pECFP-Mem encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin, also called GAP-43 (1), and a cyan fluorescent variant of the enhanced green fluorescent protein (EGFP). The neuromodulin fragment contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets ECFP to cellular membranes. The ECFP gene contains amino acid substitutions that give ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501 nm) spectra similar to other cyan emission variants (2, 3). These substitutions also enhance the brightness and solubility of the protein (2, 4, 5). In addition to the chromophore mutations, ECFP contains over 190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (6, 7). Furthermore, upstream sequences flanking the ECFP-Mem fusion protein have been converted to a Kozak consensus translation initiation site (8). These changes increase the translational efficiency of the fusion protein and consequently its expression in mammalian cells.

Expression of ECFP-Mem is driven by the immediate early promoter of CMV (P_{CMV IE}). The vector contains an SV40 origin of replication and a neomycin resistance (Neo^r) gene for selection in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in?E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in?E. coli?and an f1 origin for single-stranded DNA production.

載體應(yīng)用

pECFP-Mem can be transfected into mammalian cells using any standard method. If required, stable transformants can be selected using G418 (9). Expression of ECFP-Mem in mammalian cells results in strong labeling of the plasma membrane and allows easy tracking of individual cells in a population. This membrane labeling also permits study of fine cellular processes such as neuronal axons (10), leading edges of migrating cells, filopodia, or microvilli on cell surfaces. pECFP-Mem cannot be used as an exclusive plasma membrane marker because it also partially labels intracellular membranes.

