pGL4.42[luc2P/HRE/Hygro]

价格:1500元

联系方式:I47-825O-882O

买家导航

pGL4.42载体基本信息

出品公司: Promega
载体名称: pGL4.42 ;  pGL4.42[luc2P/HRE/Hygro]
质粒类型: 信号通路报告载体;哺乳动物载体;萤火虫荧光素酶报告载体
高拷贝/低拷贝: --
克隆方法: 限制性内切酶,多克隆位点
启动子: Minimal Promotor
载体大小: 6080 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: luc2P
载体抗性: 氨苄青霉素
筛选标记: 潮霉素(Hygromycin)
克隆菌株: TOP10等常规菌株
宿主细胞(系): HEK293等
备注: pGL4.42[luc2P/HRE/Hygro]载体是信号通路报告载体;含氧缺乏应答元件HRE;含萤火 虫荧光素酶报告基因luc2P。
产品目录号: E4001
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.42载体质粒图谱和多克隆位点信息

pGL4.42载体图谱



pGL4.42 载体特征

pGL4.42载体简介

pGL4.42载体介绍
The pGL4.42[luc2P/HRE/Hygro] Vector contains four copies of a hypoxia response element (HRE) that drives transcription of the luciferase reporter gene luc2P (Photinus pyralis). luc2P is a synthetically-derived luciferase sequence with humanized codon optimization that is designed for high expression and reduced anomalous transcription. The luc2P gene contains hPEST, a protein destabilization sequence, which allows luc2P protein levels to respond more quickly than those of luc2 to induction of transcription. The vector backbone contains an ampicillin resistance gene to allow selection in E. coli and a gene for hygromycin resistance to allow selection of stably transfected mammalian cell lines.

Example Protocol
In this example protocol, the pGL4.42[luc2P/HRE/Hygro] Vector is used to measure
activation of the HRE in HEK293 cells upon treatment with 1,10-phenanthroline. The
pGL4.75 Vector (encoding Renilla luciferase) is used as a normalization control. In
designing such experiments, it is important that the chosen cell type can be transfected
efficiently and that it expresses the proper components of the signaling pathway of
interest in order to generate the biological response. Protocol optimization may be
required for your particular cell type and assay conditions.

实验材料
 Dulbecco’s PBS (DPBS; Life Technologies Cat.# 14190)
 0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
 DMEM (Life Technologies Cat.# 11995)
 complete medium DMEM supplemented with 10% fetal bovine serum (DMEM/FBS;
Life Technologies Cat.# 16000) and 1X NEAA (Life Technologies Cat.# 11140)
 Opti-MEM I (Life Technologies Cat.# 31985)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 DMSO (Sigma Cat.# D2650)
 1,10-phenanthroline (Sigma Cat.# 131377))
 Dual-Glo Luciferase Assay System (Cat.# E2940)
 HEK293 cells
 pGL4.75[hRluc/CMV] Vector (Cat.# E6931)

实验流程
Day 1: Reverse Transfection
Preparation of Cells
1. Grow HEK293 cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash
with DPBS and treat with one volume of 0.05% trypsin-EDTA. Resuspend cells in
four volumes of complete medium.
2. Pellet the cells by centrifugation at 233 x g for 5 minutes in a swinging-bucket rotor.
Resuspend in complete medium at a concentration of 1 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.42[luc2P/HRE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
vector constructs in a 10:1 mass ratio, respectively, to 10ng total DNA/μl in
Opti-MEM I.
2. Add FuGENE HD to a 3:1 lipid:DNA ratio. Mix by pipetting. Incubate at room
temperature for 30 minutes.
3. Dilute lipid:DNA mixture 20-fold with 1 × 105 cells/ml cell suspension. Mix by
pipetting.
4. Plate 100μl per well into a solid, white 96-well plate (Corning Cat.# 3917).
5. Incubate for 24 hours in a 37°C, 5% CO2 incubator.

