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请高手帮忙设计引物

丁香园论坛

958
拟扩增408位至3533位,插入真核表达载体pCAGGS扩增,请高手帮忙设计引物,指点一下不胜感激!
1 agaagtttat ctgtgtgaac ttcttggctt agtatcgttg agaagaatcg agagattagt
61 gcagtttaaa cagtttttta gaacggaaga taaccatgac taaaaaacca ggagggcccg
121 gtaaaaaccg ggctatcaat atgctgaaac gcggcctacc ccgcgtattc ccactagtgg
181 gagtgaagag ggtagtaatg agcttgttgg acggcagagg gccagtacgt ttcgtgctgg
241 ctcttatcac gttcttcaag tttacagcat tagccccgac caaggcgctt ttaggccgat
301 ggaaagcagt ggaaaagagt gtggcaatga aacatcttac tagtttcaaa cgagaacttg
361 gaacactcat tgacgccgtg aacaagcggg gcagaaaaca aaacaaaaga ggaggaaatg
421 aaggctcaat catgtggctc gcaagcttgg cagttgtcat agcttacgca ggagcaatga
481 agttgtcgaa tttccagggg aagcttttga tgaccatcaa caacacggac attgcagacg
541 ttatcgtgat tcccacctca aaaggagaga acagatgctg ggtccgggca atagacgtcg
601 gctacatgtg tgaggacact atcacgtacg aatgtcctaa gctcaccatg ggcaatgatc
661 cagaggatgt ggattgctgg tgtgacaacc aagaagtcta cgtccaatat ggacggtgca
721 cgcggaccag gcattccaag cgaagcagga gatccgtgtc ggtccaaaca catggggaga
781 gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cggtatctca
841 tgaaaactga gaactggatc ataaggaatc ctggctatgc tttcctggcg gcgacacttg
901 gctggatgct tggcagtaac aacggtcaac gcgtggtatt taccatcctc ctgctgttgg
961 tcgctccggc ttacagtttt aattgtctgg gaatgggcaa tcgtgacttc atagaaggag
1021 ccagtggagc cacttgggtg gacttggtgc tagaaggaga tagctgcttg acaatcatgg
1081 caaacgacaa accaacattg gacgtccgca tgattaacat cgaagctagc caacttgctg
1141 aggtcagaag ttactgctat catgcttcag tcactgacat ctcgacggtg gctcggtgcc
1201 ccacgactgg agaagctcac aacgagaagc gagctgatag tagctatgtg tgcaaacaag
1261 gcttcactga tcgtgggtgg ggcaacggat gtggactttt cgggaaggga agcattgaca
1321 catgtgcaaa attctcctgc accagcaaag cgattggaag aacaatccag ccagaaaaca
1381 tcaaatacga agttggcatt tttgtgcatg gaaccaccac ttcggaaaac catgggaatt
1441 attcagcgca agttggggcg tcccaggcgg caaagtttac aataacaccc aatgctcctt
1501 cgataaccct caaacttggt gactacggag aagtcacgct ggactgtgag ccaaggagtg
1561 gactgaacac tgaagcgttt tacgtcatga ccgtggggtc aaagtcattt ctggtccata
1621 gggaatggtt tcatgacctc gctctcccct ggacgtcccc ttcgagcaca gcgtggagaa
1681 acagagaact cctcatggaa tttgaagagg cgcacgccac aaaacagtcc gttgttgctc
1741 ttgggtcaca ggaaggaggc ctccatcagg cgttggcagg agccatcgtg gtggagtact
1801 caagctcagt gaagttaaca tcaggccacc tgaaatgtag gctgaaaatg gacaaactgg
1861 ctctgaaagg cacaacctat ggcatgtgta cagaaaaatt ctcgttcgcg aaaaatccgg
1921 cggacactgg ccacggaaca gttgtcattg aactatccta ctctgggagt gatggcccct
1981 gcaaaattcc gattgtctcc gttgcgagcc tcaatgacat gacccccgtt gggcggctgg
2041 tgacagtgaa ccctttcgtc gcgacttcca gtgccaactc aaaggtgctg gtcgagatgg
2101 aacccccctt cggagactcc tacatcgtgg ttgggagggg agacaagcag atcaaccacc
