3rd Generation Packaging System Mix
| Cat. No. | LV053 | ||
| Name | 3rd Generation Packaging System Mix | ||
| Unit | 200µl | ||
| Description |
For the production of lentiviral particles, three components are generally required: 1) a lentiviral vector containing your inserts of interest, 2) one or two packaging vectors which contain all necessary viral structure proteins, 3) an envelope vector expressing Vesicular Stomatitis Virus (VSV) glycoprotein (G). The 3rd generation packaging system offers maximal biosafety as the lentiviral Rev gene is supplied as an independent vector from other structure genes, further eliminating the possibility of reverse recombination of vectors into replication competent viral particles. The 3rd generation lentiviral packaging mix will only support lentiviral expression vectors with a chimeric 5' LTR in which the HIV promoter is replaced with CMV or RSV, thus making it TAT-independent. The 3rd generation lentiviral vectors will not support the production of 2nd generation lentiviral particle productions. All of the lentiviral vectors marketed by ABM are TAT-independent.
Components (200ul mix of 3 plasmids):
*User Manual Refer to website for downloadable user manual |
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| Additional Information |
Shelf Life: Up to 1 year (when stored at -20°C in a frost-free freezer). |
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| Note |
NOT FOR RESALE without prior written consent of ABM. This product is distributed for laboratory research only. |
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| Promotions |
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| Material Citation | If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. LV053 |
Datasheet
| What is the usual survival rate of cells after initial infection? I transduced two types of cells and after 72 hour incubation, one cell line had 50% dead, the other had just about 85-90% dead. Any pointers? | |
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Each cell line is different; like two different people, one with a drug sensitivity and one without, there is no means of comparison between cell lines. There are variations in the membrane transporters for the selection marker as well as cell transduction efficiency, so each cell line must be treated differently and their optimal killing curves must be determined independently.
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| Are there any safety issues to consider if using lentiviral vectors in the same laboratory where replication competent adenovirus work is being carried out? For instance, can elements of one become incorporated into another under certain conditions (e.g . giving rise to a replication competent retrovirus)? | |
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If the adenovirus does not code for the viral packaging genes of the lentivirus, there is no safety issue. To remove all safety concerns, we recommend not to clone the viral packaging genes of the lentivirus into the adenoviral genome.
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| Which cell line is better for high-titer lentivirus production - 293T or 293FT cells? | |
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Our 293T was clonally selected to be fast-growing and transfected easily and thus also supports high-level expression of viral proteins. We routinely use them to produce very high titer (10^10) lentivirus. Both cell lines are capable for proficient lentivirus production.
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| What is the ratio of the three packaging plasmids when packaging via 293 cells? | |
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The three plasmids are present in equal molarity and in equal ratio.
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| Is it possible to grow the plasmids up when they are mixed? | |
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No, it is not possible to grow up the plasmids from the product.
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| Which system should I use to package my TAT dependent vector? | |
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TAT dependent vectors will need to be packaged using a 2nd generation system (abm Cat LV003). 3rd generation packaging systems (abm Cat. LV053) will not be compatible with this type of vector.
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| What is the titer for viruses made by Cat# LV053 with 293T cells? | |
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For both our 2nd or 3rd generation packaging mixes, the crude viral supernatant (i.e. not concentrated nor purified) titer will be around 10^6IU/ml.
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| Is RRE present? | |
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Yes, it is present after Pol in pLenti-P3A.
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- Ordelheide, AM et al. "Nor-1, a novel incretin-responsive regulator of insulin genes and insulin secretion" Mol Metab 2(3):243-55 (2013). DOI: 10.1016/j.molmet.2013.06.003. PubMed: 24044104.
- Kim, D et al. "Peroxisomal dysfunction is associated with up-regulation of apoptotic cell death via miR-223 induction in knee osteoarthritis patients with type 2 diabetes mellitus" Bone 64C:124-131 (2014). DOI: 10.1016/j.bone.2014.04.001. PubMed: 24727161.
- Kim, D et al. " Two non-coding RNAs, MicroRNA-101 and HOTTIP contribute cartilage integrity by epigenetic and homeotic regulation of integrin-α1" Cell. Signal. 25:2878 - 87 (2013). DOI: 10.1016/j.cellsig.2013.08.034. PubMed: 24018042. Application: Viral Packaging.
