pEYFP-Actin encodes a fusion of the enhanced yellow fluorescent protein (EYFP) and human cytoplasmic b-actin (1). EYFP is an enhanced yellow-green variant of the Aequorea victoria green fluorescent protein (GFP; 2–4). The EYFP gene contains the four amino acid substitutions previously published as GFP-10C (5): Ser-65 to Gly; Val-68 to Leu; Ser-72 to Ala; and Thr-203 to Tyr. It also contains the Phe-64 to Leu mutation of GFPmut1 (6). 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 (5), resulting in a bright fluorescent signal. The fluorescence observed is roughly equivalent to that from EGFP.

In addition to the chromophore mutations, EYFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (7). Furthermore, upstream sequences flanking EYFP have been converted to a Kozak consensus translation initiation site (8). These changes increase the translational efficiency of the EYFP mRNA and consequently the expression of EYFP in mammalian and plant cells. The vector contains an SV40 origin for replication and a neomycin resistance (Neor) gene for selection (using G418) in eukaryotic cells (9). A bacterial promoter (P) upstream of Neor expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

載體應用

This vector is designed for the expression of the EYFP-Actin fusion protein in mammalian cells. The protein is incorporated into growing actin filaments and allows for visualization of actin-containing subcellular structures in living and fixed cells (10, 11). With the appropriate filter sets, pEYFP-Actin can be used for dual labeling experiments with any of CLONTECH's vectors encoding enhanced cyan fluorescent protein (ECFP). pEYFP-Actin is not intended as a cloning vector; however, there are unique restriction sites at the 5' end of EYFP, at the EYFP-Actin fusion junction, and at the 3' end of the b-actin sequences for excision of the sequences encoding the EYFP-Actin protein fusion. Note that the sites at the 3' end of the b-actin sequences are downstream of the stop codon. pEYFP-Actin can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (9).

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 ATCTCGAGCT ATGGATGATG ATATCGCCGC GCTCGTCGTC 
1381 GACAACGGCT CCGGCATGTG CAAGGCCGGC TTCGCGGGCG ACGATGCCCC CCGGGCCGTC 
1441 TTCCCCTCCA TCGTGGGGCG CCCCAGGCAC CAGGGCGTGA TGGTGGGCAT GGGTCAGAAG 
1501 GATTCCTATG TGGGCGACGA GGCCCAGAGC AAGAGAGGCA TCCTCACCCT GAAGTACCCC 
1561 ATCGAGCACG GCATCGTCAC CAACTGGGAC GACATGGAGA AAATCTGGCA CCACACCTTC 
1621 TACAATGAGC TGCGTGTGGC TCCCGAGGAG CACCCCGTGC TGCTGACCGA GGCCCCCCTG 
1681 AACCCCAAGG CCAACCGCGA GAAGATGACC CAGATCATGT TTGAGACCTT CAACACCCCA 
1741 GCCATGTACG TTGCTATCCA GGCTGTGCTA TCCCTGTACG CCTCTGGCCG TACCACTGGC 
1801 ATCGTGATGG ACTCCGGTGA CGGGGTCACC CACACTGTGC CCATCTACGA GGGGTATGCC 
1861 CTCCCCCATG CCATCCTGCG TCTGGACCTG GCTGGCCGGG ACCTGACTGA CTACCTCATG 
1921 AAGATCCTCA CCGAGCGCGG CTACAGCTTC ACCACCACGG CCGAGCGGGA AATCGTGCGT 
1981 GACATTAAGG AGAAGCTGTG CTACGTCGCC CTGGACTTCG AGCAAGAGAT GGCCACGGCT 
2041 GCTTCCAGCT CCTCCCTGGA GAAGAGCTAC GAGCTGCCTG ACGGCCAGGT CATCACCATT 
2101 GGCAATGAGC GGTTCCGCTG CCCTGAGGCA CTCTTCCAGC CTTCCTTCCT GGGCATGGAG 
2161 TCCTGTGGCA TCCACGAAAC TACCTTCAAC TCCATCATGA AGTGTGACGT GGACATCCGC 
2221 AAAGACCTGT ACGCCAACAC AGTGCTGTCT GGCGGCACCA CCATGTACCC TGGCATTGCC 
2281 GACAGGATGC AGAAGGAGAT CACTGCCCTG GCACCCAGCA CAATGAAGAT CAAGATCATT 
2341 GCTCCTCCTG AGCGCAAGTA CTCCGTGTGG ATCGGCGGCT CCATCCTGGC CTCGCTGTCC 
2401 ACCTTCCAGC AGATGTGGAT CAGCAAGCAG GAGTATGACG AGTCCGGCCC CTCCATCGTC 
2461 CACCGCAAAT GCTTCTAGGG ATCCACCGGA TCTAGATAAC TGATCATAAT CAGCCATACC 
2521 ACATTTGTAG AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA 
2581 CATAAAATGA ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA 
2641 TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT 
2701 GGTTTGTCCA AACTCATCAA TGTATCTTAA GGCGTAAATT GTAAGCGTTA ATATTTTGTT 
2761 AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG 
2821 CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG 
2881 GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA 
2941 TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG 
3001 CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA 
3061 GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT 
3121 GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT 
3181 ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT 
3241 TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA 
3301 ATAATATTGA AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG 
3361 TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC 
3421 AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA 
3481 AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC 
3541 CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT 
3601 GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT 
3661 GGAGGCCTAG GCTTTTGCAA AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT 
3721 TGAACAAGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA 
3781 TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA 
3841 GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA 
3901 CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA 
3961 CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT 
4021 CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG 
4081 GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA 
4141 GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA 
4201 TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC CCGACGGCGA 
4261 GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG 
4321 CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC 
4381 GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT 
4441 GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA 
4501 GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA 
4561 TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC 
4621 CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC 
4681 CCTAGGGGGA GGCTAACTGA AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG 
4741 ACGGCAATAA AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG 
4801 GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG GGGCCAATAC 
4861 GCCCGCGTTT CTTCCTTTTC CCCACCCCAC CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC 
4921 GCAGCCAACG TCGGGGCGGC AGGCCCTGCC ATAGCCTCAG GTTACTCATA TATACTTTAG 
4981 ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT 
5041 CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA 
5101 AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA 
5161 AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT 
5221 CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTCCTTCT AGTGTAGCCG 
5281 TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC 
5341 CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA 
5401 CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC 
5461 AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC 
5521 GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA 
5581 GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG 
5641 TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA 
5701 TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT 
5761 CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCATGCAT