ptdTomato-C1

ptdTomato-C1

ptdTomato-C1载体基本信息

ptdTomato-C1载体质粒图谱和多克隆位点信息

ptdTomato-C1载体简介

ptdTomato-C1 is a mammalian expression vector designed to express a protein of interest fused to the C-terminus of tdTomato. td Tomato is 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). Expression of td Tomato as a tandem dimer prevents the fused protein of interest from being forced into a dimeric complex. Fusions that retain the fluorescence properties of tdTomato can be monitored by flow cytometry and localized by fluorescence microscopy. The multiple cloning site (MCS) in ptdTomato-C1 is positioned downstream of the tdTomato coding sequence. A Kozak consensus sequence, located immediately upstream of the tdTomato coding sequence, enhances translational efficiency in eukaryotic cells (3). SV40 polyadenylation signals downstream of the tdTomato coding sequence and the MCS direct proper processing of the 3' end of the tdTomato (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. This vector also has a neomycin resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells. This cassette consists of the SV40 early promoter, the Tn5 neomycin/kanamycin resistance gene, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette confers kanamycin resistance in E. Coli.

The gene of interest must be cloned into ptdTomato-C1 so that it is in-frame with the tdTomato coding sequence; it should also contain a proper stop codon at the 3' end of its coding region.

ptdTomato-C1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). ptdTomato-C1 can also be used as a cotransfection marker, as the unmodified vector will express tdTomato in mammalian cells.

For Western analysis, either the Living Colors DsRed Polyclonal Antibody or the DsRed Monoclonal Antibody can be used to detect the tdTomato protein.

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) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: high

 Plasmid incompatibility group: pMB1/Col E1

Excitation and emission maxima of tdTomato

 Excitation maximum = 554 nm

 Emission maximum = 581 nm

ptdTomato-C1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 CCGGTCGCCA CCATGGTGAG CAAGGGCGAG GAGGTCATCA AAGAGTTCAT GCGCTTCAAG

