented for 6 hours, 11 hours, and 26 hours were taken respectively, then performing centrifuge to remove the supernatant, the cell pellet was resuspended and diluted appropriately after washed with deionized water to measure the OD.sub.600 value of the bacterial suspension.

[0093] The growth curves of three recombinant strains BS168N/PEM21, BS168N/PEM21/ydeO, and BS168N/PEM21/yjoB are shown in FIG. 2.

(2) Extraction and Detection of CDPC

[0094] 30 mL of 26 hour fermentation broth was taken, conducting centrifuge at 8000 rpm and 4 C. for 10 minutes, and 1 mL of supernatant was taken for measuring the extracellular CDPC content. The remaining cell pellet was washed once with phosphate buffer solution at pH 7.4, then added to the cell pellet into 5 mL of phosphate buffer solution to resuspend, and then cell lysis was conducted by an ultrasonic cell disruptor, performing centrifuge at 8000 rpm and 4 C. for 10 minutes, and 1 mL of supernatant was taken to determine the intracellular CDPC content.

[0095] The HPLC detection conditions are detailed in patent CN116790466B.

[0096] FIGS. 3-5 show the high-performance liquid chromatography chromatograms of the fermentation supernatant of recombinant strain BS168N/PEM21, BS168N/PEM21/ydeO, and BS168N/PEM21/yjoB, respectively.

[0097] FIGS. 6-8 show the high-performance liquid chromatography chromatograms (all concentrated 6-fold) of the supernatant obtained from lysed cell pellets of recombinant strain BS168N/PEM21, BS168N/PEM21/ydeO, and BS168N/PEM21/yjoB, respectively.

(3) the Effect of Integrating PEM2-PEM1 and CCT-CKI Expression on CDPC Synthesis

[0098] B. Subtilis lacks a synthetic pathway from PE to PC and cannot synthesize choline from scratch, and lacks a synthetic pathway from choline to CDPC. While there is a complete CDPC pathway in S. cerevisiae. Therefore, in this embodiment, BS168N was used as the starting strain, and firstly, integrate the phosphatidylethanolamine N-methyltransferase gene PEM1 and the phosphatidylethanolamine/phosphatidyl-N-methylethanolamine N-methyltransferase gene PEM2 of S. cerevisiae with the xylose induced promoter P.sub.xyl to form an artificial operon P.sub.xyl-PEM2-PEM1, which was integrated into the genome of Bacillus subtilis 168N to obtain the recombinant strain BS168N/PEM21, aiming to open up the synthesis pathway from glucose to choline. Subsequently, the choline kinase gene CKI, the phosphocholine cytidylyltransferase gene CCT were combined with the constitutive strong promoter TP2 to form an artificial operon TP2-CCT-CKI, which was integrated into the ydeO and yjoB sites of the BS168N/PEM21 genome, respectively, to obtain recombinant strain BS168N/PEM21/ydeO and BS168N/PEM21/yjoB, aiming to open up the synthetic pathway from choline to CDPC. Due to the simultaneous integrant expression of four genes, namely PEM1, PEM2, CKI, and CCT in the BS168N/PEM21/ydeO and BS168N/PEM21/yjoB strains, thus, the synthetic pathway from glucose to CDPC has theoretically been opened up.

[0099] BS168N/PEM21, BS168N/PEM21/ydeO, and BS168N/PEM21/yjoB were subjected to shake flask fermentation culture. The growth results of the strains are shown in Table 3. The total synthesis of extracellular, intracellular, and CDPC of different recombinant strains after 26 hours of fermentation is shown in Table 4 and FIG. 9.

TABLE-US-00003 TABLE 3 Growth of strains Fermentation OD.sub.600 OD.sub.600 OD.sub.600 time (h) (BS168N/PEM21) (BS168N/PEM21/ydeO) (BS168N/PEM21/yjoB) 6 4.11 0.66 3.43 0.19 3.38 0.51 11 7.69 0.07 7.63 0.03 6.82 0.19 26 9.51 0.31 7.24* 0.27 7.27* 0.30 Note: *indicates there is a significant difference (P < 0.05) compared to BS168N/PEM21.

TABLE-US-00004 TABLE 4 Total synthesis of extracellular, intracellular, and CDPC of different recombinant strains after 26 hours of fermentation Extracellular CDPC Intracellular CDPC CDPC total synthesis Strains production (mg/L) production (mg/L) amount (mg/L) BS168N/PEM21 BS168N/PEM21/ydeO 50.5.sup.# 3.4 25.5.sup.# 2.1 76.0.sup.# 5.5 BS168N/PEM21/yjoB 49.7.sup.# 3.1 24.7.sup.# 4.5 74.4.sup.# 7.6 Note: .sup.#indicates a highly significant difference (P < 0.01) compared to BS168N/PEM21.

[0100] The results in Table 3 show that during the period of 12-26 hours, the growth of strains BS168N/PEM21/ydeO and BS168N/PEM21/yjoB decreased compared to BS168N/PEM21.

[0101] In addition, the total synthesis of extracellular, intracellular, and CDPC by different recombinant strains in Table 4 showed that after 26 hours of fermentation, no CDPC was detected in the supernatant and cell pellet of BS168N/PEM21; CDPC was detected in the supernatant and cell pellet of BS168N/PEM21/ydeO and BS168N/PEM21/yjoB.

[0102] Moreover, the extracellular CDPC content of recombinant strain BS168N/PEM21/ydeO was 50.53.4 mg/L, the intracellular CDPC content was 25.52.1 mg/L, and the total CDPC synthesis was 76.05.5 mg/L. The extracellular CDPC content of recombinant strain BS168N/PEM21/yjoB was 49.73.1 mg/L, the intracellular CDPC content was 24.74.5 mg/L, and the total CDPC synthesis was 74.47.6 mg/L.

[0103] The above results indicate that introducing PEM2-PEM1 and CCT-CKI into the genome of B. subtilis for expression indeed opens up the CDPC synthesis pathway starting from glucose, and approximately 66% of CDPC is secreted extracellular; the expression of CCT-CKI is not related to the gene locus (ydeO or yjoB) integrated and inserted, resulting in little difference in yield between the two recombinant strains; and the growth of the last two strains decreased after 26 hours, this is because some glucose is used to synthesize CDPC, resulting in a decrease in glucose used for growth.

[0104] The present disclosure is the first to utilize the engineered B. Subtilis uses glucose as a substrate and can synthesize CDPC from scratch without the need to add precursors choline chloride or choline phosphate, laying a research foundation for reducing the production cost of CDPC in the future.

