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.
하이그로마이신 B 유전자를 어디에서 가져올건지 정해졌으면 알려주세요..
promoter 부위가 어디인지 알려드리겠습니다.
ATG 상류쪽에 promoter가 있을 겁니다.
-
레벨2
옷궤짝이다
(대학생)
-
19.08.13 09:36
pCambia vector에서 hyg 를 PCR로 증폭한 후, T-vector에 삽입해서 사용할 예정입니다 ㅜㅜㅜㅜ도와주셔서 감사합니다 ㅜㅠ
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레벨2
옷궤짝이다
(대학생)
-
19.08.13 09:39
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
//
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
-
레벨2
옷궤짝이다
(대학생)
-
19.08.14 09:42
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