pCMV-tdTomato

pCMV-tdTomato

pCMV-tdTomato载体基本信息

pCMV-tdTomato载体质粒图谱和多克隆位点信息

pCMV-tdTomato载体描述

pCMV-tdTomato encodes tdTomato, a member of the family of fruit fluorescent proteins (1) derived from the Discosoma sp. red fluorescent protein, DsRed (2). Because the Tomato protein has a tendency to dimerize, the vector was designed with two copies of the Tomato coding region linked together to allow intramolecular dimerization. As a result, each tdTomato RNA transcript encodes a tandem dimer of the Tomato protein (excitation and emission maxima equal 554nm and 581nm, respectively). The tdTomato coding sequence is positioned just downstream of the constitutively active cytomegalovirus immediate early promoter (PCMV IE). As a result, mammalian cells transfected with this vector will constitutively express tdTomato. A Kozak consensus sequence is located immediately upstream of the tdTomato coding sequence to enhance translational efficiency in eukaryotic cells (3). SV40 polyadenylation signals downstream of the tdTomato coding sequence direct proper processing of the 3' end of the tdTomato 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, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.

pCMV-tdTomato is designed for use as a marker for cotransfection or cell tracking. The red fluorescence of tdTomato can be detected by fluorescence microscopy, allowing direct visual imaging. In addition, flow cytometry can be used to enrich transfected cell populations. pCMV-tdTomato can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4).

Propagation in E. coli

 Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires

a host containing an F plasmid such as JM109 or XL1-Blue.

 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ~500

 Plasmid incompatibility group: pMB1/Col E1

Excitation and emission maxima of tdTomato

 Excitation maximum = 554 nm

 Emission maximum = 581 nm

References

1. Shaner, N. C., et al. (2004) Nature Biotech. 22(12):1567-1572.

2. Bevis, B. J. & Glick, B. S. (2002) Nature Biotech. 20:83–87.

3. Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.