TABLE-US-00005 Sequencelist SEQIDNo.35 Caataacgctggatacatctgcccctttgataaaatccttgttcatcccctctacttttttaacaaaaagatcctctgccc gaagctctgacacgtgcttctccttctcaatggcagcggcatatcctgttgaactacatgcactccacacgatggcagccgc aaagaacattttcactttgcttttcatgatttcatccccccaagacttagcaagcgttttcattctatagaaaaaagaatccgccc atatcgagcggagcatcagcctaatgtgtgtttacgacaattctcacttcatacttttccatcgtcaggtcgcctgacaatatgtc tcctgtcattatgtccttcacactctgatcaaacgtgaccagctgtttttcttccgtgaaattcatgacaaaaatataatcattgtcc tgatcctgcctcgcttgtacggagacgccttttccgtgccgaaccggaaaaactggagagagagacaggtctgtgatcaga ccctcatagaaatcacgctgaaattgatcctccaaacgcgcgccgataaaatacgccttgccctgctgatactcatggcttgt gaccgctggcgtgcgcgcataaaaatcttcttgatacaccgcttccactgaagctgtctttacatcaatcacggttgcataatc cttcatttcatatatttggctgcggtagctgacagcgtttcgatccttcggatacagggtgtccgtttcaagaggctcaactcca aatatagcttgaagatccggatgccatccgcctgtgtatgttaagtcatgctcattcacaaccccgctgatatacgtcatgact aaggtgccgccgtcagccgtaaacgcttttaaacgggaaacggtgtcctcgctgattaaatacagcatcgggacgatcagc agtttatatggtgaaaagtcttgttctttcgtgatgacgtcgacagggatatcgtgttcccagaatgtgcggtaatgctgctgaa gcgtttgcggataacgttttgtcgccttcgcaaacccctgagcatcctcgagcgcccaatgattttcccagtcatataaaatcg cggtttgagccggcctcttcgttccgacaacttcggacagccgttccaatgtctcgcctaccttggccacttcttgaaagacg cggttcttcgggctattgtcatgatccacaaccgctccgtgtaatttttctgatgacccccgtgatttgcggtattggaaataga gaacgctgtccgagccgtgggcaatcatttgcatggatgacagcagattca. SEQIDNo.36 Cagtgtatttacttgagaggagacagtagacttgtttaatcctgtaatctcagagagagttgccctggagacagggg agttcttcaaaatttcatctaatattaatttttgattcattttttttactaaagcttgatctgcaatttgaataataaccactcctttgttt atccaccgaactaagttggtgttttttgaagcttgaattagatatttaaaagtatcatatctaatattataactaaattttctaaaaaaa acattgaaataaacatttattttgtatatgatgagataaagttagtttattggataaacaaactaactcaattaagatagttgatgg ataaacttgttcacttaaatcaaagggggaaatgacaaatggtccaaactagtgatatctaaaaatcaaagggggaaatg. SEQIDNo.37 Gaattgaacctctaccacaaaagaaaaacgaaatgatacaccaatcagtgcaaaaaaagatataatgggagataa gacggttcgtgttcgtgctgacttgcaccatatcataaaaatcgaaacagcaaagaatggcggaaacgtaaaagaagttatg gaaataagacttagaagcaaacttaagagtgtgttgatagtgcagtatcttaaaattttgtataataggaattgaagttaaattag atgctaaaaatttgtaattaagaaggagtgattacatgaacaaaaatataaaatattctcaaaactttttaacgagtgaaaaagt actcaaccaaataataaaacaattgaatttaaaagaaaccgataccgtttacgaaattggaacaggtaaagggcatttaacga cgaaactggctaaaataagtaaacaggtaacgtctattgaattagacagtcatctattcaacttatcgtcagaaaaattaaaact gaatactcgtgtcactttaattcaccaagatattctacagtttcaattccctaacaaacagaggtataaaattgttgggagtattc cttaccatttaagcacacaaattattaaaaaagtggtttttgaaagccatgcgtctgacatctatctgattgttgaagaaggattc tacaagcgtaccttggatattcaccgaacactagggttgctcttgcacactcaagtctcgattcagcaattgcttaagctgcca gcggaatgctttcatcctaaaccaaaagtaaacagtgtcttaataaaacttacccgccataccacagatgttccagataaatat tggaagctatatacgtactttgtttcaaaatgggtcaatcgagaatatcgtcaactgtttactaaaaatcagtttcatcaagcaat gaaacacgccaaagtaaacaatttaagtaccgttacttatgagcaagtattgtctatttttaatagttatctattatttaacgggag gaaataattctatgagtcgcttttgtaaatttggaaagttacacgttactaaagggaatgtagataaattattaggtatactactga cagcttccaaggagctaaagaggtccctagactctagacccggggatctctgcagtgagatctggtaatgactctctagctt gaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgag taggacaaatccgccgctctagctaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactcttgttaactct agagctgcctgccgcgtttcggtgatgaagatcttcccgatgattaattaattcagaacgctcggttgccgccgggcgtttttt atgcagcaatggcaagaacgttgctctagagcggccgcatcgattcacagtggcaatctcccccgtattcgtttgaaatgtg ccacattaacagcgccgggtgatgtccgtatcgttctgctaataagcggttgatgtgccgtgttttttctcggtagactttagat gtgaggcagtggttgtgccttccgccgtgcagctgtttgacgcgggaggcattgacgcgcaaaacttccggataggtttgc gacagccaggccggacgggctccgctcggcgttgctaatatgacccggccgcctatactgtgaatccgctcaaaaatatc atccagccattcaaattgatatacgccctcctccggctcaagtgcgctccatgcaaaaatgccgacagaaaacgtattcgtat gagaaagcttcatcagtttgatatcgtcagctaaaatatcgggccgatccagccactgatcggggttgtagtctcccccatgg agcataaattttgcttttgttacgtgcgttttttcaaccttagacatcaccc. SEQIDNo.38 Aaggagtcagtccaagagatcatccagcaactcatccacagtgtcgatttacagtcttccaagttccagctggccatt gtgtgcacgatgttcaatcctatcttttggaacatcgttgcaagaatggaataccacaagcattctctcaccaagatgtgtggt ggggccagaaagggctgttacatgttggcggcgaccatattttcgctaggtatcgtcagagacatggtgtacgagtctgcat tgcgtgaacagcctacgtgttctctgatcacgggcgagaactggaccaagctgggtgtggctctctttggtttggggcaagt gcttgttttgagttccatgtacaagctgggtatcacagggacgtacttgggtgactatttcggcatcctgatggatgagagagt caccggcttccccttcaacgtttccaacaaccccatgtaccagggttccactttgtccttcttgggcatagccctttacaaagg aaagcctgcggggctggttgtttctgccgtagtttacttcatgtacaagatcgctcttcgttgggaagaaccttttactgccatg atctacgctaaccgtgataaggccaaaaagaatatgtaagttctctatttct. SEQIDNo.39 aaaggaggaataaaaaatgtccagttgtaaaaccactttgtctgaaatggttggttctgtgacaaaagataggggcac tatcaacgtcgaagccagaacacgttcgagtaatgtaactttcaaaccacctgtaacccacgatatggtgcgttcgcttttcga tccaactttgaagaaatctctgctggaaaaatgcatcgcgctagctattatatcgaattttttcatttgttattgggtcttccaaag gtttggcttacagtttaccaaatacttttttctggtacagtatttattttggagaattgcttataatttaggtattggactggttctgcat taccagtcacattatgaaacactaacgaattgtgctaaaactcatgcgattttcagcaaaataccacagaataaagatgctaat tcgaatttctcaacaaattccaattccttttcagaaaaattttggaattttattaggaagttctgtcaatatgagattaggtctaaaat gccaaaggagtatgatttatttgcctatccagaagagatcaacgtctggttgatatttcgccagtttgtcgatttaatcttgatgc aagattttgtgacttacattatttacgtttacctttctattccatatagctgggttcaaatcttcaactggagatctttgctaggtgtta ttttgattttatttaacatctgggtcaagttagatgcgcatcgcgtggttaaagattatgcttggtactggggtgatttcttctttttg gaagaatctgaattgatttttgatggtgtcttcaacatctctccacatccaatgtattctattggttatttgggttattatggcctatc attgatttgtaatgactacaaggttcttctggtgtccgtatttggacattattctcaatttttgtttctgaaatacgtcgaaaatcctc atattgaaagaacgtatggtgatggcactgattccgattctcaaatgaatagcagaattgatgacttgatatcaaaagagaatt acgattattcaagacctctaattaacatgggtctttcttttaacaacttcaataagctaagatttactgactatttcacaattggtac cgtagcggcacttatgttaggaacaatcatgaatgctaggtttatcaatttaaattacctctttattaccgtttttgtaacaaaactg gtttcatggttatttatctcgacaatattgtacaagcaatctcagtctaaatggttcacgagattgtttttggagaatggttacaca caagtttattcctatgagcaatggcaatttatttacaattattatttggtattaacctacacacttatgataattcatacagggcttca aatttggagtaatttttccaacataaacaacagtcaattaatttttggtttaattcttgtggctttacaaacatggtgtgataaggaa acgagactagcaatctctgattttggttggttttatggtgattttttcttgagcaactatatctcaactagaaagctgacttcccaa ggtatctacagatatttgaatcacccagaagcggtattaggcgttgttggagtttggggtactgtattgatgacgaattttgccg ttacaaatattatcttggctgttttatggacattgacaaattttattcttgtcaagtttattgaaacacctcatgttaataagatatatg gtaaaactaaacgagttagtggtgttggaaaaactttactaggcttgaaacctctaagacaagtttcagatattgtcaatagaat tgaaaatattataatcaaatcattagttgatgaaagcaagaactccaatggtggggcagaacttttgcccaaaaattatcaaga