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pDsRed2-N1

pDsRed2-N1
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  • 产品名称：pDsRed2-N1
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简单介绍
pDsRed2-N1编码DsRed2，是一种DsRed变体，其设计用于更快的成熟和较低的非特异性聚集。衍生自Discosoma sp。红色荧光蛋白DsRed2，如其祖细胞DsRed1，含有一系列沉默的碱基对变化，对应于哺乳动物细胞中高��达的人类密码子使用偏好。pDsRed2-N1除了这些变化外，DsRed2含有六个氨基酸取代：V105A，I161T和S197A，导致转染细胞系中红色荧光的出现更加快速;和R2A，K5E和K9T，其阻止蛋白质聚集。（DsRed2可能与DsRed1形成相同的四聚体结构。）
产品描述

pDsRed2-N1

  • 产品信息
  • 基本信息
  • 质粒简介
  • 质粒图谱
  • 质粒序列

产品信息

产品货号 产品名称 产品规格 优惠价
XY0139 pDsRed2-N1

5μg

¥请询价

使用说明

信裕质粒平台的各批次质粒菌株发货前均经过严格的多重验证，如存在质量问题，请在收到产品的三个月内通知我司。收到质粒后请短暂离心，加入20μl ddH2O溶解质粒，取2μl转化至对应感受态中，挑取单克隆重新提取质粒后使用。如需获取其他详细信息请登录我司官网查询。

基本信息

启动子: CMV promoter
复制子: pUC ori,f1 ori
终止子: SV40 poly(A) signal
质粒分类: 哺乳系列质粒；哺乳荧光质粒；哺乳红色质粒
质粒大小: 4689bp
质粒标签: C-DsRed2
原核抗性: 卡那霉素Kan（50μg/ml）
筛选标记: 新霉素Neo/G418
克隆菌株: DH5α等大肠杆菌
培养条件: 37℃，有氧 LB
表达宿主: 293T等哺乳细胞
培养条件: 37℃，5%CO2
诱导方式: 无须诱导，瞬时表达
5'测序引物: CMV-F（CGCAAATGGGCGGTAGGCGTG）
3'测序引物: Sv40-polyA-R (GAAATTTGTGATGCTATTGC)
备注: 哺乳细胞红色荧光表达载体

质粒简介

        pDsRed2-N1编码DsRed2，是一种DsRed变体，其设计用于更快的成熟和较低的非特异性聚集。衍生自Discosoma sp。红色荧光蛋白DsRed2，如其祖细胞DsRed1，含有一系列沉默的碱基对变化，对应于哺乳动物细胞中高表达的人类密码子使用偏好。除了这些变化外，DsRed2含有六个氨基酸取代：V105A，I161T和S197A，导致转染细胞系中红色荧光的出现更加快速;和R2A，K5E和K9T，其阻止蛋白质聚集。（DsRed2可能与DsRed1形成相同的四聚体结构。）在DsRed2组成型表达的哺乳动物细胞培养物中，转染后24小时内可通过荧光显微镜检测发红细胞。在表达DsRed1的细胞和哺乳动物细胞系统中经常观察到的蛋白质的大不溶性聚集体在表达DsRed2的细胞中显着降低。更快成熟，更可溶性的红色荧光蛋白也被宿主细胞耐受良好;用DsRed2转染的哺乳动物细胞培养物没有显示生存力降低的明显迹象，在测试的那些细胞系中，表达DsRed2的细胞显示与非转染对照相同的形态（例如粘附，光折射）和生长特征。
       pDsRed2-N1 encodes DsRed2, a DsRed variant that has been engineered for faster maturation and lower non-specific aggregation. Derived from the Discosoma sp. red fluorescent protein (drFP583; 1), DsRed2, like its progenitor DsRed1, contains a series of silent base-pair changes that correspond to human codon-usage preferences for high expression in mammalian cells (2). In addition to these changes, DsRed2 contains six amino acid substitutions: V105A, I161T, and S197A, which result in the more rapid appearance of red fluorescence in transfected cell lines; and R2A, K5E, and K9T, which prevent the protein from aggregating. (DsRed2 may, however, form the same tetrameric structure as DsRed1 [3].) In mammalian cell cultures when DsRed2 is expressed constitutively, red-emitting cells can be detected by fluorescence microscopy within 24 hours of transfection. Large insoluble aggregates of protein, often observed in bacterial and mammalian cell systems expressing DsRed1, are dramatically reduced in cells expressing DsRed2. The faster-maturing, more soluble red fluorescent protein is also well tolerated by host cells; mammalian cell cultures transfected with DsRed2 show no obvious signs of reduced viability—in those cell lines tested, cells expressing DsRed2 display the same morphology (e.g., adherence, light-refraction) and growth characteristics as non-transfected controls.
       The multiple cloning site (MCS) in pDsRed2-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed2 coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed2. Sequences upstream of DsRed2 have been converted to a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells (4). SV40 polyadenylation signals  ownstream of the DsRed2 gene direct proper processing of the 3' end of the DsRed2 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing 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 (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette confers kanamycin resistance to E. coli. 
      pDsRed2-N1 can be used to construct fusions to the N-terminus of DsRed2. If a fusion construct retains the fluorescent properties of the native DsRed2 protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene should be cloned into pDsRed2-N1 so that it is in frame with the DsRed2 coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed2-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5). Unmodified pDsRed2-N1 can also be used to express DsRed2 in a cell line of interest (e.g., for use as a transfection marker).

