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  • pHcRed1-DR

    價格:2500元

    聯系方式:I47-825O-882O

    買家導航

    載體基本信息

    出品公司: Clontech
    載體名稱: pHcRed1-DR
    質粒類型: 無啟動子載體;熒光報告載體
    高拷貝/低拷貝: 高拷貝
    克隆方法: 限制性內切酶,多克隆位點
    啟動子: 無啟動子
    載體大小: 4.3 kb
    5' 測序引物及序列: --
    3' 測序引物及序列: --
    載體標簽: 紅色熒光蛋白HcRed1-DR(C-端)
    載體抗性: 卡那霉素
    篩選標記: 新霉素(Neomycin)
    克隆菌株: TOP10, DH5α, HB101
    宿主細胞(系): 真核細胞
    備注: pHcRed1-DR載體含有紅色熒光報告基因;
    不含啟動子,將待研究的目的啟動子插入MCS區,用于研究啟動子及順式轉錄元件在細胞內的表達水 平和活性
    ;HcRed1-DR與HcRed1相比,半衰期短,適用于需要報告基因逆轉速度快的研究領域。
    產品目錄號: 632422
    穩定性: 瞬表達 或 穩表達
    組成型/誘導型: --
    病毒/非病毒: 非病毒

    載體質粒圖譜和多克隆位點信息

    pHcRed1-DR載體圖譜



    pHcRed1-DR 多克隆位點

    pHcRed1-DR 載體特征1
    pHcRed1-DR 載體特征2

    載體簡介

    pHcRed1-DR載體描述
    pHcRed1-DR is a promoterless vector that encodes HcRed1-DR, a destabilized variant of Living Colors far-red fluorescent protein, HcRed1 (1). In contrast to the original protein, HcRed1-DR has a short half-life, making it well suited for studies that require rapid reporter turnover. HcRed1-DR was generated from HcRed1 by fusing the C-terminus of the protein to amino acid residues 422–461 of mouse ornithine decarboxylase (MODC), one of the most short-lived proteins in mammalian cells (2). This region of MODC contains a PEST sequence that targets the protein for degradation, resulting in rapid protein turnover (2, 3). The original protein, HcRed1, whose excitation and emission maxima occur at 588 nm and 618 nm ±4 nm, was generated by mutagenesis of a non-fluorescent chromoprotein from the reef coral Heteractis crispa (4; HcRed-2A). The HcRed1-DR coding sequence has been human codon-optimized for higher expression in mammalian cells (5). Sequences upstream of the HcRed1 coding sequence have been converted to a Kozak consensus translation initiation site (6) to increase the translation efficiency in eukaryotic cells.
    pHcRed1-DR can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS), located upstream of the HcRed1-DR coding sequence. SV40 polyadenylation signals downstream of the HcRed1-DR gene direct proper processing of the 3' end of the HcRed1-DR mRNA. The vector also contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418 (4). The Neor 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 this cassette confers kanamycin resistance in E. coli. pHcRed1-DR also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
    
    HcRed1-DR can be used as an in vivo reporter of gene expression. Because of its rapid turnover rate, its expression from a promoter of interest provides a more accurate assessment of the promoter's activity over time than does the more stable HcRed1. Promoter/enhancer elements should be inserted into the MCS upstream of the HcRed1-DR coding sequences. Without the addition of a functional promoter, this vector will not express HcRed1-DR. The recombinant pHcRed1-DR vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7).
    
    pHcRed1-DR載體含有以下元件:
     MCS: 12–89
     Destabilized Far-red Fluorescent Protein (HcRed1-DR) gene
    Kozak consensus translation initiation site: 90–100
    Start codon (ATG): 97–99; Stop codon: 913–915
    Insertion of Val at position 2: 100–102
    Ala-3 to Ser mutation: 103–105
    Thr-37 to Ala mutation: 205–207
    Leu-123 to His mutation: 463–465
    Cys-144 to Ser mutation: 526–528
    Arg-169 to His mutation: 601–603
    Leu-174 to His mutation: 616–618
    Pro-202 to Leu mutation: 700–702
    Mouse ornithine decarboxylase PEST sequence: 793–915
     SV40 early mRNA polyadenylation signal
    Polyadenylation signals: 1068–1073 & 1097–1102
    mRNA 3' ends: 1105 & 1118
     f1 single-strand DNA origin: 1165–1620
    (Packages noncoding strand of HcRed1.)
     Ampicillin resistance (β-lactamase) promoter
    –35 region: 1682–1687; –10 region: 1705–1710
    Transcription start point: 1717
     SV40 origin of replication: 1961–2096
     SV40 early promoter
    Enhancer (72-bp tandem repeats): 1792–1865 & 1866–1937
    21-bp repeats: 1941–1961, 1962–1982 & 1984–2004
    Early promoter element: 2017–2023
    Major transcription start points: 2013, 2051, 2057 & 2062
     Kanamycin/neomycin resistance gene
    Neomycin phosphotransferase coding sequences:
    Start codon (ATG): 2145–2147; stop codon: 2937–2939
    G→A mutation to remove Pst I site: 2327
    C→A (Arg→Ser) mutation to remove BssH II site: 2673
     Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
    Polyadenylation signals: 3175–3180 & 3188–3193
     pUC plasmid replication origin: 3524–4170
    

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