2161 attggcacaa agctggaagc acgctaggca aggccttttc aacaactttg aagggagctc
2221 aaagactggc agcgttgggc gacacagcct gggactttgg ctccattgga ggggtcttca
2281 actccatagg aaaagccgtt caccaagtgt ttggtggtgc cttcagaaca ctctttgggg
2341 gaatgtcttg gatcacacaa gggctaatgg gtgccctact actctggatg ggcgtcaacg
2401 cacgagaccg atcaattgct ttggccttct tagccacagg aggtgtgctc gtgttcttag
2461 cgaccaatgt gcatgctgac actggatgtg ccattgacat cacaagaaaa gagatgagat
2521 gtggaagtgg catctttgtg cacaacgacg tggaagcctg ggtggacagg tataaatact
2581 tgccagaaac gcccagatcc ctagcgaaga tcgtccacaa agcgcacaag gaaggcgtgt
2641 gcggagtcag atctgtcact agattggagc accaaatgtg ggaagccgta cgggacgaat
2701 tgaacgtcct gctcaaagag aatgcagtgg acctcagtgt ggttgtgaac aagcccgtgg
2761 gaagatatcg ctcagcccct aaacgcctat ccatgacgca agagaagttt gaaatgggct
2821 ggaaagcatg gggaaaaagc attctctttg ccccggaatt ggctaactcc acatttgtcg
2881 tagatggacc tgagacaaag gaatgccctg atgagcatag agcttggaac agcatgcaaa
2941 tcgaagactt cggctttggc atcacatcaa cccgtgtgtg gctgaaaatt agagaggaga
3001 gcactgacga gtgtgatgga gcgatcatag gcacggctgt caaaggacat gtggcagtcc
3061 atagtgactt gtcgtactgg attgagagtc gctacaacga cacatggaaa cttgagaggg
3121 cagtctttgg agaggtcaaa tcttgcactt ggccagagac acacaccctt tggggagatg
3181 gtgttgagga aagtgaactc atcattccgc ataccatagc cggaccaaaa agcaagcaca
3241 atcggaggga agggtataag acacaaaacc agggaccttg ggatgagaat ggcatagtct
3301 tggactttga ctattgccca gggacgaaag tcaccattac agaggactgt ggcaagagag
3361 gcccttcggt cagaaccact actgacagtg gaaagttgat tactgactgg tgctgtcgca
3421 gttgctccct tccgccccta cgattccgga cagaaaatgg ctgctggtac ggaatggaaa
3481 tcagacctgt caggcatgat gaaacaacac tcgttagatc acaggttgat gctttcaatg
3541 gtgaaatggt tgaccctttt cagctgggcc ttctggtgat gtttctggcc acccaggagg
3601 tccttcgcaa gaggtggacg gccagattga ccattcctgc ggttttgggg gccctacttg
3661 tgctgatgct tgggggcatc acttacactg atttggcgag gtatgtggtg ctagtcgctg
3721 ccgctttcgc agaggccaac agtggaggag acgtcctgca tcttgctttg attgccgttt
3781 ttaagatcca accagcgttt ctagtgatga acatgcttag cacgagatgg acgaaccaag
3841 aaaacgtggt cctggtccta ggggctgcct tgtttcaact ggcctcagta gatctgcaaa
3901 taggagtcca cggaatcctg aatgccgccg ctatagcatg gatgattgtc cgagcgatca
3961 ccttccccac aacctcctcc gtcaccatgc cagtcttagc gcttctaacc ccgggaatga
4021 gggctctata cctagacact tacagaatca tcctcctcgt catagggatt tgctccctgc
4081 tgcaagagag gaaaaagacc atggcaaaaa agaaaggagc tgtactcttg ggcttagcgc
4141 tcacatccac tggatggttc tcgcccacca ctatagctgc cggactaatg gtctgcaacc
4201 caaacaagaa gagagggtgg ccagccactg agtttttgtc ggcagttgga ttgatgtttg
4261 ccatcgtagg tggcttggcc gagttggata ttgaatccat gtcaataccc ttcatgctgg
4321 caggtctcat ggcagtgtcc tacgtggtgt caggaaaagc aacagatatg tggcttgaac