taacaaagaatggaatattttaattcaggaagcaatggatagcgttgcaacaagattgagtccatactgtgaattgaaaattga aaacgagcaggtagaaaccaattttgtcttgcctactccagtaactttgaattggaaaatgcccattgaactttataacggaga tgattggattggtttgtataaagtcattgatacaagggcagatcgtgaaaaaacaagagttggatcaggtggtcattggagtg cgacttctaaagattcatatatgaaccatggattaagacataaagaatctgtaacagaaataaaggcgactgaaaagtacgtt caaggtaaagtgacattcgacacgtctttgctttattttgaaaatggtatctacgaatttaggtatcattctggtaactcccataag gtgttattgatttctacaccatttgaaatttcattgccggtactaaataccacgacacctgaactgtttgagaaggatttgactga gtttttgacgaaagtcaacgttttgaaggatggtaaatttcgtccattaggaaataagttcttcgggatggacagcttgaaacaa ttgattaagaactcaattggcgtcgaactttcgtctgagtatatgaggagagtcaatggtgacgctcacgttatttcgcatcgc gcttgggatataaaacaaacgcttgatagtcttgcttga. SEQIDNo.40 caataacgctggatacatctgcccctttgataaaatccttgttcatcccctctacttttttaacaaaaagatcctctgcccg aagctctgacacgtgcttctccttctcaatggcagcggcatatcctgttgaactacatgcactccacacgatggcagccgca aagaacattttcactttgcttttcatgatttcatccccccaagacttagcaagcgttttcattctatagaaaaaagaatccgcccat atcgagcggagcatcagcctaatgtgtgtttacgacaattctcacttcatacttttccatcgtcaggtcgcctgacaatatgtctc ctgtcattatgtccttcacactctgatcaaacgtgaccagctgtttttcttccgtgaaattcatgacaaaaatataatcattgtcctg atcctgcctcgcttgtacggagacgccttttccgtgccgaaccggaaaaactggagagagagacaggtctgtgatcagacc ctcatagaaatcacgctgaaattgatcctccaaacgcgcgccgataaaatacgccttgccctgctgatactcatggcttgtga ccgctggcgtgcgcgcataaaaatcttcttgatacaccgcttccactgaagctgtctttacatcaatcacggttgcataatcctt catttcatatatttggctgcggtagctgacagcgtttcgatccttcggatacagggtgtccgtttcaagaggctcaactccaaat atagcttgaagatccggatgccatccgcctgtgtatgttaagtcatgctcattcacaaccccgctgatatacgtcatgactaag gtgccgccgtcagccgtaaacgcttttaaacgggaaacggtgtcctcgctgattaaatacagcatcgggacgatcagcagt ttatatggtgaaaagtcttgttctttcgtgatgacgtcgacagggatatcgtgttcccagaatgtgcggtaatgctgctgaagc gtttgcggataacgttttgtcgccttcgcaaacccctgagcatcctcgagcgcccaatgattttcccagtcatataaaatcgcg gtttgagccggcctcttcgttccgacaacttcggacagccgttccaatgtctcgcctaccttggccacttcttgaaagacgcg gttcttcgggctattgtcatgatccacaaccgctccgtgtaatttttctgatgacccccgtgatttgcggtattggaaatagaga acgctgtccgagccgtgggcaatcatttgcatggatgacagcagattcacagtgtatttacttgagaggagacagtagactt gtttaatcctgtaatctcagagagagttgccctggagacaggggagttcttcaaaatttcatctaatattaatttttgattcatttttt ttactaaagcttgatctgcaatttgaataataaccactcctttgtttatccaccgaactaagttggtgttttttgaagcttgaattag atatttaaaagtatcatatctaatattataactaaattttctaaaaaaaacattgaaataaacatttattttgtatatgatgagataaa gttagtttattggataaacaaactaactcaattaagatagttgatggataaacttgttcacttaaatcaaagggggaaatgacaa atggtccaaactagtgatatctaaaaatcaaagggggaaatgaaggagtcagtccaagagatcatccagcaactcatccac agtgtcgatttacagtcttccaagttccagctggccattgtgtgcacgatgttcaatcctatcttttggaacatcgttgcaagaat ggaataccacaagcattctctcaccaagatgtgtggtggggccagaaagggctgttacatgttggcggcgaccatattttcg ctaggtatcgtcagagacatggtgtacgagtctgcattgcgtgaacagcctacgtgttctctgatcacgggcgagaactgga ccaagctgggtgtggctctctttggtttggggcaagtgcttgttttgagttccatgtacaagctgggtatcacagggacgtactt gggtgactatttcggcatcctgatggatgagagagtcaccggcttccccttcaacgtttccaacaaccccatgtaccagggtt ccactttgtccttcttgggcatagccctttacaaaggaaagcctgcggggctggttgtttctgccgtagtttacttcatgtacaa gatcgctcttcgttgggaagaaccttttactgccatgatctacgctaaccgtgataaggccaaaaagaatatgtaagttctctat ttctaaaggaggaataaaaaatgtccagttgtaaaaccactttgtctgaaatggttggttctgtgacaaaagataggggcacta tcaacgtcgaagccagaacacgttcgagtaatgtaactttcaaaccacctgtaacccacgatatggtgcgttcgcttttcgatc caactttgaagaaatctctgctggaaaaatgcatcgcgctagctattatatcgaattttttcatttgttattgggtcttccaaaggtt tggcttacagtttaccaaatacttttttctggtacagtatttattttggagaattgcttataatttaggtattggactggttctgcatta ccagtcacattatgaaacactaacgaattgtgctaaaactcatgcgattttcagcaaaataccacagaataaagatgctaattc gaatttctcaacaaattccaattccttttcagaaaaattttggaattttattaggaagttctgtcaatatgagattaggtctaaaatg ccaaaggagtatgatttatttgcctatccagaagagatcaacgtctggttgatatttcgccagtttgtcgatttaatcttgatgca agattttgtgacttacattatttacgtttacctttctattccatatagctgggttcaaatcttcaactggagatctttgctaggtgttatt ttgattttatttaacatctgggtcaagttagatgcgcatcgcgtggttaaagattatgcttggtactggggtgatttcttctttttgg aagaatctgaattgatttttgatggtgtcttcaacatctctccacatccaatgtattctattggttatttgggttattatggcctatcat tgatttgtaatgactacaaggttcttctggtgtccgtatttggacattattctcaatttttgtttctgaaatacgtcgaaaatcctcat attgaaagaacgtatggtgatggcactgattccgattctcaaatgaatagcagaattgatgacttgatatcaaaagagaattac gattattcaagacctctaattaacatgggtctttcttttaacaacttcaataagctaagatttactgactatttcacaattggtaccg tagcggcacttatgttaggaacaatcatgaatgctaggtttatcaatttaaattacctctttattaccgtttttgtaacaaaactggt ttcatggttatttatctcgacaatattgtacaagcaatctcagtctaaatggttcacgagattgtttttggagaatggttacacaca agtttattcctatgagcaatggcaatttatttacaattattatttggtattaacctacacacttatgataattcatacagggcttcaaa tttggagtaatttttccaacataaacaacagtcaattaatttttggtttaattcttgtggctttacaaacatggtgtgataaggaaac gagactagcaatctctgattttggttggttttatggtgattttttcttgagcaactatatctcaactagaaagctgacttcccaaggt atctacagatatttgaatcacccagaagcggtattaggcgttgttggagtttggggtactgtattgatgacgaattttgccgtta caaatattatcttggctgttttatggacattgacaaattttattcttgtcaagtttattgaaacacctcatgttaataagatatatggta aaactaaacgagttagtggtgttggaaaaactttactaggcttgaaacctctaagacaagtttcagatattgtcaatagaattga aaatattataatcaaatcattagttgatgaaagcaagaactccaatggtggggcagaacttttgcccaaaaattatcaagataa caaagaatggaatattttaattcaggaagcaatggatagcgttgcaacaagattgagtccatactgtgaattgaaaattgaaa acgagcaggtagaaaccaattttgtcttgcctactccagtaactttgaattggaaaatgcccattgaactttataacggagatg attggattggtttgtataaagtcattgatacaagggcagatcgtgaaaaaacaagagttggatcaggtggtcattggagtgcg acttctaaagattcatatatgaaccatggattaagacataaagaatctgtaacagaaataaaggcgactgaaaagtacgttca aggtaaagtgacattcgacacgtctttgctttattttgaaaatggtatctacgaatttaggtatcattctggtaactcccataaggt gttattgatttctacaccatttgaaatttcattgccggtactaaataccacgacacctgaactgtttgagaaggatttgactgagtt tttgacgaaagtcaacgttttgaaggatggtaaatttcgtccattaggaaataagttcttcgggatggacagcttgaaacaattg attaagaactcaattggcgtcgaactttcgtctgagtatatgaggagagtcaatggtgacgctcacgttatttcgcatcgcgctt gggatataaaacaaacgcttgatagtcttgcttgagaattgaacctctaccacaaaagaaaaacgaaatgatacaccaatca gtgcaaaaaaagatataatgggagataagacggttcgtgttcgtgctgacttgcaccatatcataaaaatcgaaacagcaaa gaatggcggaaacgtaaaagaagttatggaaataagacttagaagcaaacttaagagtgtgttgatagtgcagtatcttaaa attttgtataataggaattgaagttaaattagatgctaaaaatttgtaattaagaaggagtgattacatgaacaaaaatataaaat attctcaaaactttttaacgagtgaaaaagtactcaaccaaataataaaacaattgaatttaaaagaaaccgataccgtttacga aattggaacaggtaaagggcatttaacgacgaaactggctaaaataagtaaacaggtaacgtctattgaattagacagtcat ctattcaacttatcgtcagaaaaattaaaactgaatactcgtgtcactttaattcaccaagatattctacagtttcaattccctaac aaacagaggtataaaattgttgggagtattccttaccatttaagcacacaaattattaaaaaagtggtttttgaaagccatgcgt