质粒图谱



质粒序列

LOCUS       Exported                4689 bp ds-DNA    circular SYN 18-10-2015
KEYWORDS    Untitled 4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4689)
  AUTHORS   admin
  TITLE     Direct Submission
  JOURNAL   Exported 2015-10-18  from MiaoLingPlasmid
            http://www.miaolingbio.com
FEATURES             Location/Qualifiers
     source          1..4689
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    591..671
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             679..1356
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /note="DsRed2"
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     polyA_signal    1477..1598
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1605..2060)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2087..2191
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        2193..2550
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2401..2536
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2585..3379
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3611..3658
                     /note="HSV TK poly(A) signal"
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      3987..4575
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 ggcctcctcc gagaacgtca tcaccgagtt catgcgcttc
      721 aaggtgcgca tggagggcac cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
      781 ggccgcccct acgagggcca caacaccgtg aagctgaagg tgaccaaggg cggccccctg
      841 cccttcgcct gggacatcct gtccccccag ttccagtacg gctccaaggt gtacgtgaag
      901 caccccgccg acatccccga ctacaagaag ctgtccttcc ccgagggctt caagtgggag
      961 cgcgtgatga acttcgagga cggcggcgtg gcgaccgtga cccaggactc ctccctgcag
     1021 gacggctgct tcatctacaa ggtgaagttc atcggcgtga acttcccctc cgacggcccc
     1081 gtgatgcaga agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac
     1141 ggcgtgctga agggcgagac ccacaaggcc ctgaagctga aggacggcgg ccactacctg
     1201 gtggagttca agtccatcta catggccaag aagcccgtgc agctgcccgg ctactactac
     1261 gtggacgcca agctggacat cacctcccac aacgaggact acaccatcgt ggagcagtac
     1321 gagcgcaccg agggccgcca ccacctgttc ctgtagcggc cgcgactcta gatcataatc
     1381 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     1441 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     1501 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1561 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     1621 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     1681 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     1741 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     1801 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     1861 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     1921 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     1981 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     2041 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2101 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2161 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     2221 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2281 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2341 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2401 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2461 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     2521 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     2581 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     2641 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     2701 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     2761 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     2821 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     2881 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     2941 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3001 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3061 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     3121 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3181 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3241 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3301 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3361 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     3421 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3481 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3541 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     3601 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     3661 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     3721 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     3781 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     3841 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3901 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3961 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4021 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4081 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4141 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4201 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4261 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4321 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4381 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4441 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4501 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4561 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4621 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4681 gccatgcat
//
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