- George, R et al. "A SHORT INTERFERING RNA MOLECULAR BEACON FOR THE ATTENUATION OF MYCOBACTERIAL INFECTION" American Journal of Biochemistry and Biotechnology 10:40-49 (2014). DOI: 10.3844/ajbbsp.2014.40.49. Application: Viral Packaging.
- Song, J et al. "A long non-coding RNA, GAS5, plays a critical role in the regulation of miR-21 during osteoarthritis" Journal of Orthopaedic Research 32(12):1628-1635 (2014). DOI: 10.1002/jor.22718. PubMed: 25196583.
- Song, J et al. "MicroRNA-222 regulates MMP-13 via targeting HDAC-4 during osteoarthritis pathogenesis" BBA Clin : (2014). DOI: 10.1016/j.bbacli.2014.11.009. PubMed: 26673737.
- Morgan, S et al. "β-Agonist-mediated relaxation of airway smooth muscle is protein kinase A-dependent" J Biol Chem 289(33):23065-23075 (2014). DOI: 10.1074/jbc.M114.557652. PubMed: 24973219.
- Kim, D et al. "Peroxisomal dysfunction is associated with up-regulation of apoptotic cell death via miR-223 induction in knee osteoarthritis patients with type 2 diabetes mellitus" Bone 64:124-131 (2014). DOI: 10.1016/j.bone.2014.04.001. PubMed: 24727161. Application: Viral Packaging.
- Song, J et al. "miR-370 and miR-373 regulate the pathogenesis of osteoarthritis by modulating one-carbon metabolism via SHMT-2 and MECP-2, respectively" Aging Cell 5:826-837 (2015). DOI: 10.1111/acel.12363. PubMed: 26103880.
- Song, J et al. "A long non-coding RNA, GAS5, plays a critical role in the regulation of miR-21 during osteoarthritis" J Orthop Res 32(12):1628-1635 (2014). DOI: 10.1002/jor.22718. PubMed: 25196583. Application: Gene Delivery, Lentivirus.
- Song, J et al. "MicroRNA-222 regulates MMP-13 via targeting HDAC-4 during osteoarthritis pathogenesis" BBA Clin. :79-89 (2015). DOI: 10.1016/j.bbacli.2014.11.009.
- Song, J et al. "PBMC and exosome-derived Hotair is a critical regulator and potent marker for rheumatoid arthritis" Clin Exp Med. 1:121-126 (2015). DOI: 10.1007/s10238-013-0271-4. PubMed: 24722995.
- Kang, Y et al. "PCGEM1 stimulates proliferation of osteoarthritic synoviocytes by acting as a sponge for miR-770" J Orthop Res. : (2015). DOI: 10.1002/jor.23046.
- Rong, H et al. ""Notch signaling suppresses regulatory T cell function in murine experimental autoimmune uveitis "" Immunology 149.4:447-459 (2016). DOI: 10.1111/imm.12663.
- Wang, J et al. "Radiofrequency hyperthermia-enhanced herpes simplex virus-thymidine kinase/ganciclovir direct intratumoral gene therapy of hepatocellular carcinoma" American Journal of Cancer Research 6.9:2054–2063 (2016). DOI: 10.1080/02656736.2016.1229045. Application: Animal infection.
- Kang, et al. "PCGEM1 stimulates proliferation of osteoarthritic synoviocytes by acting as a sponge for miR-770" Journal of Orthopaedic Research 34.3:412 (2016). DOI: 10.1002/jor.23046. Application: Lentiviral infection.
- Jin, G., Li, Y., Zhang, F., Li, P., Zhao, L., Zhou, Y., ... & Yang, X. "Epithelial ovarian cancer: feasibility of image-guided intratumoral radiofrequency hyperthermia-enhanced direct gene therapy" American journal of cancer research 9(2):378 (2019).
- Omura, H., Liu, F., Shimakami, T., Murai, K., Shirasaki, T., Kitabayashi, J., Funaki, M., Nishikawa, T., Nakai, R., Sumiyadorj, A., Hayashi, T., Yamashita, T., Honda, M., Kaneko, S. "Establishment and Characterization of a New Cell Line Permissive for Hepatitis C Virus Infection" Scientific Reports 9:7943 (2019). DOI: 10.1038/s41598-019-44257-5.
