pEYFP-Tub encodes a fusion protein consisting of enhanced yellow fluorescent protein (EYFP) and human a-tubulin (1, 2). EYFP is an enhanced yellow-green variant of the Aequorea victoria green fluorescent protein (GFP; 3–5). The EYFP gene contains the four amino acid substitutions previously published as GFP-10C (6): Ser-65 to Gly; Val-68 to Leu; Ser-72 to Ala; and and Thr-203 to Tyr. It also contains the Phe-64 to Leu mutation of GFPmut1 (7). The fluorescence excitation maximum of EYFP is 513 nm; the emission spectrum has a peak at 527 nm (in the yellow-green region). When excited at 513 nm, the Em of EYFP is 36,500 cm–1M–1 and the fluorescence quantum yield is 0.63 (6), resulting in a bright fluorescent signal. The fluorescence observed is roughly equivalent to that from EGFP.

In addition to the chromophore mutations, pEYFP-Tub contains more than 190 silent base changes that correspond to human codon-usage preferences (8). SV40 polyadenylation signals downstream of the EYFP-Tub fusion direct proper processing of the 3' end of the mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor) consisting of the SV40 early promoter, the neomycin/ kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV-TK) gene allows stably transfected eukaryotic cells to be selected using G418 (9). A bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli. The pEYFP-Tub backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

載體應(yīng)用

The pEYFP-Tub Vector is designed for expressing the EYFP-tubulin fusion protein in mammalian cells. pEYFP-Tub can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (9). The protein incorporates directly into microtubules and thereby allows the observation of microtubules in living or fixed cells by fluorescence microscopy (10). pEYFP-Tub is not sold as a cloning vector; however, there are unique restriction sites at the 5' end of EFYP, at the EYFP-Tub fusion junction, and at the 3' end of the a-tubulin sequences. Note that the sites at the 3' end of the a-tubulin sequences are downstream of the stop codon.

0001 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG 
0061 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT 
0121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA 
0181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC 
0241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 
0301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC 
0361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG 
0421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 
0481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT 
0541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA 
0601 CCGGTCGCCA CCATGGTGAG CAAGGGCGAG GAGCTGTTCA CCGGGGTGGT GCCCATCCTG 
0661 GTCGAGCTGG ACGGCGACGT AAACGGCCAC AAGTTCAGCG TGTCCGGCGA GGGCGAGGGC 
0721 GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCCGTG 
0781 CCCTGGCCCA CCCTCGTGAC CACCTTCGGC TACGGCCTGC AGTGCTTCGC CCGCTACCCC 
0841 GACCACATGA AGCAGCACGA CTTCTTCAAG TCCGCCATGC CCGAAGGCTA CGTCCAGGAG 
0901 CGCACCATCT TCTTCAAGGA CGACGGCAAC TACAAGACCC GCGCCGAGGT GAAGTTCGAG 
