pEYFP-ER encodes a fusion protein consisting of enhanced yellow fluorescent protein (EYFP); the endoplasmic reticulum (ER) targeting sequence of calreticulin (1), which is cloned at the 5' end; and the sequence encoding the ER retrieval sequence, KDEL (2, 3), which is cloned at the 3' end. The EYFP gene contains the four amino acid substitutions previously published as GFP-10C (4, 5): Ser- 65 to Gly; Val-68 to Leu; Ser-72 to Ala; and Thr-203 to Tyr. 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 514 nm, the Emof EYFP is 84,500 cm–1M–1 and the fluorescence quantum yield is 0.61 ( 5), resulting in a bright fluorescent signal. The fluorescence observed is roughly equivalent to that from EGFP.

In addition to the chromophore mutations, pEYFP-ER contains more than 190 silent base changes that correspond to human codon-usage preferences (6). SV40 polyadenylation signals downstream of the EYFP-ER 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 (7). A bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli. The pEYFP-ER 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

pEYFP-ER is designed for fluorescent labeling of the ER in mammalian cells (8, 9). Fluorescence can be observed in living cells by microscopy or flow cytometry. pEYFP-ER can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7). Filter sets are available for dual-color detection of EYFP and ECFP using conventional epifluoresence microscopy (10). Please refer to the Living Colors User Manual for additional information.

