ptdTomato-N1

ptdTomato-N1

ptdTomato-N1载体基本信息

ptdTomato-N1载体质粒图谱和多克隆位点信息

ptdTomato-N1载体描述

ptdTomato-N1 is a mammalian expression vector designed to express a protein of interest fused to the N-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-N1 is positioned upstream of the tdTomato coding sequence. A Kozak consensus sequence, located between the MCS and the tdTomato coding sequence, enhances translational efficiency of the unfused tdTomato protein in eukaryotic cells (3). SV40 polyadenylation signals downstream of the tdTomato coding sequence 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. The 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-N1 so that it is in-frame with the tdTomato coding sequence. The gene must include an initiation codon (ATG), and lack in-frame stop codons.

ptdTomato-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). ptdTomato-N1 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 (Cat. No. 632496) or the DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393) 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

References

1. Shaner, N. C., et al. (2004) Nature Biotechnol. 22(12):1567-72.

2. Matz, M. V., et al. (1999) Nature Biotechnol. 17(10):969–973.

3. Kozak, M. (1987) Nucleic Acids Res. 15(20):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.

ptdTomato-N1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 ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG

      661 GATCCACCGG TCGCCACCAT GGTGAGCAAG GGCGAGGAGG TCATCAAAGA GTTCATGCGC

      721 TTCAAGGTGC GCATGGAGGG CTCCATGAAC GGCCACGAGT TCGAGATCGA GGGCGAGGGC

      781 GAGGGCCGCC CCTACGAGGG CACCCAGACC GCCAAGCTGA AGGTGACCAA GGGCGGCCCC

      841 CTGCCCTTCG CCTGGGACAT CCTGTCCCCC CAGTTCATGT ACGGCTCCAA GGCGTACGTG

      901 AAGCACCCCG CCGACATCCC CGATTACAAG AAGCTGTCCT TCCCCGAGGG CTTCAAGTGG

      961 GAGCGCGTGA TGAACTTCGA GGACGGCGGT CTGGTGACCG TGACCCAGGA CTCCTCCCTG

     1021 CAGGACGGCA CGCTGATCTA CAAGGTGAAG ATGCGCGGCA CCAACTTCCC CCCCGACGGC

     1081 CCCGTAATGC AGAAGAAGAC CATGGGCTGG GAGGCCTCCA CCGAGCGCCT GTACCCCCGC

     1141 GACGGCGTGC TGAAGGGCGA GATCCACCAG GCCCTGAAGC TGAAGGACGG CGGCCACTAC

     1201 CTGGTGGAGT TCAAGACCAT CTACATGGCC AAGAAGCCCG TGCAACTGCC CGGCTACTAC

     1261 TACGTGGACA CCAAGCTGGA CATCACCTCC CACAACGAGG ACTACACCAT CGTGGAACAG

     1321 TACGAGCGCT CCGAGGGCCG CCACCACCTG TTCCTGGGGC ATGGCACCGG CAGCACCGGC

     1381 AGCGGCAGCT CCGGCACCGC CTCCTCCGAG GACAACAACA TGGCCGTCAT CAAAGAGTTC

     1441 ATGCGCTTCA AGGTGCGCAT GGAGGGCTCC ATGAACGGCC ACGAGTTCGA GATCGAGGGC

     1501 GAGGGCGAGG GCCGCCCCTA CGAGGGCACC CAGACCGCCA AGCTGAAGGT GACCAAGGGC

     1561 GGCCCCCTGC CCTTCGCCTG GGACATCCTG TCCCCCCAGT TCATGTACGG CTCCAAGGCG

     1621 TACGTGAAGC ACCCCGCCGA CATCCCCGAT TACAAGAAGC TGTCCTTCCC CGAGGGCTTC

     1681 AAGTGGGAGC GCGTGATGAA CTTCGAGGAC GGCGGTCTGG TGACCGTGAC CCAGGACTCC

     1741 TCCCTGCAGG ACGGCACGCT GATCTACAAG GTGAAGATGC GCGGCACCAA CTTCCCCCCC

     1801 GACGGCCCCG TAATGCAGAA GAAGACCATG GGCTGGGAGG CCTCCACCGA GCGCCTGTAC

     1861 CCCCGCGACG GCGTGCTGAA GGGCGAGATC CACCAGGCCC TGAAGCTGAA GGACGGCGGC

     1921 CACTACCTGG TGGAGTTCAA GACCATCTAC ATGGCCAAGA AGCCCGTGCA ACTGCCCGGC

     1981 TACTACTACG TGGACACCAA GCTGGACATC ACCTCCCACA ACGAGGACTA CACCATCGTG

     2041 GAACAGTACG AGCGCTCCGA GGGCCGCCAC CACCTGTTCC TGTACGGCAT GGACGAGCTG

     2101 TACAAGTAGG CGGCCGCGAC TCTAGATCAT AATCAGCCAT ACCACATTTG TAGAGGTTTT

     2161 ACTTGCTTTA AAAAACCTCC CACACCTCCC CCTGAACCTG AAACATAAAA TGAATGCAAT

     2221 TGTTGTTGTT AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC

     2281 AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT

     2341 CAATGTATCT TAAGGCGTAA ATTGTAAGCG TTAATATTTT GTTAAAATTC GCGTTAAATT

     2401 TTTGTTAAAT CAGCTCATTT TTTAACCAAT AGGCCGAAAT CGGCAAAATC CCTTATAAAT

     2461 CAAAAGAATA GACCGAGATA GGGTTGAGTG TTGTTCCAGT TTGGAACAAG AGTCCACTAT

