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질문 항생제 하이그로마이신(hygromycin b) 을 제가 사용하는 벡터에 넣으려 하는데요 ㅜㅜ
올챙이옷궤짝이다(대학생)  |  08.12 10:45

안녕하세요! 

제가 실험하고 있는 벡터에 하이그로마이신 b 저항성을 넣으려고 합니다!! ㅜㅜ

다름이 아니라 교수님께서 하이그로마이신에대한 프로모터가 들어가야한다고 하시는데, 하이그로 마이신 프로모터는 어떻게 알 수 있나요./??]

또는 하이그로마이신에 대한 자료 아시는 분 있으시면 도와주세요 ㅜㅜ

#하이그로마이신
답변하기
답변 본 정보는 네티즌에 의해 작성된 정보로, 내용 중 중요하다고 생각되는 부분은 추가적인 사실 확인을 반드시 하시길 바랍니다.
채택답변: 질문자가 채택한 답변입니다.

하이그로마이신 저항성 유전자를 벡터에 넣어요?

그러면 어디선가 그 저항성 유전자를 가져와야 넣을거 아닌가요? 유전자를 확보하려면 아마 다른 벡터에 들어있는 상태로 확보할 텐데, 그 벡터의 염기서열정보를 보세요.

아마도 유전자 orf와 프로모터 정보까지는 표시되어 있을겁니다. 그러면 그 정보에 따라서 프로모터와 orf를 잘라서 새 벡터에 삽입해야지요.

 

만일 어느 세균의 gDNA에서 클로닝해야 한다며 그 세균의 total genomic sequence를 보고 프로모터 부분을 확인해야 합니다. 또는 하이그로마이신 저항성 유전자를 연구한 논문(이 있을지 모르겠지만) 에서 프로모터 서열을 확인해야지요.

대왕개구리강시  |  08.12 15:19  
채택답변: 질문자가 채택한 답변입니다.

하이그로마이신 B 유전자를 어디에서 가져올건지 정해졌으면 알려주세요..

promoter 부위가 어디인지 알려드리겠습니다.

ATG 상류쪽에 promoter가 있을 겁니다.

대왕개구리SPEED  |  08.12 18:44  
첨부파일 파일첨부: Sequence of Hygromycin B, pCambia.hwp (128 KB)

