pHELLSGATE8

价格:2500元

联系方式:I47-825O-882O

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载体基本信息

作者: SV.Wesley (2001)
载体名称: pHELLSGATE8
质粒类型: 基因沉默载体;Gateway载体;植物双元表达载体
高拷贝/低拷贝: 高拷贝
克隆方法: Gateway
启动子: CaMV 35S
载体大小: 17485 bp
5' 测序引物及序列: 35S promoter:5’CTATCCTTCGCAAGACCCTTC 3’
3' 测序引物及序列: --
载体标签:
载体抗性: 壮观霉素和链霉素
筛选标记: Kanamycin.html' target='_blank'>卡那霉素
克隆菌株: DB3.1
宿主细胞(系): 植物细胞、农杆菌
备注: pHELLSGATE8载体是高效、高通量、有效的植物基因沉默载体。
产品目录号: --
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pHELLSGATE8载体图谱



载体简介

pHELLSGATE8载体简介

Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.

载体序列

LOCUS       AF489904               17485 bp    DNA     circular SYN 14-NOV-2005
DEFINITION  Cloning vector pHellsgate 8, complete sequence.
ACCESSION   AF489904
VERSION     AF489904.1  GI:33320493
KEYWORDS    .
SOURCE      Cloning vector pHellsgate 8
  ORGANISM  Cloning vector pHellsgate 8
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 17485)
  AUTHORS   Helliwell,C.A. III and Waterhouse,P.M.
  TITLE     High throughput vectors for gene silencing in plants
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 17485)
  AUTHORS   Helliwell,C.A. III and Waterhouse,P.M.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-FEB-2002) Plant Industry, CSIRO, GPO Box 1600,
            Canberra, ACT 2601, Australia
FEATURES             Location/Qualifiers
     source          1..17485
                     /organism="Cloning vector pHellsgate 8"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:198117"
                     /focus
                     /note="derivative of cloning vector pART27"
     source          1..264
                     /organism="Escherichia coli K-12"
                     /mol_type="genomic DNA"
                     /strain="K-12"
                     /db_xref="taxon:83333"
     source          265..448
                     /organism="Agrobacterium tumefaciens"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:358"
     source          449..1442
                     /organism="Escherichia coli"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:562"
     source          1443..7792
                     /organism="Agrobacterium tumefaciens"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:358"
     source          7793..9388
                     /organism="Escherichia coli"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:562"
     source          9389..11673
                     /organism="Agrobacterium tumefaciens"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:358"
     source          11674..13019
                     /organism="Cauliflower mosaic virus"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:10641"
     source          14474..16072
                     /organism="Flaveria trinervia"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:4227"
     source          16716..17485
                     /organism="Agrobacterium tumefaciens"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:358"
     regulatory      264..447
                     /regulatory_class="promoter"
                     /note="NOS promoter"
     gene            448..1269
                     /gene="nptII"
     CDS             448..1269
                     /gene="nptII"
                     /note="neomycin phosphotransferase II"
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /protein_id="AAQ05967.1"
                     /db_xref="GI:33320494"
                     /translation="MAITLSATSLPISARIRAGSPAAWVERLFGYDWAQQTIGCSDAA
                     VFRLSAQGRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLL
                     LGEVPGQDLLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAG
                     LVDQDDLDEEHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDC
                     GRLGVADRYQDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     regulatory      1443..2148
                     /regulatory_class="terminator"
                     /note="NOS terminator"
     repeat_region   2149..2706
                     /note="left border"
     mobile_element  7793..9388
                     /mobile_element_type="transposon:Tn7"
     gene            8600..9388
                     /gene="spec"
     CDS             8600..9388
                     /gene="spec"
                     /codon_start=1
                     /product="spectinomycin resistance protein"
                     /protein_id="AAQ05968.1"
                     /db_xref="GI:33320495"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKP
                     HSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYP
                     AKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQ
                     DLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPA
                     QYQPVILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"
     repeat_region   10706..11324
                     /note="right border"
     regulatory      11674..13019
                     /regulatory_class="promoter"
                     /note="35S promoter"
     gene            14474..16019
                     /gene="pdk"
     intron          14474..16019
                     /gene="pdk"
                     /note="pyruvate orthophosphate dikinase"
                     /number=2
     regulatory      16716..17485
                     /regulatory_class="terminator"
                     /note="octopine esynthase (ocs) terminator"
ORIGIN      
        1 ggccgcacta gtgatatccc gcggccatgg cggccgggag catgcgacgt cgggcccaat
       61 tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg
      121 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
      181 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
      241 gaatggaaat tgtaaacgtt aatgggtttc tggagtttaa tgagctaagc acatacgtca
      301 gaaaccatta ttgcgcgttc aaaagtcgcc taaggtcact atcagctagc aaatatttct
      361 tgtcaaaaat gctccactga cgttccataa attcccctcg gtatccaatt agagtctcat
      421 attcactctc aatccaaata atctgcaatg gcaattacct tatccgcaac ttctttacct
      481 atttccgccc ggatccgggc aggttctccg gccgcttggg tggagaggct attcggctat
      541 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
      601 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
      661 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
      721 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
      781 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
      841 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
      901 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
      961 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     1021 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     1081 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     1141 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     1201 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     1261 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     1321 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     1381 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     1441 ccgatccaac acttacgttt gcaacgtcca agagcaaata gaccacgaac gccggaaggt
     1501 tgccgcagcg tgtggattgc gtctcaattc tctcttgcag gaatgcaatg atgaatatga
     1561 tactgactat gaaactttga gggaatactg cctagcaccg tcacctcata acgtgcatca
     1621 tgcatgccct gacaacatgg aacatcgcta tttttctgaa gaattatgct cgttggagga
     1681 tgtcgcggca attgcagcta ttgccaacat cgaactaccc ctcacgcatg cattcatcaa
     1741 tattattcat gcggggaaag gcaagattaa tccaactggc aaatcatcca gcgtgattgg
     1801 taacttcagt tccagcgact tgattcgttt tggtgctacc cacgttttca ataaggacga
     1861 gatggtggag taaagaagga gtgcgtcgaa gcagatcgtt caaacatttg gcaataaagt
     1921 ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat
     1981 tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt
     2041 atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca
     2101 aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcgaat taattccagg
     2161 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accccagtac
     2221 attaaaaacg tccgcaatgt gttattaagt tgtctaagcg tcaatttgtt tacaccacaa
     2281 tatatcctgc caccagccag ccaacagctc cccgaccggc agctcggcac aaaatcacca
     2341 ctcgatacag gcagcccatc agtccgggac ggcgtcagcg ggagagccgt tgtaaggcgg
     2401 cagactttgc tcatgttacc gatgctattc ggaagaacgg caactaagct gccgggtttg
     2461 aaacacggat gatctcgcgg agggtagcat gttgattgta acgatgacag agcgttgctg
     2521 cctgtgatca aatatcatct ccctcgcaga gatccgaatt atcagccttc ttattcattt
     2581 ctcgcttaac cgtgacaggc tgtcgatctt gagaactatg ccgacataat aggaaatcgc
     2641 tggataaagc cgctgaggaa gctgagtggc gctatttctt tagaagtgaa cgttgacgat
     2701 gtcgacggat cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg
     2761 tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc
     2821 cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg
     2881 ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc
     2941 tgcgaggctg gccggctacc gccggcgtaa cagatgaggg caagcggatg gctgatgaaa