ctgacatctatctgattgttgaagaaggattctacaagcgtaccttggatattcaccgaacactagggttgctcttgcacactca agtctcgattcagcaattgcttaagctgccagcggaatgctttcatcctaaaccaaaagtaaacagtgtcttaataaaacttac ccgccataccacagatgttccagataaatattggaagctatatacgtactttgtttcaaaatgggtcaatcgagaatatcgtcaa ctgtttactaaaaatcagtttcatcaagcaatgaaacacgccaaagtaaacaatttaagtaccgttacttatgagcaagtattgt ctatttttaatagttatctattatttaacgggaggaaataattctatgagtcgcttttgtaaatttggaaagttacacgttactaaagg gaatgtagataaattattaggtatactactgacagcttccaaggagctaaagaggtccctagactctagacccggggatctct gcagtgagatctggtaatgactctctagcttgaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt atctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgctctagctaagcagaaggccatcctgacggatgg cctttttgcgtttctacaaactcttgttaactctagagctgcctgccgcgtttcggtgatgaagatcttcccgatgattaattaattc agaacgctcggttgccgccgggcgttttttatgcagcaatggcaagaacgttgctctagagcggccgcatcgattcacagt ggcaatctcccccgtattcgtttgaaatgtgccacattaacagcgccgggtgatgtccgtatcgttctgctaataagcggttga tgtgccgtgttttttctcggtagactttagatgtgaggcagtggttgtgccttccgccgtgcagctgtttgacgcgggaggcatt gacgcgcaaaacttccggataggtttgcgacagccaggccggacgggctccgctcggcgttgctaatatgacccggccg cctatactgtgaatccgctcaaaaatatcatccagccattcaaattgatatacgccctcctccggctcaagtgcgctccatgca aaaatgccgacagaaaacgtattcgtatgagaaagcttcatcagtttgatatcgtcagctaaaatatcgggccgatccagcc actgatcggggttgtagtctcccccatggagcataaattttgcttttgttacgtgcgttttttcaaccttagacatcaccc. SEQIDNo.41 Cttcatataacgcagcatcaatttgttgtgccagatccttgctgaatgaaaaaggcacaatttggtcgtccttagaagc aatgacagcacggtgcttcgcggactcgatgattttttgatgatcaaacgatccctgtgtaaattcatctaacatctgtaaagtg ggcaatgatttggcaaagccggaaacaagtatgattccgccaagctgttttcgtaattgaagatgttccaaaaatctcaaaat ggcggggcaacctaagctgtgcgcaaccaaatacgtattttcatgtaaagtgtgctgatataaagagagtgtatccagccaa tcctcaagtcttggctggagcggattaggcatgtttaaaatgtctgcttggaccccatctgcgagcagacgttttttcagccaa gggaaccaatgatttgttgaggaggctctgtagccatgaatgatatagacttgttttgtcattatttcctcctaagaaatattttcat tcagtataaaattcctctcattgggaaacaagtacgcacttttttgtaagtagctttacatatgaggccctccttgtgccattcccc acttacacatctcatctatgaggggatacaaagatcgccctctgtcagataacgaatactctactttcagtggggtgtgagaa acctcagaccgatgaataaggccgtcagtttgcaattccttaagctgtgcgcttaacattttatgggtgatgaagggcatagc ccttttcagctcgccgtatctcatcgttttttcacaccctaactcatagagaattctcattttccatttgccccctataagagataag gtgtattcaaatggggcttgtttactgcggcacaggctattcattgtaatcaaacctccttttttaagtatcatatcagaataaatta ggattttcggtgttgaagtttgagggcgattcacgatctgaagtgaaaggggccgcttgctcaacaggcggcacggtacat gaatgttctaagtcaaagagggttaccttactgttagttactttatttaaaaaaacgcatgtttttagctgaaaatatctattatggt atatttaaatataaagaaatatttgagaaatgaggaatggtatgaatacacccaaaaaacataagaaatttaaagcgaaaatg attctccaaatcatcatggttatcattggaggaatcatagcggcatatggactagaaacagtgctcattccaaacagtgtatct gatggaggggtgacgggtcttagcgtcgttggttca. SEQIDNo.42 Cggtgaaggtctgaatcacagaaataccaagggcgtcaaaacccttggtattttttctggttattagattttatcttcag ctttacgtgctgcttcgagcttatcaatcctgtctttatatttttctccccagtccttcatgtgcaggataacaggttctaatgttcttc caaactcagttaaagagtattcgacttttggagggacctgtttataaacctcgcggtgaatcacgccatcccgttccaattctct taattgtagcgtgagcatacgctgggtaatttttgggtatagttttcgaaactcattaaacctctttttaccgtcaatgagatgata aagaagaatgcctttccacttacctccgatgatatctaatgttgtctccaccggacatccttcaaagtcatcagaagaataatca atgccgagtttacgatttctcaagctgttcactcctaatagtatactttttgatactatatacattcaatgtgcgtactttttatatttta atatacatcatataatgatgtcacatctaaggaatgaaaggaatggatcatatgaaaacactcgtactcgttgtacatccgaat atagaatcctctcgtatcaataaaaagtggaaagaagccgttttaagtgaaccagatgtaactgtccatgatctttatgaaaaat atcgcgatcaaccaattgatgtggaatttgaacaacagcagctcctggcccatgaccgtatcgtttttcagtttccattatactg gtacagcagcccaccgcttttaaaacagtggtttgatgaagtgtttacgtttggctgggctcatggtcccggcggaaataaatt gaaggggaaagagtgggtaactgccatgtccatcggttcacctgaacactcttatcaagccggcggatataacttgttttcga taagcgagctgacaaaaccgttccaagcatctgcccatttagtaggcatgacctatcagccttcct. SEQIDNo.43 tcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatatttttaaaatatatatttatgttacag taatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattcactttagataaaaatttagg aggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaac gacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaattttaccctgcatttat tttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattgggataa gttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttt tatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctc tttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcagga aaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgttt atgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtactttttacagtcgg ttttctaatgtcactaacctgccccgttagttgaaggcattttctgtcaatgttttcttacaaagaacgctgtgatatactgaaattt gtccgtatacattttggaggaatggatatgttaccaaaatacgcgcaagtaaaagaagaaatcagttcttggattaatcaagg caaaatactgcccgatcaaaaaatccctaccgaaaacgaattaatgcagcaattcggcgtcagccggcataccatccgcaa agcgatcggagacctcgtatcacaaggtctgctgtacagcgtgcaaggcggaggcacctttgtcgcttcacgctctgctaa gtcagcgctgcattccaataaaacgatcggtgttttgacaacttacatatcagactatattttcccgagcatcatcagaggaatc gagtcctatttaagcgagcaggggtattctatgcttttgacaagcacaaacaacaacccggacaatgaaagaagaggctta gaaaacctgctgtcccagcatattgacggactcatcgtagaaccgacaaaaagcgcccttcaaaccccaaacatcggctat tatctgaacttggagaaaaacggcattccttttgcgatgattaacgcgtcatatgccgagcttgccgcgccaagttttaccttg gatgatgtgaaaggcgggatgatggcggcggagcatttgctttctctcggccacacgcatatgatgggtatttttaaagctga tgacacacaaggcgtgaaacggatgaacggatttatacaggcgcaccgggagcgtgagttgtttccttctccggatatgatc gtgacatttacaacggaagaaaaagaatcaaaacttctggagaaagtaaaagccacactggagaaaaacagcaagcacat gccgacagccattctttgttataacgatgaaattgcgctgaaggtgattgatatgctgagggagatggatcttaaagtgccgg aggatatgtctattgtcgggtacgatgattcacatttcgcccaaatctcagaagtgaaactaacctctgtcaaacatccgaaat cagtgcttggaaaagcagccgccaaatatgtcattgactgcttagagcataaaaagccgaagcaagaggatgtcatatttga gcctgagttgatcattcgccagtccgcacgaaaactgaatgaataa. SEQIDNo.44 Cggctaacatagaaggagataccctgttaattaccgttgtcggcgggattatccttggattcggaatggggctggcg ctgcgtaatggcggcgcattagatggaattgacatgcttgctgtgctgctatcccgaaaactgccattcggaacgagcgatc tcattctattcttaaacctatttgtgtttatttttgtttccacagtatttggcttacaaggggcattgctttcagtcattgcttactacatt gcatccaaagtgattcatgttgtggaggaaggcttaagcggctctaaaacctttcaaattattacaacccaacctgaattaatg gtagagacaatacgtgatcaattaggccgaagcgccacatataaagaagcttacgggggtttttcccacgagaaatttaaag aaatcacatgtgtcatcaaccgtttagaagaaaccaaactaaaggaaattattaatgatattgataaaactgcctttgttaccgt gtatgatgtagc. SEQIDNo.45 gtttatggttgcgttgatggttaaatctcaaaaaggtgttgacattcaaaacaaaatatggtataatgcccccaaaatcg caaaaaagtgttgacaacttaactcagatctggtataatagaaacacagaatagtcttttaagtaagtctactctgaatttttttaa aaggagagggtaaagaatggcaaacccaacaacagggaagtcctcgattagggctaagctttctaactcatcgctatcaaa