pTRV2植物干扰质粒

基本信息

启动子:	CaMV 35S promoter
复制子:	pVS1 oriV，ori
质粒分类:	植物系列，RNAi载体
质粒大小:	9663bp
原核抗性:	Kan
克隆菌株:	DH5α
培养条件:	37℃，有氧 LB
表达宿主:	植物细胞
5'测序引物:	35S:GACGCACAATCCCACTATCC
3'测序引物:	根据序列设计引物
备注:	Tobacco rattle virus RNA2-based VIGS vector

质粒简介

    Tobacco rattle virus (TRV) is a bipartite, positive-sense RNA virus. TRV1 (i.e., RNA1) encodes 134 and 194 kDa replicase proteins from the genomic RNA, 29 kDa movement protein, and 16 kDa cysteine-rich protein from subgenomic RNAs. TRV1 can replicate and move systemically without TRV2 (i.e., RNA2). TRV2 encodes coat protein from the genomic and two nonstructural proteins from subgenomic RNAs (A). To develop the TRV-based VIGS vector, a cDNA clone of TRV1 or TRV2 (k20 strain) was placed between a duplicated CaMV 35S promoter (2X35S) and nopaline synthase terminator (NOSt) in an Agrobacterium T-DNA vector. In the TRV2 cDNA construct, the nonessential structural genes were replaced with MCS for cloning the target gene sequences. Further, a self-cleaving ribozyme site was included at the 3' end of TRV2. The TRV1 (AF406990)-based viral vector, pTRV1 (linear plasmid 6.791 kb), is used along with pTRV2 for silencing. The pTRV2 has three variants. The first one is suitable for conventional cloning (AF406991, linear plasmid 9663 nucleotide). The second variant is a gateway-cloning-compatible vector. The third variant is suitable for ligation-independent cloning. Rz, self-cleaving ribozyme; MCS, multiple cloning site; CP, coat protein; MP, movement protein. These vectors were created by Dr. S.P. Dinesh-Kumar, UC Davis, USA. Conventional cloning-compatible (YL156) and ligation-independent-compatible (YY13TRV2) vector plasmid DNA can be obtained from ABRC, Stock No. CD3-1040 vector name YL156. Ligation-independent-compatible TRV2 vector plasmid DNA is ABRC Stock No. CD3-1042, vector name YY13.