Day 2: Cell Treatment and Luminescence Measurement
1. Dissolve 1,10-phenanthroline to a final concentration of 50mM in DMSO. Serially
dilute this solution using DMSO to give a range of concentrated stock solutions
(500X). Dilute each concentrated stock solution using Opti-MEM I to give a range
of dilute stock solutions (10X). Add 10μl of the 10X stocks to each well.
2. Incubate for 5 hours in a 37°C, 5% CO2 incubator.
3. Remove plates from the 37°C, 5% CO2 incubator. Allow plates to cool to room
temperature for approximately 15 minutes.
4. Add Dual-Glo Luciferase Assay System detection reagents, and measure luminescence
following the recommended protocol (Refer to the Dual-Glo Luciferase Assay
System Technical Manual, #TM058 for details).

pGL4.42载体序列

LOCUS       JQ858518                6080 bp    DNA     circular SYN 02-JUL-2012
DEFINITION  Reporter vector PGL4.42 [luc2P/HRE/Hygro], complete sequence.
ACCESSION   JQ858518
VERSION     JQ858518.1  GI:392934102
KEYWORDS    .
SOURCE      Reporter vector PGL4.42 [luc2P/HRE/Hygro]
  ORGANISM  Reporter vector PGL4.42 [luc2P/HRE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6080)
  AUTHORS   McNamara,B.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-MAR-2012) Scientific Communications, Promega
            Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA
FEATURES             Location/Qualifiers
     source          1..6080
                     /organism="Reporter vector PGL4.42 [luc2P/HRE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203012"
     regulatory      105..258
                     /regulatory_class="terminator"
                     /note="transcriptional pause site"
     regulatory      105..153
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     207..226
                     /note="RVprimer3 binding site"
     misc_feature    285..360
                     /note="HRE response element"
     regulatory      406..436
                     /regulatory_class="promoter"
                     /note="minP promoter"
     gene            469..2244
                     /gene="luc2P"
                     /note="synthetic luciferase"
     CDS             469..2244
                     /gene="luc2P"
                     /note="synthetic luciferase"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFM92242.1"
                     /db_xref="GI:392934104"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2284..2505
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     regulatory      2553..2971
                     /regulatory_class="enhancer"
                     /note="SV40 enhancer and early promoter"
     CDS             2996..4033
                     /note="hygromycin resistance"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin phosphotransferase"
                     /protein_id="AFM92243.1"
                     /db_xref="GI:392934105"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      4057..4105
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     complement(4172..4191)
                     /note="RVprimer4 binding site"
     rep_origin      4429..4465
     gene            complement(5220..6080)
                     /gene="bla"
                     /note="AmpR"
     CDS             complement(5220..6080)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFM92241.1"
                     /db_xref="GI:392934103"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
ORIGIN      
        1 actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg
       61 ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat
      121 tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc
      181 atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag
      241 gtgccagaac atttctctgg cctaactggc cggtacctga gctcgtgact acgtgctgcc
      301 taggtgacta cgtgctgcct aggtgactac gtgctgccta ggtgactacg tgctgcctag
      361 ctcgaggata tcaagatctg gcctcggcgg ccaagcttag acactagagg gtatataatg
      421 gaagctcgac ttccagcttg gcaatccggt actgttggta aagccaccat ggaagatgcc
      481 aaaaacatta agaagggccc agcgccattc tacccactcg aagacgggac cgccggcgag
      541 cagctgcaca aagccatgaa gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac
      601 gcacatatcg aggtggacat tacctacgcc gagtacttcg agatgagcgt tcggctggca
      661 gaagctatga agcgctatgg gctgaataca aaccatcgga tcgtggtgtg cagcgagaat
      721 agcttgcagt tcttcatgcc cgtgttgggt gccctgttca tcggtgtggc tgtggcccca
      781 gctaacgaca tctacaacga gcgcgagctg ctgaacagca tgggcatcag ccagcccacc
      841 gtcgtattcg tgagcaagaa agggctgcaa aagatcctca acgtgcaaaa gaagctaccg
      901 atcatacaaa agatcatcat catggatagc aagaccgact accagggctt ccaaagcatg
      961 tacaccttcg tgacttccca tttgccaccc ggcttcaacg agtacgactt cgtgcccgag
     1021 agcttcgacc gggacaaaac catcgccctg atcatgaaca gtagtggcag taccggattg
     1081 cccaagggcg tagccctacc gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac
     1141 cccatcttcg gcaaccagat catccccgac accgctatcc tcagcgtggt gccatttcac
     1201 cacggcttcg gcatgttcac cacgctgggc tacttgatct gcggctttcg ggtcgtgctc