0961 GGCGACACCC TGGTGAACCG CATCGAGCTG AAGGGCATCG ACTTCAAGGA GGACGGCAAC 
1021 ATCCTGGGGC ACAAGCTGGA GTACAACTAC AACAGCCACA ACGTCTATAT CATGGCCGAC 
1081 AAGCAGAAGA ACGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA GGACGGCAGC 
1141 GTGCAGCTCG CCGACCACTA CCAGCAGAAC ACCCCCATCG GCGACGGCCC CGTGCTGCTG 
1201 CCCGACAACC ACTACCTGAG CTACCAGTCC GCCCTGAGCA AAGACCCCAA CGAGAAGCGC 
1261 GATCACATGG TCCTGCTGGA GTTCGTGACC GCCGCCGGGA TCACTCTCGG CATGGACGAG 
1321 CTGTACAAGT CCGGACTCAG ATCTCGAGTG CGTGAGTGCA TCTCCATCCA CGTTGGCCAG 
1381 GCTGGTGTCC AGATTGGCAA TGCCTGCTGG GAGCTCTACT GCCTGGAACA CGGCATCCAG 
1441 CCCGATGGCC AGATGCCAAG TGACAAGACC ATTGGGGGAG GAGATGACTC CTTCAACACC 
1501 TTCTTCAGTG AGACGGGCGC TGGCAAGCAC GTGCCCCGGG CTGTGTTTGT AGACTTGGAA 
1561 CCCACAGTCA TTGATGAAGT TCGCACTGGC ACCTACCGCC AGCTCTTCCA CCCTGAGCAG 
1621 CTCATCACAG GCAAGGAAGA TGCTGCCAAT AACTATGCCC GAGGGCACTA CACCATTGGC 
1681 AAGGAGATCA TTGACCTTGT GTTGGACCGA ATTCGCAAGC TGGCTGACCA GTGCACCGGT 
1741 CTTCAGGGCT TCTTGGTTTT CCACAGCTTT GGTGGGGGAA CTGGTTCTGG GTTCACCTCC 
1801 CTGCTCATGG AACGTCTCTC AGTTGATTAT GGCAAGAAGT CCAAGCTGGA GTTCTCCATT 
1861 TACCCAGCAC CCCAGGTTTC CACAGCTGTA GTTGAGCCCT ACAACTCCAT CCTCACCACC 
1921 CACACCACCC TGGAGCACTC TGATTGTGCC TTCATGGTAG ACAATGAGGC CATCTATGAC 
1981 ATCTGTCGTA GAAACCTCGA TATCGAGCGC CCAACCTACA CTAACCTTAA CCGCCTTATT 
2041 AGCCAGATTG TGTCCTCCAT CACTGCTTCC CTGAGATTTG ATGGAGCCCT GAATGTTGAC 
2101 CTGACAGAAT TCCAGACCAA CCTGGTGCCC TACCCCCGCA TCCACTTCCC TCTGGCCACA 
2161 TATGCCCCTG TCATCTCTGC TGAGAAAGCC TACCATGAAC AGCTTTCTGT AGCAGAGATC 
2221 ACCAATGCTT GCTTTGAGCC AGCCAACCAG ATGGTGAAAT GTGACCCTCG CCATGGTAAA 
2281 TACATGGCTT GCTGCCTGTT GTACCGTGGT GACGTGGTTC CCAAAGATGT CAATGCTGCC 
2341 ATTGCCACCA TCAAAACCAA GCGCAGCATC CAGTTTGTGG ATTGGTGCCC CACTGGCTTC 
2401 AAGGTTGGCA TCAACTACCA GCCTCCCACT GTGGTGCCTG GTGGAGACCT GGCCAAGGTA 
2461 CAGAGAGCTG TGTGCATGCT GAGCAACACC ACAGCCATTG CTGAGGCCTG GGCTCGCCTG 
2521 GACCACAAGT TTGACCTGAT GTATGCCAAG CGTGCCTTTG TTCACTGGTA CGTGGGTGAG 
2581 GGGATGGAGG AAGGCGAGTT TTCAGAGGCC CGTGAAGATA TGGCTGCCCT TGAGAAGGAT 
2641 TATGAGGAGG TTGGTGTGGA TTCTGTTGAA GGAGAGGGTG AGGAAGAAGG AGAGGAATAC 
2701 TAAGGATCCA CCGGATCTAG ATAACTGATC ATAATCAGCC ATACCACATT TGTAGAGGTT 
2761 TTACTTGCTT TAAAAAACCT CCCACACCTC CCCCTGAACC TGAAACATAA AATGAATGCA 
2821 ATTGTTGTTG TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCATC 
2881 ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC 
2941 ATCAATGTAT CTTAAGGCGT AAATTGTAAG CGTTAATATT TTGTTAAAAT TCGCGTTAAA 
3001 TTTTTGTTAA ATCAGCTCAT TTTTTAACCA ATAGGCCGAA ATCGGCAAAA TCCCTTATAA 
3061 ATCAAAAGAA TAGACCGAGA TAGGGTTGAG TGTTGTTCCA GTTTGGAACA AGAGTCCACT 
3121 ATTAAAGAAC GTGGACTCCA ACGTCAAAGG GCGAAAAACC GTCTATCAGG GCGATGGCCC 
3181 ACTACGTGAA CCATCACCCT AATCAAGTTT TTTGGGGTCG AGGTGCCGTA AAGCACTAAA 
3241 TCGGAACCCT AAAGGGAGCC CCCGATTTAG AGCTTGACGG GGAAAGCCGG CGAACGTGGC 
3301 GAGAAAGGAA GGGAAGAAAG CGAAAGGAGC GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT 
3361 CACGCTGCGC GTAACCACCA CACCCGCCGC GCTTAATGCG CCGCTACAGG GCGCGTCAGG 
3421 TGGCACTTTT CGGGGAAATG TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC 
3481 AAATATGTAT CCGCTCATGA GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG 