  661 GTGCGCATGG AGGGCTCCAT GAACGGCCAC GAGTTCGAGA TCGAGGGCGA GGGCGAGGGC

  721 CGCCCCTACG AGGGCACCCA GACCGCCAAG CTGAAGGTGA CCAAGGGCGG CCCCCTGCCC

  781 TTCGCCTGGG ACATCCTGTC CCCCCAGTTC ATGTACGGCT CCAAGGCGTA CGTGAAGCAC

  841 CCCGCCGACA TCCCCGATTA CAAGAAGCTG TCCTTCCCCG AGGGCTTCAA GTGGGAGCGC

  901 GTGATGAACT TCGAGGACGG CGGTCTGGTG ACCGTGACCC AGGACTCCTC CCTGCAGGAC

  961 GGCACGCTGA TCTACAAGGT GAAGATGCGC GGCACCAACT TCCCCCCCGA CGGCCCCGTA

 1021 ATGCAGAAGA AGACCATGGG CTGGGAGGCC TCCACCGAGC GCCTGTACCC CCGCGACGGC

 1081 GTGCTGAAGG GCGAGATCCA CCAGGCCCTG AAGCTGAAGG ACGGCGGCCA CTACCTGGTG

 1141 GAGTTCAAGA CCATCTACAT GGCCAAGAAG CCCGTGCAAC TGCCCGGCTA CTACTACGTG

 1201 GACACCAAGC TGGACATCAC CTCCCACAAC GAGGACTACA CCATCGTGGA ACAGTACGAG

 1261 CGCTCCGAGG GCCGCCACCA CCTGTTCCTG GGGCATGGCA CCGGCAGCAC CGGCAGCGGC

 1321 AGCTCCGGCA CCGCCTCCTC CGAGGACAAC AACATGGCCG TCATCAAAGA GTTCATGCGC

 1381 TTCAAGGTGC GCATGGAGGG CTCCATGAAC GGCCACGAGT TCGAGATCGA GGGCGAGGGC

 1441 GAGGGCCGCC CCTACGAGGG CACCCAGACC GCCAAGCTGA AGGTGACCAA GGGCGGCCCC

 1501 CTGCCCTTCG CCTGGGACAT CCTGTCCCCC CAGTTCATGT ACGGCTCCAA GGCGTACGTG

 1561 AAGCACCCCG CCGACATCCC CGATTACAAG AAGCTGTCCT TCCCCGAGGG CTTCAAGTGG

 1621 GAGCGCGTGA TGAACTTCGA GGACGGCGGT CTGGTGACCG TGACCCAGGA CTCCTCCCTG

 1681 CAGGACGGCA CGCTGATCTA CAAGGTGAAG ATGCGCGGCA CCAACTTCCC CCCCGACGGC

 1741 CCCGTAATGC AGAAGAAGAC CATGGGCTGG GAGGCCTCCA CCGAGCGCCT GTACCCCCGC

 1801 GACGGCGTGC TGAAGGGCGA GATCCACCAG GCCCTGAAGC TGAAGGACGG CGGCCACTAC

 1861 CTGGTGGAGT TCAAGACCAT CTACATGGCC AAGAAGCCCG TGCAACTGCC CGGCTACTAC

 1921 TACGTGGACA CCAAGCTGGA CATCACCTCC CACAACGAGG ACTACACCAT CGTGGAACAG

 1981 TACGAGCGCT CCGAGGGCCG CCACCACCTG TTCCTGTACG GCATGGACGA GCTGTACAAG

 2041 TCCGGACTCA GATCTCGAGC TCAAGCTTCG AATTCTGCAG TCGACGGTAC CGCGGGCCCG

 2101 GGATCCACCG GATCTAGATA ACTGATCATA ATCAGCCATA CCACATTTGT AGAGGTTTTA

 2161 CTTGCTTTAA AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT GAATGCAATT

 2221 GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA

 2281 AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC

 2341 AATGTATCTT AACGCGTAAA TTGTAAGCGT TAATATTTTG TTAAAATTCG CGTTAAATTT

 2401 TTGTTAAATC AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC

 2461 AAAAGAATAG ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT

 2521 AAAGAACGTG GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT

 2581 ACGTGAACCA TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG

 2641 GAACCCTAAA GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG

 2701 AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC

 2761 GCTGCGCGTA ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG CGTCAGGTGG

 2821 CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA TACATTCAAA

 2881 TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT GAAAAAGGAA

 2941 GAGTCCTGAG GCGGAAAGAA CCAGCTGTGG AATGTGTGTC AGTTAGGGTG TGGAAAGTCC

 3001 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG

 3061 TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG

 3121 TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC

 3181 GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC

 3241 TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC

 3301 AAAGATCGAT CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG ATGGATTGCA

 3361 CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG CACAACAGAC

 3421 AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT

 3481 TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAA GACGAGGCAG CGCGGCTATC

 3541 GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG

 3601 AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC

 3661 TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA CGCTTGATCC

 3721 GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC GTACTCGGAT

 3781 GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC TCGCGCCAGC

 3841 CGAACTGTTC GCCAGGCTCA AGGCGAGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA

 3901 TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA

 3961 CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA CCCGTGATAT

 4021 TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG GTATCGCCGC

 4081 TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT GAGCGGGACT

 4141 CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGC CATCACGAGA TTTCGATTCC

 4201 ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC CGGCTGGATG

 4261 ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCTAGGGG GAGGCTAACT

 4321 GAAACACGGA AGGAGACAAT ACCGGAAGGA ACCCGCGCTA TGACGGCAAT AAAAAGACAG

 4381 AATAAAACGC ACGGTGTTGG GTCGTTTGTT CATAAACGCG GGGTTCGGTC CCAGGGCTGG

 4441 CACTCTGTCG ATACCCCACC GAGACCCCAT TGGGGCCAAT ACGCCCGCGT TTCTTCCTTT

 4501 TCCCCACCCC ACCCCCCAAG TTCGGGTGAA GGCCCAGGGC TCGCAGCCAA CGTCGGGGCG

 4561 GCAGGCCCTG CCATAGCCTC AGGTTACTCA TATATACTTT AGATTGATTT AAAACTTCAT

 4621 TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC CAAAATCCCT

 4681 TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA AGGATCTTCT

 4741 TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA

 4801 GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC

 4861 AGCAGAGCGC AGATACCAAA TACTGTTCTT CTAGTGTAGC CGTAGTTAGG CCACCACTTC

 4921 AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC AGTGGCTGCT

 4981 GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT ACCGGATAAG

 5041 GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC

 5101 TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG

 5161 AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG CACGAGGGAG

 5221 CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA CCTCTGACTT

 5281 GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA CGCCAGCAAC

 5341 GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG

 5401 TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCATGC AT

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