pCambia vector에서 hyg 를 PCR로 증폭한 후, T-vector에 삽입해서 사용할 예정입니다 ㅜㅜㅜㅜ도와주셔서 감사합니다 ㅜㅠ

올챙이옷궤짝이다  |  08.13 09:36  
     pCambia 1300 vector시퀀스입니다! ㅜㅜ

  1 aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca
       61 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
      121 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
      181 ccttcccaac agttgcgcag cctgaatggc gaatgctaga gcagcttgag cttggatcag
      241 attgtcgttt cccgccttca gtttaaacta tcagtgtttg acaggatata ttggcgggta
      301 aacctaagag aaaagagcgt ttattagaat aacggatatt taaaagggcg tgaaaaggtt
      361 tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg gatcaaagta
      421 ctttgatcca acccctccgc tgctatagtg cagtcggctt ctgacgttca gtgcagccgt
      481 cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg acaggctgcc gccctgccct
      541 tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa gtagaatact tgcgactaga
      601 accggagaca ttacgccatg aacaagagcg ccgccgctgg cctgctgggc tatgcccgcg
      661 tcagcaccga cgaccaggac ttgaccaacc aacgggccga actgcacgcg gccggctgca
      721 ccaagctgtt ttccgagaag atcaccggca ccaggcgcga ccgcccggag ctggccagga
      781 tgcttgacca cctacgccct ggcgacgttg tgacagtgac caggctagac cgcctggccc
      841 gcagcacccg cgacctactg gacattgccg agcgcatcca ggaggccggc gcgggcctgc
      901 gtagcctggc agagccgtgg gccgacacca ccacgccggc cggccgcatg gtgttgaccg
      961 tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat cgaccgcacc cggagcgggc
     1021 gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg ccctaccctc accccggcac
     1081 agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg caccgtgaaa gaggcggctg
     1141 cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact tgagcgcagc gaggaagtga
     1201 cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga cgcattgacc gaggccgacg
     1261 ccctggcggc cgccgagaat gaacgccaag aggaacaagc atgaaaccgc accaggacgg
     1321 ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg gagatgatcg cggccgggta
     1381 cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg catgaaatcc tggccggttt
     1441 gtctgatgcc aagctggcgg cctggccggc cagcttggcc gctgaagaaa ccgagcgccg
     1501 ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg cgtcatgcgg tcgctgcgta
     1561 tatgatgcga tgagtaaata aacaaatacg caaggggaac gcatgaaggt tatcgctgta
     1621 cttaaccaga aaggcgggtc aggcaagacg accatcgcaa cccatctagc ccgcgccctg
     1681 caactcgccg gggccgatgt tctgttagtc gattccgatc cccagggcag tgcccgcgat
     1741 tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg gcatcgaccg cccgacgatt
     1801 gaccgcgacg tgaaggccat cggccggcgc gacttcgtag tgatcgacgg agcgccccag
     1861 gcggcggact tggctgtgtc cgcgatcaag gcagccgact tcgtgctgat tccggtgcag
     1921 ccaagccctt acgacatatg ggccaccgcc gacctggtgg agctggttaa gcagcgcatt
     1981 gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt cgcgggcgat caaaggcacg
     2041 cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg agctgcccat tcttgagtcc
     2101 cgtatcacgc agcgcgtgag ctacccaggc actgccgccg ccggcacaac cgttcttgaa
     2161 tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc tggccgctga aattaaatca
     2221 aaactcattt gagttaatga ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg
     2281 ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc
     2341 cagccatgaa gcgggtcaac tttcagttgc cggcggagga tcacaccaag ctgaagatgt
     2401 acgcggtacg ccaaggcaag accattaccg agctgctatc tgaatacatc gcgcagctac
     2461 cagagtaaat gagcaaatga ataaatgagt agatgaattt tagcggctaa aggaggcggc
     2521 atggaaaatc aagaacaacc aggcaccgac gccgtggaat gccccatgtg tggaggaacg
     2581 ggcggttggc caggcgtaag cggctgggtt gtctgccggc cctgcaatgg cactggaacc
     2641 cccaagcccg aggaatcggc gtgagcggtc gcaaaccatc cggcccggta caaatcggcg
     2701 cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac
     2761 gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca
     2821 aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg
     2881 acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca
     2941 gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga
     3001 tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca
     3061 gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc
     3121 gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg
     3181 tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct
     3241 gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc
     3301 gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg
     3361 aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca
     3421 cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg
     3481 gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat
     3541 ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt
     3601 tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg
     3661 cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat
     3721 ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag
     3781 gtcgaaaagg tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg
     3841 ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt
     3901 aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta
     3961 ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag
     4021 agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc
     4081 ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag
     4141 ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc
     4201 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     4261 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     4321 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     4381 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     4441 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     4501 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4561 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4621 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4681 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4741 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4801 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4861 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4921 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4981 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5041 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5101 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5161 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5221 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5281 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact
     5341 aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca
     5401 gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga
     5461 cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc
     5521 cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga
     5581 caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa
     5641 atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt
     5701 aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga
     5761 tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt
     5821 catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc
     5881 agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata
     5941 gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca
     6001 tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc
     6061 cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc
     6121 tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga
     6181 agctaattat aacaagacga actccaattc actgttcctt gcattctaaa accttaaata
     6241 ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga
     6301 gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca
     6361 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg
     6421 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg
     6481 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg
     6541 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa
     6601 cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt aattcggggg
     6661 atctggattt tagtactgga ttttggtttt aggaattaga aattttattg atagaagtat
     6721 tttacaaata caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct
     6781 ataggaaccc taattccctt atctgggaac tactcacaca ttattatgga gaaactcgag
     6841 cttgtcgatc gacagatccg gtcggcatct actctatttc tttgccctcg gacgagtgct
     6901 ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc
     6961 gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc
     7021 gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag
     7081 ttggtcaaga ccaatgcgga gcatatacgc ccggagtcgt ggcgatcctg caagctccgg
     7141 atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa
     7201 gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac
     7261 cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag
     7321 gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac
     7381 ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat
     7441 cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc
     7501 gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatagcct ccgcgaccgg
     7561 ttgtagaaca gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg
     7621 gcgggagatg caataggtca ggctctcgct aaactcccca atgtcaagca cttccggaat
     7681 cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc
     7741 gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga
     7801 ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa
     7861 cttctcgaca gacgtcgcgg tgagttcagg ctttttcata tctcattgcc ccccgggatc
     7921 tgcgaaagct cgagagagat agatttgtag agagagactg gtgatttcag cgtgtcctct
     7981 ccaaatgaaa tgaacttcct tatatagagg aaggtcttgc gaaggatagt gggattgtgc
     8041 gtcatccctt acgtcagtgg agatatcaca tcaatccact tgctttgaag acgtggttgg
     8101 aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc
     8161 ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc
     8221 accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag
     8281 gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac
     8341 tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tatcacatca
     8401 atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg
     8461 tgggggtcca tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt
     8521 atcgcaatga tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg
     8581 acagatagct gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt
     8641 ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt
     8701 gtcgtgctcc accatgttgg caagctgctc tagccaatac gcaaaccgcc tctccccgcg
     8761 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     8821 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     8881 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     8941 agctatgacc atgattacg
//
올챙이옷궤짝이다  |  08.13 09:39  
채택답변: 질문자가 채택한 답변입니다.