     2521 TAAAGAACGT GGACTCCAAC GTCAAAGGGC GAAAAACCGT CTATCAGGGC GATGGCCCAC

     2581 TACGTGAACC ATCACCCTAA TCAAGTTTTT TGGGGTCGAG GTGCCGTAAA GCACTAAATC

     2641 GGAACCCTAA AGGGAGCCCC CGATTTAGAG CTTGACGGGG AAAGCCGGCG AACGTGGCGA

     2701 GAAAGGAAGG GAAGAAAGCG AAAGGAGCGG GCGCTAGGGC GCTGGCAAGT GTAGCGGTCA

     2761 CGCTGCGCGT AACCACCACA CCCGCCGCGC TTAATGCGCC GCTACAGGGC GCGTCAGGTG

     2821 GCACTTTTCG GGGAAATGTG CGCGGAACCC CTATTTGTTT ATTTTTCTAA ATACATTCAA

     2881 ATATGTATCC GCTCATGAGA CAATAACCCT GATAAATGCT TCAATAATAT TGAAAAAGGA

     2941 AGAGTCCTGA GGCGGAAAGA ACCAGCTGTG GAATGTGTGT CAGTTAGGGT GTGGAAAGTC

     3001 CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG

     3061 GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA

     3121 GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC CGCCCAGTTC

     3181 CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT TATGCAGAGG CCGAGGCCGC

     3241 CTCGGCCTCT GAGCTATTCC AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG

     3301 CAAAGATCGA TCAAGAGACA GGATGAGGAT CGTTTCGCAT GATTGAACAA GATGGATTGC

     3361 ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG CTATGACTGG GCACAACAGA

     3421 CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT

     3481 TTGTCAAGAC CGACCTGTCC GGTGCCCTGA ATGAACTGCA AGACGAGGCA GCGCGGCTAT

     3541 CGTGGCTGGC CACGACGGGC GTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG

     3601 GAAGGGACTG GCTGCTATTG GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG

     3661 CTCCTGCCGA GAAAGTATCC ATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC

     3721 CGGCTACCTG CCCATTCGAC CACCAAGCGA AACATCGCAT CGAGCGAGCA CGTACTCGGA

     3781 TGGAAGCCGG TCTTGTCGAT CAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG

     3841 CCGAACTGTT CGCCAGGCTC AAGGCGAGCA TGCCCGACGG CGAGGATCTC GTCGTGACCC

     3901 ATGGCGATGC CTGCTTGCCG AATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG

     3961 ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA

     4021 TTGCTGAAGA GCTTGGCGGC GAATGGGCTG ACCGCTTCCT CGTGCTTTAC GGTATCGCCG

     4081 CTCCCGATTC GCAGCGCATC GCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAGCGGGAC

     4141 TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCAACCTG CCATCACGAG ATTTCGATTC

     4201 CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT TTCCGGGACG CCGGCTGGAT

     4261 GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC CACCCTAGGG GGAGGCTAAC

     4321 TGAAACACGG AAGGAGACAA TACCGGAAGG AACCCGCGCT ATGACGGCAA TAAAAAGACA

     4381 GAATAAAACG CACGGTGTTG GGTCGTTTGT TCATAAACGC GGGGTTCGGT CCCAGGGCTG

     4441 GCACTCTGTC GATACCCCAC CGAGACCCCA TTGGGGCCAA TACGCCCGCG TTTCTTCCTT

     4501 TTCCCCACCC CACCCCCCAA GTTCGGGTGA AGGCCCAGGG CTCGCAGCCA ACGTCGGGGC

     4561 GGCAGGCCCT GCCATAGCCT CAGGTTACTC ATATATACTT TAGATTGATT TAAAACTTCA

     4621 TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC

     4681 TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC

     4741 TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC

     4801 AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT

     4861 CAGCAGAGCG CAGATACCAA ATACTGTTCT TCTAGTGTAG CCGTAGTTAG GCCACCACTT

     4921 CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC

     4981 TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA

     5041 GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC

     5101 CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG

     5161 GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA

     5221 GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT

     5281 TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA

     5341 CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC

     5401 GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCATG CAT

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