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 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG 
0661 GATCCACCGG TCGCCACCAT GCTGTGCTGT ATGAGAAGAA CCAAACAGGT TGAAAAGAAT 
0721 GATGAGGACC AAAAGATCAT GGTGAGCAAG GGCGAGGAGC TGTTCACCGG GGTGGTGCCC 
0781 ATCCTGGTCG AGCTGGACGG CGACGTAAAC GGCCACAAGT TCAGCGTGTC CGGCGAGGGC 
0841 GAGGGCGATG CCACCTACGG CAAGCTGACC CTGAAGTTCA TCTGCACCAC CGGCAAGCTG 
0901 CCCGTGCCCT GGCCCACCCT CGTGACCACC CTGACCTGGG GCGTGCAGTG CTTCAGCCGC 
0961 TACCCCGACC ACATGAAGCA GCACGACTTC TTCAAGTCCG CCATGCCCGA AGGCTACGTC 
1021 CAGGAGCGCA CCATCTTCTT CAAGGACGAC GGCAACTACA AGACCCGCGC CGAGGTGAAG 
1081 TTCGAGGGCG ACACCCTGGT GAACCGCATC GAGCTGAAGG GCATCGACTT CAAGGAGGAC 
1141 GGCAACATCC TGGGGCACAA GCTGGAGTAC AACTACATCA GCCACAACGT CTATATCACC 
1201 GCCGACAAGC AGAAGAACGG CATCAAGGCC AACTTCAAGA TCCGCCACAA CATCGAGGAC 
1261 GGCAGCGTGC AGCTCGCCGA CCACTACCAG CAGAACACCC CCATCGGCGA CGGCCCCGTG 
1321 CTGCTGCCCG ACAACCACTA CCTGAGCACC CAGTCCGCCC TGAGCAAAGA CCCCAACGAG 
1381 AAGCGCGATC ACATGGTCCT GCTGGAGTTC GTGACCGCCG CCGGGATCAC TCTCGGCATG 
1441 GACGAGCTGT ACAAGTAAAG CGGCCGCGAC TCTAGATCAT AATCAGCCAT ACCACATTTG 
1501 TAGAGGTTTT ACTTGCTTTA AAAAACCTCC CACACCTCCC CCTGAACCTG AAACATAAAA 
1561 TGAATGCAAT TGTTGTTGTT AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA 
1621 ATAGCATCAC AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT 
1681 CCAAACTCAT CAATGTATCT TAAGGCGTAA ATTGTAAGCG TTAATATTTT GTTAAAATTC 
1741 GCGTTAAATT TTTGTTAAAT CAGCTCATTT TTTAACCAAT AGGCCGAAAT CGGCAAAATC 
1801 CCTTATAAAT CAAAAGAATA GACCGAGATA GGGTTGAGTG TTGTTCCAGT TTGGAACAAG 
1861 AGTCCACTAT TAAAGAACGT GGACTCCAAC GTCAAAGGGC GAAAAACCGT CTATCAGGGC 
1921 GATGGCCCAC TACGTGAACC ATCACCCTAA TCAAGTTTTT TGGGGTCGAG GTGCCGTAAA 
1981 GCACTAAATC GGAACCCTAA AGGGAGCCCC CGATTTAGAG CTTGACGGGG AAAGCCGGCG 
2041 AACGTGGCGA GAAAGGAAGG GAAGAAAGCG AAAGGAGCGG GCGCTAGGGC GCTGGCAAGT 
2101 GTAGCGGTCA CGCTGCGCGT AACCACCACA CCCGCCGCGC TTAATGCGCC GCTACAGGGC 
2161 GCGTCAGGTG GCACTTTTCG GGGAAATGTG CGCGGAACCC CTATTTGTTT ATTTTTCTAA 
2221 ATACATTCAA ATATGTATCC GCTCATGAGA CAATAACCCT GATAAATGCT TCAATAATAT 
2281 TGAAAAAGGA AGAGTCCTGA GGCGGAAAGA ACCAGCTGTG GAATGTGTGT CAGTTAGGGT 
2341 GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT 
2401 CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC 
2461 ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC 
2521 CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT TATGCAGAGG 
2581 CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC 
2641 TAGGCTTTTG CAAAGATCGA TCAAGAGACA GGATGAGGAT CGTTTCGCAT GATTGAACAA 
2701 GATGGATTGC ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG CTATGACTGG 
2761 GCACAACAGA CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTGTCAGC GCAGGGGCGC 
2821 CCGGTTCTTT TTGTCAAGAC CGACCTGTCC GGTGCCCTGA ATGAACTGCA AGACGAGGCA 
2881 GCGCGGCTAT CGTGGCTGGC CACGACGGGC GTTCCTTGCG CAGCTGTGCT CGACGTTGTC 
2941 ACTGAAGCGG GAAGGGACTG GCTGCTATTG GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA 
3001 TCTCACCTTG CTCCTGCCGA GAAAGTATCC ATCATGGCTG ATGCAATGCG GCGGCTGCAT 
3061 ACGCTTGATC CGGCTACCTG CCCATTCGAC CACCAAGCGA AACATCGCAT CGAGCGAGCA 
3121 CGTACTCGGA TGGAAGCCGG TCTTGTCGAT CAGGATGATC TGGACGAAGA GCATCAGGGG 
3181 CTCGCGCCAG CCGAACTGTT CGCCAGGCTC AAGGCGAGCA TGCCCGACGG CGAGGATCTC 
3241 GTCGTGACCC ATGGCGATGC CTGCTTGCCG AATATCATGG TGGAAAATGG CCGCTTTTCT 
3301 GGATTCATCG ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT AGCGTTGGCT 
3361 ACCCGTGATA TTGCTGAAGA GCTTGGCGGC GAATGGGCTG ACCGCTTCCT CGTGCTTTAC 
3421 GGTATCGCCG CTCCCGATTC GCAGCGCATC GCCTTCTATC GCCTTCTTGA CGAGTTCTTC 
3481 TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCAACCTG CCATCACGAG 
3541 ATTTCGATTC CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT TTCCGGGACG 
3601 CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC CACCCTAGGG 
3661 GGAGGCTAAC TGAAACACGG AAGGAGACAA TACCGGAAGG AACCCGCGCT ATGACGGCAA 
3721 TAAAAAGACA GAATAAAACG CACGGTGTTG GGTCGTTTGT TCATAAACGC GGGGTTCGGT 
3781 CCCAGGGCTG GCACTCTGTC GATACCCCAC CGAGACCCCA TTGGGGCCAA TACGCCCGCG 
3841 TTTCTTCCTT TTCCCCACCC CACCCCCCAA GTTCGGGTGA AGGCCCAGGG CTCGCAGCCA 
3901 ACGTCGGGGC GGCAGGCCCT GCCATAGCCT CAGGTTACTC ATATATACTT TAGATTGATT 
3961 TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA 
4021 CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA GAAAAGATCA 
4081 AAGGATCTTC TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA ACAAAAAAAC 
4141 CACCGCTACC AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT TTTCCGAAGG 
4201 TAACTGGCTT CAGCAGAGCG CAGATACCAA ATACTGTCCT TCTAGTGTAG CCGTAGTTAG 
4261 GCCACCACTT CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA ATCCTGTTAC 
4321 CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA AGACGATAGT 
4381 TACCGGATAA GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG CCCAGCTTGG 
4441 AGCGAACGAC CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA AGCGCCACGC 
4501 TTCCCGAAGG GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC 
4561 GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC GGGTTTCGCC 
4621 ACCTCTGACT TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC CTATGGAAAA 
4681 ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT GCTCACATGT 
4741 TCTTTCCTGC GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCATG CAT