pmCherry-C1

pmCherry-C1 is a mammalian expression vector designed to express a protein of interest fused to the C-terminus of mCherry, a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). The excitation and emission maxima of the native mCherry protein are 587 nm and 610 nm, respectively. Expression of fusion proteins that retain the fluorescent properties of the unmodified mCherry protein can be monitored by flow cytometry and their localization in vivo can be determined by fluorescence microscopy.

The multiple cloning site (MCS) in pmCherry-C1 is positioned downstream of the mCherry coding sequence. A Kozak consensus sequence is located immediately upstream of the mCherry gene to enhance translational efficiency in eukaryotic systems (2). SV40 polyadenylation signals downstream of the mCherry gene and the MCS direct proper processing of the 3' end of the mCherry (or fusion gene) mRNA.

The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter (PSV40 e), the Tn5 neomycin/kanamycin resistance gene, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter (PKanr) upstream of the cassette confers kanamycin resistance in E.coli.

载体应用

The gene of interest must be cloned into pmCherry-C1 so that it is in-frame with the mCherry coding sequence, with no intervening stop codons.

The pmCherry-C1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (3). pmCherry-C1 can also be used as a cotransfection marker, as the unmodified vector will express mCherry in mammalian cells.

For Western analysis, either the Living Colors? DsRed Polyclonal Antibody (Cat. No. 632496) or the DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393) can be used to detect the mCherry protein.

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 GAGGATAACA TGGCCATCAT CAAGGAGTTC 
0661 ATGCGCTTCA AGGTGCACAT GGAGGGCTCC GTGAACGGCC ACGAGTTCGA GATCGAGGGC 
0721 GAGGGCGAGG GCCGCCCCTA CGAGGGCACC CAGACCGCCA AGCTGAAGGT GACCAAGGGT 
0781 GGCCCCCTGC CCTTCGCCTG GGACATCCTG TCCCCTCAGT TCATGTACGG CTCCAAGGCC 
0841 TACGTGAAGC ACCCCGCCGA CATCCCCGAC TACTTGAAGC TGTCCTTCCC CGAGGGCTTC 
0901 AAGTGGGAGC GCGTGATGAA CTTCGAGGAC GGCGGCGTGG TGACCGTGAC CCAGGACTCC 
0961 TCCCTGCAGG ACGGCGAGTT CATCTACAAG GTGAAGCTGC GCGGCACCAA CTTCCCCTCC 
1021 GACGGCCCCG TAATGCAGAA GAAGACCATG GGCTGGGAGG CCTCCTCCGA GCGGATGTAC 
1081 CCCGAGGACG GCGCCCTGAA GGGCGAGATC AAGCAGAGGC TGAAGCTGAA GGACGGCGGC 
1141 CACTACGACG CTGAGGTCAA GACCACCTAC AAGGCCAAGA AGCCCGTGCA GCTGCCCGGC 
1201 GCCTACAACG TCAACATCAA GTTGGACATC ACCTCCCACA ACGAGGACTA CACCATCGTG 
1261 GAACAGTACG AACGCGCCGA GGGCCGCCAC TCCACCGGCG GCATGGACGA GCTGTACAAG 
1321 TCCGGACTCA GATCTCGAGC TCAAGCTTCG AATTCTGCAG TCGACGGTAC CGCGGGCCCG 
1381 GGATCCACCG GATCTAGATA ACTGATCATA ATCAGCCATA CCACATTTGT AGAGGTTTTA 
1441 CTTGCTTTAA AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT GAATGCAATT 
1501 GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA 
1561 AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC 
1621 AATGTATCTT AACGCGTAAA TTGTAAGCGT TAATATTTTG TTAAAATTCG CGTTAAATTT 
1681 TTGTTAAATC AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC 
1741 AAAAGAATAG ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT 
1801 AAAGAACGTG GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT 
1861 ACGTGAACCA TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG 
1921 GAACCCTAAA GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG 
1981 AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC 
2041 GCTGCGCGTA ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG CGTCAGGTGG 
2101 CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA TACATTCAAA 
2161 TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT GAAAAAGGAA 
2221 GAGTCCTGAG GCGGAAAGAA CCAGCTGTGG AATGTGTGTC AGTTAGGGTG TGGAAAGTCC 
2281 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG 
2341 TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG 
2401 TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC 
2461 GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC 
2521 TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC 
2581 AAAGATCGAT CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG ATGGATTGCA 
2641 CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG CACAACAGAC 
2701 AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT 
2761 TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAA GACGAGGCAG CGCGGCTATC 
2821 GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG 
2881 AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC 
2941 TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA CGCTTGATCC 
3001 GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC GTACTCGGAT 
3061 GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC TCGCGCCAGC 
3121 CGAACTGTTC GCCAGGCTCA AGGCGAGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA 
3181 TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA 
3241 CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA CCCGTGATAT 
3301 TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG GTATCGCCGC 
3361 TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT GAGCGGGACT 
3421 CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGC CATCACGAGA TTTCGATTCC 
3481 ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC CGGCTGGATG 
3541 ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCTAGGGG GAGGCTAACT 
3601 GAAACACGGA AGGAGACAAT ACCGGAAGGA ACCCGCGCTA TGACGGCAAT AAAAAGACAG 
3661 AATAAAACGC ACGGTGTTGG GTCGTTTGTT CATAAACGCG GGGTTCGGTC CCAGGGCTGG 
3721 CACTCTGTCG ATACCCCACC GAGACCCCAT TGGGGCCAAT ACGCCCGCGT TTCTTCCTTT 
3781 TCCCCACCCC ACCCCCCAAG TTCGGGTGAA GGCCCAGGGC TCGCAGCCAA CGTCGGGGCG 
3841 GCAGGCCCTG CCATAGCCTC AGGTTACTCA TATATACTTT AGATTGATTT AAAACTTCAT 
3901 TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC CAAAATCCCT 
3961 TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA AGGATCTTCT 
4021 TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA 
4081 GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC 
4141 AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC CGTAGTTAGG CCACCACTTC 
4201 AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC AGTGGCTGCT 
4261 GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT ACCGGATAAG 
4321 GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC 
4381 TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG 
4441 AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG CACGAGGGAG 
4501 CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA CCTCTGACTT 
4561 GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA CGCCAGCAAC 
4621 GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG 
4681 TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCATGC AT