cctatttaaaaaaaataaaaataaaagacagcgtgaggaaacggaagagcaggacaatgaggataaggatgagagtaag aaccaggatgaaaataaggacacacagctcactccccgcaagcgtcgccggttgacgaaggagtttgaagagaaggag gctcgttacaccaacgagttgcccaaggaactgcgcaagtatcgtcctaaaggtttcagattcaatttgcctccaacggatag acccatcaggatatatgcagatggtgtttttgatcttttccatcttggccacatgaagcaactggaacagtgtaagaaggctttc cccaatgtaacactgatagttggtgtgcctagcgacaaaatcactcacaaactaaaaggtttgactgtgctgaccgataagc agcgttgtgaaactttaacgcactgcagatgggttgacgaagtcgtgcccaacgctccctggtgtgtcaccccagaatttcta ctagaacacaagattgactacgtggcacatgacgatattccttacgttagcgccgacagcgacgatatctacaagccaataa aggagatgggaaaattcttgactacccaaagaaccaatggtgtctctacaagtgatattatcacaaagatcatcagagattat gacaaatatttgatgagaaactttgcaaggggtgctaccagacaggagctgaacgtttcttggttgaagaaaaacgaactgg agttcaaaaaacacatcaatgaattcaggtcatatttcaagaaaaaccagacaaatttgaataacgcctccagagacttgtact tcgaagtccgtgaaatcttgctaaagaaaacgttgggcaaaaaactctactccaagttaataggcaatgaattaaagaaaca aaatcaacgacaaagaaaacagaattttttggatgatccgtttactaggaagctaatcagggaggcctctccggctacagag tttgccaacgaatttacgggcgaaaactctaccgctaaatcaccggatgacaatggaaatcttttcagtcaggaagatgatga agacacaaattctaacaacacgaatacaaattcagattcagattcaaacactaactcaacgcctcccagtgaagatgacgac gacaacgacaggttaactttggaaaacctaacacagaagaagaaacaatcagcgaactaagtaaggtcaaaaggaggaa ttaaaaatggtacaagaatcacgtccagggagtgtaagaagttactcggtcggttaccaagcaaggtccagatcgagttctc aaagaagacattcgttaacacgccaacgttcctcgcaaagactgattagaaccatcagtatcgagtctgatgtgtctaatatta ctgacgatgacgatttgagagctgtcaatgagggagtagcgggtgtgcaactggacgtctctgaaaccgcaaataaggga ccaagaagagcatcagcaactgatgtcacagatagtttgggttcgacttcgtcggaatatattgagattccctttgttaaggaa acattggatgcaagtttaccttcggattatctgaagcaggacatattaaatctcattcagagtttgaagatatccaaatggtataa caacaagaaaatccaaccggtagcacaagatatgaacttagtcaagatctctggtgcgatgacaaacgcaattttcaaagtt gaataccctaagttaccatcgttgctattgagaatatacggaccgaatattgataatatcattgacagggaatatgaattgcag attttggctaggctttcattgaaaaatataggtccttccctttacggctgttttgtaaacggtagatttgagcagtttctggagaatt ctaagactttaacaaaagacgacattagaaactggaagaactctcaaaggattgcaaggagaatgaaggagttacatgtag gtgttcctctcttgagttcagaaaggaagaacgggtcggcttgttggcaaaagattaaccagtggttgcgcacgattgagaa agtcgaccaatgggtgggggatcctaaaaacattgaaaactctttattatgtgagaattggtccaagtttatggatattgtcgat agatatcacaagtggcttatttctcaagaacagggtatagagcaagtcaacaaaaatcttatattctgccataatgatgcccaa tacggcaatttacttttcactgctcctgtgatgaacacaccgagcctatacactgcaccttcgtctacatcattgacttcccaatc aagttccttatttccttcgagctccaatgtcattgtagatgatataatcaacccgccaaagcaggagcaaagccaagattccaa attggtcgtcattgattttgaatatgcaggtgccaatcccgccgcatatgatttagcgaatcatctttccgagtggatgtatgatt acaacaatgctaaggccccacatcagtgccacgctgatagatatcccgataaagaacaggttttgaatttcttatactcttatgt ttcgcatctaaggggtggtgctaaggaacccatagatgaagaggttcaaagactctataagtcaatcattcaatggagaccc actgtacaactattttggtcgctctgggccatcctacaaagtggtaaattagagaaaaaagaagcctccactgccatcactag agaagaaattggacccaatggaaaaaaatatatcatcaagactgaacccgaatcccctgaagaagactttgttgaaaatgac gacgagcctgaagctggcgtcagcattgacacgttcgattatatggcttatggtcgtgacaagattgcggtcttttggggcga cctcattggcttaggcataatcaccgaagaagaatgcaaaaatttcagctctttcaagttcctcgatactagttatttgtaatacg tatacgaattccttcaacaaaggccaaggaaataaagcaaataacaataacaccattattttaattttttttctattactgtcgcta acatct. SEQIDNo.46 cttcatataacgcagcatcaatttgttgtgccagatccttgctgaatgaaaaaggcacaatttggtcgtccttagaagca atgacagcacggtgcttcgcggactcgatgattttttgatgatcaaacgatccctgtgtaaattcatctaacatctgtaaagtgg gcaatgatttggcaaagccggaaacaagtatgattccgccaagctgttttcgtaattgaagatgttccaaaaatctcaaaatg gcggggcaacctaagctgtgcgcaaccaaatacgtattttcatgtaaagtgtgctgatataaagagagtgtatccagccaat cctcaagtcttggctggagcggattaggcatgtttaaaatgtctgcttggaccccatctgcgagcagacgttttttcagccaag ggaaccaatgatttgttgaggaggctctgtagccatgaatgatatagacttgttttgtcattatttcctcctaagaaatattttcatt cagtataaaattcctctcattgggaaacaagtacgcacttttttgtaagtagctttacatatgaggccctccttgtgccattcccc acttacacatctcatctatgaggggatacaaagatcgccctctgtcagataacgaatactctactttcagtggggtgtgagaa acctcagaccgatgaataaggccgtcagtttgcaattccttaagctgtgcgcttaacattttatgggtgatgaagggcatagc ccttttcagctcgccgtatctcatcgttttttcacaccctaactcatagagaattctcattttccatttgccccctataagagataag gtgtattcaaatggggcttgtttactgcggcacaggctattcattgtaatcaaacctccttttttaagtatcatatcagaataaatta ggattttcggtgttgaagtttgagggcgattcacgatctgaagtgaaaggggccgcttgctcaacaggcggcacggtacat gaatgttctaagtcaaagagggttaccttactgttagttactttatttaaaaaaacgcatgtttttagctgaaaatatctattatggt atatttaaatataaagaaatatttgagaaatgaggaatggtatgaatacacccaaaaaacataagaaatttaaagcgaaaatg attctccaaatcatcatggttatcattggaggaatcatagcggcatatggactagaaacagtgctcattccaaacagtgtatct gatggaggggtgacgggtcttagcgtcgttggttcagtttatggttgcgttgatggttaaatctcaaaaaggtgttgacattca aaacaaaatatggtataatgcccccaaaatcgcaaaaaagtgttgacaacttaactcagatctggtataatagaaacacaga atagtcttttaagtaagtctactctgaatttttttaaaaggagagggtaaagaatggcaaacccaacaacagggaagtcctcg attagggctaagctttctaactcatcgctatcaaacctatttaaaaaaaataaaaataaaagacagcgtgaggaaacggaag agcaggacaatgaggataaggatgagagtaagaaccaggatgaaaataaggacacacagctcactccccgcaagcgtc gccggttgacgaaggagtttgaagagaaggaggctcgttacaccaacgagttgcccaaggaactgcgcaagtatcgtcct aaaggtttcagattcaatttgcctccaacggatagacccatcaggatatatgcagatggtgtttttgatcttttccatcttggcca catgaagcaactggaacagtgtaagaaggctttccccaatgtaacactgatagttggtgtgcctagcgacaaaatcactcac aaactaaaaggtttgactgtgctgaccgataagcagcgttgtgaaactttaacgcactgcagatgggttgacgaagtcgtgc ccaacgctccctggtgtgtcaccccagaatttctactagaacacaagattgactacgtggcacatgacgatattccttacgtta gcgccgacagcgacgatatctacaagccaataaaggagatgggaaaattcttgactacccaaagaaccaatggtgtctcta caagtgatattatcacaaagatcatcagagattatgacaaatatttgatgagaaactttgcaaggggtgctaccagacaggag ctgaacgtttcttggttgaagaaaaacgaactggagttcaaaaaacacatcaatgaattcaggtcatatttcaagaaaaacca gacaaatttgaataacgcctccagagacttgtacttcgaagtccgtgaaatcttgctaaagaaaacgttgggcaaaaaactct actccaagttaataggcaatgaattaaagaaacaaaatcaacgacaaagaaaacagaattttttggatgatccgtttactagg aagctaatcagggaggcctctccggctacagagtttgccaacgaatttacgggcgaaaactctaccgctaaatcaccggat gacaatggaaatcttttcagtcaggaagatgatgaagacacaaattctaacaacacgaatacaaattcagattcagattcaaa cactaactcaacgcctcccagtgaagatgacgacgacaacgacaggttaactttggaaaacctaacacagaagaagaaac aatcagcgaactaagtaaggtcaaaaggaggaattaaaaatggtacaagaatcacgtccagggagtgtaagaagttactc ggtcggttaccaagcaaggtccagatcgagttctcaaagaagacattcgttaacacgccaacgttcctcgcaaagactgatt agaaccatcagtatcgagtctgatgtgtctaatattactgacgatgacgatttgagagctgtcaatgagggagtagcgggtgt gcaactggacgtctctgaaaccgcaaataagggaccaagaagagcatcagcaactgatgtcacagatagtttgggttcga cttcgtcggaatatattgagattccctttgttaaggaaacattggatgcaagtttaccttcggattatctgaagcaggacatatta aatctcattcagagtttgaagatatccaaatggtataacaacaagaaaatccaaccggtagcacaagatatgaacttagtcaa