     3001 ccaagccaac caggggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga
     3061 ggtgctccag tggcttctgt ttctatcagc tgtccctcct gttcagctac tgacggggtg
     3121 gtgcgtaacg gcaaaagcac cgccggacat cagcgctatc tctgctctca ctgccgtaaa
     3181 acatggcaac tgcagttcac ttacaccgct tctcaacccg gtacgcacca gaaaatcatt
     3241 gatatggcca tgaatggcgt tggatgccgg gcaacagccc gcattatggg cgttggcctc
     3301 aacacgattt tacgtcactt aaaaaactca ggccgcagtc ggtaacctcg cgcatacagc
     3361 cgggcagtga cgtcatcgtc tgcgcggaaa tggacgaaca gtggggctat gtcggggcta
     3421 aatcgcgcca gcgctggctg ttttacgcgt atgacagtct ccggaagacg gttgttgcgc
     3481 acgtattcgg tgaacgcact atggcgacgc tggggcgtct tatgagcctg ctgtcaccct
     3541 ttgacgtggt gatatggatg acggatggct ggccgctgta tgaatcccgc ctgaagggaa
     3601 agctgcacgt aatcagcaag cgatatacgc agcgaattga gcggcataac ctgaatctga
     3661 ggcagcacct ggcacggctg ggacggaagt cgctgtcgtt ctcaaaatcg gtggagctgc
     3721 atgacaaagt catcgggcat tatctgaaca taaaacacta tcaataagtt ggagtcatta
     3781 cccaaccagg aagggcagcc cacctatcaa ggtgtactgc cttccagacg aacgaagagc
     3841 gattgaggaa aaggcggcgg cggccggcat gagcctgtcg gcctacctgc tggccgtcgg
     3901 ccagggctac aaaatcacgg gcgtcgtgga ctatgagcac gtccgcgagc tggcccgcat
     3961 caatggcgac ctgggccgcc tgggcggcct gctgaaactc tggctcaccg acgacccgcg
     4021 cacggcgcgg ttcggtgatg ccacgatcct cgccctgctg gcgaagatcg aagagaagca
     4081 ggacgagctt ggcaaggtca tgatgggcgt ggtccgcccg agggcagagc catgactttt
     4141 ttagccgcta aaacggccgg ggggtgcgcg tgattgccaa gcacgtcccc atgcgctcca
     4201 tcaagaagag cgacttcgcg gagctggtat tcgtgcaggg caagattcgg aataccaagt
     4261 acgagaagga cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatc
     4321 tggacaccaa ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgag
     4381 tcggggcaat cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggc
     4441 aagaactgat cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccg
     4501 tcatgcgtgc gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacgg
     4561 ccaagatcga gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccg
     4621 ccgtggagcg ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgacca
     4681 tcgacacgcg aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaa
     4741 aacaggtcag cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaagg
     4801 aaatgcagct ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaa
     4861 acgacacggc ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgc
     4921 tgcaaaacaa ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcg
     4981 agctgcgggc cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgca
     5041 cccctatcgg cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggt
     5101 cgatcaatgg ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacgg
     5161 cgatgggctt cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgct
     5221 tccgcgtcct ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaa
     5281 tcgtcgtgct gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagc
     5341 tgtcgccgac ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgc
     5401 tcaagctgga aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggc
     5461 gcgagcaggt cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcct
     5521 gggtcaatga tgacctggtg cattgcaaac gctagggcct tgtggggtca gttccggctg
     5581 ggggttcagc agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgca
     5641 cttgcttcgc tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacag
     5701 aggattaaaa ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgt
     5761 gcaggatttc cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtc
     5821 cgtttacgag cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgc
     5881 cgtggcattc ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacagga
     5941 ggacggcccc aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaaca
     6001 gcgaggccga ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgt
     6061 gatgatcgtc cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgc
     6121 acttaatatt tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcgg
     6181 ggtcgcggcg acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctct
     6241 gctaggtagc ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgt
     6301 ggcgctgttg gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcct
     6361 ggcgggggcg gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgt
     6421 gcctctgctc acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagt
     6481 agctttagtg tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggc
     6541 gtggctcggc ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcg
     6601 actcgaacct acagttgttt ccttactggg ctttctcagc cgggatggcg ctaagaagct
     6661 attgccgccg atcttcatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     6721 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     6781 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     6841 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     6901 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     6961 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     7021 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     7081 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     7141 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     7201 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     7261 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     7321 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     7381 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     7441 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatat
     7501 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     7561 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     7621 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     7681 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     7741 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     7801 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     7861 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7921 aaacaagtgg cagcaacgga ttcgcaaacc tgtcacgcct tttgtgccaa aagccgcgcc
     7981 aggtttgcga tccgctgtgc caggcgttag gcgtcatatg aagatttcgg tgatccctga
     8041 gcaggtggcg gaaacattgg atgctgagaa ccatttcatt gttcgtgaag tgttcgatgt
     8101 gcacctatcc gaccaaggct ttgaactatc taccagaagt gtgagcccct accggaagga
     8161 ttacatctcg gatgatgact ctgatgaaga ctctgcttgc tatggcgcat tcatcgacca
     8221 agagcttgtc gggaagattg aactcaactc aacatggaac gatctagcct ctatcgaaca
     8281 cattgttgtg tcgcacacgc accgaggcaa aggagtcgcg cacagtctca tcgaatttgc
     8341 gaaaaagtgg gcactaagca gacagctcct tggcatacga ttagagacac aaacgaacaa
     8401 tgtacctgcc tgcaatttgt acgcaaaatg tggctttact ctcggcggca ttgacctgtt
     8461 cacgtataaa actagacctc aagtctcgaa cgaaacagcg atgtactggt actggttctc
     8521 gggagcacag gatgacgcct aacaattcat tcaagccgac accgcttcgc ggcgcggctt
     8581 aattcaggag ttaaacatca tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc
     8641 agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg tacatttgta
     8701 cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc tggttacggt
     8761 gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc
     8821 ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga
     8881 cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag aatggcagcg
     8941 caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc tggctatctt
     9001 gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg aggaactctt
     9061 tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa cgctatggaa
     9121 ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg
     9181 gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga
     9241 gcgcctgccg gcccagtatc agcccgtcat acttgaagct aggcaggctt atcttggaca
     9301 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgttcact acgtgaaagg
     9361 cgagatcacc aaggtagtcg gcaaataatg tctaacaatt cgttcaagcc gacgccgctt
     9421 cgcggcgcgg cttaactcaa gcgttagaga gctggggaag actatgcgcg atctgttgaa
     9481 ggtggttcta agcctcgtac ttgcgatggc atcggggcag gcacttgctg acctgccaat
     9541 tgttttagtg gatgaagctc gtcttcccta tgactactcc ccatccaact acgacatttc
     9601 tccaagcaac tacgacaact ccataagcaa ttacgacaat agtccatcaa attacgacaa
     9661 ctctgagagc aactacgata atagttcatc caattacgac aatagtcgca acggaaatcg
     9721 taggcttata tatagcgcaa atgggtctcg cactttcgcc ggctactacg tcattgccaa
     9781 caatgggaca acgaacttct tttccacatc tggcaaaagg atgttctaca ccccaaaagg
     9841 ggggcgcggc gtctatggcg gcaaagatgg gagcttctgc ggggcattgg tcgtcataaa
     9901 tggccaattt tcgcttgccc tgacagataa cggcctgaag atcatgtatc taagcaacta
     9961 gcctgctctc taataaaatg ttaggagctt ggctgccatt tttggggtga ggccgttcgc
    10021 ggccgagggg cgcagcccct ggggggatgg gaggcccgcg ttagcgggcc gggagggttc
    10081 gagaaggggg ggcacccccc ttcggcgtgc gcggtcacgc gccagggcgc agccctggtt
    10141 aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc
    10201 gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa
    10261 tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc
    10321 ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag
    10381 gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga
    10441 ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc
    10501 gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa
    10561 gttttccgcg aggtatccac aacgccggcg gccggccgcg gtgtctcgca cacggcttcg
    10621 acggcgtttc tggcgcgttt gcagggccat agacggccgc cagcccagcg gcgagggcaa
    10681 ccagcccggt gagcgtcgga aagggtcgac atcttgctgc gttcggatat tttcgtggag
    10741 ttcccgccac agacccggat tgaaggcgag atccagcaac tcgcgccaga tcatcctgtg
    10801 acggaacttt ggcgcgtgat gactggccag gacgtcggcc gaaagagcga caagcagatc
    10861 acgattttcg acagcgtcgg atttgcgatc gaggattttt cggcgctgcg ctacgtccgc
    10921 gaccgcgttg agggatcaag ccacagcagc ccactcgacc ttctagccga cccagacgag
    10981 ccaagggatc tttttggaat gctgctccgt cgtcaggctt tccgacgttt gggtggttga
    11041 acagaagtca ttatcgtacg gaatgccagc actcccgagg ggaaccctgt ggttggcatg
    11101 cacatacaaa tggacgaacg gataaacctt ttcacgccct tttaaatatc cgttattcta
    11161 ataaacgctc ttttctctta ggtttacccg ccaatatatc ctgtcaaaca ctgatagttt
    11221 aaactgaagg cgggaaacga caatctgatc atgagcggag aattaaggga gtcacgttat
    11281 gacccccgcc gatgacgcgg gacaagccgt tttacgtttg gaactgacag aaccgcaacg
    11341 attgaaggag ccactcagcc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
    11401 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
    11461 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
    11521 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
    11581 attacgccaa gctatttagg tgacactata gaatactcaa gctatgcatc caacgcgttg
    11641 ggagctctcc catatcgacc tgcaggcggc cgctcgacga attaattcca atcccacaaa
    11701 aatctgagct taacagcaca gttgctcctc tcagagcaga atcgggtatt caacaccctc
    11761 atatcaacta ctacgttgtg tataacggtc cacatgccgg tatatacgat gactggggtt
    11821 gtacaaaggc ggcaacaaac ggcgttcccg gagttgcaca caagaaattt gccactatta
    11881 cagaggcaag agcagcagct gacgcgtaca caacaagtca gcaaacagac aggttgaact
    11941 tcatccccaa aggagaagct caactcaagc ccaagagctt tgctaaggcc ctaacaagcc
    12001 caccaaagca aaaagcccac tggctcacgc taggaaccaa aaggcccagc agtgatccag
    12061 ccccaaaaga gatctccttt gccccggaga ttacaatgga cgatttcctc tatctttacg
    12121 atctaggaag gaagttcgaa ggtgaaggtg acgacactat gttcaccact gataatgaga
    12181 aggttagcct cttcaatttc agaaagaatg ctgacccaca gatggttaga gaggcctacg
    12241 cagcaggtct catcaagacg atctacccga gtaacaatct ccaggagatc aaataccttc
    12301 ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ttgcatcaag aacacagaga
    12361 aagacatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa ggcttgcttc
    12421 ataaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttcct actgaatcta
    12481 aggccatgca tggagtctaa gattcaaatc gaggatctaa cagaactcgc cgtgaagact
    12541 ggcgaacagt tcatacagag tcttttacga ctcaatgaca agaagaaaat cttcgtcaac
    12601 atggtggagc acgacactct ggtctactcc aaaaatgtca aagatacagt ctcagaagac
    12661 caaagggcta ttgagacttt tcaacaaagg ataatttcgg gaaacctcct cggattccat
    12721 tgcccagcta tctgtcactt catcgaaagg acagtagaaa aggaaggtgg ctcctacaaa
    12781 tgccatcatt gcgataaagg aaaggctatc attcaagatc tctctgccga cagtggtccc
    12841 aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct
    12901 tcaaagcaag tggattgatg tgacatctcc actgacgtaa gggatgacgc acaatcccac
    12961 tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga gaggacacgc
    13021 tcgaggacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat
    13081 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
    13141 cagtcactat gaatcaacta cttagatggt attagtgacc tgtagtcgac cgacagcctt
    13201 ccaaatgttc ttcgggtgat gctgccaact tagtcgaccg acagccttcc aaatgttctt
    13261 ctcaaacgga atcgtcgtat ccagcctact cgctattgtc ctcaatgccg tattaaatca
    13321 taaaaagaaa taagaaaaag aggtgcgagc ctcttttttg tgtgacaaaa taaaaacatc
    13381 tacctattca tatacgctag tgtcatagtc ctgaaaatca tctgcatcaa gaacaatttc
    13441 acaactctta tacttttctc ttacaagtcg ttcggcttca tctggatttt cagcctctat
    13501 acttactaaa cgtgataaag tttctgtaat ttctactgta tcgacctgca gactggctgt
    13561 gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt
    13621 cattttcgcg gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg
    13681 ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt
    13741 cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc
    13801 cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt
    13861 tataggtgta aaccttaaac tgcatttcac cagtccctgt tctcgtcagc aaaagagccg
    13921 ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt
    13981 tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg
    14041 acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct
    14101 gcttcatagc acacctcttt ttgacatact tcgggtagtg ccgatcaacg tctcattttc
    14161 gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt atttattctg
    14221 cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca
    14281 acttagtcga ctacaggtca ctaataccat ctaagtagtt gattcatagt gactggatat
    14341 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
    14401 tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtc tcgaggaatt
    14461 cggtacccca gcttggtaag gaaataatta ttttcttttt tccttttagt ataaaatagt
    14521 taagtgatgt taattagtat gattataata atatagttgt tataattgtg aaaaaataat
    14581 ttataaatat attgtttaca taaacaacat agtaatgtaa aaaaatatga caagtgatgt
    14641 gtaagacgaa gaagataaaa gttgagagta agtatattat ttttaatgaa tttgatcgaa
    14701 catgtaagat gatatactag cattaatatt tgttttaatc ataatagtaa ttctagctgg
    14761 tttgatgaat taaatatcaa tgataaaata ctatagtaaa aataagaata aataaattaa
    14821 aataatattt ttttatgatt aatagtttat tatataatta aatatctata ccattactaa
    14881 atattttagt ttaaaagtta ataaatattt tgttagaaat tccaatctgc ttgtaattta
    14941 tcaataaaca aaatattaaa taacaagcta aagtaacaaa taatatcaaa ctaatagaaa
    15001 cagtaatcta atgtaacaaa acataatcta atgctaatat aacaaagcgc aagatctatc
    15061 attttatata gtattatttt caatcaacat tcttattaat ttctaaataa tacttgtagt
    15121 tttattaact tctaaatgga ttgactatta attaaatgaa ttagtcgaac atgaataaac
    15181 aaggtaacat gatagatcat gtcattgtgt tatcattgat cttacatttg gattgattac
    15241 agttgggaaa ttgggttcga aatcgataag cttggatcct ctagaccact ttgtacaaga
    15301 aagctgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata
    15361 aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatga atcaactact
    15421 tagatggtat tagtgacctg tagtcgacta agttggcagc atcacccgac gcactttgcg
    15481 ccgaataaat acctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg
    15541 ttgataccgg gaagccctgg gccaactttt ggcgaaaatg agacgttgat cggcactacc
    15601 catttcacaa ctcttatact tttctcttac aagtcgttcg gcttcatctg gattttcagc
    15661 ctctatactt actaaacgtg ataaagtttc tgtaatttct actgtatcga cctgcagact
    15721 ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt
    15781 tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gagaccggca
    15841 cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt
    15901 aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc
    15961 gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct
    16021 ctcttttata ggtgtaaacc ttaaactgca tttcaccagt ccctgttctc gtcagcaaaa
    16081 gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc
    16141 cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag
    16201 atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa
    16261 tacgctgctt catagcacac ctctttttga catacttctg ttcttgatgc agatgatttt
    16321 caggactatg acactagcgt atatgaatag gtagatgttt ttattttgtc acacaaaaaa
    16381 gaggctcgca cctctttttc ttatttcttt ttatgattta atacggcatt gaggacaata
    16441 gcgagtaggc tggatacgac gattccgttt gagaagaaca tttggaaggc tgtcggtcga
    16501 ctaagttggc agcatcaccc gaagaacatt tggaaggctg tcggtcgact acaggtcact
    16561 aataccatct aagtagttga ttcatagtga ctggatatgt tgtgttttac agtattatgt
    16621 agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc
    16681 tcgttcagct tttttgtaca aacttgtcta gagtcctgct ttaatgagat atgcgagacg
    16741 cctatgatcg catgatattt gctttcaatt ctgttgtgca cgttgtaaaa aacctgagca
    16801 tgtgtagctc agatccttac cgccggtttc ggttcattct aatgaatata tcacccgtta
    16861 ctatcgtatt tttatgaata atattctccg ttcaatttac tgattgtacc ctactactta
    16921 tatgtacaat attaaaatga aaacaatata ttgtgctgaa taggtttata gcgacatcta
    16981 tgatagagcg ccacaataac aaacaattgc gttttattat tacaaatcca attttaaaaa
    17041 aagcggcaga accggtcaaa cctaaaagac tgattacata aatcttattc aaatttcaaa
    17101 aggccccagg ggctagtatc tacgacacac cgagcggcga actaataacg ttcactgaag
    17161 ggaactccgg ttccccgccg gcgcgcatgg gtgagattcc ttgaagttga gtattggccg
    17221 tccgctctac cgaaagttac gggcaccatt caacccggtc cagcacggcg gccgggtaac
    17281 cgacttgctg ccccgagaat tatgcagcat ttttttggtg tatgtgggcc ccaaatgaag
    17341 tgcaggtcaa accttgacag tgacgacaaa tcgttgggcg ggtccagggc gaattttgcg
    17401 acaacatgtc gaggctcagc aggacctgca ggcatgcaag ctagcttact agtgatgcat
    17461 attctatagt gtcacctaaa tctgc
//

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