ptdTomato-N1

价格:2300元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Clontech
载体名称: ptdTomato-N1
质粒类型: 哺乳动物细胞表达载体;荧光报告载体
高拷贝/低拷贝: 高拷贝
克隆方法: 限制性内切酶,多克隆位点
启动子: CMV IE
载体大小: 5443 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: tdTomato (C-端)
载体抗性: 卡那霉素
筛选标记: Neomycin_1.html' target='_blank'>新霉素Neomycin
克隆菌株: DH5α, HB101
宿主细胞(系): 常规细胞系(293、CV-1、CHO等)
备注: 哺乳动物载体ptdTomato-N1组成型表达C端pAsRed2融合蛋白。
产品目录号: 632532
稳定性: 稳表达 或 瞬表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

ptdTomato-N1载体图谱



ptdTomato-N1 多克隆位点

ptdTomato-N1 载体特征

载体简介

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.

载体序列

LOCUS       ptd-Tomato-N1   5443 bp    DNA   circular             8-NOV-2009
COMMENT     Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT     Cat. No. 632532
FEATURES             Location/Qualifiers
     promoter        1..589
                     /label=CMV\IE\Promoter
     misc_feature    591..671
                     /label=MCS
     CDS             3339..4133
                     /label=Kan(R)/Neo(R)
     rep_origin      4718..5361
                     /label=pUC\origin
     rep_origin      complement(2359..2814)
                     /label=f1\origin
     rep_origin      3155..3293
                     /label=SV40\origin
     polyA_signal    2262..2267
                     /label=SV40\early\mRNA\Poly\A\signal
     polyA_signal    2291..2296
                     /label=SV40\early\mRNA\Poly\A\signal
     polyA_signal    4369..4374
                     /label=HSV\TK\polyA\signal
     polyA_signal    4382..4387
                     /label=HSV\TK\polyA\signal
     misc_feature    3211..3217
                     /label=SV40\early\Promoter
     CDS             679..2106
                     /label=td-Tomato
     promoter        2876..2904
                     /label=Kan(R)\Promoter
BASE COUNT     1281 a      1521 c      1475 g      1166 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 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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