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        请高手帮忙设计引物

        丁香园论坛

        1111
        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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