Production of Dictyostelium discoideum Hybrid Type Enzyme SteelyA in the Diatom Phaeodactylum tricornutum
by
, ,
Nicolas Sene
, 
Basanta Lamichhane
,
Sarah-Eve Gélinas
,
Alexandre Custeau
,
Natacha Merindol
,
Fatma Meddeb-Mouelhi
and
Isabel Desgagné-Penix
* Department of Biochemistry, Chemistry, Physics and Forensic Science, Université du Québec à Trois-Rivières, Trois-Rivières, QC G8Z 4M3, Canada
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(21), 11679; https://doi.org/10.3390/app152111679
Submission received: 16 September 2025
/
Revised: 15 October 2025
/
Accepted: 22 October 2025
/
Published: 31 October 2025
Abstract
The bioproduction of high-value molecules offers a sustainable and cost-effective alternative to traditional extraction and chemical synthesis, particularly for complex metabolites like cannabinoids (CBs), which have therapeutic potential for neurodegenerative diseases. The marine diatom Phaeodactylum tricornutum presents a promising chassis for CB biosynthesis due to its high lipid content, essential building blocks to biosynthesize CBs. In this study, we explored the feasibility of producing olivetolic acid (OA), the key CB precursor, using a hybrid-type polyketide synthase, SteelyA, from Dictyostelium discoideum. Unlike the native Cannabis sativa enzymes—tetraketide synthase and olivetolic acid cyclase—which exhibit low productivity and stability in diatoms, SteelyA was expected to offer an alternative biosynthetic route. Heterologous production in P. tricornutum resulted in a C-terminal fragment of the SteelyA enzyme, suggesting partial expression or processing of the very high-molecular-weight (352 kDa) SteelyA protein over six months without affecting cellular growth. However, HPLC-MS analysis did not detect intracellular OA or its derivatives in vivo and in vitro, suggesting enzymatic inactivity or metabolic limitations. These negative findings highlight the need for further investigation into the metabolic and proteomic requirements for CB precursor biosynthesis in diatoms, guiding future optimization strategies for sustainable cannabinoid production.
1. Introduction
Cannabinoids (CBs), including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), are coveted for the effects stemming from their interaction with the human endocannabinoid system [1,2]. They are naturally produced in the glandular trichomes of Cannabis plants. Tetraketide synthase (TKS) and olivetolic acid cyclase (OAC) from hexanoyl-CoA and malonyl-CoA lead to the biosynthesis of olivetolic acid (OA), a common intermediate of most cannabinoids [3]. The current cannabinoid supply relies on the extraction from cultivated Cannabis sativa; however, the cost of cultivation and difficult separation processes led to the search for alternatives [4,5].
In recent years, bioengineering has emerged as a powerful alternative to conventional extraction and chemical synthesis for obtaining a variety of valuable compounds. Based on using and enhancing natural reactions to produce complex molecules, bioproduction converts the need for costly, energy-intensive, and low-yield extraction processes into more accessible organism cultivation and metabolite purification [6,7,8]. Biofuels, solvents, pharmaceuticals, and nutraceuticals are now abundantly produced using bioengineering [9,10,11]. Achieving the bioproduction of molecules of diverse nature and size demands mastery of knowledge and techniques for each suitable model organism.
With an endogenously high lipid content, the microalgae Phaeodactylum tricornutum is a prominent choice as a green platform to produce biofuels, nutraceuticals, and even pharmaceuticals [12,13,14,15,16,17]. The toolbox and data available to work on the diatom are rapidly growing and are therefore gaining increasing interest in the bioproduction industry [18]. As it naturally produces malonyl-CoA and is predicted to produce hexanoyl-CoA, two substrates involved in the biosynthesis of fatty acids, it has been tested as a heterologous host to support the synthesis of cannabinoids [12,16,19,20,21]. The in vitro production of cannabigerolic acid by enzymatic assay using protein extracts has been successfully achieved in P. tricornutum using geranyl pyrophosphate and OA [12]. However, the heterologous expression of C. sativa tetraketide synthase and olivetolic acid cyclase cassettes to yield OA showed limited productivity and stability in the diatom expression systems [16]. As the metabolism of P. tricornutum is still being actively investigated, it is possible that the 3,5,7-trioxododecanoyl-CoA intermediate produced by the CsTKS enzyme is either unstable or channeled in other pathways instead of serving as a substrate for the CsOAC. Thus, alternative enzymes, notably from other organisms, have been investigated to circumvent this necessary step of the cannabinoid biosynthesis pathway.
One potential solution is the utilization of enzymes from Dictyostelium discoideum, a unicellular eukaryote that undergoes multicellular aggregation under nutrient deprivation, forming a fruiting body-like structure reminiscent of fungal development [22,23]. During this differentiation process, D. discoideum produces SteelyA, a hybrid enzyme combining features of type I fatty acid synthases and type III polyketide synthases. In its native host, SteelyA catalyzes the biosynthesis of 4-methyl-5-pentylbenzene-1,3-diol (MPBD) and methyl-olivetol from acyl-CoA precursors (Figure 1) [24,25]. Interestingly, deletion of the enzyme’s methyltransferase domain results in the production of olivetol, the direct precursor to OA and other cannabinoids [25,26,27]. When produced heterologously in Saccharomyces cerevisiae, SteelyA primarily produces methyl-olivetol [28] (Figure 1), with additional side products such as acylpyrones when two malonyl-CoA moieties are incorporated into the reaction. As SteelyA is the only hybrid polyketide synthase and fatty acid synthase identified to have produced the modified cannabinoid precursor methyl-olivetol in heterologous organisms, it stands out for genetic engineering in diatoms.
To address the limitations of TKS/OAC expression and assess an alternative strategy for OA biosynthesis, we investigated the heterologous production of SteelyA in P. tricornutum. The presence of olivetolic acid’s precursor in the diatom’s metabolism and the success in producing methyl-olivetol in yeast build a promising strategy to produce olivetol analogs in P. tricornutum. This study represents a significant milestone in diatom-based metabolic engineering, as it involves the production of the largest heterologous protein ever produced in P. tricornutum to date, a 352 kDa multifunctional enzyme. We inserted a genetic cassette containing the steelyA gene into P. tricornutum by conjugation [29] and evaluated both the production and enzymatic activity of the produced protein. By assessing the feasibility of producing such a large polyketide synthase/fatty acid synthase hybrid, this work provides new insights into the potential of P. tricornutum as a chassis for the biosynthesis of complex metabolites, expanding its applications in synthetic biology and metabolic engineering.
2. Materials and Methods
2.1. Microbial Strains and Growth Conditions
E. coli strains 10β (New England Biolab®, Ipswich, MA, USA) and Epi300 (TransforMax Epi300 Biosearch™ Technologies, Novato, CA, USA) were grown in Luria Broth (LB) supplemented with chloramphenicol (35 μg · mL−1) (Thermo Fisher Scientific, Waltham, MA, USA) when harboring the pPtGE30 plasmid and gentamicin (50 μg · mL−1) (Thermo Fisher Scientific, Waltham, MA, USA) when containing the pTA-MOB plasmid. All these strains were grown at 37 °C and under agitation at 220 rpm when in liquid culture. P. tricornutum strain (CCAP 1055/1, Culture Collection of Algae and Protozoa) was grown in L1 medium [30] at 18 °C under white lights (75 μE m−2 s−1) at 130 rpm with a photoperiod of 16 h of light. Each strain containing the pPtGE30 backbone was grown with 50 μg · mL−1 of zeocin (Invitrogen, Waltham, MA, USA). Each culture carried under antibiotic selection has been maintained in those conditions until experimentation.
2.2. Plasmid Constructions
For the expression plasmid constructs, two parts of the steelyA cassettes were synthesized by Genewiz® from Azenta Life Sciences (Waltham, MA, USA) in fragments 1MN and 1Q using various primers (Appendix A—Table A1 and Table A2) and assembled with the pPtGE30 vector (named EV hereafter) by Gibson assembly using the NEBuilder® HiFi DNA Assembly Bundle for Large Fragments (New England Biolabs, Ipswich, MA, USA) and then transformed into the E. coli 10β strain. The final assembled construct contains a Sh Ble bleomycin resistance gene used for the selection, a highly abundant secreted protein 1 (Hasp1) promoter, and an FcpA terminator used to drive the expression of the steelyA gene from the patent [28]. The Hasp1 promoter has been selected as it has a strong stationary expression, and it was inferred that the production of the protein could be very slow due to its unusual size and could require the strain to remain in the stationary phase to permit sufficient protein accumulation for detection and extraction. A myc tag was introduced into the C-terminal of the steelyA gene. Plasmid DNA sequence integrity was verified by Next Generation Sequencing (NGS) (Appendix A—Table A2).
2.3. Phaeodactylum tricornutum Conjugation and Screening
To introduce the steelyA constructs into P. tricornutum, each plasmid was transformed into the E. coli Epi 300 strain with the pTA-MOB plasmid. Conjugation was performed as described in [31], following exactly the steps described in [12] up to the final liquid culture of the transconjugants in L1 with zeocin.
Transconjugants were restreaked on a square plate and left to grow for seven more days. Colonies were then taken using a Fisherbrand® Loop from selective plates and then used to inoculate 150 µL of autoclaved deionized water. The cells were mixed by pipetting and vortexing for 30 s. Samples were heated at 95 °C for 12 min and vortexed a second time before being centrifuged at 13,700 rpm at 4 °C for 1 min. The supernatant was then transferred to a new Eppendorf tube. A volume of 5 µL supernatant was used for PCR reactions and followed by the Taq polymerase protocol for amplification, and then migrated by agarose electrophoresis.
2.4. Growth Kinetic Strains and Growth Conditions
Each P. tricornutum PCR-positive transconjugant was cultured in triplicate for 21 days in L1 medium with zeocin. Every two days, 250 µL of cell culture was used to measure the OD at 730 nm using a black Greiner microplate at the Synergy H1 BioTek microplate reader (Agilent, Santa Clara, CA, USA). Curves were obtained using GraphPad Prism software (Ver, 10.3.0.507). A t-test was performed using a Compare Groups of Growth Curves (CGGC) permutation test from Walter and Eliza Hall Institute of Medical Research, with 10,000 permutations [32].
2.5. Plasmid Rescue
To extract plasmid DNA from P. tricornutum transconjugants, 5 mL of P. tricornutum culture at day 7 was pelleted by centrifugation at 4000× g for 10 min. The cell pellets were resuspended in 235 µL of PDL1 Large Plasmid Extraction Kit with 5 µL lysozymes (100 mg · mL−1) and 5 µL of hemicellulase (25 mg · mL−1). The mixed solutions were incubated at 37 °C for 30 min, then 250 µL of PDL2 solution was added and mixed by inverting five to ten times at room temperature. The lysed cells were neutralized by the addition of 375 µL of PDL3 buffer, mixed by inversion five to ten times, and incubated for 2 min at room temperature. The solution mixture was then centrifuged for 3 min on a microcentrifuge at maximal speed at room temperature. The manufacturing protocol of the Large Plasmid Extraction Kit (Geneaid™, New Taipei City, Taiwan) was followed, starting from step 4 until elution in 50 µL of elution buffer preheated at 80 °C. DNA concentrations were measured using the N60/N50 Implen NanoPhotometer®. Purified DNA plasmid was further used for transformation in E. coli 10β chemicompetent cells following NEB protocol. Colonies were selected on LB agar with chloramphenicol and then put in liquid LB with chloramphenicol overnight. DNA was extracted using the Large Plasmid Extraction Kit (Geneaid™, New Taipei City, Taiwan), and then the concentration was measured at the N60/N50 NanoPhotometer® (Implen, Munich, Germany). The DNA sample was sent for NGS.
2.6. Western Blot Analysis
For each clone, a 50 mL culture was left to grow for 21 days and was then pelleted at 4000× g for 1 h at 4 °C. The supernatant was removed, and the pellets were weighed. Cell pellets were then resuspended in cell lysis buffer (SDS 0.75 mM, glycerol 10%, Tris-HCl pH 7.2 51.4 mM, EDTA 0.02 mM, urea 8 M) with a ratio of 2 µL per mg of pellet. A volume of 50 µL of PMSF and 5 µL of protein inhibitor cocktail per sample was added before protein extraction. Proteins were then extracted by sonication (amplitude 35%, ON time 15 s, OFF 30 s, run time 2 min). An aliquot of 500 µL of total protein extract was taken at this step. The rest of the protein solution was centrifuged at 15,000× g at 4 °C for 30 min before transferring the supernatant into a new tube. This step was repeated twice. The soluble protein fraction was quantified using the Bio-Rad DC protein assay reagent package. A 50 µg of total soluble protein, 5 µL of total protein fraction, and 5 µL of Invitrogen™ HiMark™ Pre-stained Protein Standard (Fisher Scientific, Waltham, MA, USA) were loaded on a SurePage Bris-Tris gel 4–12% (GenScript, Piscataway, NJ, USA). Protein migration was performed at 80 V for 25 min, then at 120 V for 2 h 45 min. The 0.2 μm polyvinylidene fluoride (PVDF) membrane was first activated in methanol and equilibrated in transfer buffer (0.6% Tris, 2.88% Glycine, 0.01% SDS, 20% methanol). Protein transfer was performed on ice using a wet transfer system at 120 V for 1 h 50 min. Following transfer, the membrane was blocked using 5% milk prepared in TBST (Tris Buffer Saline with 0.1% Tween 20) at room temperature for 2 h. The membrane was then washed three times with TBST for 10 min each and incubated overnight at 4 °C with anti-myc tag monoclonal antibody from EMD Millipore (dilution 1:500) in 5% milk. The membrane was again washed in TBST and then incubated for 1 h (dilution 1:10,000) in 5% milk of Immun-Star Goat Anti-Rabbit (GAR)-HRP from Bio-Rad (Bio-Rad, Hercules, CA, USA). Finally, the membrane was washed three more times with TBST and then dipped in Clarity Max Western ECL Substrate-Luminol solution from Bio-Rad before revelation. Total protein transferred onto the membrane was visualized using the Ponceau Red staining by incubating the membrane for one day at room temperature and then rinsing with distilled water. The images taken for protein detection, Luminol and Ponceau Red, were acquired using the ChemiDoc Imaging System with Image Lab™ Software (Ver 3.0.1),Bio-Rad, Hercules, CA, USA).
2.7. Phaeodactylum tricornutum Metabolite Extraction
For each sample, after 21 days of growth, 10 mL of culture was centrifuged at 4000× g for 50 min. Approximately 90 mg of cell pellets were obtained and resuspended in 200 μL of mobile phase (i.e., MilliQ water and methanol, both containing 0.1% formic acid (30:70)) and stored at −80 °C. Upon analysis, the sample was centrifuged at 4000× g for 3 min. An aliquot was diluted 50-fold in the same mobile phase mentioned above.
2.8. SteelyA In Vitro Enzymatic Assay
The enzymatic assay was performed in triplicate using 14 µg of total soluble protein extract in a 100 µL volume reaction in the presence of PBS 1X, DTT 5 mM, malonyl-CoA 0.75 mM, hexanoyl-CoA 0.24 mM, and repeated at least three times. The enzymatic reaction was incubated for 16 h at 22 °C and stopped using 10 µL of TCA 20%. A first extraction was performed by adding 300 µL ethyl acetate to the reaction mix. The organic phase was taken and filtered using Agilent Captiva Econofilter polytetrafluoroethylene (PTFE) 13 mm 0.2 µm. The aqueous phase of the enzymatic reaction was diluted in 300 µL of methanol and filtered too. All samples were dried in a Thermo Scientific SPD1010 Speedvac concentrator and then resuspended in 100 µL methanol prior to the HPLC-MS analysis.
2.9. HPLC-MS Analysis
Malonyl-CoA and hexanoyl-CoA authentic standards were purchased from Millipore Sigma Canada Ltd. (Oakville, ON, Canada), while the olivetolic acid and olivetol standards were purchased from Santa Cruz biotechnologies (Dallas, TX, USA).
Metabolite detection was performed using high-performance liquid chromatography (Agilent, 1260 Infinity II) coupled to mass spectrometry (Agilent, MSD iQ). Metabolite separation was performed on an InfinityLab Poroshell 120 EC-C18 (2.1 × 50 mm, 2.7 μm) analytical column equipped with an Agilent EC-C18 (4.6 × 5 mm, 2.7 μm) guard column maintained at 30 °C. An injection volume of 5 µL was set. The flow was set at 0.5 mL · min−1 with an elution program as follows: 0 min, 30% A; 7.0 min, 0% A; 12.0 to 20.1 min, 30% A. The MS source parameters were set as follows: Gas temperature: 220 °C, Gas flow: 8.0 mL · min−1, Nebulizer (psi): 55. Sample analyses were acquired in Scan mode in ESI+ and ESI− between m/z 100 to 500 with a fragmentation voltage of 135 V. Data were acquired using Agilent OpenLab CDC Acquisition (version 2) and Agilent OpenLab CDC Data Analysis (version 2.7) software was used for data acquisition and processing, respectively. A set of m/z for target compounds was analyzed (Table 1).
3. Results
3.1. Engineering P. tricornutum Strains to Produce SteelyA and Screening of Strains
To heterologously produce the D. discoideum polyketide synthase SteelyA in P. tricornutum, we constructed the expression episomal vector pSteely (Figure 2) using pPtGE30 (EV). The vector harbored the steelyA coding sequence of 9441 bp under the control of the native constitutive Hasp1 promoter, with the FcpA as a terminator and a C-terminal myc tag for protein detection (Figure 2). Plasmid construction was achieved via Gibson assembly using six primer sets (Appendix A—Table A1) and subsequently transformed into E. coli. Five SteelyA transformants and one EV were selected for E. coli-mediated conjugation with P. tricornutum.
Following conjugation, transconjugants were selected on L1 agar plates supplemented with zeocin. After two weeks, 36 colonies emerged, of which 15 were randomly selected for colony PCR to verify plasmid integration (Appendix A—Figure A1). Positive clones underwent plasmid rescue, and DNA integrity was confirmed by next-generation sequencing (Appendix A—Table A2). Sequencing analysis revealed varying degrees of mutations among the transconjugants. We retained three transconjugants for further study: pSteelyA-1, which contained a 129 bp insertion in the EV backbone but no mutation in steelyA; pSteelyA-2, which exhibited two 129 bp and a 563 bp insertion, a 129 deletion in a repetitive backbone region, and a silent mutation in steelyA; and pSteelyA-3, which carried an alanine-to-aspartic acid substitution in the methyltransferase domain, a methionine-to-isoleucine substitution, and a 984 bp deletion in the vector backbone (Appendix A—Table A2).
The three selected transconjugants, pSteelyA-1, pSteelyA-2, and pSteelyA-3, along with the EV control, were cultured for 21 days, and their growth was monitored by measuring optical density at 730 nm. No significant differences in growth were observed among the transconjugants or between the transconjugants and the EV control (Figure 3A), indicating that the presence of the large plasmid (26,190 pb) did not adversely affect cell proliferation. However, microscopic analysis revealed a distinct morphological difference, with pSteelyA-expressing transconjugants exhibiting a shorter cell phenotype compared to the EV control, with a significant reduction in cell line length; however, no changes in width (Figure 3B,C and Appendix A—Figure A2).
3.2. SteelyA Production and Activity
To assess SteelyA production, we performed a Western blot analysis on total protein extracts and soluble fractions of three transconjugants six months after conjugation (Figure 4). A band is observable around 40 kDa in the total fraction of samples pSteelyA-2 and pSteelyA-3 and in the soluble fraction of all samples. The Ponceau Red reveals an equivalent load of all the soluble fraction samples, but a variable content of protein in 5 μL of the total fraction between each sample. It shows the band at 40 kDA, and an additional band around 55 kDa is visible in all SteelyA samples except for the pSteelyA-1 total fraction.
To evaluate SteelyA enzymatic activity in vitro, total protein extracts were incubated with hexanoyl-CoA and malonyl-CoA under varying reaction conditions, including different pH levels (7, 7.5 and 8), temperatures (22 °C and 25 °C), and buffer systems (HEPES and Tris). LC-MS analysis of reaction products revealed no detectable levels of the expected compounds (m/z 100–500 and Table 1) in any transconjugants compared to the EV control (Figure 5). Similarly, no target metabolites from Table 1 were identified in total cell extracts under the same analytical conditions. These results suggest that despite successful expression and accumulation, SteelyA either lacked enzymatic activity or required additional factors for functional catalysis in P. tricornutum.
4. Discussion
Previous studies have demonstrated that large DNA insertions can be stably maintained in P. tricornutum via conjugation, such as the successful integration of a 49 kb construct [31]. Additionally, the production of heterologous proteins, including a 160 kDa antibody heavy chain, has been achieved in this diatom without adversely affecting growth [13]. In this study, we successfully introduced a 26 kb plasmid DNA into P. tricornutum, and the obtained transconjugant cell lines were stable over time under our experimental conditions. The Western blot analysis displays bands present in the total fraction pSteelyA-2 and pSteelyA-3 and in the soluble fraction of all pSteelyA samples and absent from the empty vector. All samples present a band around 40 kDa. No band is detectable in the empty vector samples, although a band is visible there in the Ponceau. Visible bands still hint towards the heterologous expression of the D. discoideum hybrid polyketide synthase SteelyA, displaying a 40 kDa fragment likely corresponding to its C-terminal region. Since the tag is located at the C-terminus, the detection of this smaller fragment could result from cleavage of a larger precursor or from alternative molecular events such as incomplete translation, internal translation initiation, splicing events, or species–species processing. These findings show that P. tricornutum is capable of the stable production over six months of a detectable portion of Steely A protein. The proteolytic cleaving of the SteelyA hypothesis remains plausible and may be linked to endogenous protease activity, such as a thrombin-like enzyme recently identified in P. tricornutum, although its exact sequence has not yet been characterized [33]. A long time kinetic of protein production and integrity verification could inform us on whether the cleavage of SteelyA is occurring progressively through subculturing due to progressive silencing mechanisms or instantaneously. One possible option to obtain catalytic activity is to identify the cutting or internal translation initiation or splicing sites and mutate them to increase the proportion of the complete form. Additionally, confirming full-length expression in P. tricornutum may require codon optimization or alternative expression strategies to improve translation efficiency and reduce potential processing issues. The use of a chaperone could also protect the protein from degradation. The inhibition of the endogenous thrombin could also be a viable option to increase SteelyA’s stability and accumulation. It is observed in SteelyA’s structure that it has an ACP site capable of binding with phosphopantetheine, and as P. tricornutum produces the molecule [21], the lack of a potential cofactor cannot be the cause of a lack of activity. A control over glycosylation patterns could increase protein stability, however, P. tricornutum’s protein glycosylation patterns are still being investigated [34,35,36,37,38].
Interestingly, the presence of SteelyA resulted in a distinct morphological change, with transconjugant cells displaying an average of 25% reduction in length compared to the EV control cells, while maintaining a consistent width (Figure 3B,C and Appendix A—Figure A2). This suggests that the metabolic burden of producing such a large protein might have influenced cell morphology. However, despite the considerable size of SteelyA, more than twice the weight of previously expressed proteins, the overall growth of transconjugants remained unaffected (Figure 3A). This finding reinforces P. tricornutum‘s potential as a suitable host for the heterologous production of large proteins.
In conclusion, this study achieved expression of a C-terminal fragment of the 352 kDa recombinant SteelyA protein in P. tricornutum, highlighting remaining obstacles for diatoms’ potential as a host for large heterologous proteins [39]. Future efforts should focus on optimizing expression conditions, thrombin’s activity and inhibition, and targeting specific cellular compartments to enhance SteelyA’s stability and activity to enable functional cannabinoid biosynthesis in this host. In all, this provides valuable negative data, which are important for guiding future engineering strategies to achieve functional production of complex, large enzymes in diatoms.
Author Contributions
N.S.: conceptualization, data curation, formal analysis, investigation, methodology, writing—original draft, writing—review and editing. B.L.: formal analysis, data curation, investigation, writing—review and editing. S.-E.G.: formal analysis, writing—review and editing. A.C.: formal analysis, validation. N.M.: conceptualization, formal analysis, investigation, writing—review and editing. F.M.-M.: formal analysis, investigation, methodology, writing—review and editing. I.D.-P.: conceptualization, funding acquisition, project administration, resources, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the Natural Sciences and Engineering Research Council of Canada through the Alliance Program (award nos. ALLRP 554429-20) to I.D.-P and by the Université du Québec à Trois-Rivières—Annexe C to IDP.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data generated or analyzed during this study are provided in full within the published article and from the corresponding author upon reasonable request.
Acknowledgments
The authors want to acknowledge the help and support of Hugo Germain and Melodie B. Plourde during this study, as well as feedback from all lab members. We also extend our appreciation to the Université du Québec à Trois-Rivières for its invaluable support in facilitating this research. We sincerely appreciate the provision of laboratory space, essential infrastructure, and utilities, which were critical for the successful completion of this work. Additionally, during the preparation of this manuscript, the authors used ChatGPT-4.0, a free AI language model, to refine grammatical accuracy and enhance readability. All content was subsequently reviewed and edited by the authors, who take full responsibility for the final version of this publication.
Conflicts of Interest
The authors declare no conflicts of interest.
Appendix A
Figure A1.
P. tricornutum SteelyA transconjugant colony PCR. Agarose gel (1%) of five colony PCR from five independent P. tricornutum SteelyA clones. Each number under the steely clone represents a different colony picked. Amplicons were obtained using primers binding in the methyltransferase domain and were expected to amplify a 403 bp fragment. Transconjugant pSteelyA** is DNA extracted from a previous assembly verified by NGS that displayed a premature stop codon. I, II, III, IV and V represented different clones.
Figure A1.
P. tricornutum SteelyA transconjugant colony PCR. Agarose gel (1%) of five colony PCR from five independent P. tricornutum SteelyA clones. Each number under the steely clone represents a different colony picked. Amplicons were obtained using primers binding in the methyltransferase domain and were expected to amplify a 403 bp fragment. Transconjugant pSteelyA** is DNA extracted from a previous assembly verified by NGS that displayed a premature stop codon. I, II, III, IV and V represented different clones.
Figure A2.
Optic microscope images of P. tricornutum SteelyA transconjugants. Phenotype observation showed a decrease of 25% on average compared to pPtGE30. Observation through the optical microscope, magnification ×10.
Figure A2.
Optic microscope images of P. tricornutum SteelyA transconjugants. Phenotype observation showed a decrease of 25% on average compared to pPtGE30. Observation through the optical microscope, magnification ×10.
Table A1.
Table of all primers used in this study.
| Primer | Sequences |
|---|---|
| E30-Stshble-F | TCATTAGTTGCAGTCACTCCGCTTTGGTTTCGTAACTATAACGGTCCTAAGGTAGCGAAC |
| E30-Stshble-R | GTTCGCTACCTTAGGACCGTTATAGTTACGAAACCAAAGCGGAGTGACTGCAACTAATGA |
| Steely-E30-F | AGGTGTTCTTATGTAGTTTTACACAGGAGTCTGGACTTGACATACAGTGAATGTAACTTT |
| Steely-E30-R | TAAGAAGAGCACATACCTCAGTCACTTATTATCACTAGCGAAACCAAAGCGGAGTGACTG |
| F-St-met-seq | GAGAAAAGAGGGTGTTCAGA |
| R-St-met-seq | GACTTAGGTTCAATACACAA |
| StlP1-R | CAATTTGTTGATTGGCGTAAACGTGAGTAGCAGACAAATC |
| StlP2-F | TCTAATTTCAAGTCCTTGAGATCCTTCGGTAGATTGATTG |
| StlP1-R 30 | ATTGGCGTAAACGTGAGTAGCAGACAAATC |
| StlP2-F 30 | AGTCCTTGAGATCCTTCGGTAGATTGATTG |
| St-E30shble-F | TCATTAGTTGCAGTCACTCCGCTTTGGTTTCATACAGTGAATGTAACTTTCGAATTGACA |
| St-E30shble-R | GTTCGCTACCTTAGGACCGTTATAGTTACGAAACCAAAGCGGAGTGACTGCAACTAATGA |
Table A2.