gatctctggtgcgatgacaaacgcaattttcaaagttgaataccctaagttaccatcgttgctattgagaatatacggaccgaa tattgataatatcattgacagggaatatgaattgcagattttggctaggctttcattgaaaaatataggtccttccctttacggctg ttttgtaaacggtagatttgagcagtttctggagaattctaagactttaacaaaagacgacattagaaactggaagaactctca aaggattgcaaggagaatgaaggagttacatgtaggtgttcctctcttgagttcagaaaggaagaacgggtcggcttgttgg caaaagattaaccagtggttgcgcacgattgagaaagtcgaccaatgggtgggggatcctaaaaacattgaaaactctttat tatgtgagaattggtccaagtttatggatattgtcgatagatatcacaagtggcttatttctcaagaacagggtatagagcaagt caacaaaaatcttatattctgccataatgatgcccaatacggcaatttacttttcactgctcctgtgatgaacacaccgagccta tacactgcaccttcgtctacatcattgacttcccaatcaagttccttatttccttcgagctccaatgtcattgtagatgatataatca acccgccaaagcaggagcaaagccaagattccaaattggtcgtcattgattttgaatatgcaggtgccaatcccgccgcata tgatttagcgaatcatctttccgagtggatgtatgattacaacaatgctaaggccccacatcagtgccacgctgatagatatcc cgataaagaacaggttttgaatttcttatactcttatgtttcgcatctaaggggtggtgctaaggaacccatagatgaagaggtt caaagactctataagtcaatcattcaatggagacccactgtacaactattttggtcgctctgggccatcctacaaagtggtaaa ttagagaaaaaagaagcctccactgccatcactagagaagaaattggacccaatggaaaaaaatatatcatcaagactgaa cccgaatcccctgaagaagactttgttgaaaatgacgacgagcctgaagctggcgtcagcattgacacgttcgattatatgg cttatggtcgtgacaagattgcggtcttttggggcgacctcattggcttaggcataatcaccgaagaagaatgcaaaaatttc agctctttcaagttcctcgatactagttatttgtaatacgtatacgaattccttcaacaaaggccaaggaaataaagcaaataac aataacaccattattttaattttttttctattactgtcgctaacatctcggtgaaggtctgaatcacagaaataccaagggcgtcaa aacccttggtattttttctggttattagattttatcttcagctttacgtgctgcttcgagcttatcaatcctgtctttatatttttctcccc agtccttcatgtgcaggataacaggttctaatgttcttccaaactcagttaaagagtattcgacttttggagggacctgtttataa acctcgcggtgaatcacgccatcccgttccaattctcttaattgtagcgtgagcatacgctgggtaatttttgggtatagttttcg aaactcattaaacctctttttaccgtcaatgagatgataaagaagaatgcctttccacttacctccgatgatatctaatgttgtctc caccggacatccttcaaagtcatcagaagaataatcaatgccgagtttacgatttctcaagctgttcactcctaatagtatacttt ttgatactatatacattcaatgtgcgtactttttatattttaatatacatcatataatgatgtcacatctaaggaatgaaaggaatgg atcatatgaaaacactcgtactcgttgtacatccgaatatagaatcctctcgtatcaataaaaagtggaaagaagccgttttaa gtgaaccagatgtaactgtccatgatctttatgaaaaatatcgcgatcaaccaattgatgtggaatttgaacaacagcagctcc tggcccatgaccgtatcgtttttcagtttccattatactggtacagcagcccaccgcttttaaaacagtggtttgatgaagtgttt acgtttggctgggctcatggtcccggcggaaataaattgaaggggaaagagtgggtaactgccatgtccatcggttcacct gaacactcttatcaagccggcggatataacttgttttcgataagcgagctgacaaaaccgttccaagcatctgcccatttagta ggcatgacctatcagccttccttcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatatttttaaa atatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattca ctttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatc attatttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggata taaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggag agttaggttattgggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaa gaatgacttcaaagagttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctat acctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttcta cccattattacagcaggaaaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatg gttatcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccg actgtactttttacagtcggttttctaatgtcactaacctgccccgttagttgaaggcattttctgtcaatgttttcttacaaagaac gctgtgatatactgaaatttgtccgtatacattttggaggaatggatatgttaccaaaatacgcgcaagtaaaagaagaaatca gttcttggattaatcaaggcaaaatactgcccgatcaaaaaatccctaccgaaaacgaattaatgcagcaattcggcgtcag ccggcataccatccgcaaagcgatcggagacctcgtatcacaaggtctgctgtacagcgtgcaaggcggaggcacctttg tcgcttcacgctctgctaagtcagcgctgcattccaataaaacgatcggtgttttgacaacttacatatcagactatattttcccg agcatcatcagaggaatcgagtcctatttaagcgagcaggggtattctatgcttttgacaagcacaaacaacaacccggaca atgaaagaagaggcttagaaaacctgctgtcccagcatattgacggactcatcgtagaaccgacaaaaagcgcccttcaaa ccccaaacatcggctattatctgaacttggagaaaaacggcattccttttgcgatgattaacgcgtcatatgccgagcttgcc gcgccaagttttaccttggatgatgtgaaaggcgggatgatggcggcggagcatttgctttctctcggccacacgcatatga tgggtatttttaaagctgatgacacacaaggcgtgaaacggatgaacggatttatacaggcgcaccgggagcgtgagttgtt tccttctccggatatgatcgtgacatttacaacggaagaaaaagaatcaaaacttctggagaaagtaaaagccacactggag aaaaacagcaagcacatgccgacagccattctttgttataacgatgaaattgcgctgaaggtgattgatatgctgagggaga tggatcttaaagtgccggaggatatgtctattgtcgggtacgatgattcacatttcgcccaaatctcagaagtgaaactaacct ctgtcaaacatccgaaatcagtgcttggaaaagcagccgccaaatatgtcattgactgcttagagcataaaaagccgaagc aagaggatgtcatatttgagcctgagttgatcattcgccagtccgcacgaaaactgaatgaataacggctaacatagaagga gataccctgttaattaccgttgtcggcgggattatccttggattcggaatggggctggcgctgcgtaatggcggcgcattaga tggaattgacatgcttgctgtgctgctatcccgaaaactgccattcggaacgagcgatctcattctattcttaaacctatttgtgtt tatttttgtttccacagtatttggcttacaaggggcattgctttcagtcattgcttactacattgcatccaaagtgattcatgttgtgg aggaaggcttaagcggctctaaaacctttcaaattattacaacccaacctgaattaatggtagagacaatacgtgatcaatta ggccgaagcgccacatataaagaagcttacgggggtttttcccacgagaaatttaaagaaatcacatgtgtcatcaaccgttt agaagaaaccaaactaaaggaaattattaatgatattgataaaactgcctttgttaccgtgtatgatgtagc. SEQIDNo.47 Gacgataaacccaaacacaacaccaataagaattgtcagcgccctcttattttcaagcggcatcagctgccagcgg ttggggcgaagttttctatccaaaaacaaacgaacgatgattgaaatggcaaccagcgttaacacatatcctagagcgtgctt aataatttcttcctgatgctggtgaaaggcggggaacaaatgcaatatgccaatggctagcgaagcactcggaatactgcc gatggctaagtattttaccaatttaaagttaatcgttttctgccgccaatgggaggcaacgccaaacagctttgtgatcgagtta tatacaagatccgtaccgaccgcgatggaaggattaattccaagcacaattaaaaggggagttaataaggccgctccaccc actcccgtcaatccgactaaggctccgacaaacaaacccatcaatatgatgcttacactcatgataaaggcctcctgaaaac ccgactatttttatcagattatatatgattaggacgcgttaatcaattaaattttttgaatttttgcacgttagttgcttgaaattttggt atgctcaatatctctattcatttgccctataatgagtgtctggacgaaatcgagaagtactaaaggcgcaaaaaaaagatacct cttaacaggtatcttttttcttgtttacgctacatgcgctgctgatagaaaggaagctcagtgtggcccatttcccgcacataca cgaacaggttgcctttatggtgaatttcatgctccattgcaagctgaagcagcgcgcggcctgtgacttttctgccgaaagcg gatgtcagatcaatttctctgtccagctgctcttcagtcagctgttcaaggatagcgaccgttttttctgtatatgtttttgccagaa cattgagatctgtttctgtttcttcctgcttgttttgaaatggtgaggcattgccttccttaatgacatttgcgaacaagtggaaag acgtaagaatgtgtttgaccagctcttctgctgacattgatgtttctgccggtttgtagctgtaatggtctttgctgattttttcggc aagctcattggtcacgtttcgatgggataaaaaatggctgacaatttgattggattggcacattatgatctcctccatatgtaaa aataaatggtgcaatgaaatcctatatcaacggttatgaattcacaagtaatatgctgtgggatgaaacaaaatgctatgtcaa tcgtatatataacgttcacaaaacatgaaatggagtataaacatgactaacatacctttcatttatcagtacgaagaaaaagag aatgaaagagttgcg. SEQIDNo.48 Tcattggcacgaagaagggaatcaccatattgaaaccatggtgaaagacatgacaggagagcaaagaaaaagcc agcggggaagctggatggaaagaaacaaagtcggttttcactaaaagaaagcacgggtgtttgaaaaacccgtgcttttttg