- Takemoto, K., Kobatake, K., Miura, K., Fukushima, T., Babasaki, T., Miyamoto, S., Sekino, Y., Kitano, H., Goto, K., Ikeda, K., Hieda, K., Hayashi, T., Hinata, N., & Kaminuma, O. (2022). <scp>BACH1</scp> promotes clear cell renal cell carcinoma progression by upregulating oxidative stress‐related tumorigenicity. Cancer Science, 114(2), 436–448. https://doi.org/10.1111/cas.15607
- Hung, C.-N., Chen, M., DeArmond, D. T., Chiu, C. H.-L., Limboy, C. A., Tan, X., Kusi, M., Chou, C.-W., Lin, L.-L., Zhang, Z., Wang, C.-M., Chen, C.-L., Mitsuya, K., Osmulski, P. A., Gaczynska, M. E., Kirma, N. B., Vadlamudi, R. K., Gibbons, D. L., Warner, S., … Taverna, J. A. (2023). AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages. Cell Reports, 42(9), 113067. https://doi.org/10.1016/j.celrep.2023.113067
- Liu, P., Nadeef, S., Serag, M. F., Paytuví-Gallart, A., Abadi, M., Della Valle, F., Radío, S., Roda, X., Dilmé Capó, J., Adroub, S., Hosny El Said, N., Fallatah, B., Celii, M., Messa, G. M., Wang, M., Li, M., Tognini, P., Aguilar-Arnal, L., Habuchi, S., … Orlando, V. (2024). PRC2-EZH1 contributes to circadian gene expression by orchestrating chromatin states and RNA polymerase II complex stability. The EMBO Journal, 43(23), 6052–6075. https://doi.org/10.1038/s44318-024-00267-2
- Maurin, M., Ranjouri, M., Megino-Luque, C., Newberg, J. Y., Du, D., Martin, K., Miner, R. E., Prater, M. S., Wee, D. K. B., Centeno, B., Pruett-Miller, S. M., Stewart, P., Fleming, J. B., Yu, X., Bravo-Cordero, J. J., Guccione, E., Black, M. A., & Mann, K. M. (2023). RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing. Nature Communications, 14(1). https://doi.org/10.1038/s41467-023-44126-w
- Ryplida, B., Kim, D., Jin, E.-J., & Park, S. Y. (2023). Coenzyme a-responsive hydrogel sensor for wireless monitoring and treatment of osteoarthritis. Chemical Engineering Journal, 476, 146479. https://doi.org/10.1016/j.cej.2023.146479
- Yang, L., Pham, K., Xi, Y., Wu, Q., Liu, D., Robertson, K., & Liu, C. (2024). Exploring Glypican-3 Targeted CAR-NK Treatment and Potential Therapy Resistance in Hepatocellular Carcinoma. https://doi.org/10.22541/au.170664301.11370201/v1
- Shirasaki, T., González-López, O., McKnight, K. L., Xie, L., Shiota, T., Chen, X., Feng, H., & Lemon, S. M. (2022). Nonlytic Quasi-Enveloped Hepatovirus Release Is Facilitated by pX Protein Interaction with the E3 Ubiquitin Ligase ITCH. Journal of Virology, 96(21). https://doi.org/10.1128/jvi.01195-22
- Lai, T. H., Ahmed, M., Hwang, J. S., Zada, S., Pham, T. M., Elashkar, O., & Kim, D. R. (2021). Transcriptional Repression of Raf Kinase Inhibitory Protein Gene by Metadherin during Cancer Progression. International Journal of Molecular Sciences, 22(6), 3052–3052. https://doi.org/10.3390/ijms22063052
- Chan, C.-H., Chiou, L.-W., Lee, T.-Y., Liu, Y.-R., Hsieh, T.-H., Yang, C.-Y., & Jeng, Y.-M. (2022). PAK and PI3K pathway activation confers resistance to KRASG12C inhibitor sotorasib. British Journal of Cancer, 128(1), 148–159. https://doi.org/10.1038/s41416-022-02032-w