4381 gggccgccga catcagctgg gagatggatg ctgcaatcac aggaagtagt cggaggctgg
4441 atgtgaaact ggatgatgac ggagattttc acttgattga tgatcccggt gttccatgga
4501 aggtctgggt cctgcgcatg tcttgtattg gcttagccgc cctcacgcct tgggccatcg
4561 ttcccgccgc tttcggttat tggctcactt taaaaacaac aaaaagaggg ggcgtgtttt
4621 gggacacgcc atccccaaaa ccttgctcaa aaggagacac tactacagga gtctaccgaa
4681 ttatggctag agggattctt ggcacttacc aggccggcgt cggagtcatg tacgagaatg
4741 ttttccacac actatggcac acaactagag gagcagccat tatgagtgga gaaggaaaat
4801 tgacgccata ctggggtagt gtgaaagaag accgcatagc ttacggaggc ccatggaggt
4861 ttgaccgaaa atggaatgga acagatgacg tgcaagtgat cgtggtagaa ccggggaagg
4921 ctgcagtaaa catccagaca aaaccaggag tgtttcggac tcccttcggg gaggttgggg
4981 ctgttagtct ggattacccg cgaggaacat ccggctcacc cattctggat tctaatggag
5041 acatcatagg cctatacggc aatggagttg agcttggcga tggctcatac gtcagcgcca
5101 tcgtgcaggg tgaccgtcag gaggaaccag tcccagaagc ttacacccca aacatgttga
5161 gaaagagaca gatgactgtg ctagatttgc accctggctc agggaagacc aggaaaattc
5221 tgccacagat aatcaaggac gccatccagc agcgcctaag aacagctgtg ctggcaccga
5281 cgcgggtggt agcagcagaa atggcagaag ctttgagagg gctcccagtg cgatatcaaa
5341 cttcagcagt gcagagagag caccaaggga atgaaatagt ggatgtgatg tgccacgcca
5401 ctctgaccca tagactgatg tcaccgaaca gagtgcccaa ctacaaccta tttgtcatgg
5461 atgaagctca tttcactgac ccagccagca tagccgcacg aggatacatt gctaccaagg
5521 tggaattagg ggaggcagca gccatcttca tgacagcgac cccgcctgga accacggatc
5581 cttttcctga ctcaaatgcc ccaatccatg atttgcaaga tgagatacca gacagggcat
5641 ggagcagtgg atacgaatgg atcacagaat atgcgggcaa aaccgtgtgg tttgtggcga
5701 gcgtaaaaat ggggaatgag attgcaatgt gcctccaaag agcggggaaa aaggtcatcc
5761 aactcaaccg caagtcctat gatacagaat acccaaaatg taagaatgga gactgggact
5821 ttgtcattac caccgacatc tctgaaatgg gggccaactt cggtgcgagc agggtcatcg
5881 actgtagaaa gagcgtgaaa cccaccatct tagaagaggg agaaggcaga gtcatcctcg
5941 gaaacccatc tcccataacc agtgcaagcg cagctcaacg gaggggcaga gtaggcagaa
6001 accccaacca agttggagat gaatatcact atgggggggc taccagtgaa gatgacagta
6061 acctagccca ttggacagag gcaaagatca tgttagacaa catacacatg cccaatggac
6121 tggtggccca gctctatgga ccagagaggg aaaaggcttt tacaatggat ggtgaatacc
6181 gtctcagagg tgaagaaaag aaaaacttct tggagctgct taggacggct gacctcccgg
6241 tgtggctggc ctacaaggtg gcgtccaatg gcattcagta caccgacaga aggtggtgtt
6301 ttgatgggcc gcgcacgaat gccatactgg aggacaacac cgaggtagag atagtcaccc
6361 ggatgggtga gaggaaaatc ctcaagccga gatggcttga tgcaagagtt tatgcagatc
6421 accaagccct caagtggttc aaagactttg cagcagggaa gagatcagcc attagcttca
6481 tagaggtgct cggtcgcatg cctgagcatt tcatgggaaa gacgcgggaa gctttagaca
6541 ccatgtactt ggttgcaacg gctgagaaag gcgggaaagc acaccgaatg gctctcgaag
