出品公司: | Clontech |
---|---|
载体名称: | pCMV-DsRed-Express2 |
质粒类型: | 慢病毒载体;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 |
载体大小: | 4638 bp |
5' 测序引物及序列: | -- |
3' 测序引物及序列: | -- |
载体标签: | -- |
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: |
pCMV-DsRed-Express2组成型表达DsRed-Express2; 在DsRed-Express2的上下游留有酶切位点,便于转移到其它载体上去; DsRed-Express2是DsRed的突变体,与DsRed相比,DsRed-Express2细胞毒性低,尤其适用于原代细胞和干细胞的荧光标记。 |
产品目录号: | 632539 |
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
pCMV-DsRed-Express2载体质粒基本信息
pCMV-DsRed-Express2质粒图谱载体图谱和pCMV-DsRed-Express2载体序列质粒序列多克隆位点信息
pCMV-DsRed-Express2质粒载体简介
pCMV-DsRed-Express2载体描述
pCMV DsRed-Express2 is a mammalian expression vector designed to be used for whole cell labeling or as a marker of cotransfection. In mammalian cells, the vector constitutively expresses DsRed-Express2, a variant of the Discosoma sp. red fluorescent protein, DsRed (1). DsRed-Express2 retains the fast maturation and high photostability characteristic of its predecessor, DsRed-Express (2), and has been engineered (through additional amino acid substitutions) for increased solubility (3). Although it most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express2 displays a greatly reduced tendency to aggregate, resulting in reduced cyto- and phototoxicity, and making DsRed-Express2 much better suited for in vivo applications involving sensitive cells, such as primary or stem cells. In fact, DsRed-Express2 has been shown to be the best red fluorescent protein for whole cell labeling applications (3). DsRed-Express2 also exhibits extremely low residual green fluorescence, which allows cells expressing the protein to be effectively separated from other fluorescently labeled cell populations by flow cytometry. The DsRed-Express2 gene is positioned just downstream of the constitutively active human cytomegalovirus immediate early promoter (PCMV IE). As a result, mammalian cells transfected with this vector will constitutively express the red fluorescent protein. A Kozak consensus sequence (4) has been placed immediately upstream of the DsRed-Express2 coding sequence to enhance translational efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed-Express2 gene direct proper processing of the 3' end of the DsRed-Express2 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. A neomycin resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418 (5). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.
pCMV DsRed-Express2 can be used for whole cell labeling or as a cotransfection marker. The vector can be transfected into mammalian cells using any standard transfection method. Cells expressing DsRed-Express2 (excitation and emission maxima: 554 nm and 591 nm, respectively) can be detected by fluorescence microscopy or flow cytometry 8-12 hours after transfection. If required, stable transfectants can be selected using G418. After cotransfection with pCMV DsRed-Express2 and an expression construct of interest, cells can also be sorted by flow cytometry to enrich for transfected cells.
Propagation in E. coli
Recommended host strain: 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/ColE1
Excitation and emission maxima of DsRed-Express2
Excitation maximum = 554 nm
Emission maximum = 591 nm
References
1. Matz, M. V. et al. (1999) Nat. Biotechnol. 17(10):969-973.
2. Bevis, B. J. & Glick, B. S. (2002) Nat. Biotechnol. 20(1):83–87. Erratum in Nat. Biotechnol. (2002) 20(11):1159
3. Strack, R. L. et al. (2008) Nat. Methods 5(11):955–957.
4. Kozak, M. (1987) Nucleic Acids Res. 15(20): 8125-8148
5. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.
pCMV-DsRed-Express2质粒序列载体序列
LOCUS pCMV DsRed-Express2 4638 bp DNA circular 8-NOV-2009 COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com COMMENT Cat. No. 632539 FEATURES Location/Qualifiers promoter 1..589 /label=CMV\IE\Promoter /note="Human cytomegalovirus (CMV) immediate early promoter" CDS 2534..3328 /label=Kan(R)/Neo(R) rep_origin 3913..4556 /label=pUC\ori CDS 628..1302 /label=DsRed-Express2 BASE COUNT 1102 a 1252 c 1229 g 1055 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 atccaccggt cgccaccatg gatagcactg agaacgtcat caagcccttc 661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc 721 gagggcgagg gcaagcccta cgagggcacc cagaccgcca agctgcaggt gaccaagggc 781 ggccccctgc ccttcgcctg ggacatcctg tccccccagt tccagtacgg ctccaaggtg 841 tacgtgaagc accccgccga catccccgac tacaagaagc tgtccttccc cgagggcttc 901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc 961 tccctgcagg acggcacctt catctaccac gtgaagttca tcggcgtgaa cttcccctcc 1021 gacggccccg taatgcagaa gaagactctg ggctgggagc cctccaccga gcgcctgtac 1081 ccccgcgacg gcgtgctgaa gggcgagatc cacaaggcgc tgaagctgaa gggcggcggc 1141 cactacctgg tggagttcaa gtcaatctac atggccaaga agcccgtgaa gctgcccggc 1201 tactactacg tggactccaa gctggacatc acctcccaca acgaggacta caccgtggtg 1261 gagcagtacg agcgcgccga ggcccgccac cacctgttcc agtagcggcc gcgactctag 1321 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 1381 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 1441 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 1501 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt 1561 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 1621 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt 1681 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa 1741 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag 1801 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt 1861 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg 1921 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc 1981 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg 2041 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 2101 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag 2161 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 2221 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 2281 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 2341 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 2401 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 2461 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg 2521 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt 2581 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt 2641 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc 2701 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 2761 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 2821 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 2881 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 2941 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 3001 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc 3061 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat 3121 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga 3181 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg 3241 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt 3301 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa 3361 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg 3421 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 3481 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg 3541 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg 3601 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga 3661 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg 3721 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt 3781 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 3841 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 3901 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 3961 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 4021 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 4081 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 4141 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 4201 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 4261 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 4321 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 4381 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 4441 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 4501 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 4561 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 4621 cgtattaccg ccatgcat //
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