- Chou, C.-W., Hung, C.-N., Chiu, C. H.-L., Tan, X., Chen, M., Chen, C.-C., Saeed, M., Hsu, C.-W., Liss, M. A., Wang, C.-M., Lai, Z., Alvarez, N., Osmulski, P. A., Gaczynska, M. E., Lin, L.-L., Ortega, V., Kirma, N. B., Xu, K., Liu, Z., … Huang, T. H.-M. (2023). Phagocytosis-initiated tumor hybrid cells acquire a c-Myc-mediated quasi-polarization state for immunoevasion and distant dissemination. Nature Communications, 14(1). https://doi.org/10.1038/s41467-023-42303-5
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col-md-4\u0022\u003E\n\u003Cdiv class=\u0022col-sm-1 col-md-1\u0022 style=\u0022padding-left: 0px; padding-bottom: 20px;\u0022\u003E\u003Cspan class=\u0022number-highlight\u0022\u003E\u003Ca class=\u0022orange-link\u0022 href=\u0022https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/?term=10.1038\/ncomms9371\u0022 rel=\u0022noopener\u0022\u003E01\u003C\/a\u003E\u003C\/span\u003E\u003C\/div\u003E\n\u003Cdiv class=\u0022col-sm-11 col-md-11\u0022 style=\u0022padding-left: 20px;\u0022\u003E\n\u003Cp\u003EMatrix mechanics and water permeation regulate extracellular vesicle transport.\u003Cbr \/\u003E\u003Cbr \/\u003E\u003Cem\u003ELenzini S. et al. \u003Cbr \/\u003ENature Nanotechnology (2020)\u003C\/em\u003E\u003Cbr \/\u003E\u003Cbr \/\u003E\u003Cspan style=\u0022color: #ef6331;\u0022\u003E\u003Ca class=\u0022orange-link\u0022 href=\u0022https:\/\/doi.org\/10.1038\/s41565-020-0636-2\u0022 style=\u0022color: #ef6331;\u0022\u003Edoi: 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class=\u0022orange-link\u0022 href=\u0022https:\/\/doi.org\/10.1016\/j.celrep.2023.113067\u0022 style=\u0022color: #ef6331;\u0022\u003Edoi: 10.1016\/j.celrep.2023.113067\u003C\/a\u003E\u003C\/span\u003E\u003C\/p\u003E\n\u003C\/div\u003E\n\u003C\/div\u003E\n\u003Cdiv class=\u0022col-sm-4 col-md-4\u0022\u003E\n\u003Cdiv class=\u0022col-sm-1 col-md-1\u0022 style=\u0022padding-left: 0px; padding-bottom: 20px;\u0022\u003E\u003Cspan class=\u0022number-highlight\u0022\u003E\u003Ca class=\u0022orange-link\u0022 href=\u0022https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/?term=10.1136\/gutjnl-2015-309359\u0022 rel=\u0022noopener\u0022\u003E03\u003C\/a\u003E\u003C\/span\u003E\u003C\/div\u003E\n\u003Cdiv class=\u0022col-sm-11 col-md-11\u0022 style=\u0022padding-left: 20px;\u0022\u003E\n\u003Cp\u003ERBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing.\u003Cbr \/\u003E\u003Cbr \/\u003E\u003Cem\u003EMaurin M. et al.\u003Cbr \/\u003ENature Communications (2023) \u003C\/em\u003E \u003Cbr \/\u003E\u003Cbr \/\u003E\u003Cspan style=\u0022color: #ef6331;\u0022\u003E\u003Ca class=\u0022orange-link\u0022 href=\u0022https:\/\/doi.org\/10.1038\/s41467-023-44126-w\u0022 style=\u0022color: #ef6331;\u0022\u003Edoi: 10.1038\/s41467-023-44126-w\u003C\/a\u003E\u003C\/span\u003E\u003C\/p\u003E\n\u003C\/div\u003E\n\u003C\/div\u003E\n\u003Cdiv class=\u0022col-sm-4 col-md-4\u0022\u003E\n\u003Cdiv class=\u0022col-sm-1 col-md-1\u0022 style=\u0022padding-left: 0px; padding-bottom: 20px;\u0022\u003E\u003C\/div\u003E\n\u003C\/div\u003E\n\u003C\/div\u003E\n\u003C\/div\u003E\n\u003Cp\u003E\u003C\/p\u003E","meta_title":"Virus Packaging Mixes","meta_keywords":"Virus Packaging Mixes, qPCR Virus Titer Kits, Virus Transduction Enhancer, Virus Purification Kits","meta_description":"abm\u2019s proprietary packaging mixes and highly efficient protocols based on years of experience permit rapid production of recombinant