6601 agctgccaga tgcactggaa accatcacac ttattgttgc cattactgtg atgacaggag
6661 gattcttcct actaatgatg cagcgaaagg gtatagggaa gatgggtctt ggagctctgg
6721 tgctcacgct agctaccttc ttcctgtggg cggcagaggt tcctggaact aaaatagcag
6781 ggaccctgct gatcgccctg ctgctgatgg tggtgctcat cccagaaccg gaaaaacaga
6841 ggtcacagac agataaccaa ctggcggtgt ttctcatctg cgtcttgacc gtggttggag
6901 tggtggcagc aaacgagtac gggatgctag agaaaaccaa agcagatctc aagagcatgt
6961 ttgtcggaaa gacgcaggca tcagggctga ctggattgcc aagcatggca ctggacctgc
7021 gtccagccac agcctgggca ctgtatgggg ggagcacagt cgtgctaacc cctcttctga
7081 aacacctgat cacgtcggaa tacgtcacca catcgctagc ctcaattaac tcacaagctg
7141 gctcattatt cgtcttgcca cgaggcgtgc cttttaccga cctagacttg accgttggcc
7201 tcgtcttcct tggctgttgg ggtcaaatca cccttacaac gtttctgaca gccatggttc
7261 tggcgacact tcattatggg tacatgctcc ctggatggca agcagaagca ctcagggctg
7321 cccagagaag gacagcggct ggaataatga agaatgccgt tgttgacgga atggtcgcca
7381 ctgatgtgcc tgaactggaa aggaccaccc ctctgatgca aaagaaagtc ggacaggtgc
7441 tcctcatagg ggtaagcgta gcagcgttcc tcgtcaaccc taatgtcacc actgtgagag
7501 aagcaggggt attggtgacg gcggctacgc tcactttgtg ggacaatgga gccagtgccg
7561 tctggaattc caccacagcc acgggactct gccatgttat gcgaggtagc tacctggctg
7621 gaggttccat tgcttggact ctcatcaaga acgcggataa gccctccttg aaaaggggaa
7681 ggcccggggg caggacgcta ggggagcaat ggaaggaaaa actaaacgct atgagcagag
7741 aagagttttt taaataccga agagaggcca taatcgaggt ggaccgcact gaagcacgca
7801 gggctagacg tgaaaacaac atagtgggag gacatccggt ttcgcgaggc tcagcaaaac
7861 tccgttggct cgtggagaaa ggatttgtct cgccaatagg aaaagtcatt gatctagggt
7921 gtgggcgtgg aggatggagc tactacgcag caaccctgaa gaaggtccag gaagtcagag
7981 gatacacgaa aggtggggcg ggacatgaag aaccgatgct catgcagagc tatggccgga
8041 atctggtctc cttgaaaagt ggagtggacg tgttttacaa accttcagaa cccagtgaca
8101 ccctgttctg tgacataggg gaatcctctc caagtccaga agtagaagaa caacgcacac
8161 tacgcgtcct agagatgaca tctgactggt tgcaccgagg acctagagag ttctgcataa
8221 aagttctctg cccttacatg cccaaggtta tagaaaaaat ggaagttctg cagcgccgct
8281 tcggaggtgg gctagtgcgt ctccccctgt cccggaactc caatcacgag atgtattggg
8341 ttagtggagc cgctggcaat gtggtgcacg ctgtgaacat gaccagccag gtactactgg
8401 ggcgaatgga tcgcacagtg tggagagggc caaagtatga ggaagatgtc aacctaggga
8461 gcggaacaag agccgtggga aagggagaag tccatagcaa tcaggagaaa atcaagaaga
8521 gaatccagaa gctcaaagaa gaattcgcca caacgtggca caaagaccct gagcatccat
8581 accgcacttg gacataccat ggaagctatg aagtgaaggc tactggctca gccagctctc
8641 tcgtcaacgg agtggtgaag ctcatgagca aaccttggga cgccattgcc aacgtcacca
8701 ccatggccat gactgacacc accccttttg gacagcaaag agtcttcaag gagaaagttg
8761 acacgaaggc tcctgagcca ccagctggag ccaaggaagt gctcaacgag accaccaact
