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 requiresa 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 E1Excitation and emission maxima of tdTomato Excitation maximum = 554 nm Emission maximum = 581 nmReferences1. 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.
LOCUS pCMV-td-Tomato 5392 bp DNA circular 28-JUL-2008COMMENT Created by Clontech Laboratories Inc. http://www.clontech.comCOMMENT Cat. No. 632534FEATURES Location/Qualifiers misc_feature 3288..4082 /label=KanR/NeoR CDS 628..2055 /label=td-Tomato promoter 1..589 /label=pCMV\IEBASE COUNT 1272 a 1505 c 1459 g 1156 t 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