pDsRED2-Mito

pDsRED2-Mito

载体基本信息

出品公司:	Ybscience
载体名称:	pDsRED2-Mito
质粒类型:	哺乳细胞表达载体；荧光报告载体；亚细胞定位载体
高拷贝/低拷贝:	高拷贝
启动子:	CMV
克隆方法:	多克隆位点，限制性内切酶
载体大小:	4715bp
5' 测序引物及序列:	--
3' 测序引物及序列:	--
载体标签:	红色荧光蛋白DsRed2
载体抗性:	卡纳霉素（Kanamycin）
筛选标记:	新霉素（Neomycin）
备注:	pDsRED2-Mito载体是亚细胞定位载体，含有线粒体定位信号。
稳定性:	瞬表达 或 稳表达
组成型:	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

载体简介

 

pDsRed2-Mito载体描述

fluorescent protein (DsRed2; 1, 2) and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (Mito; 3, 4). The Mito sequence is fused to the 5'-end of DsRed2, a human codon-optimized DsRed variant that is engineered for faster maturation and lower nonspecific aggregation (1, 5). The Mito sequence targets the Mito-DsRed2 fusion protein to the host cell’s mitochondria.
To drive expression of Mito-DsRed2, this vector contains the immmediate early promoter of cytomegalovirus (PCMV IE ). SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3'-end of the Mito-DsRed2 mRNA. This vector also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 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—consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK poly A) gene—allow stably transfected eukaryotic cells to be selected using G418 (6). A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.

pDsRed2-Mito is designed for fluorescent labeling of mitochondria. The vector can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6). The Mito-DsRed2 fusion (excitation/emission maxima: 558 nm/ 583 nm) can be detected by fluorescence microscopy and by flow cytometry. Filter sets optimized for detecting DsRed by microscopy are available from Chroma Technology Corporation and Omega Optical Inc. Please see their websites (www.chroma.com and www.omegafilters.com) and the Living Colors Vol. II User Manual, provided with this vector, for more information. To detect Mito- DsRed2-expressing cells by flow cytometry, use the instrument’s argon-ion laser to excite the fluorophore at 488 nm and the FL-2 channel to detect the fluorophore’s emission at 583 nm.

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) to E. coli hosts.
 E. coli replication origin: pUC
 Copy number: ~500
 Plasmid incompatibility group: pMB1/Col E1

References
1. Living Colors DsRed2 (July 2001) Clontechniques XVI(3):2–3.
2. Matz, M. V., et al. (1999) Nature Biotech. 17:969–973.
3. Rizzuto, R., et al. (1995) Curr. Biol. 5:635–642.
4. Rizzuto, R., et al. (1989) J. Biol. Chem. 264:10595–10600.
5. Haas, J., et al. (1996) Curr. Biol. 6:315–324.
6. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK) pp. 143–190.

 

载体序列

LOCUS       pDsRed2-Mito   4715 bp    DNA   circular             8-JAN-2010
COMMENT     Created by Clontech Laboratories Inc. http://www.biofeng.com/
COMMENT     Cat. No. 632421            
COMMENT     VNTAUTHORNAME|biofeng.com|
FEATURES             Location/Qualifiers
     misc_feature    1..589
                     /label=CMV\IE\Promoter
     misc_feature    597..683
                     /label=Mitochondrial\targeting\sequence
     misc_feature    705..1382
                     /label=DsRed2
     misc_feature    1534..1539
                     /label=SV40\early\polyA\signal
     misc_feature    1563..1568
                     /label=SV40\early\polyA\signal
     misc_feature    1631..2086
                     /label=f1\origin
     misc_feature    2148..2176
                     /label=Bacterial\promoter\for\Kan(R)
     misc_feature    2260..2489
                     /label=SV40\early\Promoter/Enhancer
     misc_feature    2427..2562
                     /label=SV40\origin
     misc_feature    2611..3405
                     /label=Kan(R)/Neo(R)
     misc_feature    3641..3646
                     /label=HSV\TK\polyA\signal
     misc_feature    3654..3659
                     /label=HSV\TK\polyA\signal
     misc_feature    3990..4693
                     /label=pUC\origin
BASE COUNT     1105 a      1282 c      1260 g      1068 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 gctagcatgt 
      601 ccgtcctgac gccgctgctg ctgcggggct tgacaggctc ggcccggcgg ctcccagtgc 
      661 cgcgcgccaa gatccattcg ttgggggatc caccggtcgc caccatggcc tcctccgaga 
      721 acgtcatcac cgagttcatg cgcttcaagg tgcgcatgga gggcaccgtg aacggccacg 
      781 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggccacaac accgtgaagc 
      841 tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttcc 
      901 agtacggctc caaggtgtac gtgaagcacc ccgccgacat ccccgactac aagaagctgt 
      961 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggcga 
     1021 ccgtgaccca ggactcctcc ctgcaggacg gctgcttcat ctacaaggtg aagttcatcg 
     1081 gcgtgaactt cccctccgac ggccccgtga tgcagaagaa gaccatgggc tgggaggcct 
     1141 ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagacccac aaggccctga 
     1201 agctgaagga cggcggccac tacctggtgg agttcaagtc catctacatg gccaagaagc 
     1261 ccgtgcagct gcccggctac tactacgtgg acgccaagct ggacatcacc tcccacaacg 
     1321 aggactacac catcgtggag cagtacgagc gcaccgaggg ccgccaccac ctgttcctgt 
     1381 agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt 
     1441 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg 
     1501 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 
     1561 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 
     1621 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 
     1681 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 
     1741 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 
     1801 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 
     1861 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 
     1921 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 
     1981 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 
     2041 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt 
     2101 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 
     2161 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct 
     2221 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 
     2281 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 
     2341 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 
     2401 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 
     2461 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct 
     2521 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc 
     2581 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt 
     2641 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc 
     2701 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag 
     2761 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg 
     2821 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac 
     2881 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc 
     2941 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc 
     3001 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc 
     3061 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 
     3121 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat 
     3181 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc 
     3241 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa 
     3301 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat 
     3361 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt 
     3421 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg 
     3481 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc 
     3541 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac 
     3601 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa 
     3661 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg 
     3721 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac 
     3781 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc 
     3841 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat 
     3901 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 
     3961 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 
     4021 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 
     4081 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 
     4141 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 
     4201 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 
     4261 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 
     4321 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 
     4381 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 
     4441 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 
     4501 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 
     4561 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 
     4621 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 
     4681 ctgattctgt ggataaccgt attaccgcca tgcat