8821 ggctgtgggc ccacttgtca cgggaaaaaa gaccccgctt gtgcaccaag gaagaattta
8881 taaagaaagt caacagcaac gcggctcttg gagcagtgtt cgctgaacag aatcaatgga
8941 gcacggctcg tgaggctgtg gatgacccgc ggttctggga gatggttgat gaagagaggg
9001 aaaaccatct gcgaggagag tgtcatacat gtatctacaa catgatggga aaaagagaga
9061 agaagcctgg agagtttgga aaagctaaag gaagcagggc catttggttc atgtggcttg
9121 gagcacggta tctagagttt gaagctttgg ggttcctgaa tgaagaccat tggctgagcc
9181 gagagaattc aggaggtgga gtggaaggct caggcgtcca aaagctggga tacatcctcc
9241 gtgacatagc aggaaagcaa ggagggaaaa tgtacgctga tgacaccgcc ggatgggaca
9301 ctagaattac cagaactgat ttagaaaatg aagccaaggt gctggagctc ctagacggtg
9361 aacaccgcat gctcgctcga gccataattg aactgactta caggcacaaa gtggtcaagg
9421 tcatgagacc tgcagcagaa ggaaagaccg taatggacgt gatatcaaga gaagatcaaa
9481 gggggagtgg acaggtggtc acttatgccc ttaacacttt cacgaacatt gctgtccagc
9541 tcgtcaggct gatggaggct gagggggtca ttggaccaca acacttggaa cagctaccta
9601 gaaaaaccaa gatagctgtc aggacctggc tctttgagaa tggagaggag agagtgacca
9661 ggatggcgat cagcggagat gactgtgtcg tcaagccgct ggatgacaga ttcgccacag
9721 ccctccactt cctcaacgca atgtcaaagg tcagaaaaga catccaggaa tggaagcctt
9781 cgcatggctg gcacgactgg cagcaagttc ccttctgctc taaccatttt caggagattg
9841 tgatgaaaga tggaaggagc atagttgtcc cgtgcagagg acaggatgag ctgataggca
9901 gggctcgcat ctctccagga gctggatgga atgtgaagga cacagcttgt ctggccaaag
9961 catatgcaca gatgtggcta ctcctatact tccatcgtag ggacttgcgt ctcatggcaa
10021 atgcgatttg ctcagcagtg ccagtggatt gggtgcccac aggcaggacg tcctggtcaa
10081 tacactcgaa aggagagtgg atgaccacgg aagacatgct gcaggtctgg aacagagtct
10141 ggattgaaga aaatgaatgg atgatggaca agactccaat cacaagctgg acagacgttc
10201 cgtatgtggg aaagcgtgag gacatctggt gtggtagcct catcggaacg cgatccagag
10261 caacctgggc tgagaacatc tatgcggcga taaaccaggt cagagctgtc atagggaaag
10321 aaaattatgt tgactacatg acctcactca ggagatacga agacgtcctg atccaggaag
10381 acagggtcat ctagtgtgat ttaaagtaga aaagtagact atgtaaataa tgtaaatgag
10441 aaaatgcatg catatggagt caggccagca aaagctgcca ccggatactg ggtagacggt
10501 gctgtctgcg tctcagtccc aggaggactg ggttaacaaa tctgacaaca gaaagtgaga
10561 aagccctcag aaccgtctcg gaagtaggtc cctgctcact ggaagttgaa agaccaacgt
10621 caggccacaa atttgtgcca ccccgctagg gggtgcggcc tgcgcagccc caggaggact
10681 gggttaccaa agccgttgag cccccacggc ccaagcctcg tctaggatgc aatagacgag
10741 gtgtaaggac tagaggttag aggagacccc gtggaaacaa caatatgcgg cccaagcccc
10801 ctcgaagctg tagaggaggt ggaaggacta gaggttagag gagaccccgc atttgcatca
10861 aacagcatat tgacacctgg gaatagactg ggagatcttc tgctctatct caacatcagc
10921 tactaggcac agagcgccga agtatgtagc tggtggtgag gaagaacaca ggatct
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