All DNA sequences used during the study.
| DNA Sequence | |
|---|---|
| Genewiz fragment 1 (1MN) | CATACAGTGAATGTAACTTTCGAATTGACAGTATTAGTAGTCGTATTGACAGTGAGGCACGCCCCTCAATGTGCGAGGTGGAAAATATACCAGCATGACAATGAATCTTGGAGATTCTTTTGCTGTCATCAAGATTCACCGCCAAATCTTCAGGAACCTATCACGTCCACAGGCGATGTTAATTCTTGAGTCGTCAAAACAAAGTCCTGTCCTACCTGTAGAAGTTGACAGCGAGCAATTGTATGCAAACTTCTGACTTTGTTATAATAACATTAAAGGTAATTAAGTATCTTCAATTAGGCATTTTGTCACTGTCAGTCCGTTCCGACAATATAGGTAGATTTGGAATGAATCTTTTCTATGCTGCTGCGAATCTTGTACACCTTTGAGGCCGTAGATTCTGTCCGACGAAGCGATAATTATTGCAAAATACATGGACTCATTATTTTGATTCGATTTCTTTTTGGTATCCGACTCGAAAAGATCCATCACGGCGAGCGCCACCATGAACAAGAACTCCAAAATCCAGTCCCCAAACTCTTCTGATGTTGCTGTTATTGGTGTTGGTTTTAGATTCCCAGGTAACTCTAATGACCCAGAATCTTTGTGGAACAACTTGTTGGATGGTTTCGATGCTATTACCCAAGTCCCAAAAGAAAGATGGGCTACTTCTTTTAGAGAGATGGGTTTGATCAAGAACAAGTTCGGTGGTTTCTTGAAGGATTCTGAATGGAAGAATTTCGACCCTTTGTTCTTTGGTATCGGTCCAAAAGAAGCTCCATTCATTGATCCACAACAAAGGTTGTTGTTGTCCATCGTTTGGGAATCTTTGGAAGATGCTTACATCAGACCAGATGAATTGAGAGGTTCTAACACTGGTGTTTTCATCGGTGTTTCTAACAACGATTACACCAAGTTGGGTTTCCAAGACAACTACTCTATTTCTCCATACACTATGACCGGCTCTAACTCTTCATTGAACTCCAACAGAATTTCCTACTGCTTCGATTTTAGAGGTCCATCCATTACTGTTGATACCGCTTGTTCTTCTTCCTTGGTTTCTGTTAATTTGGGTGTCCAATCCATCCAAATGGGTGAATGTAAGATTGCTATTTGCGGTGGTGTTAACGCTTTGTTTGATCCATCTACATCTGTTGCCTTTTCCAAGTTGGGTGTTTTGTCTGAAAATGGCAGATGCAACTCTTTTAGTGATCAAGCCTCTGGTTACGTTAGATCTGAAGGTGCTGGTGTTGTTGTTTTGAAGTCTTTGGAACAAGCTAAGTTGGATGGTGATAGAATCTACGGTGTTATCAAGGGTGTTTCCTCTAATGAAGATGGTGCTTCTAATGGTGACAAGAACTCTTTGACTACTCCATCTTGTGAAGCCCAATCCATTAACATTTCTAAGGCTATGGAAAAGGCCTCCTTGTCTCCATCTGATATCTATTACATTGAAGCCCATGGTACTGGTACTCCAGTTGGTGATCCAATTGAAGTTAAGGCCTTGTCCAAGATCTTCTCCAACTCTAACAACAACCAGTTGAACAACTTCTCTACCGATGGTAATGATAACGATGATGATGATGACGATAACACCTCTCCAGAACCATTATTGATTGGCTCATTCAAGTCCAACATCGGTCATTTGGAATCTGCTGCTGGTATTGCTTCTTTGATTAAGTGTTGCTTGATGTTGAAGAACAGGATGTTGGTTCCATCCATTAACTGCTCTAATTTGAACCCATCCATTCCATTCGATCAGTACAACATCTCCGTTATCAGAGAAATCAGACAATTCCCAACCGATAAGTTGGTTAACATCGGTATCAATTCTTTCGGTTTCGGTGGTTCTAACTGCCATTTGATTATTCAAGAGTACAACAACAACTTCAAGAACAACTCTACCATCTGCAATAACAACAACAACAACAATAACAACATCGACTACTTGATCCCAATCTCCTCTAAGACTAAGAAGTCCTTGGATAAGTACTTGATTTTGATCAAGACCAACTCCAACTACCACAAGGATATTTCTTTCGATGACTTCGTCAAGTTCCAAATCAAGTCTAAGCAGTACAACTTGTCCAACAGAATGACTACCATTGCTAACGATTGGAACTCCTTCATTAAGGGTTCTAACGAATTCCACAACTTGATCGAATCTAAGGATGGTGAAGGTGGTTCTTCATCTTCTAACAGAGGTATTGATTCCGCCAATCAAATCAACACTACTACTACCTCTACCATCAACGATATCGAACCTTTGTTGGTTTTCGTTTTCTGTGGTCAAGGTCCACAATGGAATGGTATGATTAAGACCTTGTACAACTCCGAGAACGTTTTCAAGAACACCGTTGATCATGTTGACAGCATCTTGTACAAGTACTTCGGTTACTCCATTTTGAACGTCTTGTCTAAGATCGATGATAACGACGATTCCATCAACCATCCAATAGTTGCTCAACCATCTTTGTTCTTGTTGCAAATTGGTTTGGTCGAGTTGTTTAAGTACTGGGGTATCTACCCATCTATCTCTGTTGGTCATTCTTTCGGTGAAGTCTCTTCTTATTACTTGTCCGGTATCATCTCTTTGGAAACCGCTTGTAAAATCGTCTACGTCAGATCCTCTAATCAGAACAAAACTATGGGTTCCGGTAAGATGTTGGTTGTTTCTATGGGTTTTAAGCAATGGAACGATCAATTCTCTGCTGAATGGTCCGATATTGAAATTGCTTGTTACAACGCTCCAGATTCCATAGTTGTTACTGGTAACGAAGAAAGATTGAAAGAATTGTCCATCAAGTTGTCCGACGAATCCAATCAAATTTTCAACACCTTCTTGAGGTCCCCATGTTCTTTTCATTCTTCCCATCAAGAAGTCATCAAGGGTTCTATGTTCGAAGAGTTGTCTAACTTGCAATCTACTGGTGAAACCGAAATCCCTTTGTTCTCTACTGTTACTGGTAGACAAGTTTTGTCTGGTCATGTTACTGCTCAACACATCTACGATAATGTTAGAGAACCAGTCTTGTTCCAAAAGACGATTGAATCCATTACCTCCTACATCAAGTCTCACTACCCATCCAATCAAAAGGTTATCTACGTTGAAATTGCTCCACACCCAACCTTGTTTTCATTGATCAAAAAGTCCATCCCATCCTCCAACAAGAATTCCTCTTCTGTTTTGTGTCCATTGAACAGAAAAGAAAACTCCAACAACTCCTACAAGAAGTTCGTTTCTCAGTTGTACTTCAACGGTGTTAACGTTGACTTCAACTTCCAGTTGAACTCCATTTGCGATAACGTTAACAACGATCACCATTTGAACAACGTCAAGCAAAACTCCTTCAAAGAGACTACCAATTCCTTGCCAAGATACCAATGGGAACAAGATGAATATTGGTCCGAACCATTGATCTCCAGAAAGAATAGATTGGAAGGTCCAACTACTTCCTTGTTGGGTCATAGAATTATCTACAGCTTCCCAGTTTTCCAATCCGTTTTGGACTTGCAATCTGACAACTACAAATACTTGTTGGACCACTTGGTTAACGGTAAGCCAGTTTTTCCAGGTGCTGGTTATTTGGATATCATCATCGAATTCTTCGACTACCAAAAGCAGCAGTTGAATTCCTCTGATTCCTCTAACTCCTACATCATCAACGTTGACAAGATCCAATTCTTGAACCCAATTCACTTGACCGAAAACAAGTTGCAAACCTTGCAATCTTCTTTCGAACCTATCGTTACTAAGAAGTCTGCCTTCTCTGTTAACTTCTTCATCAAGGATACCGTCGAGGATCAATCTAAGGTTAAGTCTATGTCTGACGAAACTTGGACTAACACTTGTAAGGCTACCATTTCCTTGGAACAACAACAGCCATCTCCATCTTCTACTTTGACTTTGTCTAAGAAGCAAGACTTGCAGATCTTGAGAAACAGATGCGATATTAGCAAGCTAGACAAGTTTGAGTTGTACGACAAGATCTCTAAGAATTTGGGCTTGCAGTACAACTCCTTGTTTCAAGTTGTTGATACCATCGAAACTGGTAAGGATTGCTCTTTTGCTACTTTGTCTTTGCCAGAAGATACTTTGTTCACCACCATTTTGAACCCATGCTTGTTGGATAACTGTTTCCATGGTTTGTTGACCTTGATCAACGAAAAGGGTTCTTTCGTTGTCGAGTCCATTTCTTCTGTTTCTATCTACTTGGAGAACATCGGTTCCTTCAATCAAACTTCTGTTGGTAACGTCCAGTTCTACTTGTACACCACTATTTCTAAAGCCACCTCCTTTAGTTCTGAAGGTACTTGTAAGTTGTTCACCAAGGATGGTTCCTTGATTTTGTCTATCGGTAAGTTCATCATCAAGTCCACCAATCCAAAGTCTACTAAGACCAACGAAACTATCGAATCTCCATTGGACGAAACCTTCTCTATTGAATGGCAATCTAAGGATTCTCCAATTCCAACCCCACAACAAATCCAACAACAATCTCCATTGAACTCTAACCCATCCTTCATTAGATCTACCATCTTGAAGGACATCCAGTTCGAACAATACTGCTCCTCCATTATCCACAAAGAATTGATCAACCACGAAAAGTACAAGAACCAGCAATCCTTCGATATCAACTCCTTGGAAAACCACTTGAACGATGACCAATTGATGGAATCCTTGTCCATCTCCAAAGAATACTTGAGATTCTTCACCAGGATCATCTCCATCATTAAGCAATACCCAAAGATCTTGAACGAAAAAGAGCTAAAAGAATTGAAAGAAATCATCGAATTGAAGTACCCATCCGAAGTTCAGTTGTTGGAATTCGAAGTTATCGAGAAGGTGTCCATGATTATCCCAAAGTTGTTGTTCGAAAACGACAAGCAATCTTCCATGACCTTGTTCCAAGATAACTTGTTGACCAGGTTCTACTCCAATTCTAACTCTACCAGATTCTACTTGGAAAGGGTTTCCGAAATGGTCTTGGAATCTATTAGACCAATCGTCAGAGAAAAGAGGGTGTTCAGAATTTTGGAAATTGGTGCTGATACAGCCTCTTTGTCTAATGTTGTTTTGACTAAGTTGAACACCTACTTGTCCACCTTGAATTCTAATGGTGGTTCTGGTTACAACATCATCATTGAGTACACCTTCACCGATATTTCCGCCAACTTCATTATTGGTGAAATCCAAGAAACCATGTGCAACTTGTACCCAAACGTTACTTTCAAGTTCTCCGTCTTGGACTTGGAGAAAGAGATTATTAACTCCTCCGATTTCTTGATGGGTGATTACGATATAGTTTTGATGGCCTACGTTATCCATGCCGTTTCTAACATTAAGTTCTCCATCGAACAGTTGTACAAGTTGTTGTCTCCAAGAGGTTGGTTGTTGTGTATTGAACCTAAGTCCAACGTTGTGTTCTCCGATTTGGTTTTCGGTTGTTTTAATCAGTGGTGGAACTACTACGATGATATTAGAACTACCCACTGCTCCTTGTCTGAATCTCAATGGAATCAGTTGTTGTTGAACCAGTCCTTGAACAACGAATCCTCTTCTTCTTCTAACTGTTACGGTGGTTTCTCCAACGTTTCTTTTATTGGTGGTGAAAAGGATGTCGACTCCCATTCTTTCATATTGCACTGCCAAAAAGAATCCATCTCCCAAATGAAGTTAGCCACCACTATTAACAACGGTTTGTCATCTGGTTCCATCGTTATCGTTTTGAACTCTCAACAATTGACCAACATGAAGTCCTACCCAAAGGTTATTGAGTATATTCAAGAGGCTACCTCTTTGTGCAAGACCATTGAAATTATCGATTCCAAGGACGTCTTGAACTCTACCAATTCAGTTTTGGAAAAGATCCAAAAGTCCTTGTTGGTGTTCTGTTTGTTGGGTTATGACTTGTTGGAGAACAACTACCAAGAACAGTCTTTCGAATACGTTAAGTTGTTGAACTTGATCTCTACTACCGCCTCTTCATCTAATGATAAGAAACCACCAAAGGTCTTGTTGATCACCAAGCAATCTGAAAGAATCTCCAGGTCTTTCTACTCCAGATCCTTGATTGGTATTTCCAGAACCTCTATGAACGAGTACCCAAATTTGTCCATTACCTCTATCGATTTGGATACCAACGACTACTCATTGCAGTCTTTGTTGAAGCCAATCTTCAGCAACTCTAAGTTTTCCGACAACGAGTTCATCTTCAAAAAGGGCTTGATGTTCGTGTCCAGGATCTTTAAGAACAAGCAGTTGCTAGAATCCTCCAACGCTTTTGAAACTGACTCTTCTAACTTGTACTGTAAGGCCTCTTCTGACTTGTCTTACAAGTACGCTATTAAGCAGTCTATGTTGACCGAAAATCAGATCGAAATCAAGGTTGAATGCGTCGGTATTAACTTCAAGGACAACCTATTCTACAAGGGCTTGTTGCCACAAGAAATTTTCAGAATGGGTGACATCTACAATCCACCATATGGTTTGGAATGCTCTGGTGTTATTACCAGAATTGGTTCTAACGTCACCGAATACTCAGTTGGTCAAAATGTTTTTGGTTTCGCCAGACATTCTTTGGGTTCTCATGTTGTTACCAACAAGGATTTGGTTATCTTGAAGCCAGATACCATCTCATTTTCTGAAGCTGCTTCTATCCCAGTTGTTTACTGTACTGCTTGGTACTCCTTGTTCAACATTGGTCAGTTGTCTAACGAAGAATCCATCCTAATTCATTCTGCTACTGGTGGTGTAGGTTTGGCTTCTTTGAATTTGTTGAAAATGAAGAATCAGCAACAGCAACCATTGACCAATGTTTATGCTACTGTTGGCTCTAACGAGAAGAAGAAGTTCTTGATCGATAACTTCAACAACTTGTTCAAAGAGGACGGCGAAAACATTTTCTCTACCAGAGACAAAGAATACTCCAACCAGTTGGAATCCAAGATCGATGTTATTTTGAACACCTTGTCCGGTGAATTCGTCGAATCTAATTTCAAGTCCTTGAGATCCTTCGGTAGATTGATTGATTTGTCTGCTACTCACGTTTGGATCCGGAAC |
| Genewiz Fragment 2 (1Q) | ATCCTTCGGTAGATTGATTGATTTGTCTGCTACTCACGTTTACGCCAATCAACAAATTGGTCTAGGTAACTTCAAGTTCGACCACTTGTATTCTGCTGTTGACTTGGAAAGATTGATCGACGAAAAACCTAAGTTGTTGCAGTCCATCTTGCAAAGAATTACCAACTCTATCGTCAACGGTTCCTTGGAAAAAATTCCAATTACCATCTTCCCATCCACCGAAACTAAGGATGCTATCGAATTATTGTCCAAGAGATCCCATATCGGTAAAGTTGTTGTAGATTGCACCGATATCTCTAAGTGTAATCCTGTTGGTGATGTGATCACCAACTTCTCTATGAGATTGCCAAAGCCAAACTACCAGTTGAATTTGAACTCCACCTTGTTGATTACTGGTCAGTCTGGTTTGTCTATCCCTTTGTTGAATTGGTTGTTGTCTAAGTCTGGTGGTAACGTTAAGAACGTTGTCATCATTTCTAAGTCCACCATGAAGTGGAAGTTGCAGACTATGATTTCCCATTTCGTTTCCGGTTTCGGTATCCATTTTAACTACGTTCAAGTCGACATCTCCAACTACGATGCTTTGTCTGAAGCTATTAAGCAATTGCCATCTGATTTGCCACCAATCACCTCTGTTTTTCATTTGGCTGCTATCTACAACGATGTTCCAATGGATCAAGTTACCATGTCTACCGTTGAATCTGTTCATAACCCTAAAGTTTTGGGTGCCGTTAACTTGCATAGAATCTCTGTTTCTTTTGGTTGGAAGTTGAACCACTTCGTCTTGTTCTCTTCTATTACTGCTATTACCGGTTACCCAGACCAATCTATCTACAATTCTGCCAACTCTATTTTGGACGCTTTGTCCAACTTTAGAAGGTTTATGGGTTTGCCATCCTTCTCCATTAACTTGGGTCCAATGAAGGATGAAGGTAAGGTTTCTACCAACAAGAGCATCAAGAAGCTATTCAAGTCTAGAGGTTTGCCAAGCCTATCCTTGAACAAGTTATTTGGTTTGTTGGAGGTCGTCATCAACAACCCATCTAATCATGTTATCCCATCCCAATTGATTTGCTCCCCAATCGATTTCAAGACCTACATCGAATCTTTCTCAACTATGAGGCCAAAGTTGTTACACTTGCAACCTACCATTTCCAAGCAGCAATCTTCTATCATTAACGATTCTACCAAGGCTTCCTCCAACATTTCATTGCAAGATAAGATCACCTCCAAGGTGTCTGATTTGTTGTCCATTCCAATCTCCAAGATCAACTTCGATCATCCATTGAAACACTACGGCTTGGATTCTTTGTTGACCGTTCAATTCAAATCCTGGATCGACAAAGAATTCGAAAAGAACTTGTTCACCCATATCCAATTGGCCACCATCTCTATTAACTCATTCTTGGAAAAGGTGAACGGCTTGTCTACAAACAATAACAACAACAACAATTCCAACGTCAAGTCCTCTCCATCCATTGTCAAAGAAGAAATCGTTACCTTGGACAAGGATCAACAACCATTGCTATTGAAAGAACACCAGCACATTATCATCTCCCCAGATATTAGAATCAACAAGCCAAAGAGGGAATCCTTGATTAGAACCCCAATCTTGAACAAATTCAACCAGATCACCGAATCCATTATCACTCCATCTACACCATCTTTGTCCCAATCCGATGTTTTGAAAACTCCACCAATCAAGTCTTTGAACAACACTAAGAACTCCAGCTTGATTAACACCCCACCAATTCAATCTGTCCAACAACATCAAAAGCAACAACAAAAGGTCCAAGTCATCCAACAACAGCAACAACCATTATCCAGATTGTCCTACAAGAGCAACAACAACTCTTTCGTTTTGGGTATCGGTATTTCTGTTCCAGGTGAACCTATTTCCCAACAATCCTTGAAAGACTCCATCTCCAATGACTTTTCTGATAAGGCTGAAACTAACGAGAAGGTCAAGAGAATCTTTGAGCAATCTCAAATCAAGACCAGACACTTGGTTAGAGATTACACTAAGCCAGAGAACTCCATCAAGTTCAGACATTTGGAAACCATTACCGATGTGAACAACCAGTTCAAGAAAGTTGTTCCAGATTTGGCTCAACAAGCCTGTTTGAGAGCTTTGAAAGATTGGGGTGGTGATAAGGGTGATATTACCCATATAGTTTCTGTTACCTCCACCGGTATTATCATCCCAGATGTTAATTTCAAGTTGATCGACTTGTTGGGCTTGAACAAGGATGTTGAAAGAGTGTCTTTGAACCTAATGGGTTGTTTGGCTGGTTTGAGTTCTTTGAGAACTGCTGCTTCTTTGGCTAAGGCTTCTCCAAGAAATAGAATTTTGGTTGTCTGTACCGAAGTCTGCTCCTTGCATTTTTCTAATACTGATGGTGGTGATCAAATGGTCGCCTCTTCTATTTTTGCTGATGGTTCTGCTGCTTACATTATTGGTTGTAACCCAAGAATTGAAGAAACCCCATTATACGAAGTCATGTGCTCCATTAACAGATCTTTCCCAAATACCGAAAACGCCATGGTTTGGGATTTGGAAAAAGAAGGTTGGAACTTGGGTTTGGATGCTTCTATTCCAATTGTCATTGGTTCTGGTATTGAAGCCTTCGTTGATACTTTGTTGGATAAGGCTAAGTTGCAAACTTCCACTGCTATTTCTGCTAAGGATTGCGAATTCTTGATTCATACTGGTGGCAAGTCCATCTTGATGAACATCGAAAATTCCTTGGGTATCGACCCAAAGCAAACTAAGAATACTTGGGATGTTTACCATGCCTACGGCAATATGTCATCTGCCTCTGTTATTTTCGTTATGGATCATGCCAGAAAGTCCAAGTCTTTGCCAACTTACTCAATTTCTTTGGCTTTTGGTCCAGGTTTGGCTTTTGAAGGTTGTTTCTTGAAGAACGTCGTCGAACAGAAGCTCATTTCCGAAGAGGACTTGTAACCGCAACAACTACCTCGACTTTGGCTGGGACACTTTCAGTGAGGACAAGAAGCTTCAGAAGCGTGCTATCGAACTCAACCAGGGACGTGCGGCACAAATGGGCATCCTTGCTCTCATGGTGCACGAACAGTTGGGAGTCTCTATCCTTCCTTAAAAATTTAATTTTCATTAGTTGCAGTCACTCCGCTTTGGTTT |
| pPTGE30 sequencing | ACTAAGGGTACTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAGAGACATGGGTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGTGGGTTTAGATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGATGTGGTCTCTACAGGATCTGACATTATTATTGTTGGAAGAGGACTATTTGCAAAGGGAAGGGATGCTAAGGTAGAGGGTGAACGTTACAGAAAAGCAGGCTGGGAAGCATATTTGAGAAGATGCGGCCAGCAAAACTAAAAAACTGTATTATAAGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCGGCCGGGAATCTCGGTCGTAATGATTTTTATAATGACGAAAAAAAAAAAAATTGGAAAGAAAACCCCCCCCCCCCCCGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGAGTTACGCTAGGGATAACAGGGTAATATAGATCTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGGGTTCGTGTAGACTTTCCTTGGTGTATCCAACGGCGTCAGCCGGGCAGGATAGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCCGGCTACCGCCGGCGTAACAGATGAGGGCAAGCGGATGGCTGATGAAACCAAGCCAACCAGGAAGGGCAGCCCACCTATCAAGGTGTACTGCCTTCCAGACGAACGAAGAGCGATTGAGGAAAAGGCGGCGGCGGCCGGCATGAGCCTGTCGGCCTACCTGCTGGCCGTCGGCCAGGGCTACAAAATCACGGGCGTCGTGGACTATGAGCACGTCCGCGAGCTGGCCCGCATCAATGGCGACCTGGGCCGCCTGGGCGGCCTGCTGAAACTCTGGCTCACCGACGACCCGCGCACGGCGCGGTTCGGTGATGCCACGATCCTCGCCCTGCTGGCGAAGATCGAAGAGAAGCAGGACGAGCTTGGCAAGGTCATGATGGGCGTGGTCCGCCCGAGGGCAGAGCCATGACTTTTTTAGCCGCTAAAACGGCCGGGGGGTGCGCGTGATTGCCAAGCACGTCCCCATGCGCTCCATCAAGAAGAGGCACTTCGAGCTGTAAGTACATCACCGACGAGCAAGGCAAGACGATCCGCGCCTGTTTTATTGAGAACGTTGTTCGTGTTGGCCTCAATGGTAGCGATGCGTCATTCAGCGAAGTTCTGGTGCTGATGATGTGGTTCGCTTTGCCACTGGTCAATGTGGTAAGCCCGTGTAATGTCAGTAACCTTTTTACTGATCTCAGCTTGAGCACGGTCGCTGATGAGCTTATCCATGGCCCCACGGTAACGGATATGATCCTCTAGGGCGTTGACAAATTCTTTGTCGGTTTTCATGGGGTAGACGTCAGTGACCAGGGAACGTCTCCCTACAAAAAGTTGCGCATACTTTGCTCCGTTGTCGACGGCAGGGGTGTCGGACCAGATTGTATCTGTGGCAACGGCCTCATTGCGTCAATGGACCTTTAAAGCCGGGAAACGAGACTTGAAATGTTTACGTAGAGGTGCATTATAGACCTCACGCGCATATTGAGTGGTTGCAAAAATGGTTTTGCGAACGGTATCACTGGAGACCCATGCCAGGCAAGGACGGAGGGCATCATAATCATGTTCATTGCGTTGGACAGCATGCTTGTTACACGTAAGTATACGGTCGAGAGCATCGTGACGTTGAGAATTACAGATGGCAACGTGTCGGTGCATGTATTGGCCGAGATTGTCAAATCGTGGCTCAATATATGGCATGCCAGGTAAGTCATGGATGTCGGTATGCCATTCAGCCTTCATGTCGATTTCGTTGTCGAGAACTGATGGGTCCCAGTCAACGTCAGAAGTAAGAACAACATGGGGAAGGGAATGAAGTTCATGACTGGTGGGTGCGCGCATATCCATGTACGGAAGACCCTGTCGTATGTTTAGTGGGATGATGTAATCATCCAGGGTAACAATACGTTGGTTGCCGCCCACGGTACGAGACCGATCTTGAACACAATTGTGGCAATGTTCAAGTTGGGCGCTGGAATGGATGGTTTTACCTTTACCAAGATGCGCGTATTGATGCATGATAACAACAATGGGCCCTCGTTGCGATTCGACAAGACCGGCAGCCGTGACAATGTCTAGATTGGACAAGGTATGTTCATTTATACCGGTTATGTTGGCTGAACGTCCAGTTTTGTGCAGTACGGTAACATCACTCCCCGCAAGTCCACCGTTGGCACCGCGATCGACAAGTGCAGATGTGGTATTTTGTACTGTGTGTCGGGAGACTTGGTACTGTAACACATTTGACTGAAGTGACTTGGTGGTACCATGAGGACGGAGGGAACCCGTTGATACGGTGTTGCGTGACGCTGCAAGTACCTTACGTATGTCGCCTGGATCCATACGTCCAACCCGATCAGAAAGATGTGCTAGGAGTTCCGTTTCTGGTGCACAATCATGAAAGGTATCCACGGGAGTTCCATTGTTGTCGTCCGGGGCTGCGTCGCGACTGCCAGTTGCATGTACATGCGCCATCAAGGGACGTGCCAATCCATTACTAACCGGGAGAACCTTAGCTGGGGCAGCCAATCCCTGAAGTATAGCCTTAGCATCATCTGAGAGCTGGTTCCACATATCTCTAGGGATATAAGGTCGTTCACGGTTAGTGGGCGTACTATGATGTCGGTTATTAGTACAATTCTCCCTGCGGGCATGCGCATTGGCTTCATAGAGTACGGAAGGTGACAAGTCAATATTGTAGTTAACATCCGGATCAGTGTCAAAGTCTGTAGGATGGTAAGAAAGATCAGTAGAATGAATACTACGCTTTTCCTTGGGACTACGGGAATTAGAGAAGTTGTTTCCTTTATTGTAGAGTGACGCTGAAGCAAGTAGAAGACTAAGGTAACTCTCGTAGCTAATAGGATTACCTCCTTTGGCTAGGTCAAGAGTGGCTGTGATCTTCACTTGACAAAGTTCCGGTACATTGTGGACAGCATTCTCCAAAAGACTAAGACACAGTTGCTTTGGGAGTTGCTCAGCCATTGGTACAGTATTGTGGTAGATACAAAGGTGGTTCTTCCAATGAAGGATAAATCCTTCTGCTGTGCCTGTCCATGAGGATCCATATTTCGCCGTAGTTAGGTAACCAAGCGTGGTGGCTGAACTGATCTTCGCACTTGCTGATTCCGTATAGTGTTTGACAACTTTACAAAACACTTCTTGCGCAGTTCGCTCTAGCCTGGACAGAGAAGGGACAATCTTCGTCGTTAAGACTCGTGCGAATAGCAAAAGATCACAAAATAGCACATCGGCACCGACCAACGATTATTTCCAAGGAAAAAAAGAATGCTTCACTACAAGAAATTGTGTCATCCCTATACAGAGTCTTGTTACTGTGACAGAAAATTGATGGAAGATGTGGCGGATTGCCTTTACACTAGCCAACTTGTTCGACTAATTGCAGCTTCTTCTGAGAGGCTTCACCGAGTAACGCGAAGAACACCGGTGTCTCGTACATGCTCGTCGGTGAACGCTCGTCCAATGACACCCCCCACTTTGTATCAATATCCCAACTTGGTAGTGAACTGGAATGATACATGCAATTTCGCGCCGCATCAACAGCCACGGGCACCATCGACGAATAGACTCGGTCGAGCTGGTTGCCCTCGCCGCTGGGCTGGCGGCCGTCTATGGCCCTGCAAACGCGCCAGAAACGCCGTCGAAGCCGTGTGCGAGACACCGCGGCCGGCCGCCGGCGTTGTGGATACCTCGCGGAAAACTTGGCCCTCACTGACAGATGAGGGGCGGACGTTGACACTTGAGGGGCCGACTCACCCGGCGCGGCGTTGACAGATGAGGGGCAGGCTCGATTTCGGCCGGCGACGTGGAGCTGGCCAGCCTCGCAAATCGGCGAAAACGCCTGATTTTACGCGAGTTTCCCACAGATGATGTGGACAAGCCTGGGGATAAGTGCCCTGCGGTATTGACACTTGAGGGGCGCGACTACTGACAGATGAGGGGCGCGATCCTTGACACTTGAGGGGCAGAGTGCTGACAGATGAGGGGCGCACCTATTGACATTTGAGGGGCTGTCCACAGGCAGAAAATCCAGCATTTGCAAGGGTTTCCGCCCGTTTTTCGGCCACCGCTAACCTGTCTTTTAACCTGCTTTTAAACCAATATTTATAAACCTTGTTTTTAACCAGGGCTGCGCCCTGTGCGCGTGACCGCGCACGCCGAAGGGGGGTGCCCCCCCTTCTCGAACCCTCCCGGTCGAGTGAGCGAGGAAGCACCAGGGAACAGCACTTATATATTCTGCTTACACACGATGCCTGAAAAAACTTCCCTTGGGGTTATCCACTTATCCACGGGGATATTTTTATAATTATTTTTTTTATAGTTTTTAGATCTTCTTTTTTAGAGCGCCTTGTAGGCCTTTATCCATGCTGGTTCTAGAGAAGGTGTTGTGACAAATTGCCCTTTCAGTGTGACAAATCACCCTCAAATGACAGTCCTGTCTGTGACAAATTGCCCTTAACCCTGTGACAAATTGCCCTCAGAAGAAGCTGTTTTTTCACAAAGTTATCCCTGCTTATTGACTCTTTTTTATTTAGTGTGACAATCTAAAAACTTGTCACACTTCACATGGATCTGTCATGGCGGAAACAGCGGTTATCAATCACAAGAAACGTAAAAATAGCCCGCGAATCGTCCAGTCAAACGACCTCACTGAGGCGGCATATAGTCTCTCCCGGGATCAAAAACGTATGCTGTATCTGTTCGTTGACCAGATCAGAAAATCTGATGGCACCCTACAGGAACATGACGGTATCTGCGAGATCCATGTTGCTAAATATGCTGAAATATTCGGATTGACCTCTGCGGAAGCCAGTAAGGATATACGGCAGGCATTGAAGAGTTTCGCGGGGAAGGAAGTGGTTTTTTATCGCCCTGAAGAGGATGCCGGCGATGAAAAAGGCTATGAATCTTTTCCTTGGTTTATCAAACGTGCGCACAGTCCATCCAGAGGGCTTTACAGTGTACATATCAACCCATATCTCATTCCCTTCTTTATCGGGTTACAGAACCGGTTTACGCAGTTTCGGCTTAGTGAAACAAAAGAAATCACCAATCCGTATGCCATGCGTTTATACGAATCCCTGTGTCAGTATCGTAAGCCGGATGGCTCAGGCATCGTCTCTCTGAAAATCGACTGGATCATAGAGCGTTACCAGCTGCCTCAAAGTTACCAGCGTATGCCTGACTTCCGCCGCCGCTTCCTGCAGGTCTGTGTTAATGAGATCAACAGCAGAACTCCAATGCGCCTCTCATACATTGAGAAAAAGAAAGGCCGCCAGACGACTCATATCGTATTTTCCTTCCGCGATATCACTTCCATGACGACAGGATAGTCTGAGGGTTATCTGTCACAGATTTGAGGGTGGTTCGTCACATTTGTTCTGACCTACTGAGGGTAATTTGTCACAGTTTTGCTGTTTCCTTCAGCCTGCATGGATTTTCTCATACTTTTTGAACTGTAATTTTTAAGGAAGCCAAATTTGAGGGCAGTTTGTCACAGTTGATTTCCTTCTCTTTCCCTTCGTCATGTGACCTGATATCGGGGGTTAGTTCGTCATCATTGATGAGGGTTGATTATCACAGTTTATTACTCTGAATTGGCTATCCGCGTGTGTACCTCTACCTGGAGTTTTTCCCACGGTGGATATTTCTTCTTGCGCTGAGCGTAAGAGCTATCTGACAGAACAGTTCTTCTTTGCTTCCTCGCCAGTTCGCTCGCTATGCTCGGTTACACGGCTGCGGCGAGCATCACGTGCTATAAAAATAATTATAATTTAAATTTTTTAATATAAATATATAAATTAAAAATAGAAAGTAAAAAAAGAAATTAAAGAAAAAATAGTTTTTGTTTTCCGAAGATGTAAAAGACTCTAGGGGGATCGCCAACAAATACTACCTTTTACCTTGCTCTTCCTGCTCTCAGGTATTAATGCCGAATTGTTTCATCTTGTCTGTGTAGAAGACCACACACGAAAATCCTGTGATTTTACATTTTACTTATCGTTAATCGAATGTATATCTATTTAATCTGCTTTTCTTGTCTAATAAATATATATGTAAAGTACGCTTTTTGTTGAAATTTTTTAAACCTTTGTTTATTTTTTTTTCTTCATTCCGTAACTCTTCTACCTTCTTTATTTACTTTCTAAAATCCAAATACAAAACATAAAAATAAATAAACACAGAGTAAATTCCCAAATTATTCCATCATTAAAAGATACGAGGCGCGTGTAAGTTACAGGCAAGCGATCCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTTTAAAGAGGCCCTAGGGGCCGTGCGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACAGGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTTTTACACAGGAGTCTGGACTTGACGCTAGTGATAATAAGTGACTGAGGTATGTGCTCTTCTTATCTCCTTTTGTAGTGTTGCTCTTATTTTAAACAACTTTGCGGTTTTTTGATGACTTTGCGATTTTGTTGTTGCTTTGCAGTAAATTGCAAGATTTAATAAAAAAACGCAAAGCAATGATTAAAGGATGTTCAGAATGAAACTCATGGAAACACTTAACCAGTGCATAAACGCTGGTCATGAAATGACGAAGGCTATCGCCATTGCACAGTTTAATGATGACAGCCCGGAAGCGAGGAAAATAACCCGGCGCTGGAGAATAGGTGAAGCAGCGGATTTAGTTGGGGTTTCTTCTCAGGCTATCAGAGATGCCGAGAAAGCAGGGCGACTACCGCACCCGGATATGGAAATTCGAGGACGGGTTGAGCAACGTGTTGGTTATACAATTGAACAAATTAATCATATGCGTGATGTGTTTGGTACGCGATTGCGACGTGCTGAAGACGTATTTCCACCGGTGATCGGGGTTGCTGCCCATAAAGGTGGCGTTTACAAAACCTCAGTTTCTGTTCATCTTGCTCAGGATCTGGCTCTGAAGGGGCTACGTGTTTTGCTCGTGGAAGGTAACGACCCCCAGGGAACAGCCTCAATGTATCACGGATGGGTACCAGATCTTCATATTCATGCAGAAGACACTCTCCTGCCTTTCTATCTTGGGGAAAAGGACGATGTCACTTATGCAATAAAGCCCACTTGCTGGCCGGGGCTTGACATTATTCCTTCCTGTCTGGCTCTGCACCGTATTGAAACTGAGTTAATGGGCAAATTTGATGAAGGTAAACTGCCCACCGATCCACACCTGATGCTCCGACTGGCCATTGAAACTGTTGCTCATGACTATGATGTCATAGTTATTGACAGCGCGCCTAACCTGGGTATCGGCACGATTAATGTCGTATGTGCTGCTGATGTGCTGATTGTTCCCACGCCTGCTGAGTTGTTTGACTACACCTCCGCACTGCAGTTTTTCGATATGCTTCGTGATCTGCTCAAGAACGTTGATCTTAAAGGGTTCGAGCCTGATGTACGTATTTTGCTTACCAAATACAGCAATAGCAATGGCTCTCAGTCCCCGTGGATGGAGGAGCAAATTCGGGATGCCTGGGGAAGCATGGTTCTAAAAAATGTTGTACGTGAAACGGATGAAGTTGGTAAAGGTCAGATCCGGATGAGAACTGTTTTTGAACAGGCCATTGATCAACGCTCTTCAACTGGTGCCTGGAGAAATGCTCTTTCTATTTGGGAACCTGTCTGCAATGAAATTTTCGATCGTCTGATTAAACCACGCTGGGAGATTAGATAATGAAGCGTGCGCCTGTTATTCCAAAACATACGCTCAATACTCAACCGGTTGAAGATACTTCGTTATCGACACCAGCTGCCCCGATGGTGGATTCGTTAATTGCGCGCGTAGGAGTAATGGCTCGCGGTAATGCCATTACTTTGCCTGTATGTGGTCGGGATGTGAAGTTTACTCTTGAAGTGCTCCGGGGTGATAGTGTTGAGAAGACCTCTCGGGTATGGTCAGGTAATGAACGTGACCAGGAGCTGCTTACTGAGGACGCACTGGATGATCTCATCCCTTCTTTTCTACTGACTGGTCAACAGACACCGGCGTTCGGTCGAAGAGTATCTGGTGTCATAGAAATTGCCGATGGGAGTCGCCGTCGTAAAGCTGCTGCACTTACCGAAAGTGATTATCGTGTTCTGGTTGGCGAGCTGGATGATGAGCAGATGGCTGCATTATCCAGATTGGGTAACGATTATCGCCCAACAAGTGCTTATGAACGTGGTCAGCGTTATGCAAGCCGATTGCAGAATGAATTTGCTGGAAATATTTCTGCGCTGGCTGATGCGGAAAATATTTCACGTAAGATTATTACCCGCTGTATCAACACCGCCAAATTGCCTAAATCAGTTGTTGCTCTTTTTTCTCACCCCGGTGAACTATCTGCCCGGTCAGGTGATGCACTTCAAAAAGCCTTTACAGATAAAGAGGAATTACTTAAGCAGCAGGCATCTAACCTTCATGAGCAGAAAAAAGCTGGGGTGATATTTGAAGCTGAAGAAGTTATCACTCTTTTAACTTCTGTGCTTAAAACGTCATCTGCATCAAGAACTAGTTTAAGCTCACGACATCAGTTTGCTCCTGGAGCGACAGTATTGTATAAGGGCGATAAAATGGTGCTTAACCTGGACAGGTCTCGTGTTCCAACTGAGTGTATAGAGAAAATTGAGGCCATTCTTAAGGAACTTGAAAAGCCAGCACCCTGATGCGACCTCGTTTTAGTCTACGTTTATCTGTCTTTACTTAATGTCCTTTGTTACAGGCCAGAAAGCATAACTGGCCTGAATATTCTCTCTGGGCCCACTGTTCCACTTGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCATGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGAACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGATCCCACTCGTGTTGTCGGTCTGATTATCGGTCTGGGACCACGGTCCCACTTGTATTGTCGATCAGACTATCAGCGTGAGACTACGATTCCATCAATGCCTGTCAAGGGCAAGTATTGACATGTCGTCGTAACCTGTAGAACGGAGTAACCTCGGTGTGCGGTTGTATGCCTGCTGTGGATTGCTGCTGTGTCCTGCTTATCCACAACATTTTGCGCACGGTTATGTGGACAAAATACCTGGTTACCCAGGCCGTGCCGGCACGTTAACCGGGCTGCATCCGATGCAAGTGTGTCGCTGTCGACGAGCTCGCGAGCTCGGACATGAGGTTGCCCCGTATTCAGTGTCGCTGATTTGTATTGTCTGAAGTTGTTTTTACGTTAAGTTGATGCAGATCAATTAATACGATACCTGCGTCATAATTGATTATTTGACGTGGTTTGATGGCCTCCACGCACGTTGTGATATGTAGATGATAATCATTATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTACGGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGCTTTTTGGCCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATACATTATACGAAGTTATATTCGATGCGGCCGCAAGGGGTTCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCACACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGAGTATTCTATAGTCTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGAACCCCTTGCGGCCGCCCGGGCCGTCGACCAATTCTCATGTTTGACAGCTTATCATCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGCGATAAGCTCATGGAGCGGCGTAACCGTCGCACAGGAAGGACAGAGAAAGCGCGGATCTGGGAAGTGACGGACAGAACGGTCAGGACCTGGATTGGGGAGGCGGTTGCCGCCGCTGCTGCTGACGGTGTGACGTTCTCTGTTCCGGTCACACCACATACGTTCCGCCATTCCTATGCGATGCACATGCTGTATGCCGGTATACCGCTGAAAGTTCTGCAAAGCCTGATGGGACATAAGTCCATCAGTTCAACGGAAGTCTACACGAAGGTTTTTGCGCTGGATGTGGCTGCCCGGCACCGGGTGCAGTTTGCGATGCCGGAGTCTGATGCGGTTGCGATGCTGAAACAATTATCCTGAGAATAAATGCCTTGGCCTTTATATGGAAATGTGGAACTGAGTGGATATGCTGTTTTTGTCTGTTAAACAGAGAAGCTGGCTGTTATCCACTGAGAAGCGAACGAAACAGTCGGGAAAATCTCCCATTATCGTAGAGATCCGCATTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATTTGAATATGCCTTCAGGAACAATAGAAATCTTCGTGCGGTGTTACGTTGAAGTGGAGCGGATTATGTCAGCAATGGACAGAACAACCTAATGAACACAGAACCATGATGTGGTCTGTCCTTTTACAGCCAGTAGTGCTCGCCGCAGTCGAGCGACAGGGCGAAGCCCATCGATACTAGCTTGATTGGGATATCTCGCTCGTGCTTGTCGCGTGCTATGTCTTTTTAGGGTACTTTGAACCTACGTTCGTACTTGTGTAATATGATCATCGTCGTCATCGTATTATCGTTTTTCATCCGTCCAGCGCAAAATGCATTAGCAGCTAGTCCTAGCGTGCGGAGCTACCTGTACAGGTGCATGACGGATGCGTGTCCTTAAGTGAGTTTCTAATTAACAGTAACTTCTTTACTTATGTTTCAGTTTGTAAGAAGCGGGATGCGCTCGTCGCTTGACATCTGATTGGACTGCGTCGGCACGTGAAAACTACATTGTGAAATCTGCTAAAACTCCGGGTATCTCTGACACAAAACGATTCGGCTTCGCAATTTCAACATTACGGTCAAGGCTAACGTATCTTTCTCGGTCAACTTCAGATTATGCCGATTAAATTGTCGTAGCTTTCAAGGCGTTTTGAGTACTGCGGCAGTTGTTGAACCTGCAAGGAGAAGATCTCGACAACAGAATAAAGCGAAAAATGGGTCTCATGCACTAACACTCAGGCCTCCCTCATAATCTCTGTTTGAGTTTACCAACAACACATATATACATTTCGACAAAATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCAGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACCGACGCCGACCAACACCGCCGGTCCGACGCGGCCCGACGGGTCCGAGGCCTCGGAGATCTGGGCCCATGCGGCCGCAACAACTACCTCGACTTTGGCTGGGACACTTTCAGTGAGGACAAGAAGCTTCAGAAGCGTGCTATCGAACTCAACCAGGGACGTGCGGCACAAATGGGCATCCTTGCTCTCATGGTGCACGAACAGTTGGGAGTCTCTATCCTTCCTTAAAAATTTAATTTTCATTAGTTGCAGTCACTCCGCTTTGGTTTCGTAACTATAACGGTCCTAAGGTAGCGAACGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCGGGGGGGGGGGGGTTTCAATTCATCATTTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCCTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATATAAGGAACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAAGCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAATTTGTTTACTAAAAACACATGTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAAAGGCATTATCCGCCAAGTACAATTTTTTACTCTTCGAAGACAGAAAATTTGCTGACATTGGTAATACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAGAATGGGCAGACATTACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGTTTGAAGCAGGCGGCGGAAGAAGTAACAAAGGAACCTAGAGGCCTTTTGATGTTAGCAGAATTGTCATGCAAGGGCTCCCTAGCTACTGGAGAATAT |
| pSteelyA-1 sequencing | TTCGATGCGGCCGCAAGGGGTTCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCACACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGAGTATTCTATAGTCTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGAACCCCTTGCGGCCGCCCGGGCCGTCGACCAATTCTCATGTTTGACAGCTTATCATCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGCGATAAGCTCATGGAGCGGCGTAACCGTCGCACAGGAAGGACAGAGAAAGCGCGGATCTGGGAAGTGACGGACAGAACGGTCAGGACCTGGATTGGGGAGGCGGTTGCCGCCGCTGCTGCTGACGGTGTGACGTTCTCTGTTCCGGTCACACCACATACGTTCCGCCATTCCTATGCGATGCACATGCTGTATGCCGGTATACCGCTGAAAGTTCTGCAAAGCCTGATGGGACATAAGTCCATCAGTTCAACGGAAGTCTACACGAAGGTTTTTGCGCTGGATGTGGCTGCCCGGCACCGGGTGCAGTTTGCGATGCCGGAGTCTGATGCGGTTGCGATGCTGAAACAATTATCCTGAGAATAAATGCCTTGGCCTTTATATGGAAATGTGGAACTGAGTGGATATGCTGTTTTTGTCTGTTAAACAGAGAAGCTGGCTGTTATCCACTGAGAAGCGAACGAAACAGTCGGGAAAATCTCCCATTATCGTAGAGATCCGCATTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATTTGAATATGCCTTCAGGAACAATAGAAATCTTCGTGCGGTGTTACGTTGAAGTGGAGCGGATTATGTCAGCAATGGACAGAACAACCTAATGAACACAGAACCATGATGTGGTCTGTCCTTTTACAGCCAGTAGTGCTCGCCGCAGTCGAGCGACAGGGCGAAGCCCATCGATACTAGCTTGATTGGGATATCTCGCTCGTGCTTGTCGCGTGCTATGTCTTTTTAGGGTACTTTGAACCTACGTTCGTACTTGTGTAATATGATCATCGTCGTCATCGTATTATCGTTTTTCATCCGTCCAGCGCAAAATGCATTAGCAGCTAGTCCTAGCGTGCGGAGCTACCTGTACAGGTGCATGACGGATGCGTGTCCTTAAGTGAGTTTCTAATTAACAGTAACTTCTTTACTTATGTTTCAGTTTGTAAGAAGCGGGATGCGCTCGTCGCTTGACATCTGATTGGACTGCGTCGGCACGTGAAAACTACATTGTGAAATCTGCTAAAACTCCGGGTATCTCTGACACAAAACGATTCGGCTTCGCAATTTCAACATTACGGTCAAGGCTAACGTATCTTTCTCGGTCAACTTCAGATTATGCCGATTAAATTGTCGTAGCTTTCAAGGCGTTTTGAGTACTGCGGCAGTTGTTGAACCTGCAAGGAGAAGATCTCGACAACAGAATAAAGCGAAAAATGGGTCTCATGCACTAACACTCAGGCCTCCCTCATAATCTCTGTTTGAGTTTACCAACAACACATATATACATTTCGACAAAATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCAGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACCGACGCCGACCAACACCGCCGGTCCGACGCGGCCCGACGGGTCCGAGGCCTCGGAGATCTGGGCCCATGCGGCCGCAACAACTACCTCGACTTTGGCTGGGACACTTTCAGTGAGGACAAGAAGCTTCAGAAGCGTGCTATCGAACTCAACCAGGGACGTGCGGCACAAATGGGCATCCTTGCTCTCATGGTGCACGAACAGTTGGGAGTCTCTATCCTTCCTTAAAAATTTAATTTTCATTAGTTGCAGTCACTCCGCTTTGGTTTCGTAACTATAACGGTCCTAAGGTAGCGAACGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCGGGGGGGGGGGGGGGGTTTCAATTCATCATTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCCTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATATAAGGAACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAAGCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAATTTGTTTACTAAAAACACATGTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAAAGGCATTATCCGCCAAGTACAATTTTTTACTCTTCGAAGACAGAAAATTTGCTGACATTGGTAATACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAGAATGGGCAGACATTACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGTTTGAAGCAGGCGGCGGAAGAAGTAACAAAGGAACCTAGAGGCCTTTTGATGTTAGCAGAATTGTCATGCAAGGGCTCCCTAGCTACTGGAGAATATACTAAGGGTACTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAGAGACATGGGTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGTGGGTTTAGATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGATGTGGTCTCTACAGGATCTGACATTATTATTGTTGGAAGAGGACTATTTGCAAAGGGAAGGGATGCTAAGGTAGAGGGTGAACGTTACAGAAAAGCAGGCTGGGAAGCATATTTGAGAAGATGCGGCCAGCAAAACTAAAAAACTGTATTATAAGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCGGCCGGGAATCTCGGTCGTAATGATTTTTATAATGACGAAAAAAAAAAAAATTGGAAAGAAAACCCCCCCCCCCCCCCCGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGAGTTACGCTAGGGATAACAGGGTAATATAGATCTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGGGTTCGTGTAGACTTTCCTTGGTGTATCCAACGGCGTCAGCCGGGCAGGATAGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCCGGCTACCGCCGGCGTAACAGATGAGGGCAAGCGGATGGCTGATGAAACCAAGCCAACCAGGAAGGGCAGCCCACCTATCAAGGTGTACTGCCTTCCAGACGAACGAAGAGCGATTGAGGAAAAGGCGGCGGCGGCCGGCATGAGCCTGTCGGCCTACCTGCTGGCCGTCGGCCAGGGCTACAAAATCACGGGCGTCGTGGACTATGAGCACGTCCGCGAGCTGGCCCGCATCAATGGCGACCTGGGCCGCCTGGGCGGCCTGCTGAAACTCTGGCTCACCGACGACCCGCGCACGGCGCGGTTCGGTGATGCCACGATCCTCGCCCTGCTGGCGAAGATCGAAGAGAAGCAGGACGAGCTTGGCAAGGTCATGATGGGCGTGGTCCGCCCGAGGGCAGAGCCATGACTTTTTTAGCCGCTAAAACGGCCGGGGGGTGCGCGTGATTGCCAAGCACGTCCCCATGCGCTCCATCAAGAAGAGGCACTTCGAGCTGTAAGTACATCACCGACGAGCAAGGCAAGACGATCCGCGCCTGTTTTATTGAGAACGTTGTTCGTGTTGGCCTCAATGGTAGCGATGCGTCATTCAGCGAAGTTCTGGTGCTGATGATGTGGTTCGCTTTGCCACTGGTCAATGTGGTAAGCCCGTGTAATGTCAGTAACCTTTTTACTGATCTCAGCTTGAGCACGGTCGCTGATGAGCTTATCCATGGCCCCACGGTAACGGATATGATCCTCTAGGGCGTTGACAAATTCTTTGTCGGTTTTCATGGGGTAGACGTCAGTGACCAGGGAACGTCTCCCTACAAAAAGTTGCGCATACTTTGCTCCGTTGTCGACGGCAGGGGTGTCGGACCAGATTGTATCTGTGGCAACGGCCTCATTGCGTCAATGGACCTTTAAAGCCGGGAAACGAGACTTGAAATGTTTACGTAGAGGTGCATTATAGACCTCACGCGCATATTGAGTGGTTGCAAAAATGGTTTTGCGAACGGTATCACTGGAGACCCATGCCAGGCAAGGACGGAGGGCATCATAATCATGTTCATTGCGTTGGACAGCATGCTTGTTACACGTAAGTATACGGTCGAGAGCATCGTGACGTTGAGAATTACAGATGGCAACGTGTCGGTGCATGTATTGGCCGAGATTGTCAAATCGTGGCTCAATATATGGCATGCCAGGTAAGTCATGGATGTCGGTATGCCATTCAGCCTTCATGTCGATTTCGTTGTCGAGAACTGATGGGTCCCAGTCAACGTCAGAAGTAAGAACAACATGGGGAAGGGAATGAAGTTCATGACTGGTGGGTGCGCGCATATCCATGTACGGAAGACCCTGTCGTATGTTTAGTGGGATGATGTAATCATCCAGGGTAACAATACGTTGGTTGCCGCCCACGGTACGAGACCGATCTTGAACACAATTGTGGCAATGTTCAAGTTGGGCGCTGGAATGGATGGTTTTACCTTTACCAAGATGCGCGTATTGATGCATGATAACAACAATGGGCCCTCGTTGCGATTCGACAAGACCGGCAGCCGTGACAATGTCTAGATTGGACAAGGTATGTTCATTTATACCGGTTATGTTGGCTGAACGTCCAGTTTTGTGCAGTACGGTAACATCACTCCCCGCAAGTCCACCGTTGGCACCGCGATCGACAAGTGCAGATGTGGTATTTTGTACTGTGTGTCGGGAGACTTGGTACTGTAACACATTTGACTGAAGTGACTTGGTGGTACCATGAGGACGGAGGGAACCCGTTGATACGGTGTTGCGTGACGCTGCAAGTACCTTACGTATGTCGCCTGGATCCATACGTCCAACCCGATCAGAAAGATGTGCTAGGAGTTCCGTTTCTGGTGCACAATCATGAAAGGTATCCACGGGAGTTCCATTGTTGTCGTCCGGGGCTGCGTCGCGACTGCCAGTTGCATGTACATGCGCCATCAAGGGACGTGCCAATCCATTACTAACCGGGAGAACCTTAGCTGGGGCAGCCAATCCCTGAAGTATAGCCTTAGCATCATCTGAGAGCTGGTTCCACATATCTCTAGGGATATAAGGTCGTTCACGGTTAGTGGGCGTACTATGATGTCGGTTATTAGTACAATTCTCCCTGCGGGCATGCGCATTGGCTTCATAGAGTACGGAAGGTGACAAGTCAATATTGTAGTTAACATCCGGATCAGTGTCAAAGTCTGTAGGATGGTAAGAAAGATCAGTAGAATGAATACTACGCTTTTCCTTGGGACTACGGGAATTAGAGAAGTTGTTTCCTTTATTGTAGAGTGACGCTGAAGCAAGTAGAAGACTAAGGTAACTCTCGTAGCTAATAGGATTACCTCCTTTGGCTAGGTCAAGAGTGGCTGTGATCTTCACTTGACAAAGTTCCGGTACATTGTGGACAGCATTCTCCAAAAGACTAAGACACAGTTGCTTTGGGAGTTGCTCAGCCATTGGTACAGTATTGTGGTAGATACAAAGGTGGTTCTTCCAATGAAGGATAAATCCTTCTGCTGTGCCTGTCCATGAGGATCCATATTTCGCCGTAGTTAGGTAACCAAGCGTGGTGGCTGAACTGATCTTCGCACTTGCTGATTCCGTATAGTGTTTGACAACTTTACAAAACACTTCTTGCGCAGTTCGCTCTAGCCTGGACAGAGAAGGGACAATCTTCGTCGTTAAGACTCGTGCGAATAGCAAAAGATCACAAAATAGCACATCGGCACCGACCAACGATTATTTCCAAGGAAAAAAAGAATGCTTCACTACAAGAAATTGTGTCATCCCTATACAGAGTCTTGTTACTGTGACAGAAAATTGATGGAAGATGTGGCGGATTGCCTTTACACTAGCCAACTTGTTCGACTAATTGCAGCTTCTTCTGAGAGGCTTCACCGAGTAACGCGAAGAACACCGGTGTCTCGTACATGCTCGTCGGTGAACGCTCGTCCAATGACACCCCCCACTTTGTATCAATATCCCAACTTGGTAGTGAACTGGAATGATACATGCAATTTCGCGCCGCATCAACAGCCACGGGCACCATCGACGAATAGACTCGGTCGAGCTGGTTGCCCTCGCCGCTGGGCTGGCGGCCGTCTATGGCCCTGCAAACGCGCCAGAAACGCCGTCGAAGCCGTGTGCGAGACACCGCGGCCGGCCGCCGGCGTTGTGGATACCTCGCGGAAAACTTGGCCCTCACTGACAGATGAGGGGCGGACGTTGACACTTGAGGGGCCGACTCACCCGGCGCGGCGTTGACAGATGAGGGGCAGGCTCGATTTCGGCCGGCGACGTGGAGCTGGCCAGCCTCGCAAATCGGCGAAAACGCCTGATTTTACGCGAGTTTCCCACAGATGATGTGGACAAGCCTGGGGATAAGTGCCCTGCGGTATTGACACTTGAGGGGCGCGACTACTGACAGATGAGGGGCGCGATCCTTGACACTTGAGGGGCAGAGTGCTGACAGATGAGGGGCGCACCTATTGACATTTGAGGGGCTGTCCACAGGCAGAAAATCCAGCATTTGCAAGGGTTTCCGCCCGTTTTTCGGCCACCGCTAACCTGTCTTTTAACCTGCTTTTAAACCAATATTTATAAACCTTGTTTTTAACCAGGGCTGCGCCCTGTGCGCGTGACCGCGCACGCCGAAGGGGGGTGCCCCCCCTTCTCGAACCCTCCCGGTCGAGTGAGCGAGGAAGCACCAGGGAACAGCACTTATATATTCTGCTTACACACGATGCCTGAAAAAACTTCCCTTGGGGTTATCCACTTATCCACGGGGATATTTTTATAATTATTTTTTTTATAGTTTTTAGATCTTCTTTTTTAGAGCGCCTTGTAGGCCTTTATCCATGCTGGTTCTAGAGAAGGTGTTGTGACAAATTGCCCTTTCAGTGTGACAAATCACCCTCAAATGACAGTCCTGTCTGTGACAAATTGCCCTTAACCCTGTGACAAATTGCCCTCAGAAGAAGCTGTTTTTTCACAAAGTTATCCCTGCTTATTGACTCTTTTTTATTTAGTGTGACAATCTAAAAACTTGTCACACTTCACATGGATCTGTCATGGCGGAAACAGCGGTTATCAATCACAAGAAACGTAAAAATAGCCCGCGAATCGTCCAGTCAAACGACCTCACTGAGGCGGCATATAGTCTCTCCCGGGATCAAAAACGTATGCTGTATCTGTTCGTTGACCAGATCAGAAAATCTGATGGCACCCTACAGGAACATGACGGTATCTGCGAGATCCATGTTGCTAAATATGCTGAAATATTCGGATTGACCTCTGCGGAAGCCAGTAAGGATATACGGCAGGCATTGAAGAGTTTCGCGGGGAAGGAAGTGGTTTTTTATCGCCCTGAAGAGGATGCCGGCGATGAAAAAGGCTATGAATCTTTTCCTTGGTTTATCAAACGTGCGCACAGTCCATCCAGAGGGCTTTACAGTGTACATATCAACCCATATCTCATTCCCTTCTTTATCGGGTTACAGAACCGGTTTACGCAGTTTCGGCTTAGTGAAACAAAAGAAATCACCAATCCGTATGCCATGCGTTTATACGAATCCCTGTGTCAGTATCGTAAGCCGGATGGCTCAGGCATCGTCTCTCTGAAAATCGACTGGATCATAGAGCGTTACCAGCTGCCTCAAAGTTACCAGCGTATGCCTGACTTCCGCCGCCGCTTCCTGCAGGTCTGTGTTAATGAGATCAACAGCAGAACTCCAATGCGCCTCTCATACATTGAGAAAAAGAAAGGCCGCCAGACGACTCATATCGTATTTTCCTTCCGCGATATCACTTCCATGACGACAGGATAGTCTGAGGGTTATCTGTCACAGATTTGAGGGTGGTTCGTCACATTTGTTCTGACCTACTGAGGGTAATTTGTCACAGTTTTGCTGTTTCCTTCAGCCTGCATGGATTTTCTCATACTTTTTGAACTGTAATTTTTAAGGAAGCCAAATTTGAGGGCAGTTTGTCACAGTTGATTTCCTTCTCTTTCCCTTCGTCATGTGACCTGATATCGGGGGTTAGTTCGTCATCATTGATGAGGGTTGATTATCACAGTTTATTACTCTGAATTGGCTATCCGCGTGTGTACCTCTACCTGGAGTTTTTCCCACGGTGGATATTTCTTCTTGCGCTGAGCGTAAGAGCTATCTGACAGAACAGTTCTTCTTTGCTTCCTCGCCAGTTCGCTCGCTATGCTCGGTTACACGGCTGCGGCGAGCATCACGTGCTATAAAAATAATTATAATTTAAATTTTTTAATATAAATATATAAATTAAAAATAGAAAGTAAAAAAAGAAATTAAAGAAAAAATAGTTTTTGTTTTCCGAAGATGTAAAAGACTCTAGGGGGATCGCCAACAAATACTACCTTTTACCTTGCTCTTCCTGCTCTCAGGTATTAATGCCGAATTGTTTCATCTTGTCTGTGTAGAAGACCACACACGAAAATCCTGTGATTTTACATTTTACTTATCGTTAATCGAATGTATATCTATTTAATCTGCTTTTCTTGTCTAATAAATATATATGTAAAGTACGCTTTTTGTTGAAATTTTTTAAACCTTTGTTTATTTTTTTTTCTTCATTCCGTAACTCTTCTACCTTCTTTATTTACTTTCTAAAATCCAAATACAAAACATAAAAATAAATAAACACAGAGTAAATTCCCAAATTATTCCATCATTAAAAGATACGAGGCGCGTGTAAGTTACAGGCAAGCGATCCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTTTAAAGAGGCCCTAGGGGCCGTGCGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACAGGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTTTTACACAGGAGTCTGGACTTGACATACAGTGAATGTAACTTTCGAATTGACAGTATTAGTAGTCGTATTGACAGTGAGGCACGCCCCTCAATGTGCGAGGTGGAAAATATACCAGCATGACAATGAATCTTGGAGATTCTTTTGCTGTCATCAAGATTCACCGCCAAATCTTCAGGAACCTATCACGTCCACAGGCGATGTTAATTCTTGAGTCGTCAAAACAAAGTCCTGTCCTACCTGTAGAAGTTGACAGCGAGCAATTGTATGCAAACTTCTGACTTTGTTATAATAACATTAAAGGTAATTAAGTATCTTCAATTAGGCATTTTGTCACTGTCAGTCCGTTCCGACAATATAGGTAGATTTGGAATGAATCTTTTCTATGCTGCTGCGAATCTTGTACACCTTTGAGGCCGTAGATTCTGTCCGACGAAGCGATAATTATTGCAAAATACATGGACTCATTATTTTGATTCGATTTCTTTTTGGTATCCGACTCGAAAAGATCCATCACGGCGAGCGCCACCATGAACAAGAACTCCAAAATCCAGTCCCCAAACTCTTCTGATGTTGCTGTTATTGGTGTTGGTTTTAGATTCCCAGGTAACTCTAATGACCCAGAATCTTTGTGGAACAACTTGTTGGATGGTTTCGATGCTATTACCCAAGTCCCAAAAGAAAGATGGGCTACTTCTTTTAGAGAGATGGGTTTGATCAAGAACAAGTTCGGTGGTTTCTTGAAGGATTCTGAATGGAAGAATTTCGACCCTTTGTTCTTTGGTATCGGTCCAAAAGAAGCTCCATTCATTGATCCACAACAAAGGTTGTTGTTGTCCATCGTTTGGGAATCTTTGGAAGATGCTTACATCAGACCAGATGAATTGAGAGGTTCTAACACTGGTGTTTTCATCGGTGTTTCTAACAACGATTACACCAAGTTGGGTTTCCAAGACAACTACTCTATTTCTCCATACACTATGACCGGCTCTAACTCTTCATTGAACTCCAACAGAATTTCCTACTGCTTCGATTTTAGAGGTCCATCCATTACTGTTGATACCGCTTGTTCTTCTTCCTTGGTTTCTGTTAATTTGGGTGTCCAATCCATCCAAATGGGTGAATGTAAGATTGCTATTTGCGGTGGTGTTAACGCTTTGTTTGATCCATCTACATCTGTTGCCTTTTCCAAGTTGGGTGTTTTGTCTGAAAATGGCAGATGCAACTCTTTTAGTGATCAAGCCTCTGGTTACGTTAGATCTGAAGGTGCTGGTGTTGTTGTTTTGAAGTCTTTGGAACAAGCTAAGTTGGATGGTGATAGAATCTACGGTGTTATCAAGGGTGTTTCCTCTAATGAAGATGGTGCTTCTAATGGTGACAAGAACTCTTTGACTACTCCATCTTGTGAAGCCCAATCCATTAACATTTCTAAGGCTATGGAAAAGGCCTCCTTGTCTCCATCTGATATCTATTACATTGAAGCCCATGGTACTGGTACTCCAGTTGGTGATCCAATTGAAGTTAAGGCCTTGTCCAAGATCTTCTCCAACTCTAACAACAACCAGTTGAACAACTTCTCTACCGATGGTAATGATAACGATGATGATGATGACGATAACACCTCTCCAGAACCATTATTGATTGGCTCATTCAAGTCCAACATCGGTCATTTGGAATCTGCTGCTGGTATTGCTTCTTTGATTAAGTGTTGCTTGATGTTGAAGAACAGGATGTTGGTTCCATCCATTAACTGCTCTAATTTGAACCCATCCATTCCATTCGATCAGTACAACATCTCCGTTATCAGAGAAATCAGACAATTCCCAACCGATAAGTTGGTTAACATCGGTATCAATTCTTTCGGTTTCGGTGGTTCTAACTGCCATTTGATTATTCAAGAGTACAACAACAACTTCAAGAACAACTCTACCATCTGCAATAACAACAACAACAACAATAACAACATCGACTACTTGATCCCAATCTCCTCTAAGACTAAGAAGTCCTTGGATAAGTACTTGATTTTGATCAAGACCAACTCCAACTACCACAAGGATATTTCTTTCGATGACTTCGTCAAGTTCCAAATCAAGTCTAAGCAGTACAACTTGTCCAACAGAATGACTACCATTGCTAACGATTGGAACTCCTTCATTAAGGGTTCTAACGAATTCCACAACTTGATCGAATCTAAGGATGGTGAAGGTGGTTCTTCATCTTCTAACAGAGGTATTGATTCCGCCAATCAAATCAACACTACTACTACCTCTACCATCAACGATATCGAACCTTTGTTGGTTTTCGTTTTCTGTGGTCAAGGTCCACAATGGAATGGTATGATTAAGACCTTGTACAACTCCGAGAACGTTTTCAAGAACACCGTTGATCATGTTGACAGCATCTTGTACAAGTACTTCGGTTACTCCATTTTGAACGTCTTGTCTAAGATCGATGATAACGACGATTCCATCAACCATCCAATAGTTGCTCAACCATCTTTGTTCTTGTTGCAAATTGGTTTGGTCGAGTTGTTTAAGTACTGGGGTATCTACCCATCTATCTCTGTTGGTCATTCTTTCGGTGAAGTCTCTTCTTATTACTTGTCCGGTATCATCTCTTTGGAAACCGCTTGTAAAATCGTCTACGTCAGATCCTCTAATCAGAACAAAACTATGGGTTCCGGTAAGATGTTGGTTGTTTCTATGGGTTTTAAGCAATGGAACGATCAATTCTCTGCTGAATGGTCCGATATTGAAATTGCTTGTTACAACGCTCCAGATTCCATAGTTGTTACTGGTAACGAAGAAAGATTGAAAGAATTGTCCATCAAGTTGTCCGACGAATCCAATCAAATTTTCAACACCTTCTTGAGGTCCCCATGTTCTTTTCATTCTTCCCATCAAGAAGTCATCAAGGGTTCTATGTTCGAAGAGTTGTCTAACTTGCAATCTACTGGTGAAACCGAAATCCCTTTGTTCTCTACTGTTACTGGTAGACAAGTTTTGTCTGGTCATGTTACTGCTCAACACATCTACGATAATGTTAGAGAACCAGTCTTGTTCCAAAAGACGATTGAATCCATTACCTCCTACATCAAGTCTCACTACCCATCCAATCAAAAGGTTATCTACGTTGAAATTGCTCCACACCCAACCTTGTTTTCATTGATCAAAAAGTCCATCCCATCCTCCAACAAGAATTCCTCTTCTGTTTTGTGTCCATTGAACAGAAAAGAAAACTCCAACAACTCCTACAAGAAGTTCGTTTCTCAGTTGTACTTCAACGGTGTTAACGTTGACTTCAACTTCCAGTTGAACTCCATTTGCGATAACGTTAACAACGATCACCATTTGAACAACGTCAAGCAAAACTCCTTCAAAGAGACTACCAATTCCTTGCCAAGATACCAATGGGAACAAGATGAATATTGGTCCGAACCATTGATCTCCAGAAAGAATAGATTGGAAGGTCCAACTACTTCCTTGTTGGGTCATAGAATTATCTACAGCTTCCCAGTTTTCCAATCCGTTTTGGACTTGCAATCTGACAACTACAAATACTTGTTGGACCACTTGGTTAACGGTAAGCCAGTTTTTCCAGGTGCTGGTTATTTGGATATCATCATCGAATTCTTCGACTACCAAAAGCAGCAGTTGAATTCCTCTGATTCCTCTAACTCCTACATCATCAACGTTGACAAGATCCAATTCTTGAACCCAATTCACTTGACCGAAAACAAGTTGCAAACCTTGCAATCTTCTTTCGAACCTATCGTTACTAAGAAGTCTGCCTTCTCTGTTAACTTCTTCATCAAGGATACCGTCGAGGATCAATCTAAGGTTAAGTCTATGTCTGACGAAACTTGGACTAACACTTGTAAGGCTACCATTTCCTTGGAACAACAACAGCCATCTCCATCTTCTACTTTGACTTTGTCTAAGAAGCAAGACTTGCAGATCTTGAGAAACAGATGCGATATTAGCAAGCTAGACAAGTTTGAGTTGTACGACAAGATCTCTAAGAATTTGGGCTTGCAGTACAACTCCTTGTTTCAAGTTGTTGATACCATCGAAACTGGTAAGGATTGCTCTTTTGCTACTTTGTCTTTGCCAGAAGATACTTTGTTCACCACCATTTTGAACCCATGCTTGTTGGATAACTGTTTCCATGGTTTGTTGACCTTGATCAACGAAAAGGGTTCTTTCGTTGTCGAGTCCATTTCTTCTGTTTCTATCTACTTGGAGAACATCGGTTCCTTCAATCAAACTTCTGTTGGTAACGTCCAGTTCTACTTGTACACCACTATTTCTAAAGCCACCTCCTTTAGTTCTGAAGGTACTTGTAAGTTGTTCACCAAGGATGGTTCCTTGATTTTGTCTATCGGTAAGTTCATCATCAAGTCCACCAATCCAAAGTCTACTAAGACCAACGAAACTATCGAATCTCCATTGGACGAAACCTTCTCTATTGAATGGCAATCTAAGGATTCTCCAATTCCAACCCCACAACAAATCCAACAACAATCTCCATTGAACTCTAACCCATCCTTCATTAGATCTACCATCTTGAAGGACATCCAGTTCGAACAATACTGCTCCTCCATTATCCACAAAGAATTGATCAACCACGAAAAGTACAAGAACCAGCAATCCTTCGATATCAACTCCTTGGAAAACCACTTGAACGATGACCAATTGATGGAATCCTTGTCCATCTCCAAAGAATACTTGAGATTCTTCACCAGGATCATCTCCATCATTAAGCAATACCCAAAGATCTTGAACGAAAAAGAGCTAAAAGAATTGAAAGAAATCATCGAATTGAAGTACCCATCCGAAGTTCAGTTGTTGGAATTCGAAGTTATCGAGAAGGTGTCCATGATTATCCCAAAGTTGTTGTTCGAAAACGACAAGCAATCTTCCATGACCTTGTTCCAAGATAACTTGTTGACCAGGTTCTACTCCAATTCTAACTCTACCAGATTCTACTTGGAAAGGGTTTCCGAAATGGTCTTGGAATCTATTAGACCAATCGTCAGAGAAAAGAGGGTGTTCAGAATTTTGGAAATTGGTGCTGATACAGCCTCTTTGTCTAATGTTGTTTTGACTAAGTTGAACACCTACTTGTCCACCTTGAATTCTAATGGTGGTTCTGGTTACAACATCATCATTGAGTACACCTTCACCGATATTTCCGCCAACTTCATTATTGGTGAAATCCAAGAAACCATGTGCAACTTGTACCCAAACGTTACTTTCAAGTTCTCCGTCTTGGACTTGGAGAAAGAGATTATTAACTCCTCCGATTTCTTGATGGGTGATTACGATATAGTTTTGATGGCCTACGTTATCCATGCCGTTTCTAACATTAAGTTCTCCATCGAACAGTTGTACAAGTTGTTGTCTCCAAGAGGTTGGTTGTTGTGTATTGAACCTAAGTCCAACGTTGTGTTCTCCGATTTGGTTTTCGGTTGTTTTAATCAGTGGTGGAACTACTACGATGATATTAGAACTACCCACTGCTCCTTGTCTGAATCTCAATGGAATCAGTTGTTGTTGAACCAGTCCTTGAACAACGAATCCTCTTCTTCTTCTAACTGTTACGGTGGTTTTTCCAACGTTTCTTTTATTGGTGGTGAAAAGGATGTCGACTCCCATTCTTTCATATTGCACTGCCAAAAAGAATCCATCTCCCAAATGAAGTTAGCCACCACTATTAACAACGGTTTGTCATCTGGTTCCATCGTTATCGTTTTGAACTCTCAACAATTGACCAACATGAAGTCCTACCCAAAGGTTATTGAGTATATTCAAGAGGCTACCTCTTTGTGCAAGACCATTGAAATTATCGATTCCAAGGACGTCTTGAACTCTACCAATTCAGTTTTGGAAAAGATCCAAAAGTCCTTGTTGGTGTTCTGTTTGTTGGGTTATGACTTGTTGGAGAACAACTACCAAGAACAGTCTTTCGAATACGTTAAGTTGTTGAACTTGATCTCTACTACCGCCTCTTCATCTAATGATAAGAAACCACCAAAGGTCTTGTTGATCACCAAGCAATCTGAAAGAATCTCCAGGTCTTTCTACTCCAGATCCTTGATTGGTATTTCCAGAACCTCTATGAACGAGTACCCAAATTTGTCCATTACCTCTATCGATTTGGATACCAACGACTACTCATTGCAGTCTTTGTTGAAGCCAATCTTCAGCAACTCTAAGTTTTCCGACAACGAGTTCATCTTCAAAAAGGGCTTGATGTTCGTGTCCAGGATCTTTAAGAACAAGCAGTTGCTAGAATCCTCCAACGCTTTTGAAACTGACTCTTCTAACTTGTACTGTAAGGCCTCTTCTGACTTGTCTTACAAGTACGCTATTAAGCAGTCTATGTTGACCGAAAATCAGATCGAAATCAAGGTTGAATGCGTCGGTATTAACTTCAAGGACAACCTATTCTACAAGGGCTTGTTGCCACAAGAAATTTTCAGAATGGGTGACATCTACAATCCACCATATGGTTTGGAATGCTCTGGTGTTATTACCAGAATTGGTTCTAACGTCACCGAATACTCAGTTGGTCAAAATGTTTTTGGTTTCGCCAGACATTCTTTGGGTTCTCATGTTGTTACCAACAAGGATTTGGTTATCTTGAAGCCAGATACCATCTCATTTTCTGAAGCTGCTTCTATCCCAGTTGTTTACTGTACTGCTTGGTACTCCTTGTTCAACATTGGTCAGTTGTCTAACGAAGAATCCATCCTAATTCATTCTGCTACTGGTGGTGTAGGTTTGGCTTCTTTGAATTTGTTGAAAATGAAGAATCAGCAACAGCAACCATTGACCAATGTTTATGCTACTGTTGGCTCTAACGAGAAGAAGAAGTTCTTGATCGATAACTTCAACAACTTGTTCAAAGAGGACGGCGAAAACATTTTCTCTACCAGAGACAAAGAATACTCCAACCAGTTGGAATCCAAGATCGATGTTATTTTGAACACCTTGTCCGGTGAATTCGTCGAATCTAATTTCAAGTCCTTGAGATCCTTCGGTAGATTGATTGATTTGTCTGCTACTCACGTTTACGCCAATCAACAAATTGGTCTAGGTAACTTCAAGTTCGACCACTTGTATTCTGCTGTTGACTTGGAAAGATTGATCGACGAAAAACCTAAGTTGTTGCAGTCCATCTTGCAAAGAATTACCAACTCTATCGTCAACGGTTCCTTGGAAAAAATTCCAATTACCATCTTCCCATCCACCGAAACTAAGGATGCTATCGAATTATTGTCCAAGAGATCCCATATCGGTAAAGTTGTTGTAGATTGCACCGATATCTCTAAGTGTAATCCTGTTGGTGATGTGATCACCAACTTCTCTATGAGATTGCCAAAGCCAAACTACCAGTTGAATTTGAACTCCACCTTGTTGATTACTGGTCAGTCTGGTTTGTCTATCCCTTTGTTGAATTGGTTGTTGTCTAAGTCTGGTGGTAACGTTAAGAACGTTGTCATCATTTCTAAGTCCACCATGAAGTGGAAGTTGCAGACTATGATTTCCCATTTCGTTTCCGGTTTCGGTATCCATTTTAACTACGTTCAAGTCGACATCTCCAACTACGATGCTTTGTCTGAAGCTATTAAGCAATTGCCATCTGATTTGCCACCAATCACCTCTGTTTTTCATTTGGCTGCTATCTACAACGATGTTCCAATGGATCAAGTTACCATGTCTACCGTTGAATCTGTTCATAACCCTAAAGTTTTGGGTGCCGTTAACTTGCATAGAATCTCTGTTTCTTTTGGTTGGAAGTTGAACCACTTCGTCTTGTTCTCTTCTATTACTGCTATTACCGGTTACCCAGACCAATCTATCTACAATTCTGCCAACTCTATTTTGGACGCTTTGTCCAACTTTAGAAGGTTTATGGGTTTGCCATCCTTCTCCATTAACTTGGGTCCAATGAAGGATGAAGGTAAGGTTTCTACCAACAAGAGCATCAAGAAGCTATTCAAGTCTAGAGGTTTGCCAAGCCTATCCTTGAACAAGTTATTTGGTTTGTTGGAGGTCGTCATCAACAACCCATCTAATCATGTTATCCCATCCCAATTGATTTGCTCCCCAATCGATTTCAAGACCTACATCGAATCTTTCTCAACTATGAGGCCAAAGTTGTTACACTTGCAACCTACCATTTCCAAGCAGCAATCTTCTATCATTAACGATTCTACCAAGGCTTCCTCCAACATTTCATTGCAAGATAAGATCACCTCCAAGGTGTCTGATTTGTTGTCCATTCCAATCTCCAAGATCAACTTCGATCATCCATTGAAACACTACGGCTTGGATTCTTTGTTGACCGTTCAATTCAAATCCTGGATCGACAAAGAATTCGAAAAGAACTTGTTCACCCATATCCAATTGGCCACCATCTCTATTAACTCATTCTTGGAAAAGGTGAACGGCTTGTCTACAAACAATAACAACAACAACAATTCCAACGTCAAGTCCTCTCCATCCATTGTCAAAGAAGAAATCGTTACCTTGGACAAGGATCAACAACCATTGCTATTGAAAGAACACCAGCACATTATCATCTCCCCAGATATTAGAATCAACAAGCCAAAGAGGGAATCCTTGATTAGAACCCCAATCTTGAACAAATTCAACCAGATCACCGAATCCATTATCACTCCATCTACACCATCTTTGTCCCAATCCGATGTTTTGAAAACTCCACCAATCAAGTCTTTGAACAACACTAAGAACTCCAGCTTGATTAACACCCCACCAATTCAATCTGTCCAACAACATCAAAAGCAACAACAAAAGGTCCAAGTCATCCAACAACAGCAACAACCATTATCCAGATTGTCCTACAAGAGCAACAACAACTCTTTCGTTTTGGGTATCGGTATTTCTGTTCCAGGTGAACCTATTTCCCAACAATCCTTGAAAGACTCCATCTCCAATGACTTTTCTGATAAGGCTGAAACTAACGAGAAGGTCAAGAGAATCTTTGAGCAATCTCAAATCAAGACCAGACACTTGGTTAGAGATTACACTAAGCCAGAGAACTCCATCAAGTTCAGACATTTGGAAACCATTACCGATGTGAACAACCAGTTCAAGAAAGTTGTTCCAGATTTGGCTCAACAAGCCTGTTTGAGAGCTTTGAAAGATTGGGGTGGTGATAAGGGTGATATTACCCATATAGTTTCTGTTACCTCCACCGGTATTATCATCCCAGATGTTAATTTCAAGTTGATCGACTTGTTGGGCTTGAACAAGGATGTTGAAAGAGTGTCTTTGAACCTAATGGGTTGTTTGGCTGGTTTGAGTTCTTTGAGAACTGCTGCTTCTTTGGCTAAGGCTTCTCCAAGAAATAGAATTTTGGTTGTCTGTACCGAAGTCTGCTCCTTGCATTTTTCTAATACTGATGGTGGTGATCAAATGGTCGCCTCTTCTATTTTTGCTGATGGTTCTGCTGCTTACATTATTGGTTGTAACCCAAGAATTGAAGAAACCCCATTATACGAAGTCATGTGCTCCATTAACAGATCTTTCCCAAATACCGAAAACGCCATGGTTTGGGATTTGGAAAAAGAAGGTTGGAACTTGGGTTTGGATGCTTCTATTCCAATTGTCATTGGTTCTGGTATTGAAGCCTTCGTTGATACTTTGTTGGATAAGGCTAAGTTGCAAACTTCCACTGCTATTTCTGCTAAGGATTGCGAATTCTTGATTCATACTGGTGGCAAGTCCATCTTGATGAACATCGAAAATTCCTTGGGTATCGACCCAAAGCAAACTAAGAATACTTGGGATGTTTACCATGCCTACGGCAATATGTCATCTGCCTCTGTTATTTTCGTTATGGATCATGCCAGAAAGTCCAAGTCTTTGCCAACTTACTCAATTTCTTTGGCTTTTGGTCCAGGTTTGGCTTTTGAAGGTTGTTTCTTGAAGAACGTCGTCGAACAGAAGCTCATTTCCGAAGAGGACTTGTAACCGCAACAACTACCTCGACTTTGGCTGGGACACTTTCAGTGAGGACAAGAAGCTTCAGAAGCGTGCTATCGAACTCAACCAGGGACGTGCGGCACAAATGGGCATCCTTGCTCTCATGGTGCACGAACAGTTGGGAGTCTCTATCCTTCCTTAAAAATTTAATTTTCATTAGTTGCAGTCACTCCGCTTTGGTTTCGCTAGTGATAATAAGTGACTGAGGTATGTGCTCTTCTTATCTCCTTTTGTAGTGTTGCTCTTATTTTAAACAACTTTGCGGTTTTTTGATGACTTTGCGATTTTGTTGTTGCTTTGCAGTAAATTGCAAGATTTAATAAAAAAACGCAAAGCAATGATTAAAGGATGTTCAGAATGAAACTCATGGAAACACTTAACCAGTGCATAAACGCTGGTCATGAAATGACGAAGGCTATCGCCATTGCACAGTTTAATGATGACAGCCCGGAAGCGAGGAAAATAACCCGGCGCTGGAGAATAGGTGAAGCAGCGGATTTAGTTGGGGTTTCTTCTCAGGCTATCAGAGATGCCGAGAAAGCAGGGCGACTACCGCACCCGGATATGGAAATTCGAGGACGGGTTGAGCAACGTGTTGGTTATACAATTGAACAAATTAATCATATGCGTGATGTGTTTGGTACGCGATTGCGACGTGCTGAAGACGTATTTCCACCGGTGATCGGGGTTGCTGCCCATAAAGGTGGCGTTTACAAAACCTCAGTTTCTGTTCATCTTGCTCAGGATCTGGCTCTGAAGGGGCTACGTGTTTTGCTCGTGGAAGGTAACGACCCCCAGGGAACAGCCTCAATGTATCACGGATGGGTACCAGATCTTCATATTCATGCAGAAGACACTCTCCTGCCTTTCTATCTTGGGGAAAAGGACGATGTCACTTATGCAATAAAGCCCACTTGCTGGCCGGGGCTTGACATTATTCCTTCCTGTCTGGCTCTGCACCGTATTGAAACTGAGTTAATGGGCAAATTTGATGAAGGTAAACTGCCCACCGATCCACACCTGATGCTCCGACTGGCCATTGAAACTGTTGCTCATGACTATGATGTCATAGTTATTGACAGCGCGCCTAACCTGGGTATCGGCACGATTAATGTCGTATGTGCTGCTGATGTGCTGATTGTTCCCACGCCTGCTGAGTTGTTTGACTACACCTCCGCACTGCAGTTTTTCGATATGCTTCGTGATCTGCTCAAGAACGTTGATCTTAAAGGGTTCGAGCCTGATGTACGTATTTTGCTTACCAAATACAGCAATAGCAATGGCTCTCAGTCCCCGTGGATGGAGGAGCAAATTCGGGATGCCTGGGGAAGCATGGTTCTAAAAAATGTTGTACGTGAAACGGATGAAGTTGGTAAAGGTCAGATCCGGATGAGAACTGTTTTTGAACAGGCCATTGATCAACGCTCTTCAACTGGTGCCTGGAGAAATGCTCTTTCTATTTGGGAACCTGTCTGCAATGAAATTTTCGATCGTCTGATTAAACCACGCTGGGAGATTAGATAATGAAGCGTGCGCCTGTTATTCCAAAACATACGCTCAATACTCAACCGGTTGAAGATACTTCGTTATCGACACCAGCTGCCCCGATGGTGGATTCGTTAATTGCGCGCGTAGGAGTAATGGCTCGCGGTAATGCCATTACTTTGCCTGTATGTGGTCGGGATGTGAAGTTTACTCTTGAAGTGCTCCGGGGTGATAGTGTTGAGAAGACCTCTCGGGTATGGTCAGGTAATGAACGTGACCAGGAGCTGCTTACTGAGGACGCACTGGATGATCTCATCCCTTCTTTTCTACTGACTGGTCAACAGACACCGGCGTTCGGTCGAAGAGTATCTGGTGTCATAGAAATTGCCGATGGGAGTCGCCGTCGTAAAGCTGCTGCACTTACCGAAAGTGATTATCGTGTTCTGGTTGGCGAGCTGGATGATGAGCAGATGGCTGCATTATCCAGATTGGGTAACGATTATCGCCCAACAAGTGCTTATGAACGTGGTCAGCGTTATGCAAGCCGATTGCAGAATGAATTTGCTGGAAATATTTCTGCGCTGGCTGATGCGGAAAATATTTCACGTAAGATTATTACCCGCTGTATCAACACCGCCAAATTGCCTAAATCAGTTGTTGCTCTTTTTTCTCACCCCGGTGAACTATCTGCCCGGTCAGGTGATGCACTTCAAAAAGCCTTTACAGATAAAGAGGAATTACTTAAGCAGCAGGCATCTAACCTTCATGAGCAGAAAAAAGCTGGGGTGATATTTGAAGCTGAAGAAGTTATCACTCTTTTAACTTCTGTGCTTAAAACGTCATCTGCATCAAGAACTAGTTTAAGCTCACGACATCAGTTTGCTCCTGGAGCGACAGTATTGTATAAGGGCGATAAAATGGTGCTTAACCTGGACAGGTCTCGTGTTCCAACTGAGTGTATAGAGAAAATTGAGGCCATTCTTAAGGAACTTGAAAAGCCAGCACCCTGATGCGACCTCGTTTTAGTCTACGTTTATCTGTCTTTACTTAATGTCCTTTGTTACAGGCCAGAAAGCATAACTGGCCTGAATATTCTCTCTGGGCCCACTGTTCCACTTGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCATGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGAACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGATCCCACTCGTGTTGTCGGTCTGATTATCGGTCTGGGACCACGGTCCCACTTGTATTGTCGATCAGACTATCAGCGTGAGACTACGATTCCATCAATGCCTGTCAAGGGCAAGTATTGACATGTCGTCGTAACCTGTAGAACGGAGTAACCTCGGTGTGCGGTTGTATGCCTGCTGTGGATTGCTGCTGTGTCCTGCTTATCCACAACATTTTGCGCACGGTTATGTGGACAAAATACCTGGTTACCCAGGCCGTGCCGGCACGTTAACCGGGCTGCATCCGATGCAAGTGTGTCGCTGTCGACGAGCTCGCGAGCTCGGACATGAGGTTGCCCCGTATTCAGTGTCGCTGATTTGTATTGTCTGAAGTTGTTTTTACGTTAAGTTGATGCAGATCAATTAATACGATACCTGCGTCATAATTGATTATTTGACGTGGTTTGATGGCCTCCACGCACGTTGTGATATGTAGATGATAATCATTATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTACGGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGCTTTTTGGCCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATACATTATACGAAGTTATA |
| pSteelyA-2 sequencing | TTCGATGCGGCCGCAAGGGGTTCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCACACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGAGTATTCTATAGTCTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGAACCCCTTGCGGCCGCCCGGGCCGTCGACCAATTCTCATGTTTGACAGCTTATCATCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGCGATAAGCTCATGGAGCGGCGTAACCGTCGCACAGGAAGGACAGAGAAAGCGCGGATCTGGGAAGTGACGGACAGAACGGTCAGGACCTGGATTGGGGAGGCGGTTGCCGCCGCTGCTGCTGACGGTGTGACGTTCTCTGTTCCGGTCACACCACATACGTTCCGCCATTCCTATGCGATGCACATGCTGTATGCCGGTATACCGCTGAAAGTTCTGCAAAGCCTGATGGGACATAAGTCCATCAGTTCAACGGAAGTCTACACGAAGGTTTTTGCGCTGGATGTGGCTGCCCGGCACCGGGTGCAGTTTGCGATGCCGGAGTCTGATGCGGTTGCGATGCTGAAACAATTATCCTGAGAATAAATGCCTTGGCCTTTATATGGAAATGTGGAACTGAGTGGATATGCTGTTTTTGTCTGTTAAACAGAGAAGCTGGCTGTTATCCACTGAGAAGCGAACGAAACAGTCGGGAAAATCTCCCATTATCGTAGAGATCCGCATTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATTTGAATATGCCTTCAGGAACAATAGAAATCTTCGTGCGGTGTTACGTTGAAGTGGAGCGGATTATGTCAGCAATGGACAGAACAACCTAATGAACACAGAACCATGATGTGGTCTGTCCTTTTACAGCCAGTAGTGCTCGCCGCAGTCGAGCGACAGGGCGAAGCCCATCGATACTAGCTTGATTGGGATATCTCGCTCGTGCTTGTCGCGTGCTATGTCTTTTTAGGGTACTTTGAACCTACGTTCGTACTTGTGTAATATGATCATCGTCGTCATCGTATTATCGTTTTTCATCCGTCCAGCGCAAAATGCATTAGCAGCTAGTCCTAGCGTGCGGAGCTACCTGTACAGGTGCATGACGGATGCGTGTCCTTAAGTGAGTTTCTAATTAACAGTAACTTCTTTACTTATGTTTCAGTTTGTAAGAAGCGGGATGCGCTCGTCGCTTGACATCTGATTGGACTGCGTCGGCACGTGAAAACTACATTGTGAAATCTGCTAAAACTCCGGGTATCTCTGACACAAAACGATTCGGCTTCGCAATTTCAACATTACGGTCAAGGCTAACGTATCTTTCTCGGTCAACTTCAGATTATGCCGATTAAATTGTCGTAGCTTTCAAGGCGTTTTGAGTACTGCGGCAGTTGTTGAACCTGCAAGGAGAAGATCTCGACAACAGAATAAAGCGAAAAATGGGTCTCATGCACTAACACTCAGGCCTCCCTCATAATCTCTGTTTGAGTTTACCAACAACACATATATACATTTCGACAAAATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCAGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACCGACGCCGACCAACACCGCCGGTCCGACGCGGCCCGACGGGTCCGAGGCCTCGGAGATCTGGGCCCATGCGGCCGCAACAACTACCTCGACTTTGGCTGGGACACTTTCAGTGAGGACAAGAAGCTTCAGAAGCGTGCTATCGAACTCAACCAGGGACGTGCGGCACAAATGGGCATCCTTGCTCTCATGGTGCACGAACAGTTGGGAGTCTCTATCCTTCCTTAAAAATTTAATTTTCATTAGTTGCAGTCACTCCGCTTTGGTTTCGTAACTATAACGGTCCTAAGGTAGCGAACGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCGGGGGGGGGGGGGTTTCAATTCATCATTTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCCTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATATAAGGAACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAAGCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAATTTGTTTACTAAAAACACATGTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAAAGGCATTATCCGCCAAGTACAATTTTTTACTCTTCGAAGACAGAAAATTTGCTGACATTGGTAATACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAGAATGGGCAGACATTACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGTTTGAAGCAGGCGGCGGAAGAAGTAACAAAGGAACCTAGAGGCCTTTTGATGTTAGCAGAATTGTCATGCAAGGGCTCCCTAGCTACTGGAGAATATACTAAGGGTACTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAGAGACATGGGTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGTGGGTTTAGATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGATGTGGTCTCTACAGGATCTGACATTATTATTGTTGGAAGAGGACTATTTGCAAAGGGAAGGGATGCTAAGGTAGAGGGTGAACGTTACAGAAAAGCAGGCTGGGAAGCATATTTGAGAAGATGCGGCCAGCAAAACTAAAAAACTGTATTATAAGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCGGCCGGGAATCTCGGTCGTAATGATTTTTATAATGACGAAAAAAAAAAAATTGGAAAGAAAACCCCCCCCCCCCCCGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGAGTTACGCTAGGGATAACAGGGTAATATAGATCTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGGGTTCGTGTAGACTTTCCTTGGTGTATCCAACGGCGTCAGCCGGGCAGGATAGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCCGGCTACCGCCGGCGTAACAGATGAGGGCAAGCGGATGGCTGATGAAACCAAGCCAACCAGGAAGGGCAGCCCACCTATCAAGGTGTACTGCCTTCCAGACGAACGAAGAGCGATTGAGGAAAAGGCGGCGGCGGCCGGCATGAGCCTGTCGGCCTACCTGCTGGCCGTCGGCCAGGGCTACAAAATCACGGGCGTCGTGGACTATGAGCACGTCCGCGAGCTGGCCCGCATCAATGGCGACCTGGGCCGCCTGGGCGGCCTGCTGAAACTCTGGCTCACCGACGACCCGCGCACGGCGCGGTTCGGTGATGCCACGATCCTCGCCCTGCTGGCGAAGATCGAAGAGAAGCAGGACGAGCTTGGCAAGGTCATGATGGGCGTGGTCCGCCCGAGGGCAGAGCCATGACTTTTTTAGCCGCTAAAACGGCCGGGGGGTGCGCGTGATTGCCAAGCACGTCCCCATGCGCTCCATCAAGAAGAGGCACTTCGAGCTGTAAGTACATCACCGACGAGCAAGGCAAGACGATCCGCGCCTGTTTTATTGAGAACGTTGTTCGTGTTGGCCTCAATGGTAGCGATGCGTCATTCAGCGAAGTTCTGGTGCTGATGATGTGGTTCGCTTTGCCACTGGTCAATGTGGTAAGCCCGTGTAATGTCAGTAACCTTTTTACTGATCTCAGCTTGAGCACGGTCGCTGATGAGCTTATCCATGGCCCCACGGTAACGGATATGATCCTCTAGGGCGTTGACAAATTCTTTGTCGGTTTTCATGGGGTAGACGTCAGTGACCAGGGAACGTCTCCCTACAAAAAGTTGCGCATACTTTGCTCCGTTGTCGACGGCAGGGGTGTCGGACCAGATTGTATCTGTGGCAACGGCCTCATTGCGTCAATGGACCTTTAAAGCCGGGAAACGAGACTTGAAATGTTTACGTAGAGGTGCATTATAGACCTCACGCGCATATTGAGTGGTTGCAAAAATGGTTTTGCGAACGGTATCACTGGAGACCCATGCCAGGCAAGGACGGAGGGCATCATAATCATGTTCATTGCGTTGGACAGCATGCTTGTTACACGTAAGTATACGGTCGAGAGCATCGTGACGTTGAGAATTACAGATGGCAACGTGTCGGTGCATGTATTGGCCGAGATTGTCAAATCGTGGCTCAATATATGGCATGCCAGGTAAGTCATGGATGTCGGTATGCCATTCAGCCTTCATGTCGATTTCGTTGTCGAGAACTGATGGGTCCCAGTCAACGTCAGAAGTAAGAACAACATGGGGAAGGGAATGAAGTTCATGACTGGTGGGTGCGCGCATATCCATGTACGGAAGACCCTGTCGTATGTTTAGTGGGATGATGTAATCATCCAGGGTAACAATACGTTGGTTGCCGCCCACGGTACGAGACCGATCTTGAACACAATTGTGGCAATGTTCAAGTTGGGCGCTGGAATGGATGGTTTTACCTTTACCAAGATGCGCGTATTGATGCATGATAACAACAATGGGCCCTCGTTGCGATTCGACAAGACCGGCAGCCGTGACAATGTCTAGATTGGACAAGGTATGTTCATTTATACCGGTTATGTTGGCTGAACGTCCAGTTTTGTGCAGTACGGTAACATCACTCCCCGCAAGTCCACCGTTGGCACCGCGATCGACAAGTGCAGATGTGGTATTTTGTACTGTGTGTCGGGAGACTTGGTACTGTAACACATTTGACTGAAGTGACTTGGTGGTACCATGAGGACGGAGGGAACCCGTTGATACGGTGTTGCGTGACGCTGCAAGTACCTTACGTATGTCGCCTGGATCCATACGTCCAACCCGATCAGAAAGATGTGCTAGGAGTTCCGTTTCTGGTGCACAATCATGAAAGGTATCCACGGGAGTTCCATTGTTGTCGTCCGGGGCTGCGTCGCGACTGCCAGTTGCATGTACATGCGCCATCAAGGGACGTGCCAATCCATTACTAACCGGGAGAACCTTAGCTGGGGCAGCCAATCCCTGAAGTATAGCCTTAGCATCATCTGAGAGCTGGTTCCACATATCTCTAGGGATATAAGGTCGTTCACGGTTAGTGGGCGTACTATGATGTCGGTTATTAGTACAATTCTCCCTGCGGGCATGCGCATTGGCTTCATAGAGTACGGAAGGTGACAAGTCAATATTGTAGTTAACATCCGGATCAGTGTCAAAGTCTGTAGGATGGTAAGAAAGATCAGTAGAATGAATACTACGCTTTTCCTTGGGACTACGGGAATTAGAGAAGTTGTTTCCTTTATTGTAGAGTGACGCTGAAGCAAGTAGAAGACTAAGGTAACTCTCGTAGCTAATAGGATTACCTCCTTTGGCTAGGTCAAGAGTGGCTGTGATCTTCACTTGACAAAGTTCCGGTACATTGTGGACAGCATTCTCCAAAAGACTAAGACACAGTTGCTTTGGGAGTTGCTCAGCCATTGGTACAGTATTGTGGTAGATACAAAGGTGGTTCTTCCAATGAAGGATAAATCCTTCTGCTGTGCCTGTCCATTACTAACCGGGAGAACCTTAGCTGGGGCAGCCAATCCCTGAAGTATAGCCTTAGCATCATCTGAGAGCTGGTTCCACATATCTCTAGGGATATAAGGTCGTTCACGGTTAGTGGGCGTACTATGATGTCGGTTATTAGTACAATTCTCCCTGCGGGCATGCGCATTGGCTTCATAGAGTACGGAAGGTGACAAGTCAATATTGTAGTTAACATCCGGATCAGTGTCAAAGTCTGTAGGATGGTAAGAAAGATCAGTAGAATGAATACTACGCTTTTCCTTGGGACTACGGGAATTAGAGAAGTTGTTTCCTTTATTGTAGAGTGACGCTGAAGCAAGTAGAAGACTAAGGTAACTCTCGTAGCTAATAGGATTACCTCCTTTGGCTAGGTCAAGAGTGGCTGTGATCTTCACTTGACAAAGTTCCGGTACATTGTGGACAGCATTCTCCAAAAGACTAAGACACAGTTGCTTTGGGAGTTGCTCAGCCATTGGTACAGTATTGTGGTAGATACAAAGGTGGTTCTTCCAATGAAGGATAAATCCTTCTGCTGTGCCTGTCCATGAGGATCCATATTTCGCCGTAGTTAGGTAACCAAGCGTGGTGGCTGAACTGATCTTCGCACTTGCTGATTCCGTATAGTGTTTGACAACTTTACAAAACACTTCTTGCGCAGTTCGCTCTAGCCTGGACAGAGAAGGGACAATCTTCGTCGTTAAGACTCGTGCGAATAGCAAAAGATCACAAAATAGCACATCGGCACCGACCAACGATTATTTCCAAGGAAAAAAAGAATGCTTCACTACAAGAAATTGTGTCATCCCTATACAGAGTCTTGTTACTGTGACAGAAAATTGATGGAAGATGTGGCGGATTGCCTTTACACTAGCCAACTTGTTCGACTAATTGCAGCTTCTTCTGAGAGGCTTCACCGAGTAACGCGAAGAACACCGGTGTCTCGTACATGCTCGTCGGTGAACGCTCGTCCAATGACACCCCCCACTTTGTATCAATATCCCAACTTGGTAGTGAACTGGAATGATACATGCAATTTCGCGCCGCATCAACAGCCACGGGCACCATCGACGAATAGACTCGGTCGAGCTGGTTGCCCTCGCCGCTGGGCTGGCGGCCGTCTATGGCCCTGCAAACGCGCCAGAAACGCCGTCGAAGCCGTGTGCGAGACACCGCGGCCGGCCGCCGGCGTTGTGGATACCTCGCGGAAAACTTGGCCCTCACTGACAGATGAGGGGCGGACGTTGACACTTGAGGGGCCGACTCACCCGGCGCGGCGTTGACAGATGAGGGGCAGGCTCGATTTCGGCCGGCGACGTGGAGCTGGCCAGCCTCGCAAATCGGCGAAAACGCCTGATTTTACGCGAGTTTCCCACAGATGATGTGGACAAGCCTGGGGATAAGTGCCCTGCGGTATTGACACTTGAGGGGCGCGACTACTGACAGATGAGGGGCGCGATCCTTGACACTTGAGGGGCAGAGTGCTGACAGATGAGGGGCGCACCTATTGACATTTGAGGGGCTGTCCACAGGCAGAAAATCCAGCATTTGCAAGGGTTTCCGCCCGTTTTTCGGCCACCGCTAACCTGTCTTTTAACCTGCTTTTAAACCAATATTTATAAACCTTGTTTTTAACCAGGGCTGCGCCCTGTGCGCGTGACCGCGCACGCCGAAGGGGGGTGCCCCCCCTTCTCGAACCCTCCCGGTCGAGTGAGCGAGGAAGCACCAGGGAACAGCACTTATATATTCTGCTTACACACGATGCCTGAAAAAACTTCCCTTGGGGTTATCCACTTATCCACGGGGATATTTTTATAATTATTTTTTTTATAGTTTTTAGATCTTCTTTTTTAGAGCGCCTTGTAGGCCTTTATCCATGCTGGTTCTAGAGAAGGTGTTGTGACAAATTGCCCTTTCAGTGTGACAAATCACCCTCAAATGACAGTCCTGTCTGTGACAAATTGCCCTTAACCCTGTGACAAATTGCCCTCAGAAGAAGCTGTTTTTTCACAAAGTTATCCCTGCTTATTGACTCTTTTTTATTTAGTGTGACAATCTAAAAACTTGTCACACTTCACATGGATCTGTCATGGCGGAAACAGCGGTTATCAATCACAAGAAACGTAAAAATAGCCCGCGAATCGTCCAGTCAAACGACCTCACTGAGGCGGCATATAGTCTCTCCCGGGATCAAAAACGTATGCTGTATCTGTTCGTTGACCAGATCAGAAAATCTGATGGCACCCTACAGGAACATGACGGTATCTGCGAGATCCATGTTGCTAAATATGCTGAAATATTCGGATTGACCTCTGCGGAAGCCAGTAAGGATATACGGCAGGCATTGAAGAGTTTCGCGGGGAAGGAAGTGGTTTTTTATCGCCCTGAAGAGGATGCCGGCGATGAAAAAGGCTATGAATCTTTTCCTTGGTTTATCAAACGTGCGCACAGTCCATCCAGAGGGCTTTACAGTGTACATATCAACCCATATCTCATTCCCTTCTTTATCGGGTTACAGAACCGGTTTACGCAGTTTCGGCTTAGTGAAACAAAAGAAATCACCAATCCGTATGCCATGCGTTTATACGAATCCCTGTGTCAGTATCGTAAGCCGGATGGCTCAGGCATCGTCTCTCTGAAAATCGACTGGATCATAGAGCGTTACCAGCTGCCTCAAAGTTACCAGCGTATGCCTGACTTCCGCCGCCGCTTCCTGCAGGTCTGTGTTAATGAGATCAACAGCAGAACTCCAATGCGCCTCTCATACATTGAGAAAAAGAAAGGCCGCCAGACGACTCATATCGTATTTTCCTTCCGCGATATCACTTCCATGACGACAGGATAGTCTGAGGGTTATCTGTCACAGATTTGAGGGTGGTTCGTCACATTTGTTCTGACCTACTGAGGGTAATTTGTCACAGTTTTGCTGTTTCCTTCAGCCTGCATGGATTTTCTCATACTTTTTGAACTGTAATTTTTAAGGAAGCCAAATTTGAGGGCAGTTTGTCACAGTTGATTTCCTTCTCTTTCCCTTCGTCATGTGACCTGATATCGGGGGTTAGTTCGTCATCATTGATGAGGGTTGATTATCACAGTTTATTACTCTGAATTGGCTATCCGCGTGTGTACCTCTACCTGGAGTTTTTCCCACGGTGGATATTTCTTCTTGCGCTGAGCGTAAGAGCTATCTGACAGAACAGTTCTTCTTTGCTTCCTCGCCAGTTCGCTCGCTATGCTCGGTTACACGGCTGCGGCGAGCATCACGTGCTATAAAAATAATTATAATTTAAATTTTTTAATATAAATATATAAATTAAAAATAGAAAGTAAAAAAAGAAATTAAAGAAAAAATAGTTTTTGTTTTCCGAAGATGTAAAAGACTCTAGGGGGATCGCCAACAAATACTACCTTTTACCTTGCTCTTCCTGCTCTCAGGTATTAATGCCGAATTGTTTCATCTTGTCTGTGTAGAAGACCACACACGAAAATCCTGTGATTTTACATTTTACTTATCGTTAATCGAATGTATATCTATTTAATCTGCTTTTCTTGTCTAATAAATATATATGTAAAGTACGCTTTTTGTTGAAATTTTTTAAACCTTTGTTTATTTTTTTTTCTTCATTCCGTAACTCTTCTACCTTCTTTATTTACTTTCTAAAATCCAAATACAAAACATAAAAATAAATAAACACAGAGTAAATTCCCAAATTATTCCATCATTAAAAGATACGAGGCGCGTGTAAGTTACAGGCAAGCGATCCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTTTAAAGAGGCCCTAGGGGCCGTGCGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACAGGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTTTTACACAGGAGTCTGGACTTGACATACAGTGAATGTAACTTTCGAATTGACAGTATTAGTAGTCGTATTGACAGTGAGGCACGCCCCTCAATGTGCGAGGTGGAAAATATACCAGCATGACAATGAATCTTGGAGATTCTTTTGCTGTCATCAAGATTCACCGCCAAATCTTCAGGAACCTATCACGTCCACAGGCGATGTTAATTCTTGAGTCGTCAAAACAAAGTCCTGTCCTACCTGTAGAAGTTGACAGCGAGCAATTGTATGCAAACTTCTGACTTTGTTATAATAACATTAAAGGTAATTAAGTATCTTCAATTAGGCATTTTGTCACTGTCAGTCCGTTCCGACAAATATAGGTAGATTTGGAATGAATCTTTTCTATGCTGCTGCGAATCTTGTACACCTTTGAGGCCGTAGATTCTGTCCGACGAAGCGATAATTATTGCAAAATACATGGACTCATTATTTTGATTCGATTTCTTTTTGGTATCCGACTCGAAAAGATCCATCACGGCGAGCGCCACCATGAACAAGAACTCCAAAATCCAGTCCCCAAACTCTTCTGATGTTGCTGTTATTGGTGTTGGTTTTAGATTCCCAGGTAACTCTAATGACCCAGAATCTTTGTGGAACAACTTGTTGGATGGTTTCGATGCTATTACCCAAGTCCCAAAAGAAAGATGGGCTACTTCTTTTAGAGAGATGGGTTTGATCAAGAACAAGTTCGGTGGTTTCTTGAAGGATTCTGAATGGAAGAATTTCGACCCTTTGTTCTTTGGTATCGGTCCAAAAGAAGCTCCATTCATTGATCCACAACAAAGGTTGTTGTTGTCCATCGTTTGGGAATCTTTGGAAGATGCTTACATCAGACCAGATGAATTGAGAGGTTCTAACACTGGTGTTTTCATCGGTGTTTCTAACAACGATTACACCAAGTTGGGTTTCCAAGACAACTACTCTATTTCTCCATACACTATGACCGGCTCTAACTCTTCATTGAACTCCAACAGAATTTCCTACTGCTTCGATTTTAGAGGTCCATCCATTACTGTTGATACCGCTTGTTCTTCTTCCTTGGTTTCTGTTAATTTGGGTGTCCAATCCATCCAAATGGGTGAATGTAAGATTGCTATTTGCGGTGGTGTTAACGCTTTGTTTGATCCATCTACATCTGTTGCCTTTTCCAAGTTGGGTGTTTTGTCTGAAAATGGCAGATGCAACTCTTTTAGTGATCAAGCCTCTGGTTACGTTAGATCTGAAGGTGCTGGTGTTGTTGTTTTGAAGTCTTTGGAACAAGCTAAGTTGGATGGTGATAGAATCTACGGTGTTATCAAGGGTGTTTCCTCTAATGAAGATGGTGCTTCTAATGGTGACAAGAACTCTTTGACTACTCCATCTTGTGAAGCCCAATCCATTAACATTTCTAAGGCTATGGAAAAGGCCTCCTTGTCTCCATCTGATATCTATTACATTGAAGCCCATGGTACTGGTACTCCAGTTGGTGATCCAATTGAAGTTAAGGCCTTGTCCAAGATCTTCTCCAACTCTAACAACAACCAGTTGAACAACTTCTCTACCGATGGTAATGATAACGATGATGATGATGACGATAACACCTCTCCAGAACCATTATTGATTGGCTCATTCAAGTCCAACATCGGTCATTTGGAATCTGCTGCTGGTATTGCTTCTTTGATTAAGTGTTGCTTGATGTTGAAGAACAGGATGTTGGTTCCATCCATTAACTGCTCTAATTTGAACCCATCCATTCCATTCGATCAGTACAACATCTCCGTTATCAGAGAAATCAGACAATTCCCAACCGATAAGTTGGTTAACATCGGTATCAATTCTTTCGGTTTCGGTGGTTCTAACTGCCATTTGATTATTCAAGAGTACAACAACAACTTCAAGAACAACTCTACCATCTGCAATAACAACAACAACAACAATAACAACATCGACTACTTGATCCCAATCTCCTCTAAGACTAAGAAGTCCTTGGATAAGTACTTGATTTTGATCAAGACCAACTCCAACTACCACAAGGATATTTCTTTCGATGACTTCGTCAAGTTCCAAATCAAGTCTAAGCAGTACAACTTGTCCAACAGAATGACTACCATTGCTAACGATTGGAACTCCTTCATTAAGGGTTCTAACGAATTCCACAACTTGATCGAATCTAAGGATGGTGAAGGTGGTTCTTCATCTTCTAACAGAGGTATTGATTCCGCCAATCAAATCAACACTACTACTACCTCTACCATCAACGATATCGAACCTTTGTTGGTTTTCGTTTTCTGTGGTCAAGGTCCACAATGGAATGGTATGATTAAGACCTTGTACAACTCCGAGAACGTTTTCAAGAACACCGTTGATCATGTTGACAGCATCTTGTACAAGTACTTCGGTTACTCCATTTTGAACGTCTTGTCTAAGATCGATGATAACGACGATTCCATCAACCATCCAATAGTTGCTCAACCATCTTTGTTCTTGTTGCAAATTGGTTTGGTCGAGTTGTTTAAGTACTGGGGTATCTACCCATCTATCTCTGTTGGTCATTCTTTCGGTGAAGTCTCTTCTTATTACTTGTCCGGTATCATCTCTTTGGAAACCGCTTGTAAAATCGTCTACGTCAGATCCTCTAATCAGAACAAAACTATGGGTTCCGGTAAGATGTTGGTTGTTTCTATGGGTTTTAAGCAATGGAACGATCAATTCTCTGCTGAATGGTCCGATATTGAAATTGCTTGTTACAACGCTCCAGATTCCATAGTTGTTACTGGTAACGAAGAAAGATTGAAAGAATTGTCCATCAAGTTGTCCGACGAATCCAATCAAATTTTCAACACCTTCTTGAGGTCCCCATGTTCTTTTCATTCTTCCCATCAAGAAGTCATCAAGGGTTCTATGTTCGAAGAGTTGTCTAACTTGCAATCTACTGGTGAAACCGAAATCCCTTTGTTCTCTACTGTTACTGGTAGACAAGTTTTGTCTGGTCATGTTACTGCTCAACACATCTACGATAATGTTAGAGAACCAGTCTTGTTCCAAAAGACGATTGAATCCATTACCTCCTACATCAAGTCTCACTACCCATCCAATCAAAAGGTTATCTACGTTGAAATTGCTCCACACCCAACCTTGTTTTCATTGATCAAAAAGTCCATCCCATCCTCCAACAAGAATTCCTCTTCTGTTTTGTGTCCATTGAACAGAAAAGAAAACTCCAACAACTCCTACAAGAAGTTCGTTTCTCAGTTGTACTTCAACGGTGTTAACGTTGACTTCAACTTCCAGTTGAACTCCATTTGCGATAACGTTAACAACGATCACCATTTGAACAACGTCAAGCAAAACTCCTTCAAAGAGACTACCAATTCCTTGCCAAGATACCAATGGGAACAAGATGAATATTGGTCCGAACCATTGATCTCCAGAAAGAATAGATTGGAAGGTCCAACTACTTCCTTGTTGGGTCATAGAATTATCTACAGCTTCCCAGTTTTCCAATCCGTTTTGGACTTGCAATCTGACAACTACAAATACTTGTTGGACCACTTGGTTAACGGTAAGCCAGTTTTTCCAGGTGCTGGTTATTTGGATATCATCATCGAATTCTTCGACTACCAAAAGCAGCAGTTGAATTCCTCTGATTCCTCTAACTCCTACATCATCAACGTTGACAAGATCCAATTCTTGAACCCAATTCACTTGACCGAAAACAAGTTGCAAACCTTGCAATCTTCTTTCGAACCTATCGTTACTAAGAAGTCTGCCTTCTCTGTTAACTTCTTCATCAAGGATACCGTCGAGGATCAATCTAAGGTTAAGTCTATGTCTGACGAAACTTGGACTAACACTTGTAAGGCTACCATTTCCTTGGAACAACAACAGCCATCTCCATCTTCTACTTTGACTTTGTCTAAGAAGCAAGACTTGCAGATCTTGAGAAACAGATGCGATATTAGCAAGCTAGACAAGTTTGAGTTGTACGACAAGATCTCTAAGAATTTGGGCTTGCAGTACAACTCCTTGTTTCAAGTTGTTGATACCATCGAAACTGGTAAGGATTGCTCTTTTGCTACTTTGTCTTTGCCAGAAGATACTTTGTTCACCACCATTTTGAACCCATGCTTGTTGGATAACTGTTTCCATGGTTTGTTGACCTTGATCAACGAAAAGGGTTCTTTCGTTGTCGAGTCCATTTCTTCTGTTTCTATCTACTTGGAGAACATCGGTTCCTTCAATCAAACTTCTGTTGGTAACGTCCAGTTCTACTTGTACACCACTATTTCTAAAGCCACCTCCTTTAGTTCTGAAGGTACTTGTAAGTTGTTCACCAAGGATGGTTCCTTGATTTTGTCTATCGGTAAGTTCATCATCAAGTCCACCAATCCAAAGTCTACTAAGACCAACGAAACTATCGAATCTCCATTGGACGAAACCTTCTCTATTGAATGGCAATCTAAGGATTCTCCAATTCCAACCCCACAACAAATCCAACAACAATCTCCATTGAACTCTAACCCATCCTTCATTAGATCTACCATCTTGAAGGACATCCAGTTCGAACAATACTGCTCCTCCATTATCCACAAAGAATTGATCAACCACGAAAAGTACAAGAACCAGCAATCCTTCGATATCAACTCCTTGGAAAACCACTTGAACGATGACCAATTGATGGAATCCTTGTCCATCTCCAAAGAATACTTGAGATTCTTCACCAGGATCATCTCCATCATTAAGCAATACCCAAAGATCTTGAACGAAAAAGAGCTAAAAGAATTGAAAGAAATCATCGAATTGAAGTACCCATCCGAAGTTCAGTTGTTGGAATTCGAAGTTATCGAGAAGGTGTCCATGATTATCCCAAAGTTGTTGTTCGAAAACGACAAGCAATCTTCCATGACCTTGTTCCAAGATAACTTGTTGACCAGGTTCTACTCCAATTCTAACTCTACCAGATTCTACTTGGAAAGGGTTTCCGAAATGGTCTTGGAATCTATTAGACCAATCGTCAGAGAAAAGAGGGTGTTCAGAATTTTGGAAATTGGTGCTGATACAGCCTCTTTGTCTAATGTTGTTTTGACTAAGTTGAACACCTACTTGTCCACCTTGAATTCTAATGGTGGTTCTGGTTACAACATCATCATTGAGTACACCTTCACCGATATTTCCGCCAACTTCATTATTGGTGAAATCCAAGAAACCATGTGCAACTTGTACCCAAACGTTACTTTCAAGTTCTCCGTCTTGGACTTGGAGAAAGAGATTATTAACTCCTCCGATTTCTTGATGGGTGATTACGATATAGTTTTGATGGCCTACGTTATCCATGCCGTTTCTAACATTAAGTTCTCCATCGAACAGTTGTACAAGTTGTTGTCTCCAAGAGGTTGGTTGTTGTGTATTGAACCTAAGTCCAACGTTGTGTTCTCCGATTTGGTTTTCGGTTGTTTTAATCAGTGGTGGAACTACTACGATGATATTAGAACTACCCACTGCTCCTTGTCTGAATCTCAATGGAATCAGTTGTTGTTGAACCAGTCCTTGAACAACGAATCCTCTTCTTCTTCTAACTGTTACGGTGGTTTCTCCAACGTTTCTTTTATTGGTGGTGAAAAGGATGTCGACTCCCATTCTTTCATATTGCACTGCCAAAAAGAATCCATCTCCCAAATGAAGTTAGCCACCACTATTAACAACGGTTTGTCATCTGGTTCCATCGTTATCGTTTTGAACTCTCAACAATTGACCAACATGAAGTCCTACCCAAAGGTTATTGAGTATATTCAAGAGGCTACCTCTTTGTGCAAGACCATTGAAATTATCGATTCCAAGGACGTCTTGAACTCTACCAATTCAGTTTTGGAAAAGATCCAAAAGTCCTTGTTGGTGTTCTGTTTGTTGGGTTATGACTTGTTGGAGAACAACTACCAAGAACAGTCTTTCGAATACGTTAAGTTGTTGAACTTGATCTCTACTACCGCCTCTTCATCTAATGATAAGAAACCACCAAAGGTCTTGTTGATCACCAAGCAATCTGAAAGAATCTCCAGGTCTTTCTACTCCAGATCCTTGATTGGTATTTCCAGAACCTCTATGAACGAGTACCCAAATTTGTCCATTACCTCTATCGATTTGGATACCAACGACTACTCATTGCAGTCTTTGTTGAAGCCAATCTTCAGCAACTCTAAGTTTTCCGACAACGAGTTCATCTTCAAAAAGGGCTTGATGTTCGTGTCCAGGATCTTTAAGAACAAGCAGTTGCTAGAATCCTCCAACGCTTTTGAAACTGACTCTTCTAACTTGTACTGTAAGGCCTCTTCTGACTTGTCTTACAAGTACGCTATTAAGCAGTCTATGTTGACCGAAAATCAGATCGAAATCAAGGTTGAATGCGTCGGTATTAACTTCAAGGACAACCTATTCTACAAGGGCTTGTTGCCACAAGAAATTTTCAGAATGGGTGACATCTACAATCCACCATATGGTTTGGAATGCTCTGGTGTTATTACCAGAATTGGTTCTAACGTCACCGAATACTCAGTTGGTCAAAATGTTTTTGGTTTCGCCAGACATTCTTTGGGTTCTCATGTTGTTACCAACAAGGATTTGGTTATCTTGAAGCCAGATACCATCTCATTTTCTGAAGCTGCTTCTATCCCAGTTGTTTACTGTACTGCTTGGTACTCCTTGTTCAACATTGGTCAGTTGTCTAACGAAGAATCCATCCTAATTCATTCTGCTACTGGTGGTGTAGGTTTGGCTTCTTTGAATTTGTTGAAAATGAAGAATCAGCAACAGCAACCATTGACCAATGTTTATGCTACTGTTGGCTCTAACGAGAAGAAGAAGTTCTTGATCGATAACTTCAACAACTTGTTCAAAGAGGACGGCGAAAACATTTTCTCTACCAGAGACAAAGAATACTCCAACCAGTTGGAATCCAAGATCGATGTTATTTTGAACACCTTGTCCGGTGAATTCGTCGAATCTAATTTCAAGTCCTTGAGATCCTTCGGTAGATTGATTGATTTGTCTGCTACTCACGTTTACGCCAATCAACAAATTGGTCTAGGTAACTTCAAGTTCGACCACTTGTATTCTGCTGTTGACTTGGAAAGATTGATCGACGAAAAACCTAAGTTGTTGCAGTCCATCTTGCAAAGAATTACCAACTCTATCGTCAACGGTTCCTTGGAAAAAATTCCAATTACCATCTTCCCATCCACCGAAACTAAGGATGCTATCGAATTATTGTCCAAGAGATCCCATATCGGTAAAGTTGTTGTAGATTGCACCGATATCTCTAAGTGTAATCCTGTTGGTGATGTGATCACCAACTTCTCTATGAGATTGCCAAAGCCAAACTACCAGTTGAATTTGAACTCCACCTTGTTGATTACTGGTCAGTCTGGTTTGTCTATCCCTTTGTTGAATTGGTTGTTGTCTAAGTCTGGTGGTAACGTTAAGAACGTTGTCATCATTTCTAAGTCCACCATGAAGTGGAAGTTGCAGACTATGATTTCCCATTTCGTTTCCGGTTTCGGTATCCATTTTAACTACGTTCAAGTCGACATCTCCAACTACGATGCTTTGTCTGAAGCTATTAAGCAATTGCCATCTGATTTGCCACCAATCACCTCTGTTTTTCATTTGGCTGCTATCTACAACGATGTTCCAATGGATCAAGTTACCATGTCTACCGTTGAATCTGTTCATAACCCTAAAGTTTTGGGTGCCGTTAACTTGCATAGAATCTCTGTTTCTTTTGGTTGGAAGTTGAACCACTTCGTCTTGTTCTCTTCTATTACTGCTATTACCGGTTACCCAGACCAATCTATCTACAATTCTGCCAACTCTATTTTGGACGCTTTGTCCAACTTTAGAAGGTTTATGGGTTTGCCATCCTTCTCCATTAACTTGGGTCCAATGAAGGATGAAGGTAAGGTTTCTACCAACAAGAGCATCAAGAAGCTATTCAAGTCTAGAGGTTTGCCAAGCCTATCCTTGAACAAGTTATTTGGTTTGTTGGAGGTCGTCATCAACAACCCATCTAATCATGTTATCCCATCCCAATTGATTTGCTCCCCAATCGATTTCAAGACCTACATCGAATCTTTCTCAACTATGAGGCCAAAGTTGTTACACTTGCAACCTACCATTTCCAAGCAGCAATCTTCTATCATTAACGATTCTACCAAGGCTTCCTCCAACATTTCATTGCAAGATAAGATCACCTCCAAGGTGTCTGATTTGTTGTCCATTCCAATCTCCAAGATCAACTTCGATCATCCATTGAAACACTACGGCTTGGATTCTTTGTTGACCGTTCAATTCAAATCCTGGATCGACAAAGAATTCGAAAAGAACTTGTTCACCCATATCCAATTGGCCACCATCTCTATTAACTCATTCTTGGAAAAGGTGAACGGCTTGTCTACAAACAATAACAACAACAACAATTCCAACGTCAAGTCCTCTCCATCCATTGTCAAAGAAGAAATCGTTACCTTGGACAAGGATCAACAACCATTGCTATTGAAAGAACACCAGCACATTATCATCTCCCCAGATATTAGAATCAACAAGCCAAAGAGGGAATCCTTGATTAGAACCCCAATCTTGAACAAATTCAACCAGATCACCGAATCCATTATCACTCCATCTACACCATCTTTGTCCCAATCCGATGTTTTGAAAACTCCACCAATCAAGTCTTTGAACAACACTAAGAACTCCAGCTTGATTAACACCCCACCAATTCAATCTGTCCAACAACATCAAAAGCAACAACAAAAGGTCCAAGTCATCCAACAACAGCAACAACCATTATCCAGATTGTCCTACAAGAGCAACAACAACTCTTTCGTTTTGGGTATCGGTATTTCTGTTCCAGGTGAACCTATTTCCCAACAATCCTTGAAAGACTCCATCTCCAATGACTTTTCTGATAAGGCTGAAACTAACGAGAAGGTCAAGAGAATCTTTGAGCAATCTCAAATCAAGACCAGACACTTGGTTAGAGATTACACTAAGCCAGAGAACTCCATCAAGTTCAGACATTTGGAAACCATTACCGATGTGAACAACCAGTTCAAGAAAGTTGTTCCAGATTTGGCTCAACAAGCCTGTTTGAGAGCTTTGAAAGATTGGGGTGGTGATAAGGGTGATATTACCCATATAGTTTCTGTTACCTCCACCGGTATTATCATCCCAGATGTTAATTTCAAGTTGATCGACTTGTTGGGCTTGAACAAGGATGTTGAAAGAGTGTCTTTGAACCTAATGGGTTGTTTGGCTGGTTTGAGTTCTTTGAGAACTGCTGCTTCTTTGGCTAAGGCTTCTCCAAGAAATAGAATTTTGGTTGTCTGTACCGAAGTCTGCTCCTTGCATTTTTCTAATACTGATGGTGGTGATCAAATGGTCGCCTCTTCTATTTTTGCTGATGGTTCTGCTGCTTACATTATTGGTTGTAACCCAAGAATTGAAGAAACCCCATTATACGAAGTCATGTGCTCCATTAACAGATCTTTCCCAAATACCGAAAACGCCATGGTTTGGGATTTGGAAAAAGAAGGTTGGAACTTGGGTTTGGATGCTTCTATTCCAATTGTCATTGGTTCTGGTATTGAAGCCTTCGTTGATACTTTGTTGGATAAGGCTAAGTTGCAAACTTCCACTGCTATTTCTGCTAAGGATTGCGAATTCTTGATTCATACTGGTGGCAAGTCCATCTTGATGAACATCGAAAATTCCTTGGGTATCGACCCAAAGCAAACTAAGAATACTTGGGATGTTTACCATGCCTACGGCAATATGTCATCTGCCTCTGTTATTTTCGTTATGGATCATGCCAGAAAGTCCAAGTCTTTGCCAACTTACTCAATTTCTTTGGCTTTTGGTCCAGGTTTGGCTTTTGAAGGTTGTTTCTTGAAGAACGTCGTCGAACAGAAGCTCATTTCCGAAGAGGACTTGTAACCGCAACAACTACCTCGACTTTGGCTGGGACACTTTCAGTGAGGACAAGAAGCTTCAGAAGCGTGCTATCGAACTCAACCAGGGACGTGCGGCACAAATGGGCATCCTTGCTCTCATGGTGCACGAACAGTTGGGAGTCTCTATCCTTCCTTAAAAATTTAATTTTCATTAGTTGCAGTCACTCCGCTTTGGTTTCGCTAGTGATAATAAGTGACTGAGGTATGTGCTCTTCTTATCTCCTTTTGTAGTGTTGCTCTTATTTTAAACAACTTTGCGGTTTTTTGATGACTTTGCGATTTTGTTGTTGCTTTGCAGTAAATTGCAAGATTTAATAAAAAAACGCAAAGCAATGATTAAAGGATGTTCAGAATGAAACTCATGGAAACACTTAACCAGTGCATAAACGCTGGTCATGAAATGACGAAGGCTATCGCCATTGCACAGTTTAATGATGACAGCCCGGAAGCGAGGAAAATAACCCGGCGCTGGAGAATAGGTGAAGCAGCGGATTTAGTTGGGGTTTCTTCTCAGGCTATCAGAGATGCCGAGAAAGCAGGGCGACTACCGCACCCGGATATGGAAATTCGAGGACGGGTTGAGCAACGTGTTGGTTATACAATTGAACAAATTAATCATATGCGTGATGTGTTTGGTACGCGATTGCGACGTGCTGAAGACGTATTTCCACCGGTGATCGGGGTTGCTGCCCATAAAGGTGGCGTTTACAAAACCTCAGTTTCTGTTCATCTTGCTCAGGATCTGGCTCTGAAGGGGCTACGTGTTTTGCTCGTGGAAGGTAACGACCCCCAGGGAACAGCCTCAATGTATCACGGATGGGTACCAGATCTTCATATTCATGCAGAAGACACTCTCCTGCCTTTCTATCTTGGGGAAAAGGACGATGTCACTTATGCAATAAAGCCCACTTGCTGGCCGGGGCTTGACATTATTCCTTCCTGTCTGGCTCTGCACCGTATTGAAACTGAGTTAATGGGCAAATTTGATGAAGGTAAACTGCCCACCGATCCACACCTGATGCTCCGACTGGCCATTGAAACTGTTGCTCATGACTATGATGTCATAGTTATTGACAGCGCGCCTAACCTGGGTATCGGCACGATTAATGTCGTATGTGCTGCTGATGTGCTGATTGTTCCCACGCCTGCTGAGTTGTTTGACTACACCTCCGCACTGCAGTTTTTCGATATGCTTCGTGATCTGCTCAAGAACGTTGATCTTAAAGGGTTCGAGCCTGATGTACGTATTTTGCTTACCAAATACAGCAATAGCAATGGCTCTCAGTCCCCGTGGATGGAGGAGCAAATTCGGGATGCCTGGGGAAGCATGGTTCTAAAAAATGTTGTACGTGAAACGGATGAAGTTGGTAAAGGTCAGATCCGGATGAGAACTGTTTTTGAACAGGCCATTGATCAACGCTCTTCAACTGGTGCCTGGAGAAATGCTCTTTCTATTTGGGAACCTGTCTGCAATGAAATTTTCGATCGTCTGATTAAACCACGCTGGGAGATTAGATAATGAAGCGTGCGCCTGTTATTCCAAAACATACGCTCAATACTCAACCGGTTGAAGATACTTCGTTATCGACACCAGCTGCCCCGATGGTGGATTCGTTAATTGCGCGCGTAGGAGTAATGGCTCGCGGTAATGCCATTACTTTGCCTGTATGTGGTCGGGATGTGAAGTTTACTCTTGAAGTGCTCCGGGGTGATAGTGTTGAGAAGACCTCTCGGGTATGGTCAGGTAATGAACGTGACCAGGAGCTGCTTACTGAGGACGCACTGGATGATCTCATCCCTTCTTTTCTACTGACTGGTCAACAGACACCGGCGTTCGGTCGAAGAGTATCTGGTGTCATAGAAATTGCCGATGGGAGTCGCCGTCGTAAAGCTGCTGCACTTACCGAAAGTGATTATCGTGTTCTGGTTGGCGAGCTGGATGATGAGCAGATGGCTGCATTATCCAGATTGGGTAACGATTATCGCCCAACAAGTGCTTATGAACGTGGTCAGCGTTATGCAAGCCGATTGCAGAATGAATTTGCTGGAAATATTTCTGCGCTGGCTGATGCGGAAAATATTTCACGTAAGATTATTACCCGCTGTATCAACACCGCCAAATTGCCTAAATCAGTTGTTGCTCTTTTTTCTCACCCCGGTGAACTATCTGCCCGGTCAGGTGATGCACTTCAAAAAGCCTTTACAGATAAAGAGGAATTACTTAAGCAGCAGGCATCTAACCTTCATGAGCAGAAAAAAGCTGGGGTGATATTTGAAGCTGAAGAAGTTATCACTCTTTTAACTTCTGTGCTTAAAACGTCATCTGCATCAAGAACTAGTTTAAGCTCACGACATCAGTTTGCTCCTGGAGCGACAGTATTGTATAAGGGCGATAAAATGGTGCTTAACCTGGACAGGTCTCGTGTTCCAACTGAGTGTATAGAGAAAATTGAGGCCATTCTTAAGGAACTTGAAAAGCCAGCACCCTGATGCGACCTCGTTTTAGTCTACGTTTATCTGTCTTTACTTAATGTCCTTTGTTACAGGCCAGAAAGCATAACTGGCCTGAATATTCTCTCTGGGCCCACTGTTCCACTTGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCATGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGAACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGATCCCACTCGTGTTGTCGGTCTGATTATCGGTCTGGGACCACGGTCCCACTTGTATTGTCGATCAGACTATCAGCGTGAGACTACGATTCCATCAATGCCTGTCAAGGGCAAGTATTGACATGTCGTCGTAACCTGTAGAACGGAGTAACCTCGGTGTGCGGTTGTATGCCTGCTGTGGATTGCTGCTGTGTCCTGCTTATCCACAACATTTTGCGCACGGTTATGTGGACAAAATACCTGGTTACCCAGGCCGTGCCGGCACGTTAACCGGGCTGCATCCGATGCAAGTGTGTCGCTGTCGACGAGCTCGCGAGCTCGGACATGAGGTTGCCCCGTATTCAGTGTCGCTGATTTGTATTGTCTGAAGTTGTTTTTACGTTAAGTTGATGCAGATCAATTAATACGATACCTGCGTCATAATTGATTATTTGACGTGGTTTGATGGCCTCCACGCACGTTGTGATATGTAGATGATAATCATTATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTACGGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGCTTTTTGGCCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATACATTATACGAAGTTATA |
| pSteelyA-3 sequencing | TTCGATGCGGCCGCAAGGGGTTCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCACACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGAGTATTCTATAGTCTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGAACCCCTTGCGGCCGCCCGGGCCGTCGACCAATTCTCATGTTTGACAGCTTATCATCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGCGATAAGCTCATGGAGCGGCGTAACCGTCGCACAGGAAGGACAGAGAAAGCGCGGATCTGGGAAGTGACGGACAGAACGGTCAGGACCTGGATTGGGGAGGCGGTTGCCGCCGCTGCTGCTGACGGTGTGACGTTCTCTGTTCCGGTCACACCACATACGTTCCGCCATTCCTATGCGATGCACATGCTGTATGCCGGTATACCGCTGAAAGTTCTGCAAAGCCTGATGGGACATAAGTCCATCAGTTCAACGGAAGTCTACACGAAGGTTTTTGCGCTGGATGTGGCTGCCCGGCACCGGGTGCAGTTTGCGATGCCGGAGTCTGATGCGGTTGCGATGCTGAAACAATTATCCTGAGAATAAATGCCTTGGCCTTTATATGGAAATGTGGAACTGAGTGGATATGCTGTTTTTGTCTGTTAAACAGAGAAGCTGGCTGTTATCCACTGAGAAGCGAACGAAACAGTCGGGAAAATCTCCCATTATCGTAGAGATCCGCATTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATTTGAATATGCCTTCAGGAACAATAGAAATCTTCGTGCGGTGTTACGTTGAAGTGGAGCGGATTATGTCAGCAATGGACAGAACAACCTAATGAACACAGAACCATGATGTGGTCTGTCCTTTTACAGCCAGTAGTGCTCGCCGCAGTCGAGCGACAGGGCGAAGCCCATCGATACTAGCTTGATTGGGATATCTCGCTCGTGCTTGTCGCGTGCTATGTCTTTTTAGGGTACTTTGAACCTACGTTCGTACTTGTGTAATATGATCATCGTCGTCATCGTATTATCGTTTTTCATCCGTCCAGCGCAAAATGCATTAGCAGCTAGTCCTAGCGTGCGGAGCTACCTGTACAGGTGCATGACGGATGCGTGTCCTTAAGTGAGTTTCTAATTAACAGTAACTTCTTTACTTATGTTTCAGTTTGTAAGAAGCGGGATGCGCTCGTCGCTTGACATCTGATTGGACTGCGTCGGCACGTGAAAACTACATTGTGAAATCTGCTAAAACTCCGGGTATCTCTGACACAAAACGATTCGGCTTCGCAATTTCAACATTACGGTCAAGGCTAACGTATCTTTCTCGGTCAACTTCAGATTATGCCGATTAAATTGTCGTAGCTTTCAAGGCGTTTTGAGTACTGCGGCAGTTGTTGAACCTGCAAGGAGAAGATCTCGACAACAGAATAAAGCGAAAAATGGGTCTCATGCACTAACACTCAGGCCTCCCTCATAATCTCTGTTTGAGTTTACCAACAACACATATATACATTTCGACAAAATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCAGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACCGACGCCGACCAACACCGCCGGTCCGACGCGGCCCGACGGGTCCGAGGCCTCGGAGATCTGGGCCCATGCGGCCGCAACAACTACCTCGACTTTGGCTGGGACACTTTCAGTGAGGACAAGAAGCTTCAGAAGCGTGCTATCGAACTCAACCAGGGACGTGCGGCACAAATGGGCATCCTTGCTCTCATGGTGCACGAACAGTTGGGAGTCTCTATCCTTCCTTAAAAATTTAATTTTCATTAGTTGCAGTCACTCCGCTTTGGTTTCGTAACTATAACGGTCCTAAGGTAGCGAACGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCGGGGGGGGGGGGGGGGTTTCAATTCATCATTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCCTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATATAAGGAACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAAGCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAATTTGTTTACTAAAAACACATGTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAAAAGGCATTATCCGCCAAGTACAATTTTTTACTCTTCGAAGACAGAAAATTTGCTGACATTGGTAATACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAGAATGGGCAGACATTACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGTTTGAAGCAGGCGGCGGAAGAAGTAACAAAGGAACCTAGAGGCCTTTTGATGTTAGCAGAATTGTCATGCAAGGGCTCCCTAGCTACTGGAGAATATACTAAGGGTACTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAGAGACATGGGTGGAAGAGATGAAGGTTACCAAATAGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCCGGCTACCGCCGGCGTAACAGATGAGGGCAAGCGGATGGCTGATGAAACCAAGCCAACCAGGAAGGGCAGCCCACCTATCAAGGTGTACTGCCTTCCAGACGAACGAAGAGCGATTGAGGAAAAGGCGGCGGCGGCCGGCATGAGCCTGTCGGCCTACCTGCTGGCCGTCGGCCAGGGCTACAAAATCACGGGCGTCGTGGACTATGAGCACGTCCGCGAGCTGGCCCGCATCAATGGCGACCTGGGCCGCCTGGGCGGCCTGCTGAAACTCTGGCTCACCGACGACCCGCGCACGGCGCGGTTCGGTGATGCCACGATCCTCGCCCTGCTGGCGAAGATCGAAGAGAAGCAGGACGAGCTTGGCAAGGTCATGATGGGCGTGGTCCGCCCGAGGGCAGAGCCATGACTTTTTTAGCCGCTAAAACGGCCGGGGGGTGCGCGTGATTGCCAAGCACGTCCCCATGCGCTCCATCAAGAAGAGGCACTTCGAGCTGTAAGTACATCACCGACGAGCAAGGCAAGACGATCCGCGCCTGTTTTATTGAGAACGTTGTTCGTGTTGGCCTCAATGGTAGCGATGCGTCATTCAGCGAAGTTCTGGTGCTGATGATGTGGTTCGCTTTGCCACTGGTCAATGTGGTAAGCCCGTGTAATGTCAGTAACCTTTTTACTGATCTCAGCTTGAGCACGGTCGCTGATGAGCTTATCCATGGCCCCACGGTAACGGATATGATCCTCTAGGGCGTTGACAAATTCTTTGTCGGTTTTCATGGGGTAGACGTCAGTGACCAGGGAACGTCTCCCTACAAAAAGTTGCGCATACTTTGCTCCGTTGTCGACGGCAGGGGTGTCGGACCAGATTGTATCTGTGGCAACGGCCTCATTGCGTCAATGGACCTTTAAAGCCGGGAAACGAGACTTGAAATGTTTACGTAGAGGTGCATTATAGACCTCACGCGCATATTGAGTGGTTGCAAAAATGGTTTTGCGAACGGTATCACTGGAGACCCATGCCAGGCAAGGACGGAGGGCATCATAATCATGTTCATTGCGTTGGACAGCATGCTTGTTACACGTAAGTATACGGTCGAGAGCATCGTGACGTTGAGAATTACAGATGGCAACGTGTCGGTGCATGTATTGGCCGAGATTGTCAAATCGTGGCTCAATATATGGCATGCCAGGTAAGTCATGGATGTCGGTATGCCATTCAGCCTTCATGTCGATTTCGTTGTCGAGAACTGATGGGTCCCAGTCAACGTCAGAAGTAAGAACAACATGGGGAAGGGAATGAAGTTCATGACTGGTGGGTGCGCGCATATCCATGTACGGAAGACCCTGTCGTATGTTTAGTGGGATGATGTAATCATCCAGGGTAACAATACGTTGGTTGCCGCCCACGGTACGAGACCGATCTTGAACACAATTGTGGCAATGTTCAAGTTGGGCGCTGGAATGGATGGTTTTACCTTTACCAAGATGCGCGTATTGATGCATGATAACAACAATGGGCCCTCGTTGCGATTCGACAAGACCGGCAGCCGTGACAATGTCTAGATTGGACAAGGTATGTTCATTTATACCGGTTATGTTGGCTGAACGTCCAGTTTTGTGCAGTACGGTAACATCACTCCCCGCAAGTCCACCGTTGGCACCGCGATCGACAAGTGCAGATGTGGTATTTTGTACTGTGTGTCGGGAGACTTGGTACTGTAACACATTTGACTGAAGTGACTTGGTGGTACCATGAGGACGGAGGGAACCCGTTGATACGGTGTTGCGTGACGCTGCAAGTACCTTACGTATGTCGCCTGGATCCATACGTCCAACCCGATCAGAAAGATGTGCTAGGAGTTCCGTTTCTGGTGCACAATCATGAAAGGTATCCACGGGAGTTCCATTGTTGTCGTCCGGGGCTGCGTCGCGACTGCCAGTTGCATGTACATGCGCCATCAAGGGACGTGCCAATCCATTACTAACCGGGAGAACCTTAGCTGGGGCAGCCAATCCCTGAAGTATAGCCTTAGCATCATCTGAGAGCTGGTTCCACATATCTCTAGGGATATAAGGTCGTTCACGGTTAGTGGGCGTACTATGATGTCGGTTATTAGTACAATTCTCCCTGCGGGCATGCGCATTGGCTTCATAGAGTACGGAAGGTGACAAGTCAATATTGTAGTTAACATCCGGATCAGTGTCAAAGTCTGTAGGATGGTAAGAAAGATCAGTAGAATGAATACTACGCTTTTCCTTGGGACTACGGGAATTAGAGAAGTTGTTTCCTTTATTGTAGAGTGACGCTGAAGCAAGTAGAAGACTAAGGTAACTCTCGTAGCTAATAGGATTACCTCCTTTGGCTAGGTCAAGAGTGGCTGTGATCTTCACTTGACAAAGTTCCGGTACATTGTGGACAGCATTCTCCAAAAGACTAAGACACAGTTGCTTTGGGAGTTGCTCAGCCATTGGTACAGTATTGTGGTAGATACAAAGGTGGTTCTTCCAATGAAGGATAAATCCTTCTGCTGTGCCTGTCCATGAGGATCCATATTTCGCCGTAGTTAGGTAACCAAGCGTGGTGGCTGAACTGATCTTCGCACTTGCTGATTCCGTATAGTGTTTGACAACTTTACAAAACACTTCTTGCGCAGTTCGCTCTAGCCTGGACAGAGAAGGGACAATCTTCGTCGTTAAGACTCGTGCGAATAGCAAAAGATCACAAAATAGCACATCGGCACCGACCAACGATTATTTCCAAGGAAAAAAAGAATGCTTCACTACAAGAAATTGTGTCATCCCTATACAGAGTCTTGTTACTGTGACAGAAAATTGATGGAAGATGTGGCGGATTGCCTTTACACTAGCCAACTTGTTCGACTAATTGCAGCTTCTTCTGAGAGGCTTCACCGAGTAACGCGAAGAACACCGGTGTCTCGTACATGCTCGTCGGTGAACGCTCGTCCAATGACACCCCCCACTTTGTATCAATATCCCAACTTGGTAGTGAACTGGAATGATACATGCAATTTCGCGCCGCATCAACAGCCACGGGCACCATCGACGAATAGACTCGGTCGAGCTGGTTGCCCTCGCCGCTGGGCTGGCGGCCGTCTATGGCCCTGCAAACGCGCCAGAAACGCCGTCGAAGCCGTGTGCGAGACACCGCGGCCGGCCGCCGGCGTTGTGGATACCTCGCGGAAAACTTGGCCCTCACTGACAGATGAGGGGCGGACGTTGACACTTGAGGGGCCGACTCACCCGGCGCGGCGTTGACAGATGAGGGGCAGGCTCGATTTCGGCCGGCGACGTGGAGCTGGCCAGCCTCGCAAATCGGCGAAAACGCCTGATTTTACGCGAGTTTCCCACAGATGATGTGGACAAGCCTGGGGATAAGTGCCCTGCGGTATTGACACTTGAGGGGCGCGACTACTGACAGATGAGGGGCGCGATCCTTGACACTTGAGGGGCAGAGTGCTGACAGATGAGGGGCGCACCTATTGACATTTGAGGGGCTGTCCACAGGCAGAAAATCCAGCATTTGCAAGGGTTTCCGCCCGTTTTTCGGCCACCGCTAACCTGTCTTTTAACCTGCTTTTAAACCAATATTTATAAACCTTGTTTTTAACCAGGGCTGCGCCCTGTGCGCGTGACCGCGCACGCCGAAGGGGGGTGCCCCCCCTTCTCGAACCCTCCCGGTCGAGTGAGCGAGGAAGCACCAGGGAACAGCACTTATATATTCTGCTTACACACGATGCCTGAAAAAACTTCCCTTGGGGTTATCCACTTATCCACGGGGATATTTTTATAATTATTTTTTTTATAGTTTTTAGATCTTCTTTTTTAGAGCGCCTTGTAGGCCTTTATCCATGCTGGTTCTAGAGAAGGTGTTGTGACAAATTGCCCTTTCAGTGTGACAAATCACCCTCAAATGACAGTCCTGTCTGTGACAAATTGCCCTTAACCCTGTGACAAATTGCCCTCAGAAGAAGCTGTTTTTTCACAAAGTTATCCCTGCTTATTGACTCTTTTTTATTTAGTGTGACAATCTAAAAACTTGTCACACTTCACATGGATCTGTCATGGCGGAAACAGCGGTTATCAATCACAAGAAACGTAAAAATAGCCCGCGAATCGTCCAGTCAAACGACCTCACTGAGGCGGCATATAGTCTCTCCCGGGATCAAAAACGTATGCTGTATCTGTTCGTTGACCAGATCAGAAAATCTGATGGCACCCTACAGGAACATGACGGTATCTGCGAGATCCATGTTGCTAAATATGCTGAAATATTCGGATTGACCTCTGCGGAAGCCAGTAAGGATATACGGCAGGCATTGAAGAGTTTCGCGGGGAAGGAAGTGGTTTTTTATCGCCCTGAAGAGGATGCCGGCGATGAAAAAGGCTATGAATCTTTTCCTTGGTTTATCAAACGTGCGCACAGTCCATCCAGAGGGCTTTACAGTGTACATATCAACCCATATCTCATTCCCTTCTTTATCGGGTTACAGAACCGGTTTACGCAGTTTCGGCTTAGTGAAACAAAAGAAATCACCAATCCGTATGCCATGCGTTTATACGAATCCCTGTGTCAGTATCGTAAGCCGGATGGCTCAGGCATCGTCTCTCTGAAAATCGACTGGATCATAGAGCGTTACCAGCTGCCTCAAAGTTACCAGCGTATGCCTGACTTCCGCCGCCGCTTCCTGCAGGTCTGTGTTAATGAGATCAACAGCAGAACTCCAATGCGCCTCTCATACATTGAGAAAAAGAAAGGCCGCCAGACGACTCATATCGTATTTTCCTTCCGCGATATCACTTCCATGACGACAGGATAGTCTGAGGGTTATCTGTCACAGATTTGAGGGTGGTTCGTCACATTTGTTCTGACCTACTGAGGGTAATTTGTCACAGTTTTGCTGTTTCCTTCAGCCTGCATGGATTTTCTCATACTTTTTGAACTGTAATTTTTAAGGAAGCCAAATTTGAGGGCAGTTTGTCACAGTTGATTTCCTTCTCTTTCCCTTCGTCATGTGACCTGATATCGGGGGTTAGTTCGTCATCATTGATGAGGGTTGATTATCACAGTTTATTACTCTGAATTGGCTATCCGCGTGTGTACCTCTACCTGGAGTTTTTCCCACGGTGGATATTTCTTCTTGCGCTGAGCGTAAGAGCTATCTGACAGAACAGTTCTTCTTTGCTTCCTCGCCAGTTCGCTCGCTATGCTCGGTTACACGGCTGCGGCGAGCATCACGTGCTATAAAAATAATTATAATTTAAATTTTTTAATATAAATATATAAATTAAAAATAGAAAGTAAAAAAAGAAATTAAAGAAAAAATAGTTTTTGTTTTCCGAAGATGTAAAAGACTCTAGGGGGATCGCCAACAAATACTACCTTTTACCTTGCTCTTCCTGCTCTCAGGTATTAATGCCGAATTGTTTCATCTTGTCTGTGTAGAAGACCACACACGAAAATCCTGTGATTTTACATTTTACTTATCGTTAATCGAATGTATATCTATTTAATCTGCTTTTCTTGTCTAATAAATATATATGTAAAGTACGCTTTTTGTTGAAATTTTTTAAACCTTTGTTTATTTTTTTTTCTTCATTCCGTAACTCTTCTACCTTCTTTATTTACTTTCTAAAATCCAAATACAAAACATAAAAATAAATAAACACAGAGTAAATTCCCAAATTATTCCATCATTAAAAGATACGAGGCGCGTGTAAGTTACAGGCAAGCGATCCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTTTAAAGAGGCCCTAGGGGCCGTGCGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACAGGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTTTTACACAGGAGTCTGGACTTGACATACAGTGAATGTAACTTTCGAATTGACAGTATTAGTAGTCGTATTGACAGTGAGGCACGCCCCTCAATGTGCGAGGTGGAAAATATACCAGCATGACAATGAATCTTGGAGATTCTTTTGCTGTCATCAAGATTCACCGCCAAATCTTCAGGAACCTATCACGTCCACAGGCGATGTTAATTCTTGAGTCGTCAAAACAAAGTCCTGTCCTACCTGTAGAAGTTGACAGCGAGCAATTGTATGCAAACTTCTGACTTTGTTATAATAACATTAAAGGTAATTAAGTATCTTCAATTAGGCATTTTGTCACTGTCAGTCCGTTCCGACAATATAGGTAGATTTGGAATGAATCTTTTCTATGCTGCTGCGAATCTTGTACACCTTTGAGGCCGTAGATTCTGTCCGACGAAGCGATAATTATTGCAAAATACATGGACTCATTATTTTGATTCGATTTCTTTTTGGTATCCGACTCGAAAAGATCCATCACGGCGAGCGCCACCATGAACAAGAACTCCAAAATCCAGTCCCCAAACTCTTCTGATGTTGCTGTTATTGGTGTTGGTTTTAGATTCCCAGGTAACTCTAATGACCCAGAATCTTTGTGGAACAACTTGTTGGATGGTTTCGATGCTATTACCCAAGTCCCAAAAGAAAGATGGGCTACTTCTTTTAGAGAGATGGGTTTGATCAAGAACAAGTTCGGTGGTTTCTTGAAGGATTCTGAATGGAAGAATTTCGACCCTTTGTTCTTTGGTATCGGTCCAAAAGAAGCTCCATTCATTGATCCACAACAAAGGTTGTTGTTGTCCATCGTTTGGGAATCTTTGGAAGATGCTTACATCAGACCAGATGAATTGAGAGGTTCTAACACTGGTGTTTTCATCGGTGTTTCTAACAACGATTACACCAAGTTGGGTTTCCAAGACAACTACTCTATTTCTCCATACACTATGACCGGCTCTAACTCTTCATTGAACTCCAACAGAATTTCCTACTGCTTCGATTTTAGAGGTCCATCCATTACTGTTGATACCGCTTGTTCTTCTTCCTTGGTTTCTGTTAATTTGGGTGTCCAATCCATCCAAATGGGTGAATGTAAGATTGCTATTTGCGGTGGTGTTAACGCTTTGTTTGATCCATCTACATCTGTTGCCTTTTCCAAGTTGGGTGTTTTGTCTGAAAATGGCAGATGCAACTCTTTTAGTGATCAAGCCTCTGGTTACGTTAGATCTGAAGGTGCTGGTGTTGTTGTTTTGAAGTCTTTGGAACAAGCTAAGTTGGATGGTGATAGAATCTACGGTGTTATCAAGGGTGTTTCCTCTAATGAAGATGGTGCTTCTAATGGTGACAAGAACTCTTTGACTACTCCATCTTGTGAAGCCCAATCCATTAACATTTCTAAGGCTATGGAAAAGGCCTCCTTGTCTCCATCTGATATCTATTACATTGAAGCCCATGGTACTGGTACTCCAGTTGGTGATCCAATTGAAGTTAAGGCCTTGTCCAAGATCTTCTCCAACTCTAACAACAACCAGTTGAACAACTTCTCTACCGATGGTAATGATAACGATGATGATGATGACGATAACACCTCTCCAGAACCATTATTGATTGGCTCATTCAAGTCCAACATCGGTCATTTGGAATCTGCTGCTGGTATTGCTTCTTTGATTAAGTGTTGCTTGATGTTGAAGAACAGGATGTTGGTTCCATCCATTAACTGCTCTAATTTGAACCCATCCATTCCATTCGATCAGTACAACATCTCCGTTATCAGAGAAATCAGACAATTCCCAACCGATAAGTTGGTTAACATCGGTATCAATTCTTTCGGTTTCGGTGGTTCTAACTGCCATTTGATTATTCAAGAGTACAACAACAACTTCAAGAACAACTCTACCATCTGCAATAACAACAACAACAACAATAACAACATCGACTACTTGATCCCAATCTCCTCTAAGACTAAGAAGTCCTTGGATAAGTACTTGATTTTGATCAAGACCAACTCCAACTACCACAAGGATATTTCTTTCGATGACTTCGTCAAGTTCCAAATCAAGTCTAAGCAGTACAACTTGTCCAACAGAATGACTACCATTGCTAACGATTGGAACTCCTTCATTAAGGGTTCTAACGAATTCCACAACTTGATCGAATCTAAGGATGGTGAAGGTGGTTCTTCATCTTCTAACAGAGGTATTGATTCCGCCAATCAAATCAACACTACTACTACCTCTACCATCAACGATATCGAACCTTTGTTGGTTTTCGTTTTCTGTGGTCAAGGTCCACAATGGAATGGTATGATTAAGACCTTGTACAACTCCGAGAACGTTTTCAAGAACACCGTTGATCATGTTGACAGCATCTTGTACAAGTACTTCGGTTACTCCATTTTGAACGTCTTGTCTAAGATCGATGATAACGACGATTCCATCAACCATCCAATAGTTGCTCAACCATCTTTGTTCTTGTTGCAAATTGGTTTGGTCGAGTTGTTTAAGTACTGGGGTATCTACCCATCTATCTCTGTTGGTCATTCTTTCGGTGAAGTCTCTTCTTATTACTTGTCCGGTATCATCTCTTTGGAAACCGCTTGTAAAATCGTCTACGTCAGATCCTCTAATCAGAACAAAACTATGGGTTCCGGTAAGATGTTGGTTGTTTCTATGGGTTTTAAGCAATGGAACGATCAATTCTCTGCTGAATGGTCCGATATTGAAATTGCTTGTTACAACGCTCCAGATTCCATAGTTGTTACTGGTAACGAAGAAAGATTGAAAGAATTGTCCATCAAGTTGTCCGACGAATCCAATCAAATTTTCAACACCTTCTTGAGGTCCCCATGTTCTTTTCATTCTTCCCATCAAGAAGTCATCAAGGGTTCTATGTTCGAAGAGTTGTCTAACTTGCAATCTACTGGTGAAACCGAAATCCCTTTGTTCTCTACTGTTACTGGTAGACAAGTTTTGTCTGGTCATGTTACTGCTCAACACATCTACGATAATGTTAGAGAACCAGTCTTGTTCCAAAAGACGATTGAATCCATTACCTCCTACATCAAGTCTCACTACCCATCCAATCAAAAGGTTATCTACGTTGAAATTGCTCCACACCCAACCTTGTTTTCATTGATCAAAAAGTCCATCCCATCCTCCAACAAGAATTCCTCTTCTGTTTTGTGTCCATTGAACAGAAAAGAAAACTCCAACAACTCCTACAAGAAGTTCGTTTCTCAGTTGTACTTCAACGGTGTTAACGTTGACTTCAACTTCCAGTTGAACTCCATTTGCGATAACGTTAACAACGATCACCATTTGAACAACGTCAAGCAAAACTCCTTCAAAGAGACTACCAATTCCTTGCCAAGATACCAATGGGAACAAGATGAATATTGGTCCGAACCATTGATCTCCAGAAAGAATAGATTGGAAGGTCCAACTACTTCCTTGTTGGGTCATAGAATTATCTACAGCTTCCCAGTTTTCCAATCCGTTTTGGACTTGCAATCTGACAACTACAAATACTTGTTGGACCACTTGGTTAACGGTAAGCCAGTTTTTCCAGGTGCTGGTTATTTGGATATCATCATCGAATTCTTCGACTACCAAAAGCAGCAGTTGAATTCCTCTGATTCCTCTAACTCCTACATCATCAACGTTGACAAGATCCAATTCTTGAACCCAATTCACTTGACCGAAAACAAGTTGCAAACCTTGCAATCTTCTTTCGAACCTATCGTTACTAAGAAGTCTGCCTTCTCTGTTAACTTCTTCATCAAGGATACCGTCGAGGATCAATCTAAGGTTAAGTCTATGTCTGACGAAACTTGGACTAACACTTGTAAGGCTACCATTTCCTTGGAACAACAACAGCCATCTCCATCTTCTACTTTGACTTTGTCTAAGAAGCAAGACTTGCAGATCTTGAGAAACAGATGCGATATTAGCAAGCTAGACAAGTTTGAGTTGTACGACAAGATCTCTAAGAATTTGGGCTTGCAGTACAACTCCTTGTTTCAAGTTGTTGATACCATCGAAACTGGTAAGGATTGCTCTTTTGCTACTTTGTCTTTGCCAGAAGATACTTTGTTCACCACCATTTTGAACCCATGCTTGTTGGATAACTGTTTCCATGGTTTGTTGACCTTGATCAACGAAAAGGGTTCTTTCGTTGTCGAGTCCATTTCTTCTGTTTCTATCTACTTGGAGAACATCGGTTCCTTCAATCAAACTTCTGTTGGTAACGTCCAGTTCTACTTGTACACCACTATTTCTAAAGCCACCTCCTTTAGTTCTGAAGGTACTTGTAAGTTGTTCACCAAGGATGGTTCCTTGATTTTGTCTATCGGTAAGTTCATCATCAAGTCCACCAATCCAAAGTCTACTAAGACCAACGAAACTATCGAATCTCCATTGGACGAAACCTTCTCTATTGAATGGCAATCTAAGGATTCTCCAATTCCAACCCCACAACAAATCCAACAACAATCTCCATTGAACTCTAACCCATCCTTCATTAGATCTACCATCTTGAAGGACATCCAGTTCGAACAATACTGCTCCTCCATTATCCACAAAGAATTGATCAACCACGAAAAGTACAAGAACCAGCAATCCTTCGATATCAACTCCTTGGAAAACCACTTGAACGATGACCAATTGATGGAATCCTTGTCCATCTCCAAAGAATACTTGAGATTCTTCACCAGGATCATCTCCATCATTAAGCAATACCCAAAGATCTTGAACGAAAAAGAGCTAAAAGAATTGAAAGAAATCATCGAATTGAAGTACCCATCCGAAGTTCAGTTGTTGGAATTCGAAGTTATCGAGAAGGTGTCCATGATTATCCCAAAGTTGTTGTTCGAAAACGACAAGCAATCTTCCATGACCTTGTTCCAAGATAACTTGTTGACCAGGTTCTACTCCAATTCTAACTCTACCAGATTCTACTTGGAAAGGGTTTCCGAAATGGTCTTGGAATCTATTAGACCAATCGTCAGAGAAAAGAGGGTGTTCAGAAATTTGGAAATTGGTGCTGATACAGCCTCTTTGTCTAATGTTGTTTTGACTAAGTTGAACACCTACTTGTCCACCTTGAATTCTAATGGTGGTTCTGGTTACAACATCATCATTGAGTACACCTTCACCGATATTTCCGCCAACTTCATTATTGGTGAAATCCAAGAAACCATGTGCAACTTGTACCCAAACGTTACTTTCAAGTTCTCCGTCTTGGACTTGGAGAAAGAGATTATTAACTCCTCCGATTTCTTGATGGGTGATTACGATATAGTTTTGATGGCCTACGTTATCCATGCCGTTTCTAACATTAAGTTCTCCATCGAACAGTTGTACAAGTTGTTGTCTCCAAGAGGTTGGTTGTTGTGTATTGAACCTAAGTCCAACGTTGTGTTCTCCGATTTGGTTTTCGGTTGTTTTAATCAGTGGTGGAACTACTACGATGATATTAGAACTACCCACTGCTCCTTGTCTGAATCTCAATGGAATCAGTTGTTGTTGAACCAGTCCTTGAACAACGAATCCTCTTCTTCTTCTAACTGTTACGGTGGTTTCTCCAACGTTTCTTTTATTGGTGGTGAAAAGGATGTCGACTCCCATTCTTTCATATTGCACTGCCAAAAAGAATCCATCTCCCAAATGAAGTTAGCCACCACTATTAACAACGGTTTGTCATCTGGTTCCATCGTTATCGTTTTGAACTCTCAACAATTGACCAACATGAAGTCCTACCCAAAGGTTATTGAGTATATTCAAGAGGCTACCTCTTTGTGCAAGACCATTGAAATTATCGATTCCAAGGACGTCTTGAACTCTACCAATTCAGTTTTGGAAAAGATCCAAAAGTCCTTGTTGGTGTTCTGTTTGTTGGGTTATGACTTGTTGGAGAACAACTACCAAGAACAGTCTTTCGAATACGTTAAGTTGTTGAACTTGATCTCTACTACCGCCTCTTCATCTAATGATAAGAAACCACCAAAGGTCTTGTTGATCACCAAGCAATCTGAAAGAATCTCCAGGTCTTTCTACTCCAGATCCTTGATTGGTATTTCCAGAACCTCTATGAACGAGTACCCAAATTTGTCCATTACCTCTATCGATTTGGATACCAACGACTACTCATTGCAGTCTTTGTTGAAGCCAATCTTCAGCAACTCTAAGTTTTCCGACAACGAGTTCATCTTCAAAAAGGGCTTGATGTTCGTGTCCAGGATCTTTAAGAACAAGCAGTTGCTAGAATCCTCCAACGCTTTTGAAACTGACTCTTCTAACTTGTACTGTAAGGCCTCTTCTGACTTGTCTTACAAGTACGCTATTAAGCAGTCTATGTTGACCGAAAATCAGATCGAAATCAAGGTTGAATGCGTCGGTATTAACTTCAAGGACAACCTATTCTACAAGGGCTTGTTGCCACAAGAAATTTTCAGAATGGGTGACATCTACAATCCACCATATGGTTTGGAATGCTCTGGTGTTATTACCAGAATTGGTTCTAACGTCACCGAATACTCAGTTGGTCAAAATGTTTTTGGTTTCGCCAGACATTCTTTGGGTTCTCATGTTGTTACCAACAAGGATTTGGTTATCTTGAAGCCAGATACCATCTCATTTTCTGAAGCTGCTTCTATCCCAGTTGTTTACTGTACTGCTTGGTACTCCTTGTTCAACATTGGTCAGTTGTCTAACGAAGAATCCATCCTAATTCATTCTGCTACTGGTGGTGTAGGTTTGGCTTCTTTGAATTTGTTGAAAATGAAGAATCAGCAACAGCAACCATTGACCAATGTTTATGCTACTGTTGGCTCTAACGAGAAGAAGAAGTTCTTGATCGATAACTTCAACAACTTGTTCAAAGAGGACGGCGAAAACATTTTCTCTACCAGAGACAAAGAATACTCCAACCAGTTGGAATCCAAGATCGATGTTATTTTGAACACCTTGTCCGGTGAATTCGTCGAATCTAATTTCAAGTCCTTGAGATCCTTCGGTAGATTGATTGATTTGTCTGCTACTCACGTTTACGCCAATCAACAAATTGGTCTAGGTAACTTCAAGTTCGACCACTTGTATTCTGCTGTTGACTTGGAAAGATTGATCGACGAAAAACCTAAGTTGTTGCAGTCCATCTTGCAAAGAATTACCAACTCTATCGTCAACGGTTCCTTGGAAAAAATTCCAATTACCATCTTCCCATCCACCGAAACTAAGGATGCTATCGAATTATTGTCCAAGAGATCCCATATCGGTAAAGTTGTTGTAGATTGCACCGATATCTCTAAGTGTAATCCTGTTGGTGATGTGATCACCAACTTCTCTATGAGATTGCCAAAGCCAAACTACCAGTTGAATTTGAACTCCACCTTGTTGATTACTGGTCAGTCTGGTTTGTCTATCCCTTTGTTGAATTGGTTGTTGTCTAAGTCTGGTGGTAACGTTAAGAACGTTGTCATCATTTCTAAGTCCACCATGAAGTGGAAGTTGCAGACTATGATTTCCCATTTCGTTTCCGGTTTCGGTATCCATTTTAACTACGTTCAAGTCGACATCTCCAACTACGATGCTTTGTCTGAAGCTATTAAGCAATTGCCATCTGATTTGCCACCAATCACCTCTGTTTTTCATTTGGCTGCTATCTACAACGATGTTCCAATGGATCAAGTTACCATGTCTACCGTTGAATCTGTTCATAACCCTAAAGTTTTGGGTGCCGTTAACTTGCATAGAATCTCTGTTTCTTTTGGTTGGAAGTTGAACCACTTCGTCTTGTTCTCTTCTATTACTGCTATTACCGGTTACCCAGACCAATCTATCTACAATTCTGCCAACTCTATTTTGGACGCTTTGTCCAACTTTAGAAGGTTTATGGGTTTGCCATCCTTCTCCATTAACTTGGGTCCAATTAAGGATGAAGGTAAGGTTTCTACCAACAAGAGCATCAAGAAGCTATTCAAGTCTAGAGGTTTGCCAAGCCTATCCTTGAACAAGTTATTTGGTTTGTTGGAGGTCGTCATCAACAACCCATCTAATCATGTTATCCCATCCCAATTGATTTGCTCCCCAATCGATTTCAAGACCTACATCGAATCTTTCTCAACTATGAGGCCAAAGTTGTTACACTTGCAACCTACCATTTCCAAGCAGCAATCTTCTATCATTAACGATTCTACCAAGGCTTCCTCCAACATTTCATTGCAAGATAAGATCACCTCCAAGGTGTCTGATTTGTTGTCCATTCCAATCTCCAAGATCAACTTCGATCATCCATTGAAACACTACGGCTTGGATTCTTTGTTGACCGTTCAATTCAAATCCTGGATCGACAAAGAATTCGAAAAGAACTTGTTCACCCATATCCAATTGGCCACCATCTCTATTAACTCATTCTTGGAAAAGGTGAACGGCTTGTCTACAAACAATAACAACAACAACAATTCCAACGTCAAGTCCTCTCCATCCATTGTCAAAGAAGAAATCGTTACCTTGGACAAGGATCAACAACCATTGCTATTGAAAGAACACCAGCACATTATCATCTCCCCAGATATTAGAATCAACAAGCCAAAGAGGGAATCCTTGATTAGAACCCCAATCTTGAACAAATTCAACCAGATCACCGAATCCATTATCACTCCATCTACACCATCTTTGTCCCAATCCGATGTTTTGAAAACTCCACCAATCAAGTCTTTGAACAACACTAAGAACTCCAGCTTGATTAACACCCCACCAATTCAATCTGTCCAACAACATCAAAAGCAACAACAAAAGGTCCAAGTCATCCAACAACAGCAACAACCATTATCCAGATTGTCCTACAAGAGCAACAACAACTCTTTCGTTTTGGGTATCGGTATTTCTGTTCCAGGTGAACCTATTTCCCAACAATCCTTGAAAGACTCCATCTCCAATGACTTTTCTGATAAGGCTGAAACTAACGAGAAGGTCAAGAGAATCTTTGAGCAATCTCAAATCAAGACCAGACACTTGGTTAGAGATTACACTAAGCCAGAGAACTCCATCAAGTTCAGACATTTGGAAACCATTACCGATGTGAACAACCAGTTCAAGAAAGTTGTTCCAGATTTGGCTCAACAAGCCTGTTTGAGAGCTTTGAAAGATTGGGGTGGTGATAAGGGTGATATTACCCATATAGTTTCTGTTACCTCCACCGGTATTATCATCCCAGATGTTAATTTCAAGTTGATCGACTTGTTGGGCTTGAACAAGGATGTTGAAAGAGTGTCTTTGAACCTAATGGGTTGTTTGGCTGGTTTGAGTTCTTTGAGAACTGCTGCTTCTTTGGCTAAGGCTTCTCCAAGAAATAGAATTTTGGTTGTCTGTACCGAAGTCTGCTCCTTGCATTTTTCTAATACTGATGGTGGTGATCAAATGGTCGCCTCTTCTATTTTTGCTGATGGTTCTGCTGCTTACATTATTGGTTGTAACCCAAGAATTGAAGAAACCCCATTATACGAAGTCATGTGCTCCATTAACAGATCTTTCCCAAATACCGAAAACGCCATGGTTTGGGATTTGGAAAAAGAAGGTTGGAACTTGGGTTTGGATGCTTCTATTCCAATTGTCATTGGTTCTGGTATTGAAGCCTTCGTTGATACTTTGTTGGATAAGGCTAAGTTGCAAACTTCCACTGCTATTTCTGCTAAGGATTGCGAATTCTTGATTCATACTGGTGGCAAGTCCATCTTGATGAACATCGAAAATTCCTTGGGTATCGACCCAAAGCAAACTAAGAATACTTGGGATGTTTACCATGCCTACGGCAATATGTCATCTGCCTCTGTTATTTTCGTTATGGATCATGCCAGAAAGTCCAAGTCTTTGCCAACTTACTCAATTTCTTTGGCTTTTGGTCCAGGTTTGGCTTTTGAAGGTTGTTTCTTGAAGAACGTCGTCGAACAGAAGCTCATTTCCGAAGAGGACTTGTAACCGCAACAACTACCTCGACTTTGGCTGGGACACTTTCAGTGAGGACAAGAAGCTTCAGAAGCGTGCTATCGAACTCAACCAGGGACGTGCGGCACAAATGGGCATCCTTGCTCTCATGGTGCACGAACAGTTGGGAGTCTCTATCCTTCCTTAAAAATTTAATTTTCATTAGTTGCAGTCACTCCGCTTTGGTTTCGCTAGTGATAATAAGTGACTGAGGTATGTGCTCTTCTTATCTCCTTTTGTAGTGTTGCTCTTATTTTAAACAACTTTGCGGTTTTTTGATGACTTTGCGATTTTGTTGTTGCTTTGCAGTAAATTGCAAGATTTAATAAAAAAACGCAAAGCAATGATTAAAGGATGTTCAGAATGAAACTCATGGAAACACTTAACCAGTGCATAAACGCTGGTCATGAAATGACGAAGGCTATCGCCATTGCACAGTTTAATGATGACAGCCCGGAAGCGAGGAAAATAACCCGGCGCTGGAGAATAGGTGAAGCAGCGGATTTAGTTGGGGTTTCTTCTCAGGCTATCAGAGATGCCGAGAAAGCAGGGCGACTACCGCACCCGGATATGGAAATTCGAGGACGGGTTGAGCAACGTGTTGGTTATACAATTGAACAAATTAATCATATGCGTGATGTGTTTGGTACGCGATTGCGACGTGCTGAAGACGTATTTCCACCGGTGATCGGGGTTGCTGCCCATAAAGGTGGCGTTTACAAAACCTCAGTTTCTGTTCATCTTGCTCAGGATCTGGCTCTGAAGGGGCTACGTGTTTTGCTCGTGGAAGGTAACGACCCCCAGGGAACAGCCTCAATGTATCACGGATGGGTACCAGATCTTCATATTCATGCAGAAGACACTCTCCTGCCTTTCTATCTTGGGGAAAAGGACGATGTCACTTATGCAATAAAGCCCACTTGCTGGCCGGGGCTTGACATTATTCCTTCCTGTCTGGCTCTGCACCGTATTGAAACTGAGTTAATGGGCAAATTTGATGAAGGTAAACTGCCCACCGATCCACACCTGATGCTCCGACTGGCCATTGAAACTGTTGCTCATGACTATGATGTCATAGTTATTGACAGCGCGCCTAACCTGGGTATCGGCACGATTAATGTCGTATGTGCTGCTGATGTGCTGATTGTTCCCACGCCTGCTGAGTTGTTTGACTACACCTCCGCACTGCAGTTTTTCGATATGCTTCGTGATCTGCTCAAGAACGTTGATCTTAAAGGGTTCGAGCCTGATGTACGTATTTTGCTTACCAAATACAGCAATAGCAATGGCTCTCAGTCCCCGTGGATGGAGGAGCAAATTCGGGATGCCTGGGGAAGCATGGTTCTAAAAAATGTTGTACGTGAAACGGATGAAGTTGGTAAAGGTCAGATCCGGATGAGAACTGTTTTTGAACAGGCCATTGATCAACGCTCTTCAACTGGTGCCTGGAGAAATGCTCTTTCTATTTGGGAACCTGTCTGCAATGAAATTTTCGATCGTCTGATTAAACCACGCTGGGAGATTAGATAATGAAGCGTGCGCCTGTTATTCCAAAACATACGCTCAATACTCAACCGGTTGAAGATACTTCGTTATCGACACCAGCTGCCCCGATGGTGGATTCGTTAATTGCGCGCGTAGGAGTAATGGCTCGCGGTAATGCCATTACTTTGCCTGTATGTGGTCGGGATGTGAAGTTTACTCTTGAAGTGCTCCGGGGTGATAGTGTTGAGAAGACCTCTCGGGTATGGTCAGGTAATGAACGTGACCAGGAGCTGCTTACTGAGGACGCACTGGATGATCTCATCCCTTCTTTTCTACTGACTGGTCAACAGACACCGGCGTTCGGTCGAAGAGTATCTGGTGTCATAGAAATTGCCGATGGGAGTCGCCGTCGTAAAGCTGCTGCACTTACCGAAAGTGATTATCGTGTTCTGGTTGGCGAGCTGGATGATGAGCAGATGGCTGCATTATCCAGATTGGGTAACGATTATCGCCCAACAAGTGCTTATGAACGTGGTCAGCGTTATGCAAGCCGATTGCAGAATGAATTTGCTGGAAATATTTCTGCGCTGGCTGATGCGGAAAATATTTCACGTAAGATTATTACCCGCTGTATCAACACCGCCAAATTGCCTAAATCAGTTGTTGCTCTTTTTTCTCACCCCGGTGAACTATCTGCCCGGTCAGGTGATGCACTTCAAAAAGCCTTTACAGATAAAGAGGAATTACTTAAGCAGCAGGCATCTAACCTTCATGAGCAGAAAAAAGCTGGGGTGATATTTGAAGCTGAAGAAGTTATCACTCTTTTAACTTCTGTGCTTAAAACGTCATCTGCATCAAGAACTAGTTTAAGCTCACGACATCAGTTTGCTCCTGGAGCGACAGTATTGTATAAGGGCGATAAAATGGTGCTTAACCTGGACAGGTCTCGTGTTCCAACTGAGTGTATAGAGAAAATTGAGGCCATTCTTAAGGAACTTGAAAAGCCAGCACCCTGATGCGACCTCGTTTTAGTCTACGTTTATCTGTCTTTACTTAATGTCCTTTGTTACAGGCCAGAAAGCATAACTGGCCTGAATATTCTCTCTGGGCCCACTGTTCCACTTGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCATGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGAACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGATCCCACTCGTGTTGTCGGTCTGATTATCGGTCTGGGACCACGGTCCCACTTGTATTGTCGATCAGACTATCAGCGTGAGACTACGATTCCATCAATGCCTGTCAAGGGCAAGTATTGACATGTCGTCGTAACCTGTAGAACGGAGTAACCTCGGTGTGCGGTTGTATGCCTGCTGTGGATTGCTGCTGTGTCCTGCTTATCCACAACATTTTGCGCACGGTTATGTGGACAAAATACCTGGTTACCCAGGCCGTGCCGGCACGTTAACCGGGCTGCATCCGATGCAAGTGTGTCGCTGTCGACGAGCTCGCGAGCTCGGACATGAGGTTGCCCCGTATTCAGTGTCGCTGATTTGTATTGTCTGAAGTTGTTTTTACGTTAAGTTGATGCAGATCAATTAATACGATACCTGCGTCATAATTGATTATTTGACGTGGTTTGATGGCCTCCACGCACGTTGTGATATGTAGATGATAATCATTATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTACGGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGCTTTTTGGCCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATACATTATACGAAGTTATA |
References
- Śledziński, P.; Nowak-Terpiłowska, A.; Zeyland, J. Cannabinoids in medicine: Cancer, immunity, and microbial diseases. Int. J. Mol. Sci. 2020, 22, 263. [Google Scholar] [CrossRef]
- Crocq, M.-A. History of cannabis and the endocannabinoid system. Dialogues Clin. Neurosci. 2020, 22, 223–228. [Google Scholar] [CrossRef]
- Luo, X.; Reiter, M.A.; D’eSpaux, L.; Wong, J.; Denby, C.M.; Lechner, A.; Zhang, Y.; Grzybowski, A.T.; Harth, S.; Lin, W.; et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 2019, 567, 123–126. [Google Scholar] [CrossRef] [PubMed]
- Lewis, M.M.; Yang, Y.; Wasilewski, E.; Clarke, H.A.; Kotra, L.P. Chemical profiling of medical cannabis extracts. ACS Omega 2017, 2, 6091–6103. [Google Scholar] [CrossRef] [PubMed]
- Backer, R.; Schwinghamer, T.; Rosenbaum, P.; McCarty, V.; Bilodeau, S.E.; Lyu, D.; Ahmed, B.; Robinson, G.; Lefsrud, M.; Wilkins, O.; et al. Closing the yield gap for cannabis: A meta-analysis of factors determining cannabis yield. Front. Plant Sci. 2019, 10, 495. [Google Scholar] [CrossRef] [PubMed]
- Hara, K.Y.; Morita, T.; Mochizuki, M.; Yamamoto, K.; Ogino, C.; Kondo, A. Development of bio-based fine chemical production through synthetic bioengineering. Microb. Cell Factories 2014, 13, 173. [Google Scholar] [CrossRef]
- Clomburg, J.M.; Crumbley, A.M.; Gonzalez, R. Industrial biomanufacturing: The future of chemical production. Science 2017, 355, aag0804. [Google Scholar] [CrossRef]
- Wilding, K.M.; Schinn, S.-M.; Long, E.A.; Bundy, B.C. The emerging impact of cell-free chemical biosynthesis. Curr. Opin. Biotechnol. 2018, 53, 115–121. [Google Scholar] [CrossRef]