viral particles with high titer and purity.","deleted_at":null,"enable":"Y","parent_list":"8","table_name":null,"image":null,"independentPage":0,"top_type":1,"sort_order":null,"in_footer":1,"fid":null,"created_at":null,"updated_at":"2025-04-02 01:04:45"},"info":{"id":16621,"cat_no_base":"LV053","parent_id":596,"description":"\u003Cp\u003EFor the production of lentiviral particles, three components are generally required: 1) a lentiviral vector containing your inserts of interest, 2) one or two packaging vectors which contain all necessary viral structure proteins, 3) an envelope vector expressing Vesicular Stomatitis Virus (VSV) glycoprotein (G).\u003C\/p\u003E\n\u003Cp\u003EThe 3rd generation packaging system offers maximal biosafety as the lentiviral Rev gene is supplied as an independent vector from other structure genes, further eliminating the possibility of reverse recombination of vectors into replication competent viral particles.\u003C\/p\u003E\n\u003Cp\u003EThe 3rd generation lentiviral packaging mix will only support lentiviral expression vectors with a chimeric 5\u0027 LTR in which the HIV promoter is replaced with CMV or RSV, thus making it TAT-independent. The 3rd generation lentiviral vectors will not support the production of 2nd generation lentiviral particle productions. All of the lentiviral vectors marketed by ABM are TAT-independent.\u003C\/p\u003E\n\u003Cp\u003E\u0026nbsp;\u003C\/p\u003E\n\u003Cp\u003E\u003Cstrong\u003EComponents (200ul mix of 3 plasmids):\u003C\/strong\u003E\u003C\/p\u003E\n\u003Cp\u003E\u003Ca href=\u0022\/vector\/pLenti-P3a\u0022\u003E\u003Cstrong\u003EpLenti-P3A\u003C\/strong\u003E\u003C\/a\u003E\u003C\/p\u003E\n\u003Cp\u003E\u003Ca href=\u0022\/vector\/pLenti-P3B\u0022\u003E\u003Cstrong\u003EpLenti-P3B\u003C\/strong\u003E\u003C\/a\u003E\u003C\/p\u003E\n\u003Cp\u003E\u003Ca href=\u0022\/vector\/pLenti-P3C\u0022\u003E\u003Cstrong\u003EpLenti-P3C\u003C\/strong\u003E\u003C\/a\u003E\u003C\/p\u003E\n\u003Cul\u003E\n\u003Cli\u003E\u003Cstrong\u003ESelection:\u003C\/strong\u003E Ampicillin (Bacterial Selection)\u003C\/li\u003E\n\u003Cli\u003E\u003Cstrong\u003EAmount:\u003C\/strong\u003E 200 \u0026mu;l (100 \u0026micro;g mixture of pLenti-P3A, pLenti-P3B, and pLenti-P3C)\u003C\/li\u003E\n\u003Cli\u003E\u003Cstrong\u003EAppearance:\u003C\/strong\u003E Liquid\u003C\/li\u003E\n\u003Cli\u003E\u003Cstrong\u003EStorage:\u003C\/strong\u003E Temperature \u0026deg;20-C or below (shipped at ambient temperature)\u003C\/li\u003E\n\u003Cli\u003E\u003Cstrong\u003EShelf Life:\u003C\/strong\u003E Up to 1 year (when at \u0026deg;20-C or below in a frost-free freezer)\u003C\/li\u003E\n\u003C\/ul\u003E\n\u003Cp\u003E\u0026nbsp;\u003C\/p\u003E\n\u003Cp\u003E*User Manual Refer to website for \u003Ca href=\u0022\/uploads\/document\/A4_lenti_packaging_2023_V2.pdf\u0022\u003E\u003Cstrong\u003Edownloadable user manual\u003C\/strong\u003E\u003C\/a\u003E\u003C\/p\u003E","disclaimer":null,"application":null,"components":null,"cas9_origin":null,"concentration":null,"enzymes_size":null,"genecraft_series":null,"guarantee":null,"population":null,"qc":null,"format_general":null,"including_screening_kit":null,"expression_system_general":null,"purity":null,"image":null,"insert_size":null,"shipping_conditions":null,"source_catalog_number":null,"inactivation_protocol":null,"led_viewer_compatibility":null,"unit_definition":null,"vector":null,"reaction_buffer":null,"storage_buffer":null,"caution":"Not