4. Gorman, C. (1985) In DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.

pCMV-tdTomato载体序列

ORIGIN

        1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG

       61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT

      121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA

      181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC

      241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA

      301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC

      361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG

      421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG

      481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT

      541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA

      601 CCGGACTCAG ATCCACCGGT CGCCACCATG GTGAGCAAGG GCGAGGAGGT CATCAAAGAG

      661 TTCATGCGCT TCAAGGTGCG CATGGAGGGC TCCATGAACG GCCACGAGTT CGAGATCGAG

      721 GGCGAGGGCG AGGGCCGCCC CTACGAGGGC ACCCAGACCG CCAAGCTGAA GGTGACCAAG

      781 GGCGGCCCCC TGCCCTTCGC CTGGGACATC CTGTCCCCCC AGTTCATGTA CGGCTCCAAG

      841 GCGTACGTGA AGCACCCCGC CGACATCCCC GATTACAAGA AGCTGTCCTT CCCCGAGGGC

      901 TTCAAGTGGG AGCGCGTGAT GAACTTCGAG GACGGCGGTC TGGTGACCGT GACCCAGGAC

      961 TCCTCCCTGC AGGACGGCAC GCTGATCTAC AAGGTGAAGA TGCGCGGCAC CAACTTCCCC

     1021 CCCGACGGCC CCGTAATGCA GAAGAAGACC ATGGGCTGGG AGGCCTCCAC CGAGCGCCTG

     1081 TACCCCCGCG ACGGCGTGCT GAAGGGCGAG ATCCACCAGG CCCTGAAGCT GAAGGACGGC

     1141 GGCCACTACC TGGTGGAGTT CAAGACCATC TACATGGCCA AGAAGCCCGT GCAACTGCCC

     1201 GGCTACTACT ACGTGGACAC CAAGCTGGAC ATCACCTCCC ACAACGAGGA CTACACCATC

     1261 GTGGAACAGT ACGAGCGCTC CGAGGGCCGC CACCACCTGT TCCTGGGGCA TGGCACCGGC

     1321 AGCACCGGCA GCGGCAGCTC CGGCACCGCC TCCTCCGAGG ACAACAACAT GGCCGTCATC

     1381 AAAGAGTTCA TGCGCTTCAA GGTGCGCATG GAGGGCTCCA TGAACGGCCA CGAGTTCGAG

     1441 ATCGAGGGCG AGGGCGAGGG CCGCCCCTAC GAGGGCACCC AGACCGCCAA GCTGAAGGTG

     1501 ACCAAGGGCG GCCCCCTGCC CTTCGCCTGG GACATCCTGT CCCCCCAGTT CATGTACGGC

     1561 TCCAAGGCGT ACGTGAAGCA CCCCGCCGAC ATCCCCGATT ACAAGAAGCT GTCCTTCCCC

     1621 GAGGGCTTCA AGTGGGAGCG CGTGATGAAC TTCGAGGACG GCGGTCTGGT GACCGTGACC

     1681 CAGGACTCCT CCCTGCAGGA CGGCACGCTG ATCTACAAGG TGAAGATGCG CGGCACCAAC

     1741 TTCCCCCCCG ACGGCCCCGT AATGCAGAAG AAGACCATGG GCTGGGAGGC CTCCACCGAG

     1801 CGCCTGTACC CCCGCGACGG CGTGCTGAAG GGCGAGATCC ACCAGGCCCT GAAGCTGAAG

     1861 GACGGCGGCC ACTACCTGGT GGAGTTCAAG ACCATCTACA TGGCCAAGAA GCCCGTGCAA

     1921 CTGCCCGGCT ACTACTACGT GGACACCAAG CTGGACATCA CCTCCCACAA CGAGGACTAC

     1981 ACCATCGTGG AACAGTACGA GCGCTCCGAG GGCCGCCACC ACCTGTTCCT GTACGGCATG

     2041 GACGAGCTGT ACAAGTAGGC GGCCGCGACT CTAGATCATA ATCAGCCATA CCACATTTGT

     2101 AGAGGTTTTA CTTGCTTTAA AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT

     2161 GAATGCAATT GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA

     2221 TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC

     2281 CAAACTCATC AATGTATCTT AAGGCGTAAA TTGTAAGCGT TAATATTTTG TTAAAATTCG

     2341 CGTTAAATTT TTGTTAAATC AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC

     2401 CTTATAAATC AAAAGAATAG ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA

     2461 GTCCACTATT AAAGAACGTG GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG

     2521 ATGGCCCACT ACGTGAACCA TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG

     2581 CACTAAATCG GAACCCTAAA GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA

     2641 ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG

     2701 TAGCGGTCAC GCTGCGCGTA ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG

     2761 CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA

     2821 TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT

     2881 GAAAAAGGAA GAGTCCTGAG GCGGAAAGAA CCAGCTGTGG AATGTGTGTC AGTTAGGGTG

     2941 TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC

     3001 AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA

     3061 TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC

     3121 GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC

     3181 CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT

     3241 AGGCTTTTGC AAAGATCGAT CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG

     3301 ATGGATTGCA CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG

     3361 CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC

     3421 CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAA GACGAGGCAG

     3481 CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA

     3541 CTGAAGCGGG AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT

     3601 CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA

     3661 CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC

     3721 GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC

     3781 TCGCGCCAGC CGAACTGTTC GCCAGGCTCA AGGCGAGCAT GCCCGACGGC GAGGATCTCG

     3841 TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG

     3901 GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA

     3961 CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG

     4021 GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT

     4081 GAGCGGGACT CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGC CATCACGAGA

     4141 TTTCGATTCC ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC

     4201 CGGCTGGATG ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCTAGGGG

     4261 GAGGCTAACT GAAACACGGA AGGAGACAAT ACCGGAAGGA ACCCGCGCTA TGACGGCAAT

     4321 AAAAAGACAG AATAAAACGC ACGGTGTTGG GTCGTTTGTT CATAAACGCG GGGTTCGGTC

     4381 CCAGGGCTGG CACTCTGTCG ATACCCCACC GAGACCCCAT TGGGGCCAAT ACGCCCGCGT

     4441 TTCTTCCTTT TCCCCACCCC ACCCCCCAAG TTCGGGTGAA GGCCCAGGGC TCGCAGCCAA

     4501 CGTCGGGGCG GCAGGCCCTG CCATAGCCTC AGGTTACTCA TATATACTTT AGATTGATTT

     4561 AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC

     4621 CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA

     4681 AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA CAAAAAAACC

     4741 ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT

     4801 AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTTCTT CTAGTGTAGC CGTAGTTAGG

     4861 CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC

     4921 AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT

     4981 ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC CCAGCTTGGA

     5041 GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT

     5101 TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG

     5161 CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA

     5221 CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA

     5281 CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG CTCACATGTT

     5341 CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCATGC AT

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