- Terol, G.L.; Gallego-Jara, J.; Martínez, R.A.S.; Díaz, M.C.; Puente, T.d.D. Engineering protein production by rationally choosing a carbon and nitrogen source using E. coli BL21 acetate metabolism knockout strains. Microb. Cell Factories 2019, 18, 151. [Google Scholar] [CrossRef]
- Parapouli, M.; Vasileiadi, A.; Afendra, A.-S.; Hatziloukas, E. Saccharomyces cerevisiae and its industrial applications. AIMS Microbiol. 2020, 6, 1. [Google Scholar] [CrossRef]
- Overkamp, K.M.; Bakker, B.M.; Kotter, P.; Luttik, M.A.H.; van Dijken, J.P.; Pronk, J.T. Metabolic engineering of glycerol production in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2002, 68, 2814–2821. [Google Scholar] [CrossRef] [PubMed]
- Fantino, E.; Awwad, F.; Merindol, N.; Garza, A.M.D.; Gélinas, S.-E.; Robles, G.C.G.; Custeau, A.; Meddeb-Mouelhi, F.; Desgagné-Penix, I. Bioengineering Phaeodactylum tricornutum, a marine diatom, for cannabinoid biosynthesis. Algal Res. 2024, 77, 103379. [Google Scholar] [CrossRef]
- Hempel, F.; Lau, J.; Klingl, A.; Maier, U.G. Algae as protein factories: Expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum. PLoS ONE 2011, 6, e28424. [Google Scholar] [CrossRef] [PubMed]
- Branco-Vieira, M.; Martin, S.S.; Agurto, C.; Freitas, M.A.; Martins, A.A.; Mata, T.M.; Caetano, N.S. Biotechnological potential of Phaeodactylum tricornutum for biorefinery processes. Fuel 2020, 268, 117357. [Google Scholar] [CrossRef]
- Cui, Y.; Thomas-Hall, S.R.; Chua, E.T.; Schenk, P.M. Development of high-level omega-3 eicosapentaenoic acid (EPA) production from Phaeodactylum tricornutum. J. Phycol. 2021, 57, 258–268. [Google Scholar] [CrossRef]
- Awwad, F.; Fantino, E.I.; Héneault, M.; Diaz-Garza, A.M.; Merindol, N.; Custeau, A.; Gélinas, S.-E.; Meddeb-Mouelhi, F.; Li, J.; Lemay, J.-F.; et al. Bioengineering of the Marine Diatom Phaeodactylum tricornutum with Cannabis Genes Enables the Production of the Cannabinoid Precursor, Olivetolic Acid. Int. J. Mol. Sci. 2023, 24, 16624. [Google Scholar] [CrossRef]
- Dhaouadi, F.; Awwad, F.; Diamond, A.; Desgagné-Penix, I. Diatoms’ breakthroughs in biotechnology: Phaeodactylum tricornutum as a model for producing high-added value molecules. Am. J. Plant Sci. 2020, 11, 1632–1670. [Google Scholar] [CrossRef]
- Butler, T.O.; Padmaperuma, G.; Lizzul, A.M.; McDonald, J.; Vaidyanathan, S. Towards a Phaeodactylum tricornutum biorefinery in an outdoor UK environment. Bioresour. Technol. 2022, 344, 126320. [Google Scholar] [CrossRef]
- Cui, Y.; Thomas-Hall, S.R.; Schenk, P.M. Phaeodactylum tricornutum microalgae as a rich source of omega-3 oil: Progress in lipid induction techniques towards industry adoption. Food Chem. 2019, 297, 124937. [Google Scholar] [CrossRef]
- BioCyC. hexanoyl-CoA. 2024. Available online: https://biocyc.org/compound?orgid=PHAEO&id=HEXANOYL-COA#RXNS (accessed on 23 October 2024).
- Fortunato, A.; Queller, D.C.; Strassmann, J.E.; Santorelli, L.A. Co-occurrence in nature of different clones of the social amoeba, Dictyostelium discoideum. Mol. Ecol. 2003, 12, 1031–1038. [Google Scholar] [CrossRef]
- Aubry, L.; Firtel, R. Integration of signaling networks that regulate Dictyostelium differentiation. Annu. Rev. Cell Dev. Biol. 1999, 15, 469–517. [Google Scholar] [CrossRef]
- Narita, T.B.; Ishida, M.; Namatame, M.; Ueda, M.; Suematsu, N.; Ohishi, K.; Urushihara, H.; Fukuzawa, H.; Shimada, N.; Enomoto, K.; et al. Dictyostelium hybrid polyketide synthase, SteelyA, produces 4-methyl-5-pentylbenzene-1, 3-diol and induces spore maturation. FEMS Microbiol. Lett. 2011, 319, 82–87. [Google Scholar] [CrossRef]
- Reimer, C.; Poschmann, G.; Stühler, K.; Kruse, J.; Stork, T.; Schmitt, M.; Schulz, B.; Kayser, O.; Anjard, C.; Winckler, T. Engineering the amoeba Dictyostelium discoideum for biosynthesis of a cannabinoid precursor and other polyketides. Nat. Biotechnol. 2022, 40, 751–758. [Google Scholar] [CrossRef] [PubMed]
- Anjard, C.; Su, Y.; Loomis, W.F. The polyketide MPBD initiates the SDF-1 signaling cascade that coordinates terminal differentiation in Dictyostelium. Eukaryot. Cell 2011, 10, 956–963. [Google Scholar] [CrossRef] [PubMed]
- Ghosh, R.; Chhabra, S.; Phan, A.N.; Phan, Q.N.; Ennis, H.L.; Siu, C.H.; Alexander, S.; Eichinger, L.; Glöckner, G.; Loomis, W.F. Dissecting the functional role of polyketide synthases in Dictyostelium discoideum: Biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1, 3-diol. J. Biol. Chem. 2008, 283, 11348–11354. [Google Scholar] [CrossRef]
- Mookerjee, S.; McCafferty, J.; Nachman, R.; Keasling, J.D. Method and Cell Line for Production of Phytocannabinoids and Phytocannabinoid Analogues in Yeast. Patent WO2018148848A1, 23 August 2018. [Google Scholar]
- Diamond, A.; Desgagné-Penix, I.; Fantino, E.; Awwad, F.; Lemay, J.-F.; Meddeb-Mouelhi, F. Instability of extrachromosomal DNA transformed into the diatom Phaeodactylum tricornutum. Algal Res. 2023, 70, 102998. [Google Scholar] [CrossRef]
- Karas, B.J.; Diner, R.E.; Lefebvre, S.C.; McQuaid, J.; Phillips, A.P.R.; Noddings, C.M.; Brunson, J.K.; Valas, R.E.; Deerinck, T.J.; Jablanovic, J.; et al. Designer diatom episomes delivered by bacterial conjugation. Nat. Commun. 2015, 6, 6925. [Google Scholar] [CrossRef]
- Elso, C.M.; Roberts, L.J.; Smyth, G.K.; Thomson, R.J.; Baldwin, T.M.; Foote, S.J.; Handman, E. Leishmaniasis host response loci (lmr1-3) modify disease severity through a Th1/Th2-independent pathway. Genes Immun. 2004, 5, 93–100. [Google Scholar] [CrossRef]
- Messaabi, A.; Awwad, F.; Fantino, E.; Desgagné-Penix, I. In vivo thrombin activity in the diatom Phaeodactylum tricornutum: Biotechnological insights. Appl. Microbiol. Biotechnol. 2024, 108, 481. [Google Scholar] [CrossRef]
- Austin, M.B.; Saito, T.; Bowman, M.E.; Haydock, S.F.; Kato, A.; Moore, B.S.; Kay, R.R.; Noel, J.P. Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid–type III polyketide synthase. Nat. Chem. Biol. 2006, 2, 494–502. [Google Scholar] [CrossRef]
- Mital, S.; Christie, G.; Dikicioglu, D. Recombinant expression of insoluble enzymes in Escherichia coli: A systematic review of experimental design and its manufacturing implications. Microb. Cell Factories 2021, 20, 1–20. [Google Scholar] [CrossRef]
- Athanasakoglou, A.; Kampranis, S.C.; Degenhardt, J.; Makris, A.M. Isoprenoid biosynthesis in the diatom Haslea ostrearia. New Phytol. 2019, 222, 230–243. [Google Scholar] [CrossRef]
- Solá, R.J.; Griebenow, K. Effects of glycosylation on the stability of protein pharmaceuticals. J. Pharm. Sci. 2009, 98, 1223–1245. [Google Scholar] [CrossRef] [PubMed]
- Stanley, P. Golgi glycosylation. Cold Spring Harb. Perspect. Biol. 2011, 3, a005199. [Google Scholar] [CrossRef] [PubMed]
- Dumontier, R.; Loutelier-Bourhis, C.; Walet-Balieu, M.-L.; Burel, C.; Mareck, A.; Afonso, C.; Lerouge, P.; Bardor, M. Identification of N-glycan oligomannoside isomers in the diatom Phaeodactylum tricornutum. Carbohydr. Polym. 2021, 259, 117660. [Google Scholar] [CrossRef]
- Mathieu-Rivet, E.; Kiefer-Meyer, M.-C.; Vanier, G.; Ovide, C.; Burel, C.; Lerouge, P.; Bardor, M. Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals. Front. Plant Sci. 2014, 5, 359. [Google Scholar] [CrossRef]
- Sharma, N.; Simon, D.P.; Diaz-Garza, A.M.; Fantino, E.; Messaabi, A.; Meddeb-Mouelhi, F.; Germain, H.; Desgagn-Penix, I. Diatoms Biotechnology: Various Industrial Applications for a Greener Tomorrow. Front. Mar. Sci. 2021, 8, 636613. [Google Scholar] [CrossRef]
Figure 1.
Proposed reactions catalyzed by the SteelyA enzyme. Bolded molecules indicate products observed from SteelyA activity. Left reaction: The native reaction of SteelyA in Dictyostelium discoideum, as reported by [27], where SteelyA utilizes acetyl-CoA and five malonyl-CoA molecules to produce MPBD. Middle reaction: The reaction described by [25] in D. discoideum showing that SteelyA can use a hexanoyl-CoA starter unit along with three malonyl-CoA molecules to generate methyl-olivetol. Right reaction: represents the reaction observed by heterologous production of SteelyA in Saccharomyces cerevisiae, as described by [28].
Figure 1.
Proposed reactions catalyzed by the SteelyA enzyme. Bolded molecules indicate products observed from SteelyA activity. Left reaction: The native reaction of SteelyA in Dictyostelium discoideum, as reported by [27], where SteelyA utilizes acetyl-CoA and five malonyl-CoA molecules to produce MPBD. Middle reaction: The reaction described by [25] in D. discoideum showing that SteelyA can use a hexanoyl-CoA starter unit along with three malonyl-CoA molecules to generate methyl-olivetol. Right reaction: represents the reaction observed by heterologous production of SteelyA in Saccharomyces cerevisiae, as described by [28].
Figure 2.
Schematic representation of the construction of the episomal plasmid pSteelyA. The pPtGE30 plasmid, referred to as the Empty Vector (EV), is 16,015 bp in length. The inserted cassette consists of the SteelyA coding sequence (brown) under the control of the native highly abundant secreted protein 1 (Hasp1) promoter (light blue). The sequence is tagged with a Myc marker (dark blue) and includes a fucoxanthin-chlorophyll binding protein A (FcpA) terminator (red). The insertion cassette is 10,174 bp long, resulting in the pSteely plasmid with a total length of 26,189 bp. Abbreviations: CEN-HIS-ARS, centromere-histidine-autonomously replicating sequence region; OriT, origin of transfer; ShBle zeocin resistance gene; CmR, chloramphenicol resistance gene.
Figure 2.
Schematic representation of the construction of the episomal plasmid pSteelyA. The pPtGE30 plasmid, referred to as the Empty Vector (EV), is 16,015 bp in length. The inserted cassette consists of the SteelyA coding sequence (brown) under the control of the native highly abundant secreted protein 1 (Hasp1) promoter (light blue). The sequence is tagged with a Myc marker (dark blue) and includes a fucoxanthin-chlorophyll binding protein A (FcpA) terminator (red). The insertion cassette is 10,174 bp long, resulting in the pSteely plasmid with a total length of 26,189 bp. Abbreviations: CEN-HIS-ARS, centromere-histidine-autonomously replicating sequence region; OriT, origin of transfer; ShBle zeocin resistance gene; CmR, chloramphenicol resistance gene.
Figure 3.
Growth and morphological analysis of P. tricornutum SteelyA and empty vector (EV) control transconjugant cell lines. (A) Growth curves of SteelyA transconjugants and EV control cultured in L1 at 18 °C. Optical density (OD) at 730 nm was monitored for 21 days. Data represent the mean of three biological replicates, with error bars indicating standard deviation. t-test, p < 0.05. Part (B) represents the length of SteelyA transconjugant cell lines (different symbols have been used to ease the distinction between samples; circle is EV, square is pSteely-1, triangle is pSteely-2 and reverse-triangle is pSteely-3). Part (C) represents the width of SteelyA transconjugant cell lines. Aligned dot plot of cell length and width in pixels. (n ≤ 5 cells per condition).
Figure 3.
Growth and morphological analysis of P. tricornutum SteelyA and empty vector (EV) control transconjugant cell lines. (A) Growth curves of SteelyA transconjugants and EV control cultured in L1 at 18 °C. Optical density (OD) at 730 nm was monitored for 21 days. Data represent the mean of three biological replicates, with error bars indicating standard deviation. t-test, p < 0.05. Part (B) represents the length of SteelyA transconjugant cell lines (different symbols have been used to ease the distinction between samples; circle is EV, square is pSteely-1, triangle is pSteely-2 and reverse-triangle is pSteely-3). Part (C) represents the width of SteelyA transconjugant cell lines. Aligned dot plot of cell length and width in pixels. (n ≤ 5 cells per condition).
Figure 4.
Detection of SteelyA protein in total protein extracts. (a) Western blot analysis using anti-myc on total protein extracts (T) and soluble protein fractions (S) from pSteelyA-1, pSteelyA-2, and pSteelyA-3 transconjugant cell lines. Proteins were separated on a GenScript SurePage Bris-Tris gel 4–12%. The empty vector (EV) transconjugants serve as a negative control. The SteelyA (StlA) protein is expected to be approximately 352 kDa. (b) Total protein staining of the blot using Ponceau Red (RP) to confirm protein transfer and loading consistency.
Figure 4.
Detection of SteelyA protein in total protein extracts. (a) Western blot analysis using anti-myc on total protein extracts (T) and soluble protein fractions (S) from pSteelyA-1, pSteelyA-2, and pSteelyA-3 transconjugant cell lines. Proteins were separated on a GenScript SurePage Bris-Tris gel 4–12%. The empty vector (EV) transconjugants serve as a negative control. The SteelyA (StlA) protein is expected to be approximately 352 kDa. (b) Total protein staining of the blot using Ponceau Red (RP) to confirm protein transfer and loading consistency.
Figure 5.
Total ion chromatogram (TIC) from LC-MS analysis in single ion monitoring (SIM) mode. The chromatogram includes a mix of standards showing expected peaks (blue) and triplicate samples from the empty vector (EV) control, and pSteelyA-1, pSteelyA-2, and pSteelyA-3 transconjugant lines (various colors). No transconjugant exhibited peaks corresponding to the expected SteelyA products. The monitored m/z values are listed in Table 1.
Figure 5.
Total ion chromatogram (TIC) from LC-MS analysis in single ion monitoring (SIM) mode. The chromatogram includes a mix of standards showing expected peaks (blue) and triplicate samples from the empty vector (EV) control, and pSteelyA-1, pSteelyA-2, and pSteelyA-3 transconjugant lines (various colors). No transconjugant exhibited peaks corresponding to the expected SteelyA products. The monitored m/z values are listed in Table 1.
Table 1.
Targeted compounds and their corresponding m/z values in positive ion mode or LC-MS analysis.
Table 1.
Targeted compounds and their corresponding m/z values in positive ion mode or LC-MS analysis.
| Compound Name | Verified m/z in M + H+ |
|---|---|
| Triketide acyl pyrone (=4-hydroxy-6-propyl-pyran-2-one) | 156 |
| Olivetol | 181 |
| Triketide pyrone | 182 |
| Methyl olivetol and MPBD (4-methyl-5-pentylbenzene-1,3-diol) | 195 |
| Tetraketide acyl pyrone (=4-hydroxy-6-(2-oxo-pentyl)-pyran-2-one) | 196 |
| Olivetolic acid | 225 |
| Tetraketide pyrone | 225 |
| Naringenin chalcone | 273 |
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Sene, N.; Lamichhane, B.; Gélinas, S.-E.; Custeau, A.; Merindol, N.; Meddeb-Mouelhi, F.; Desgagné-Penix, I. Production of Dictyostelium discoideum Hybrid Type Enzyme SteelyA in the Diatom Phaeodactylum tricornutum. Appl. Sci. 2025, 15, 11679. https://doi.org/10.3390/app152111679
AMA Style
Sene N, Lamichhane B, Gélinas S-E, Custeau A, Merindol N, Meddeb-Mouelhi F, Desgagné-Penix I. Production of Dictyostelium discoideum Hybrid Type Enzyme SteelyA in the Diatom Phaeodactylum tricornutum. Applied Sciences. 2025; 15(21):11679. https://doi.org/10.3390/app152111679
Chicago/Turabian StyleSene, Nicolas, Basanta Lamichhane, Sarah-Eve Gélinas, Alexandre Custeau, Natacha Merindol, Fatma Meddeb-Mouelhi, and Isabel Desgagné-Penix. 2025. "Production of Dictyostelium discoideum Hybrid Type Enzyme SteelyA in the Diatom Phaeodactylum tricornutum" Applied Sciences 15, no. 21: 11679. https://doi.org/10.3390/app152111679
APA StyleSene, N., Lamichhane, B., Gélinas, S.-E., Custeau, A., Merindol, N., Meddeb-Mouelhi, F., & Desgagné-Penix, I. (2025). Production of Dictyostelium discoideum Hybrid Type Enzyme SteelyA in the Diatom Phaeodactylum tricornutum. Applied Sciences, 15(21), 11679. https://doi.org/10.3390/app152111679
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