for diagnostic use.","storage_condition":null,"product_volume":null,"reporter":null,"safeview_series":null,"source_catno":null,"stain_color":null,"supplier":"ABM","internal_supplier":null,"internal_note":null,"inventory_location":"Chemical\/Vector Freezer-Rack1","note":"\u003Cp\u003ENOT FOR RESALE without prior written consent of ABM. This product is distributed for laboratory research only.\u003C\/p\u003E","recommend":null,"depositor":null,"licensor_name":null,"licensor_contact_information":null,"contract_termination_date":null,"royalty_rates":null,"cas_type":null,"cas_origin":null,"cas_protein_marker":null,"source":null,"endotoxin_level":null,"additional_information":"\u003Cp\u003EShelf Life: Up to 1 year (when stored at -20\u003Cstrong\u003E\u0026deg;\u003C\/strong\u003EC in a frost-free freezer).\u003C\/p\u003E","titer":null,"nucleotide_format":null,"protocol_overview":null,"source_price":null,"created_at":"2022-09-15 07:17:34","updated_at":"2025-02-24 23:38:02","short_description":null,"reaction_definition":null,"specificity":null,"promotions":"\u003Ch5 class=\u0022abm-custom-page-p\u0022\u003E\u0026nbsp;\u003C\/h5\u003E\n\u003Cdiv class=\u0022abm-categories-text\u0022\u003E\n\u003Ctable style=\u0022background-color: #f9eae4; border-radius: 10px; vertical-align: middle; width: 99.1458%;\u0022 border=\u00220\u0022 cellpadding=\u002210\u0022\u003E\n\u003Ctbody\u003E\n\u003Ctr valign=\u0022middle\u0022\u003E\n\u003Ctd style=\u0022width: 6.65725%;\u0022\u003E\u003Cimg style=\u0022display: block; margin-left: auto; margin-right: auto;\u0022 src=\u0022\/assets\/images\/wysiwyg\/promotion-tag-v2.png\u0022 width=\u002254\u0022 height=\u002255\u0022 \/\u003E\u003C\/td\u003E\n\u003Ctd style=\u0022width: 90.3008%;\u0022\u003E\n\u003Cdiv class=\u0022abm-categories-text\u0022\u003E\u003Cstrong\u003EBundle up, save more!\u0026nbsp;\u003C\/strong\u003E\u0026nbsp;These\u0026nbsp;\u003Ca title=\u0022Lentivirus Bundles\u0022 href=\u0022\/Lentivirus-Bundles.html\u0022\u003Ecost-saving bundles\u003C\/a\u003E combine our best-selling Lentivirus Packaging Mix with reagents for transfection, virus titration, and transduction!\u003C\/div\u003E\n\u003C\/td\u003E\n\u003C\/tr\u003E\n\u003C\/tbody\u003E\n\u003C\/table\u003E\n\u003C\/div\u003E\n\u003Cdiv class=\u0022abm-categories-text\u0022\u003E\u0026nbsp;\u003C\/div\u003E\n\u003Cdiv class=\u0022abm-categories-text\u0022\u003E\u003Cspan style=\u0022color: #ffffff;\u0022\u003E.\u003C\/span\u003E\u003C\/div\u003E"},"maps":[{"id":129,"name":"pLenti-P3A","sequence":"gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctcccctcgaagcttacatgtggtaccgagctcggatcctgagaacttcagggtgagtctatgggacccttgatgttttctttccccttcttttctatggttaagttcatgtcataggaaggggagaagtaacagggtacacatattgaccaaatcagggtaattttgcatttgtaattttaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagcacgtgagatctgaattcgagatctgccgccgccatgggtgcgagagcgtcagtattaagcgggggagaattagatcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaaaaaagcacagcaagcagcagctgacacaggacacagcaatcaggtcagccaaaattaccctatagtgcagaacatccaggggcaaatggtacatcaggccatatcacctagaactttaaatgcatgggtaaaagtagtagaagagaaggctttcagcccagaagtgatacccatgttttcagcattatcagaaggagccaccccacaagatttaaacaccatgctaaacacagtggggggacatcaagcagccatgcaaatgttaaaagagaccatcaatgaggaagctgcagaatgggatagagtgcatccagtgcatgcagggcctattgcaccaggccagatgagagaaccaaggggaagtgacatagcaggaactactagtacccttcaggaacaaataggatggatgacacataatccacctatcccagtaggagaaatctataaaagatggataatcctgggattaaataaaatagtaagaatgtatagccctaccagcattctggacataagacaaggaccaaaggaaccctttagagactatgtagaccgattctataaaactctaagagccgagcaagcttcacaagaggtaaaaaattggatgacagaaaccttgttggtccaaaatgcgaacccagattgtaagactattttaaaagcattgggaccaggagcgacactagaagaaatgatgacagcatgtcagggagtggggggacccggccataaagcaagagttttggctgaagcaatgagccaagtaacaaatccagctaccataatgatacagaaaggcaattttaggaaccaaagaaagactgttaagtgtttcaattgtggcaaagaagggcacatagccaaaaattgcagggcccctaggaaaaagggctgttggaaatgtggaaaggaaggacaccaaatgaaagattgtactgagagacaggctaattttttagggaagatctggccttcccacaagggaaggccagggaattttcttcagagcagaccagagccaacagccccaccagaagagagcttcaggtttggggaagagacaacaactccctctcagaagcaggagccgatagacaaggaactgtatcctttagcttccctcagatcactctttggcagcgacccctcgtcacaataaagataggggggcaattaaaggaagctctattagatacaggagcagatgatacagtattagaagaaatgaatttgccaggaagatggaaaccaaaaatgatagggggaattggaggttttatcaaagtaagacagtatgatcagatactcatagaaatctgcggacataaagctataggtacagtattagtaggacctacacctgtcaacataattggaagaaatctgttgactcagattggctgcactttaaattttcccattagtcctattgagactgtaccagtaaaattaaagccaggaatggatggcccaaaagttaaacaatggccattgacagaagaaaaaataaaagcattagtagaaatttgtacagaaatggaaaaggaaggaaaaatttcaaaaattgggcctgaaaatccatacaatactccagtatttgccataaagaaaaaagacagtactaaatggagaaaattagtagatttcagagaacttaataagagaactcaagatttctgggaagttcaattaggaataccacatcctgcagggttaaaacagaaaaaatcagtaacagtactggatgtgggcgatgcatatttttcagttcccttagataaagacttcaggaagtatactgcatttaccatacctagtataaacaatgagacaccagggattagatatcagtacaatgtgcttccacagggatggaaaggatcaccagcaatattccagtgtagcatgacaaaaatcttagagccttttagaaaacaaaatccagacatagtcatctatcaatacatggatgatttgtatgtaggatctgacttagaaatagggcagcatagaacaaaaatagaggaactgagacaacatctgttgaggtggggatttaccacaccagacaaaaaacatcagaaagaacctccattcctttggatgggttatgaactccatcctgataaatggacagtacagcctatagtgctgccagaaaaggacagctggactgtcaatgacatacagaaattagtgggaaaattgaattgggcaagtcagatttatgcagggattaaagtaaggcaattatgtaaacttcttaggggaaccaaagcactaacagaagtagtaccactaacagaagaagcagagctagaactggcagaaaacagggagattctaaaagaaccggtacatggagtgtattatgacccatcaaaagacttaatagcagaaatacagaagcaggggcaaggccaatggacatatcaaatttatcaagagccatttaaaaatctgaaaacaggaaagtatgcaagaatgaagggtgcccacactaatgatgtgaaacaattaacagaggcagtacaaaaaatagccacagaaagcatagtaatatggggaaagactcctaaatttaaattacccatacaaaaggaaacatgggaagcatggtggacagagtattggcaagccacctggattcctgagtgggagtttgtcaatacccctcccttagtgaagttatggtaccagttagagaaagaacccataataggagcagaaactttctatgtagatggggcagccaatagggaaactaaattaggaaaagcaggatatgtaactgacagaggaagacaaaaagttgtccccctaacggacacaacaaatcagaagactgagttacaagcaattcatctagctttgcaggattcgggattagaagtaaacatagtgacagactcacaatatgcattgggaatcattcaagcacaaccagataagagtgaatcagagttagtcagtcaaataatagagcagttaataaaaaaggaaaaagtctacctggcatgggtaccagcacacaaaggaattggaggaaatgaacaagtagataaattggtcagtgctggaatcaggaaagtactatttttagatggaatagataaggcccaagaagaacatgagaaatatcacagtaattggagagcaatggctagtgattttaacctaccacctgtagtagcaaaagaaatagtagccagctgtgataaatgtcagctaaaaggggaagccatgcatggacaagtagactgtagcccag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