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ptdTomato-N1
大量
钰博生物
1ug/5ug
出品公司: | Ybscience |
---|---|
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: | 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。 |
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 |
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 E1Excitation and emission maxima of tdTomato Excitation maximum = 554 nm Emission maximum = 581 nmReferences1. 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.
LOCUS ptd-Tomato-N1 5443 bp DNA circular 8-NOV-2009COMMENT Created by Clontech Laboratories Inc. http://www.clontech.comCOMMENT Cat. No. 632532FEATURES 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)\PromoterBASE 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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