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pHcRed1-DR

pHcRed1-DR

pHcRed1-DR


 

编号

载体名称

北京华越洋VECT75329

pHcRed1-DR

 

 

pHcRed1-DR载体基本信息:

 

载体名称:

pHcRed1-DR

质粒类型:

无启动子载体;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:

无启动子

载体大小:

4.3   kb

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HcRed1-DR(C-端)

载体抗性:

卡那霉素

筛选标记:

Neomycin_1.html'   target='_blank'>新霉素(Neomycin

克隆菌株:

TOP10,   DH5α, HB101

宿主细胞(系):

真核细胞

备注:

pHcRed1-DR载体含有红色荧光报告基因;
 
不含启动子,将待研究的目的启动子插入MCS区,用于研究启动子及顺式转录元件在细胞内的表达水 平和活性
 
HcRed1-DRHcRed1相比,半衰期短,适用于需要报告基因逆转速度快的研究领域。

稳定性:

瞬表达 稳表达

组成型/诱导型:

--

病毒/非病毒:

非病毒

 

pHcRed1-DR载体质粒图谱和多克隆位点信息:

 

 

pHcRed1-DR载体描述:

 

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 422461 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载体含有以下元件:

 

MCS: 1289

 Destabilized Far-red Fluorescent Protein (HcRed1-DR) gene

Kozak consensus translation initiation site: 90100

Start codon (ATG): 9799; Stop codon: 913915

Insertion of Val at position 2: 100102

Ala-3 to Ser mutation: 103105

Thr-37 to Ala mutation: 205207

Leu-123 to His mutation: 463465

Cys-144 to Ser mutation: 526528

Arg-169 to His mutation: 601603

Leu-174 to His mutation: 616618

Pro-202 to Leu mutation: 700702

Mouse ornithine decarboxylase PEST sequence: 793915

 SV40 early mRNA polyadenylation signal

Polyadenylation signals: 10681073 & 10971102

mRNA 3' ends: 1105 & 1118

 f1 single-strand DNA origin: 11651620

(Packages noncoding strand of HcRed1.)

 Ampicillin resistance (β-lactamase) promoter

35 region: 16821687; 10 region: 17051710

Transcription start point: 1717

 SV40 origin of replication: 19612096

 SV40 early promoter

Enhancer (72-bp tandem repeats): 17921865 & 18661937

21-bp repeats: 19411961, 19621982 & 19842004

Early promoter element: 20172023

Major transcription start points: 2013, 2051, 2057 & 2062

 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:

Start codon (ATG): 21452147; stop codon: 29372939

GA mutation to remove Pst I site: 2327

CA (ArgSer) mutation to remove BssH II site: 2673

 Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal

Polyadenylation signals: 31753180 & 31883193

 pUC plasmid replication origin: 35244170

 

 

pHcRed1-DR载体其他相关的其他类型载体:

TOPFlash

pISRE-TA-Luc

TEV protease

pIL253

SuperTopFlash

pIJ8860

pZsGreen1-DR

pHY300PLK

pZsGreen1-1

pHY300

pZL507

pHT304

pYCT

pHT43

pXMJ19

pHT01

pVLT33

pHSE-Luc

pUG6

pHIS1525

pTimer-1

pHGF9050

pTimer

pHD_v2

pTA-Luc

pHcRed1-DR

pTALETF_v2 (NN)

pHcRed1-1

pTALETF_v2 (NI)

pHCMC05

pTALETF_v2 (NG)

pHCMC04

pTALETF_v2 (HD)

pGRE-luc

pTALEN_v2 (NN)

pGFP315

pTALEN_v2 (NI)

pGFP22

pTALEN_v2 (NG)

pGEM-3

pTALEN_v2 (HD)

pGAS-TA-Luc

pSRE-Luc

pE2F-TA-Luc

pSH65

pDsRed-Express-DR

pSH63

pDsRed-Express2-1

pSH47

pDsRed-Express-1

pSG1154

pDsRed2-1

pset152

pDG1363

pRK2013

pCSN44

pQE-TriSystem

pDD-tdTomato Reporter

pOS7001

pCRE-Luc

pNZ8048

pCR-Blunt

pNN_v2

pCMV-Raf-1

pNI v2

pCMV-p53

pNG_v2

pCMV-IκBα

pMUTIN4

pCMV-CREB

pMG36e

pCaspase3-sensor

pMetLuc2-Reporter

pCantab 5E

pMetLuc2-Control

pBT2

pmCherry-1

pBR322

pMA09-J

pBIG2R-HPH2

pMA09-H

pAP-1-Luc

pMA5MCS

pAN8-1

pKOR1

pAD43-25

pkc1139

pAD43-25

pK18mobSacB

pAcGFP1-1

pJLA502

HRV 3C protease

pIκB-EGFP

GG499

pAN7-1

p53-Luc

pAP1(PMA)-TA-Luc