ttgcggttagccgaaattcgacaattgcggttattttgcgttcttctttttcttgtaaatatgataaaatatgacatatctcgggtaat tcaaaaggggggattaattgaggatgaagcagacgattccgtcctcttatgtcgggcttaaaattaatgaatggtatactcata tccggcagttccacgtcgctgaagccgaacgggtcaagctcgaagtagaaagagaaattgaggatatggaagaagacca agatttgctgctgtattattctttaatggagttcaggcaccgtgtcatgctggattacattaagccttttggagaggacacgtcgc agctagagttttcagaattgttagaagacatcgaagggaatcagtacaagctgacagggcttctcgaatattactttaattttttt cgaggaatgtatgaatttaagcagaagatgtttgtcagtgccatgatgtattataaacgggcagaaaagaatcttgccctcgt ctcggatgatattgagaaagcagagtttgcttttaaaatggctgagattttttacaatttaaaacaaacctatgtttcgatgagcta cgccgttcaggcattagaaacataccaaatgtatgaaacgtacaccgtccgcagaatc. SEQIDNo.49 Ggttgattggagcctgagcatgtggcatatatatttgaaaagcttgaaaagaaaacctttgcttacgatgaagatgag aaagaaccggattataccgtccataagtctataagaaacagtgtatacgcgtatcctgaaaagggagttgcttttgccagaat tccatattttcaagatgggagcattatgagttttgattgtctgtttgcggttaacgatgaaaagatgcgtgcatttctggagggag tcagaccgcgtctttgggaaaaaagcaagcggaaagtgaccgtgtttacggatggagatggagggacttcaagagagca ggaagccattgtcagagaggttcagcggagtcaagtcatcatgaatccgctattgaaaaaagagatatacagatcaattgat cagttttttcatagtgataaatcgttttatcaaacatatgacatcccttacaagcgcggcattctgttatatggacctcctggaaac ggaaagacgacgttagtgaagtcgatcgcaggcagtatcgatgcacctgttgcttattggcaaattactgaatttacgtcgag cgagacaatagaagaagtctttcaggcagcgagacgcctcgctcctgcagttctggtcatcgaggatatagattcgatgcc ggaagatgtgcggtccttttttctcaatacgcaggacg. SEQIDNo.50 gtttatggttgcgttgatggttaaatctcaaaaaggtgttgacattcaaaacaaaatatggtataatgcccccaaaatcg caaaaaagtgttgacaacttaactcagatctggtataatagaaacacagaatagtcttttaagtaagtctactctgaatttttttaa aaggagagggtaaagaatggcaaacccaacaacagggaagtcctcgattagggctaagctttctaactcatcgctatcaaa cctatttaaaaaaaataaaaataaaagacagcgtgaggaaacggaagagcaggacaatgaggataaggatgagagtaag aaccaggatgaaaataaggacacacagctcactccccgcaagcgtcgccggttgacgaaggagtttgaagagaaggag gctcgttacaccaacgagttgcccaaggaactgcgcaagtatcgtcctaaaggtttcagattcaatttgcctccaacggatag acccatcaggatatatgcagatggtgtttttgatcttttccatcttggccacatgaagcaactggaacagtgtaagaaggctttc cccaatgtaacactgatagttggtgtgcctagcgacaaaatcactcacaaactaaaaggtttgactgtgctgaccgataagc agcgttgtgaaactttaacgcactgcagatgggttgacgaagtcgtgcccaacgctccctggtgtgtcaccccagaatttcta ctagaacacaagattgactacgtggcacatgacgatattccttacgttagcgccgacagcgacgatatctacaagccaataa aggagatgggaaaattcttgactacccaaagaaccaatggtgtctctacaagtgatattatcacaaagatcatcagagattat gacaaatatttgatgagaaactttgcaaggggtgctaccagacaggagctgaacgtttcttggttgaagaaaaacgaactgg agttcaaaaaacacatcaatgaattcaggtcatatttcaagaaaaaccagacaaatttgaataacgcctccagagacttgtact tcgaagtccgtgaaatcttgctaaagaaaacgttgggcaaaaaactctactccaagttaataggcaatgaattaaagaaaca aaatcaacgacaaagaaaacagaattttttggatgatccgtttactaggaagctaatcagggaggcctctccggctacagag tttgccaacgaatttacgggcgaaaactctaccgctaaatcaccggatgacaatggaaatcttttcagtcaggaagatgatga agacacaaattctaacaacacgaatacaaattcagattcagattcaaacactaactcaacgcctcccagtgaagatgacgac gacaacgacaggttaactttggaaaacctaacacagaagaagaaacaatcagcgaactaagtaaggtcaaaaggaggaa ttaaaaatggtacaagaatcacgtccagggagtgtaagaagttactcggtcggttaccaagcaaggtccagatcgagttctc aaagaagacattcgttaacacgccaacgttcctcgcaaagactgattagaaccatcagtatcgagtctgatgtgtctaatatta ctgacgatgacgatttgagagctgtcaatgagggagtagcgggtgtgcaactggacgtctctgaaaccgcaaataaggga ccaagaagagcatcagcaactgatgtcacagatagtttgggttcgacttcgtcggaatatattgagattccctttgttaaggaa acattggatgcaagtttaccttcggattatctgaagcaggacatattaaatctcattcagagtttgaagatatccaaatggtataa caacaagaaaatccaaccggtagcacaagatatgaacttagtcaagatctctggtgcgatgacaaacgcaattttcaaagtt gaataccctaagttaccatcgttgctattgagaatatacggaccgaatattgataatatcattgacagggaatatgaattgcag attttggctaggctttcattgaaaaatataggtccttccctttacggctgttttgtaaacggtagatttgagcagtttctggagaatt ctaagactttaacaaaagacgacattagaaactggaagaactctcaaaggattgcaaggagaatgaaggagttacatgtag gtgttcctctcttgagttcagaaaggaagaacgggtcggcttgttggcaaaagattaaccagtggttgcgcacgattgagaa agtcgaccaatgggtgggggatcctaaaaacattgaaaactctttattatgtgagaattggtccaagtttatggatattgtcgat agatatcacaagtggcttatttctcaagaacagggtatagagcaagtcaacaaaaatcttatattctgccataatgatgcccaa tacggcaatttacttttcactgctcctgtgatgaacacaccgagcctatacactgcaccttcgtctacatcattgacttcccaatc aagttccttatttccttcgagctccaatgtcattgtagatgatataatcaacccgccaaagcaggagcaaagccaagattccaa attggtcgtcattgattttgaatatgcaggtgccaatcccgccgcatatgatttagcgaatcatctttccgagtggatgtatgatt acaacaatgctaaggccccacatcagtgccacgctgatagatatcccgataaagaacaggttttgaatttcttatactcttatgt ttcgcatctaaggggtggtgctaaggaacccatagatgaagaggttcaaagactctataagtcaatcattcaatggagaccc actgtacaactattttggtcgctctgggccatcctacaaagtggtaaattagagaaaaaagaagcctccactgccatcactag agaagaaattggacccaatggaaaaaaatatatcatcaagactgaacccgaatcccctgaagaagactttgttgaaaatgac gacgagcctgaagctggcgtcagcattgacacgttcgattatatggcttatggtcgtgacaagattgcggtcttttggggcga cctcattggcttaggcataatcaccgaagaagaatgcaaaaatttcagctctttcaagttcctcgatactagttatttgtaatacg tatacgaattccttcaacaaaggccaaggaaataaagcaaataacaataacaccattattttaattttttttctattactgtggcta actcca. SEQIDNo.51 tcttcctgatgctggtgaaaggcggggaacaaatgcaatatgccaatggctagcgaagcactcggaatactgccga tggctaagtattttaccaatttaaagttaatcgttttctgccgccaatgggaggcaacgccaaacagctttgtgatcgagttatat acaagatccgtaccgaccgcgatggaaggattaattccaagcacaattaaaaggggagttaataaggccgctccacccac tcccgtcaatccgactaaggctccgacaaacaaacccatcaatatgatgcttacactcatgataaaggcctcctgaaaaccc gactatttttatcagattatatatgattaggacgcgttaatcaattaaattttttgaatttttgcacgttagttgcttgaaattttggtat gctcaatatctctattcatttgccctataatgagtgtctggacgaaatcgagaagtactaaaggcgcaaaaaaaagatacctct taacaggtatcttttttcttgtttacgctacatgcgctgctgatagaaaggaagctcagtgtggcccatttcccgcacatacacg aacaggttgcctttatggtgaatttcatgctccattgcaagctgaagcagcgcgcggcctgtgacttttctgccgaaagcgga tgtcagatcaatttctctgtccagctgctcttcagtcagctgttcaaggatagcgaccgttttttctgtatatgtttttgccagaaca ttgagatctgtttctgtttcttcctgcttgttttgaaatggtgaggcattgccttccttaatgacatttgcgaacaagtggaaagac gtaagaatgtgtttgaccagctcttctgctgacattgatgtttctgccggtttgtagctgtaatggtctttgctgattttttcggcaa gctcattggtcacgtttcgatgggataaaaaatggctgacaatttgattggattggcacattatgatctcctccatatgtaaaaat aaatggtgcaatgaaatcctatatcaacggttatgaattcacaagtaatatgctgtgggatgaaacaaaatgctatgtcaatcg tatatataacgttcacaaaacatgaaatggagtataaacatgactaacatacctttcatttatcagtacgaagaaaaagagaat gaaagagttgcggtttatggttgcgttgatggttaaatctcaaaaaggtgttgacattcaaaacaaaatatggtataatgcccc caaaatcgcaaaaaagtgttgacaacttaactcagatctggtataatagaaacacagaatagtcttttaagtaagtctactctg aatttttttaaaaggagagggtaaagaatggcaaacccaacaacagggaagtcctcgattagggctaagctttctaactcatc gctatcaaacctatttaaaaaaaataaaaataaaagacagcgtgaggaaacggaagagcaggacaatgaggataaggatg agagtaagaaccaggatgaaaataaggacacacagctcactccccgcaagcgtcgccggttgacgaaggagtttgaaga