质粒图谱

质粒序列

LOCUS       Exported                9663 bp ds-DNA     circular SYN 28-SEP-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9663)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 28, 2017 from Plasmid
    FEATURES             Location/Qualifiers
     source          1..9663
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..1639
                     /label=TRV strain k20 RNA2 5'-sequence
     misc_feature    1647..1707
                     /label=MCS
     misc_feature    1709..2103
                     /label=TRV strain k20 RNA2 3'-sequence
     CDS             2164..2181
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2216..2468
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    2490..2514
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             3814..4443
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4877..5944
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQAAGAPSIRKKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      6010..6204
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     misc_feature    6548..6688
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6874..7462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7549..8343)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    8768..8792
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     misc_feature    8895..9663
                     /label=CaMV 35S promoter from pCASS2
ORIGIN
        1 ataaaacatt gcacctatgg tgttgccctg gctggggtat gtcagtgatc gcagtagaat
       61 gtactaattg acaagttgga gaatacggta gaacgtcctt atccaacaca gcctttatcc
      121 ctctccctga cgaggttttt gtcagtgtaa tatttctttt tgaactatcc agcttagtac
      181 cgtacgggaa agtgactggt gtgcttatct ttgaaatgtt actttgggtt tcggttcttt
      241 aggttagtaa gaaagcactt gtcttctcat acaaaggaaa acctgagacg tatcgcttac
      301 gaaagtagca atgaaagaaa ggtggtggtt ttaatcgcta ccgcaaaaac gatggggtcg
      361 ttttaattaa cttctcctac gcaagcgtct aaacggacgt tggggttttg ctagtttctt
      421 tagagaaaac tagctaagtc tttaatgtta tcattagaga tggcataaat ataatacttg
      481 tgtctgctga taagatcatt ttaatttgga cgattagact tgttgaacta caggttactg
      541 aatcacttgc gctaatcaac atgggagata tgtacgatga atcatttgac aagtcgggcg
      601 gtcctgctga cttgatggac gattcttggg tggaatcagt ttcgtggaaa gatctgttga
      661 agaagttaca cagcataaaa tttgcactac agtctggtag agatgagatc actgggttac
      721 tagcggcact gaatagacag tgtccttatt caccatatga gcagtttcca gataagaagg
      781 tgtatttcct tttagactca cgggctaaca gtgctcttgg tgtgattcag aacgcttcag
      841 cgttcaagag acgagctgat gagaagaatg cagtggcggg tgttacaaat attcctgcga
      901 atccaaacac aacggttacg acgaaccaag ggagtactac tactaccaag gcgaacactg
      961 gctcgacttt ggaagaagac ttgtacactt attacaaatt cgatgatgcc tctacagctt
     1021 tccacaaatc tctaacttcg ttagagaaca tggagttgaa gagttattac cgaaggaact
     1081 ttgagaaagt attcgggatt aagtttggtg gagcagctgc tagttcatct gcaccgcctc
     1141 cagcgagtgg aggtccgata cgtcctaatc cctagggatt taaggacgtg aactctgttg
     1201 agatctctgt gaaattcaga gggtgggtga taccatattc actgatgcca ttagcgacat
     1261 ctaaataggg ctaattgtga ctaatttgag ggaatttcct ttaccattga cgtcagtgtc
     1321 gttggtagca tttgagtttc gcaatgcacg aattacttag gaagtggctt gacgacacta
     1381 atgtgttatt gttagataat ggtttggtgg tcaaggtacg tagtagagtc ccacatattc
     1441 gcacgtatga agtaattgga aagttgtcag tttttgataa ttcactggga gatgatacgc
     1501 tgtttgaggg aaaagtagag aacgtatttg tttttatgtt caggcggttc ttgtgtgtca
     1561 acaaagatgg acattgttac tcaaggaagc acgatgagct ttattattac ggacgagtgg
     1621 acttagattc tgtgagtaag gttaccgaat tctctagaag gcctccatgg ggatccggta
     1681 ccgagctcac gcgtctcgag gcccgggcat gtcccgaaga cattaaacta cggttcttta
     1741 agtagatccg tgtctgaagt tttaggttca atttaaacct acgagattga cattctcgac
     1801 tgatcttgat tgatcggtaa gtcttttgta atttaatttt ctttttgatt ttattttaaa
     1861 ttgttatctg tttctgtgta tagactgttt gagatcggcg tttggccgac tcattgtctt
     1921 accatagggg aacggacttt gtttgtgttg ttattttatt tgtattttat taaaattctc
     1981 aacgatctga aaaagcctcg cggctaagag attgttgggg ggtgagtaag tacttttaaa
     2041 gtgatgatgg ttacaaaggc aaaaggggta aaacccctcg cctacgtaag cgttattacg
     2101 cccgtctgta cttatatcag tacactgacg agtccctaaa ggacgaaacg ggagaacgct
     2161 agccaccacc accaccacca cgtgtgaatt acaggtgacc agctcgaatt tccccgatcg
     2221 ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat
     2281 tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac
     2341 gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat
     2401 agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt
     2461 actagatcgg gaattaaact atcagtgttt gacaggatat attggcgggt aaacctaaga
     2521 gaaaagagcg tttattagaa taacggatat ttaaaagggc gtgaaaaggt ttatccgttc
     2581 gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt actttgatcc
     2641 aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg tcttctgaaa
     2701 acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc ttttcctggc
     2761 gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag aaccggagac
     2821 attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg
     2881 acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc accaagctgt
     2941 tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg atgcttgacc
     3001 acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc
     3061 gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg
     3121 cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc gtgttcgccg
     3181 gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg cgcgaggccg
     3241 ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca cagatcgcgc
     3301 acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct gcactgcttg
     3361 gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg
     3421 aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac gccctggcgg
     3481 ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg gccaggacga
     3541 accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt acgtgttcga
     3601 gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc
     3661 caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa
     3721 aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg
     3781 atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag
     3841 aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct gcaactcgcc
     3901 ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga ttgggcggcc
     3961 gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac
     4021 gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca ggcggcggac
     4081 ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca gccaagccct
     4141 tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat tgaggtcacg
     4201 gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc
     4261 ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc ccgtatcacg
     4321 cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga atcagaaccc
     4381 gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc aaaactcatt
     4441 tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt gccggccgtc
     4501 cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg ccagccatga
     4561 agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg tacgcggtac
     4621 gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta ccagagtaaa
     4681 tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat
     4741 caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac gggcggttgg
     4801 ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac ccccaagccc
     4861 gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg cggcgctggg
     4921 tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc
     4981 agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca aagaatcccg
     5041 gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg acgagcaacc
     5101 agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca gcatcatgga
     5161 cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga
     5221 gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga
     5281 ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga
     5341 agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt
     5401 ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt
     5461 aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac
     5521 ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg
     5581 gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa
     5641 gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg
     5701 ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa
     5761 gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg
     5821 caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc
     5881 tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc
     5941 ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg
     6001 tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa
     6061 cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga
     6121 tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta ttaaaactct
     6181 taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag agctgcaaaa
     6241 agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc
     6301 ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag ggcgcggaca
     6361 agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt
     6421 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     6481 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     6541 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     6601 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     6661 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     6721 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6781 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6841 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6901 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6961 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7021 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7081 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7141 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7201 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7261 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     7321 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7381 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7441 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7501 gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact aaaacaattc
     7561 atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa
     7621 aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc
     7681 aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt
     7741 gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc
     7801 ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc
     7861 ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc
     7921 ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa
     7981 gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata
     8041 ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg
     8101 ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc
     8161 cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata
     8221 taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc
     8281 ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc
     8341 catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat
     8401 aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca
     8461 gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg
     8521 aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc
     8581 cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg aatagcatcg
     8641 gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg tcccggactg
     8701 atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc
     8761 tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca
     8821 cattgcggac gtttttaatg tactgaatta acgccgaatt aattcctagg ccaccatgtt
     8881 gggcccggcg cgccaagctt gcatgcctgc aggtcaacat ggtggagcac gacactctcg
     8941 tctactccaa gaatatcaaa gatacagtct cagaagacca gagggctatt gagacttttc
     9001 aacaaagggt aatatcggga aacctcctcg gattccattg cccagctatc tgtcacttca
     9061 tcgaaaggac agtagaaaag gaagatggct tctacaaatg ccatcattgc gataaaggaa
     9121 aggctatcgt tcaagatgcc tctaccgaca gtggtcccaa agatggaccc ccacccacga
     9181 ggaacatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg
     9241 atggtcaaca tggtggagca cgacactctc gtctactcca agaatatcaa agatacagtc
     9301 tcagaagacc agagggctat tgagactttt caacaaaggg taatatcggg aaacctcctc
     9361 ggattccatt gcccagctat ctgtcacttc atcgaaagga cagtagaaaa ggaagatggc
     9421 ttctacaaat gccatcattg cgataaagga aaggctatcg ttcaagatgc ctctaccgac
     9481 agtggtccca aagatggacc cccacccacg aggaacatcg tggaaaaaga agacgttcca
     9541 accacgtctt caaagcaagt ggattgatgt gatatctcca ctgacgtaag ggatgacgca
     9601 caatcccact atccttcgca agacccttcc tctatataag gaagttcatt tcatttggag
     9661 agg