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 GCTAGCATGC 
0601 TGCTATCCGT GCCGTTGCTG CTCGGCCTCC TCGGCCTGGC CGTCGCCGTG AGCAAGGGCG 
0661 AGGAGCTGTT CACCGGGGTG GTGCCCATCC TGGTCGAGCT GGACGGCGAC GTAAACGGCC 
0721 ACAAGTTCAG CGTGTCCGGC GAGGGCGAGG GCGATGCCAC CTACGGCAAG CTGACCCTGA 
0781 AGTTCATCTG CACCACCGGC AAGCTGCCCG TGCCCTGGCC CACCCTCGTG ACCACCTTCG 
0841 GCTACGGCCT GCAGTGCTTC GCCCGCTACC CCGACCACAT GAAGCAGCAC GACTTCTTCA 
0901 AGTCCGCCAT GCCCGAAGGC TACGTCCAGG AGCGCACCAT CTTCTTCAAG GACGACGGCA 
0961 ACTACAAGAC CCGCGCCGAG GTGAAGTTCG AGGGCGACAC CCTGGTGAAC CGCATCGAGC 
1021 TGAAGGGCAT CGACTTCAAG GAGGACGGCA ACATCCTGGG GCACAAGCTG GAGTACAACT 
1081 ACAACAGCCA CAACGTCTAT ATCATGGCCG ACAAGCAGAA GAACGGCATC AAGGTGAACT 
1141 TCAAGATCCG CCACAACATC GAGGACGGCA GCGTGCAGCT CGCCGACCAC TACCAGCAGA 
1201 ACACCCCCAT CGGCGACGGC CCCGTGCTGC TGCCCGACAA CCACTACCTG AGCTACCAGT 
1261 CCGCCCTGAG CAAAGACCCC AACGAGAAGC GCGATCACAT GGTCCTGCTG GAGTTCGTGA 
1321 CCGCCGCCGG GATCACTCTC GGCATGGACG AGCTGTACAA GAAGGACGAG CTGTAAAGAT 
1381 CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG ACGGTACCGC GGGCCCGGGA TCCACCGGAT 
1441 CTAGATAACT GATCATAATC AGCCATACCA CATTTGTAGA GGTTTTACTT GCTTTAAAAA 
1501 ACCTCCCACA CCTCCCCCTG AACCTGAAAC ATAAAATGAA TGCAATTGTT GTTGTTAACT 
1561 TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG CATCACAAAT TTCACAAATA 
1621 AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA ACTCATCAAT GTATCTTAAG 
1681 GCGTAAATTG TAAGCGTTAA TATTTTGTTA AAATTCGCGT TAAATTTTTG TTAAATCAGC 
1741 TCATTTTTTA ACCAATAGGC CGAAATCGGC AAAATCCCTT ATAAATCAAA AGAATAGACC 
1801 GAGATAGGGT TGAGTGTTGT TCCAGTTTGG AACAAGAGTC CACTATTAAA GAACGTGGAC 
1861 TCCAACGTCA AAGGGCGAAA AACCGTCTAT CAGGGCGATG GCCCACTACG TGAACCATCA 
1921 CCCTAATCAA GTTTTTTGGG GTCGAGGTGC CGTAAAGCAC TAAATCGGAA CCCTAAAGGG 
1981 AGCCCCCGAT TTAGAGCTTG ACGGGGAAAG CCGGCGAACG TGGCGAGAAA GGAAGGGAAG 
2041 AAAGCGAAAG GAGCGGGCGC TAGGGCGCTG GCAAGTGTAG CGGTCACGCT GCGCGTAACC 
2101 ACCACACCCG CCGCGCTTAA TGCGCCGCTA CAGGGCGCGT CAGGTGGCAC TTTTCGGGGA 
2161 AATGTGCGCG GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC 
2221 ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TCCTGAGGCG 
2281 GAAAGAACCA GCTGTGGAAT GTGTGTCAGT TAGGGTGTGG AAAGTCCCCA GGCTCCCCAG 
2341 CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC 
2401 CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCATAG 
2461 TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC CAGTTCCGCC CATTCTCCGC 
2521 CCCATGGCTG ACTAATTTTT TTTATTTATG CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC 
2581 TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA GATCGATCAA 
2641 GAGACAGGAT GAGGATCGTT TCGCATGATT GAACAAGATG GATTGCACGC AGGTTCTCCG 
2701 GCCGCTTGGG TGGAGAGGCT ATTCGGCTAT GACTGGGCAC AACAGACAAT CGGCTGCTCT 
2761 GATGCCGCCG TGTTCCGGCT GTCAGCGCAG GGGCGCCCGG TTCTTTTTGT CAAGACCGAC 
2821 CTGTCCGGTG CCCTGAATGA ACTGCAAGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG 
2881 ACGGGCGTTC CTTGCGCAGC TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG 
2941 CTATTGGGCG AAGTGCCGGG GCAGGATCTC CTGTCATCTC ACCTTGCTCC TGCCGAGAAA 
3001 GTATCCATCA TGGCTGATGC AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA 
3061 TTCGACCACC AAGCGAAACA TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT 
3121 GTCGATCAGG ATGATCTGGA CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC 
3181 AGGCTCAAGG CGAGCATGCC CGACGGCGAG GATCTCGTCG TGACCCATGG CGATGCCTGC 
3241 TTGCCGAATA TCATGGTGGA AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG 
3301 GGTGTGGCGG ACCGCTATCA GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT 
3361 GGCGGCGAAT GGGCTGACCG CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG 
3421 CGCATCGCCT TCTATCGCCT TCTTGACGAG TTCTTCTGAG CGGGACTCTG GGGTTCGAAA 
3481 TGACCGACCA AGCGACGCCC AACCTGCCAT CACGAGATTT CGATTCCACC GCCGCCTTCT 
3541 ATGAAAGGTT GGGCTTCGGA ATCGTTTTCC GGGACGCCGG CTGGATGATC CTCCAGCGCG 
3601 GGGATCTCAT GCTGGAGTTC TTCGCCCACC CTAGGGGGAG GCTAACTGAA ACACGGAAGG 
3661 AGACAATACC GGAAGGAACC CGCGCTATGA CGGCAATAAA AAGACAGAAT AAAACGCACG 
3721 GTGTTGGGTC GTTTGTTCAT AAACGCGGGG TTCGGTCCCA GGGCTGGCAC TCTGTCGATA 
3781 CCCCACCGAG ACCCCATTGG GGCCAATACG CCCGCGTTTC TTCCTTTTCC CCACCCCACC 
3841 CCCCAAGTTC GGGTGAAGGC CCAGGGCTCG CAGCCAACGT CGGGGCGGCA GGCCCTGCCA 
3901 TAGCCTCAGG TTACTCATAT ATACTTTAGA TTGATTTAAA ACTTCATTTT TAATTTAAAA 
3961 GGATCTAGGT GAAGATCCTT TTTGATAATC TCATGACCAA AATCCCTTAA CGTGAGTTTT 
4021 CGTTCCACTG AGCGTCAGAC CCCGTAGAAA AGATCAAAGG ATCTTCTTGA GATCCTTTTT 
4081 TTCTGCGCGT AATCTGCTGC TTGCAAACAA AAAAACCACC GCTACCAGCG GTGGTTTGTT 
4141 TGCCGGATCA AGAGCTACCA ACTCTTTTTC CGAAGGTAAC TGGCTTCAGC AGAGCGCAGA 
4201 TACCAAATAC TGTCCTTCTA GTGTAGCCGT AGTTAGGCCA CCACTTCAAG AACTCTGTAG 
4261 CACCGCCTAC ATACCTCGCT CTGCTAATCC TGTTACCAGT GGCTGCTGCC AGTGGCGATA 
4321 AGTCGTGTCT TACCGGGTTG GACTCAAGAC GATAGTTACC GGATAAGGCG CAGCGGTCGG 
4381 GCTGAACGGG GGGTTCGTGC ACACAGCCCA GCTTGGAGCG AACGACCTAC ACCGAACTGA 
4441 GATACCTACA GCGTGAGCTA TGAGAAAGCG CCACGCTTCC CGAAGGGAGA AAGGCGGACA 
4501 GGTATCCGGT AAGCGGCAGG GTCGGAACAG GAGAGCGCAC GAGGGAGCTT CCAGGGGGAA 
4561 ACGCCTGGTA TCTTTATAGT CCTGTCGGGT TTCGCCACCT CTGACTTGAG CGTCGATTTT 
4621 TGTGATGCTC GTCAGGGGGG CGGAGCCTAT GGAAAAACGC CAGCAACGCG GCCTTTTTAC 
4681 GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC ACATGTTCTT TCCTGCGTTA TCCCCTGATT 
4741 CTGTGGATAA CCGTATTACC GCCATGCAT