3541 GAAGAGTCCT GAGGCGGAAA GAACCAGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG 
3601 TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC 
3661 AGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT 
3721 TAGTCAGCAA CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT 
3781 TCCGCCCATT CTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC 
3841 GCCTCGGCCT CTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT 
3901 TGCAAAGATC GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATGATTGAAC AAGATGGATT 
3961 GCACGCAGGT TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT GGGCACAACA 
4021 GACAATCGGC TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT 
4081 TTTTGTCAAG ACCGACCTGT CCGGTGCCCT GAATGAACTG CAAGACGAGG CAGCGCGGCT 
4141 ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TCACTGAAGC 
4201 GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CATCTCACCT 
4261 TGCTCCTGCC GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC ATACGCTTGA 
4321 TCCGGCTACC TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG 
4381 GATGGAAGCC GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC 
4441 AGCCGAACTG TTCGCCAGGC TCAAGGCGAG CATGCCCGAC GGCGAGGATC TCGTCGTGAC 
4501 CCATGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT CTGGATTCAT 
4561 CGACTGTGGC CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG CTACCCGTGA 
4621 TATTGCTGAA GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT ACGGTATCGC 
4681 CGCTCCCGAT TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT TCTGAGCGGG 
4741 ACTCTGGGGT TCGAAATGAC CGACCAAGCG ACGCCCAACC TGCCATCACG AGATTTCGAT 
4801 TCCACCGCCG CCTTCTATGA AAGGTTGGGC TTCGGAATCG TTTTCCGGGA CGCCGGCTGG 
4861 ATGATCCTCC AGCGCGGGGA TCTCATGCTG GAGTTCTTCG CCCACCCTAG GGGGAGGCTA 
4921 ACTGAAACAC GGAAGGAGAC AATACCGGAA GGAACCCGCG CTATGACGGC AATAAAAAGA 
4981 CAGAATAAAA CGCACGGTGT TGGGTCGTTT GTTCATAAAC GCGGGGTTCG GTCCCAGGGC 
5041 TGGCACTCTG TCGATACCCC ACCGAGACCC CATTGGGGCC AATACGCCCG CGTTTCTTCC 
5101 TTTTCCCCAC CCCACCCCCC AAGTTCGGGT GAAGGCCCAG GGCTCGCAGC CAACGTCGGG 
5161 GCGGCAGGCC CTGCCATAGC CTCAGGTTAC TCATATATAC TTTAGATTGA TTTAAAACTT 
5221 CATTTTTAAT TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC 
5281 CCTTAACGTG AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT 
5341 TCTTGAGATC CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA 
5401 CCAGCGGTGG TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC 
5461 TTCAGCAGAG CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC 
5521 TTCAAGAACT CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT 
5581 GCTGCCAGTG GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT 
5641 AAGGCGCAGC GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG 
5701 ACCTACACCG AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA 
5761 GGGAGAAAGG CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG 
5821 GAGCTTCCAG GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA 
5881 CTTGAGCGTC GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC 
5941 AACGCGGCCT TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT 
6001 GCGTTATCCC CTGATTCTGT GGATAACCGT ATTACCGCCA TGCAT