gaaggaggctcgttacaccaacgagttgcccaaggaactgcgcaagtatcgtcctaaaggtttcagattcaatttgcctcca acggatagacccatcaggatatatgcagatggtgtttttgatcttttccatcttggccacatgaagcaactggaacagtgtaag aaggctttccccaatgtaacactgatagttggtgtgcctagcgacaaaatcactcacaaactaaaaggtttgactgtgctgac cgataagcagcgttgtgaaactttaacgcactgcagatgggttgacgaagtcgtgcccaacgctccctggtgtgtcacccc agaatttctactagaacacaagattgactacgtggcacatgacgatattccttacgttagcgccgacagcgacgatatctaca agccaataaaggagatgggaaaattcttgactacccaaagaaccaatggtgtctctacaagtgatattatcacaaagatcatc agagattatgacaaatatttgatgagaaactttgcaaggggtgctaccagacaggagctgaacgtttcttggttgaagaaaaa cgaactggagttcaaaaaacacatcaatgaattcaggtcatatttcaagaaaaaccagacaaatttgaataacgcctccaga gacttgtacttcgaagtccgtgaaatcttgctaaagaaaacgttgggcaaaaaactctactccaagttaataggcaatgaatta aagaaacaaaatcaacgacaaagaaaacagaattttttggatgatccgtttactaggaagctaatcagggaggcctctccgg ctacagagtttgccaacgaatttacgggcgaaaactctaccgctaaatcaccggatgacaatggaaatcttttcagtcagga agatgatgaagacacaaattctaacaacacgaatacaaattcagattcagattcaaacactaactcaacgcctcccagtgaa gatgacgacgacaacgacaggttaactttggaaaacctaacacagaagaagaaacaatcagcgaactaagtaaggtcaa aaggaggaattaaaaatggtacaagaatcacgtccagggagtgtaagaagttactcggtcggttaccaagcaaggtccag atcgagttctcaaagaagacattcgttaacacgccaacgttcctcgcaaagactgattagaaccatcagtatcgagtctgatg tgtctaatattactgacgatgacgatttgagagctgtcaatgagggagtagcgggtgtgcaactggacgtctctgaaaccgc aaataagggaccaagaagagcatcagcaactgatgtcacagatagtttgggttcgacttcgtcggaatatattgagattccct ttgttaaggaaacattggatgcaagtttaccttcggattatctgaagcaggacatattaaatctcattcagagtttgaagatatcc aaatggtataacaacaagaaaatccaaccggtagcacaagatatgaacttagtcaagatctctggtgcgatgacaaacgca attttcaaagttgaataccctaagttaccatcgttgctattgagaatatacggaccgaatattgataatatcattgacagggaata tgaattgcagattttggctaggctttcattgaaaaatataggtccttccctttacggctgttttgtaaacggtagatttgagcagttt ctggagaattctaagactttaacaaaagacgacattagaaactggaagaactctcaaaggattgcaaggagaatgaaggag ttacatgtaggtgttcctctcttgagttcagaaaggaagaacgggtcggcttgttggcaaaagattaaccagtggttgcgcac gattgagaaagtcgaccaatggggggggatcctaaaaacattgaaaactctttattatgtgagaattggtccaagtttatgga tattgtcgatagatatcacaagtggcttatttctcaagaacagggtatagagcaagtcaacaaaaatcttatattctgccataat gatgcccaatacggcaatttacttttcactgctcctgtgatgaacacaccgagcctatacactgcaccttcgtctacatcattga cttcccaatcaagttccttatttccttcgagctccaatgtcattgtagatgatataatcaacccgccaaagcaggagcaaagcc aagattccaaattggtcgtcattgattttgaatatgcaggtgccaatcccgccgcatatgatttagcgaatcatctttccgagtg gatgtatgattacaacaatgctaaggccccacatcagtgccacgctgatagatatcccgataaagaacaggttttgaatttctt atactcttatgtttcgcatctaaggggtggtgctaaggaacccatagatgaagaggttcaaagactctataagtcaatcattca atggagacccactgtacaactattttggtcgctctgggccatcctacaaagtggtaaattagagaaaaaagaagcctccact gccatcactagagaagaaattggacccaatggaaaaaaatatatcatcaagactgaacccgaatcccctgaagaagacttt gttgaaaatgacgacgagcctgaagctggcgtcagcattgacacgttcgattatatggcttatggtcgtgacaagattgcggt cttttggggcgacctcattggcttaggcataatcaccgaagaagaatgcaaaaatttcagctctttcaagttcctcgatactagt tatttgtaatacgtatacgaattccttcaacaaaggccaaggaaataaagcaaataacaataacaccattattttaattttttttcta ttactgtggctaactccatcattggcacgaagaagggaatcaccatattgaaaccatggtgaaagacatgacaggagagca aagaaaaagccagcggggaagctggatggaaagaaacaaagtcggttttcactaaaagaaagcacgggtgtttgaaaaa cccgtgcttttttgttgcggttagccgaaattcgacaattgcggttattttgcgttcttctttttcttgtaaatatgataaaatatgaca tatctcgggtaattcaaaaggggggattaattgaggatgaagcagacgattccgtcctcttatgtcgggcttaaaattaatgaa tggtatactcatatccggcagttccacgtcgctgaagccgaacgggtcaagctcgaagtagaaagagaaattgaggatatg gaagaagaccaagatttgctgctgtattattctttaatggagttcaggcaccgtgtcatgctggattacattaagccttttggag aggacacgtcgcagctagagttttcagaattgttagaagacatcgaagggaatcagtacaagctgacagggcttctcgaat attactttaatttttttcgaggaatgtatgaatttaagcagaagatgtttgtcagtgccatgatgtattataaacgggcagaaaag aatcttgccctcgtctcggatgatattgagaaagcagagtttgcttttaaaatggctgagattttttacaatttaaaacaaacctat gtttcgatgagctacgccgttcaggcattagaaacataccaaatgtatgaaacgtacaccgtccgcagaatctcttcaactaa agcacccattagttcaacaaacgaaaattggataaagtgggatatttttaaaatatatatttatgttacagtaatattgacttttaaa aaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatg aactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataac cacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaattttaccctgcatttattttcttagtgacaag ggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattgggataagttagagccacttta tacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatgatttatacctttc tgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctctttctattattccatgg acttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaag gtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgtttatgaactctattcag gaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtactttttacagtcggttttctaatgtcacta acctgccccgttagttgaaggcattttctgtcaatgttttcttacaaagaacgctgtgatatactgaaatttgtccgtatacattttg gaggaatggatatgttaccaaaatacgcgcaagtaaaagaagaaatcagttcttggattaatcaaggcaaaatactgcccga tcaaaaaatccctaccgaaaacgaattaatgcagcaattcggcgtcagccggcataccatccgcaaagcgatcggagacc tcgtatcacaaggtctgctgtacagcgtgcaaggcggaggcacctttgtcgcttcacgctctgctaagtcagcgctgcattcc aataaaacgatcggtgttttgacaacttacatatcagactatattttcccgagcatcatcagaggaatcgagtcctatttaagcg agcaggggtattctatgcttttgacaagcacaaacaacaacccggacaatgaaagaagaggcttagaaaacctgctgtccc agcatattgacggactcatcgtagaaccgacaaaaagcgcccttcaaaccccaaacatcggctattatctgaacttggagaa aaacggcattccttttgcgatgattaacgcgtcatatgccgagcttgccgcgccaagttttaccttggatgatgtgaaaggcg ggatgatggcggcggagcatttgctttctctcggccacacgcatatgatgggtatttttaaagctgatgacacacaaggcgtg aaacggatgaacggatttatacaggcgcaccgggagcgtgagttgtttccttctccggatatgatcgtgacatttacaacgg aagaaaaagaatcaaaacttctggagaaagtaaaagccacactggagaaaaacagcaagcacatgccgacagccattctt tgttataacgatgaaattgcgctgaaggtgattgatatgctgagggagatggatcttaaagtgccggaggatatgtctattgtc gggtacgatgattcacatttcgcccaaatctcagaagtgaaactaacctctgtcaaacatccgaaatcagtgcttggaaaagc agccgccaaatatgtcattgactgcttagagcataaaaagccgaagcaagaggatgtcatatttgagcctgagttgatcattc gccagtccgcacgaaaactgaatgaataaggttgattggagcctgagcatgtggcatatatatttgaaaagcttgaaaagaa aacctttgcttacgatgaagatgagaaagaaccggattataccgtccataagtctataagaaacagtgtatacgcgtatcctg aaaagggagttgcttttgccagaattccatattttcaagatgggagcattatgagttttgattgtctgtttgcggttaacgatgaa aagatgcgtgcatttctggagggagtcagaccgcgtctttgggaaaaaagcaagcggaaagtgaccgtgtttacggatgg agatggagggacttcaagagagcaggaagccattgtcagagaggttcagcggagtcaagtcatcatgaatccgctattga aaaaagagatatacagatcaattgatcagttttttcatagtgataaatcgttttatcaaacatatgacatcccttacaagcgcggc attctgttatatggacctcctggaaacggaaagacgacgttagtgaagtcgatcgcaggcagtatcgatgcacctgttgctta ttggcaaattactgaatttacgtcgagcgagacaatagaagaagtctttcaggcagcgagacgcctcgctcctgcagttctg gtcatcgaggatatagattcgatgccggaagatgtgcggtccttttttctcaatacgcaggacg.