| 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: | 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 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
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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