8,644bp -> 6,660bp를 포함해야 합니다.

promoter는 hygromycin 저항성 유전자의 promoter가 아니라 CaMV 35S promoter로

대체를 해서 만든 vector입니다.

promoter : 8,644bp -> 7,967bp

gene CDS : 7,899bp -> 6,874bp

Poly A tail : 6,834bp -> 6,660bp

대왕개구리SPEED  |  08.13 14:50  
  하이그로마이신 시퀀스

                                         ctatttcttt gccctcggac gagtgctggg
     7681 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct
     7741 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
     7801 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg
     7861 gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa gctccggatg
     7921 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
     7981 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
     8041 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
     8101 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
     8161 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
     8221 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
     8281 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg cgaccggttg
     8341 tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
     8401 ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt ccggaatcgg
     8461 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
     8521 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
     8581 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
     8641 ctcgacagac gtcgcggtga gttcaggctt tttcat

35S promoter 시퀀스

    8761 aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc
     8821 atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac
     8881 gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc
     8941 agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc
     9001 ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag
     9061 gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt
     9121 atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc
     9181 cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg
     9241 gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc
     9301 gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca
     9361 gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc
     9421 aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc
     9481 gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt

 

CaMV 3'UTR (polyA signal)

     7381 aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcgggggatc
     7441 tggattttag tactggattt tggttttagg aattagaaat tttattgata gaagtatttt
     7501 acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg aaaccctata
     7561 ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa actcgagctt
     7621 gtcgatcgac agatc

 

하이그로마이신에 대한 프라이머는 어떻게 제작해야 할까요..??ㅜㅜ

제한효소 사이트인 Xba1 서열을 넣고싶습니다

올챙이옷궤짝이다  |  08.14 09:42  
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56,90054,000
38,10036,000
36,30034,000
123,500117,000
172,100163,000
78,00074,000
245,100233,000
234,600223,000
82,80079,000
118,900113,000
45,60043,000
130,00065,000
75,50072,000
97,20092,000
36,30034,000
76,60073,000
286,700272,000
49,70047,000
668,600334,000
231,000219,000
121,700116,000
56,30053,000
100,30095,000
218,200207,000
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