     1261 atgtaccgct tcgaggagga gctattcttg cgcagcttgc aagactataa gattcaatct
     1321 gccctgctgg tgcccacact atttagcttc ttcgctaaga gcactctcat cgacaagtac
     1381 gacctaagca acttgcacga gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt
     1441 gaggccgtgg ccaaacgctt ccacctacca ggcatccgcc agggctacgg cctgacagaa
     1501 acaaccagcg ccattctgat cacccccgaa ggggacgaca agcctggcgc agtaggcaag
     1561 gtggtgccct tcttcgaggc taaggtggtg gacttggaca ccggtaagac actgggtgtg
     1621 aaccagcgcg gcgagctgtg cgtccgtggc cccatgatca tgagcggcta cgttaacaac
     1681 cccgaggcta caaacgctct catcgacaag gacggctggc tgcacagcgg cgacatcgcc
     1741 tactgggacg aggacgagca cttcttcatc gtggaccggc tgaagagcct gatcaaatac
     1801 aagggctacc aggtagcccc agccgaactg gagagcatcc tgctgcaaca ccccaacatc
     1861 ttcgacgccg gggtcgccgg cctgcccgac gacgatgccg gcgagctgcc cgccgcagtc
     1921 gtcgtgctgg aacacggtaa aaccatgacc gagaaggaga tcgtggacta tgtggccagc
     1981 caggttacaa ccgccaagaa gctgcgcggt ggtgttgtgt tcgtggacga ggtgcctaaa
     2041 ggactgaccg gcaagttgga cgcccgcaag atccgcgaga ttctcattaa ggccaagaag
     2101 ggcggcaaga tcgccgtgaa ttctcacggc ttccctcccg aggtggagga gcaggccgcc
     2161 ggcaccctgc ccatgagctg cgcccaggag agcggcatgg atagacaccc tgctgcttgc
     2221 gccagcgcca ggatcaacgt ctaaggccgc gactctagag tcggggcggc cggccgcttc
     2281 gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2341 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2401 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     2461 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc
     2521 cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca ccatggcctg aaataacctc
     2581 tgaaagagga acttggttag ctaccttctg aggcggaaag aaccagctgt ggaatgtgtg
     2641 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2701 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2761 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2821 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2881 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     2941 ttttggaggc ctaggctttt gcaaaaagct cgattcttct gacactagcg ccaccatgaa
     3001 gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc atcgagaagt tcgacagtgt
     3061 gagcgacctg atgcagttgt cggagggcga agagagccga gccttcagct tcgatgtcgg
     3121 cggacgcggc tatgtactgc gggtgaatag ctgcgctgat ggcttctaca aagaccgcta
     3181 cgtgtaccgc cacttcgcca gcgctgcact acccatcccc gaagtgttgg acatcggcga
     3241 gttcagcgag agcctgacat actgcatcag tagacgcgcc caaggcgtta ctctccaaga
     3301 cctccccgaa acagagctgc ctgctgtgtt acagcctgtc gccgaagcta tggatgctat
     3361 tgccgccgcc gacctcagtc aaaccagcgg cttcggccca ttcgggcccc aaggcatcgg
     3421 ccagtacaca acctggcggg atttcatttg cgccattgct gatccccatg tctaccactg
     3481 gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct caagccctgg acgaactgat
     3541 gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc catgccgact tcggcagcaa
     3601 caacgtcctg accgacaacg gccgcatcac cgccgtaatc gactggtccg aagctatgtt
     3661 cggggacagt cagtacgagg tggccaacat cttcttctgg cggccctggc tggcttgcat
     3721 ggagcagcag actcgctact tcgagcgccg gcatcccgag ctggccggca gccctcgtct
     3781 gcgagcctac atgctgcgca tcggcctgga tcagctctac cagagcctcg tggacggcaa
     3841 cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc atcgtccgca gcggggccgg
     3901 caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc gtatggaccg acggctgcgt
     3961 cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca cgaccgcgcg ctaaggaggt
     4021 aggtcgagtt taaactctag aaccggtcat ggccgcaata aaatatcttt attttcatta
     4081 catctgtgtg ttggtttttt gtgtgttcga actagatgct gtcgaccgat gcccttgaga
     4141 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     4201 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc
     4261 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4321 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4381 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4441 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4501 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4561 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4621 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4681 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4741 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4801 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4861 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4921 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4981 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5041 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5101 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5161 aagtatatat gagtaaactt ggtctgacag cggccgcaaa tgctaaacca ctgcagtggt
     5221 taccagtgct tgatcagtga ggcaccgatc tcagcgatct gcctatttcg ttcgtccata
     5281 gtggcctgac tccccgtcgt gtagatcact acgattcgtg agggcttacc atcaggcccc
     5341 agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc ccgatttgtc agcaatgaac
     5401 cagccagcag ggagggccga gcgaagaagt ggtcctgcta ctttgtccgc ctccatccag
     5461 tctatgagct gctgtcgtga tgctagagta agaagttcgc cagtgagtag tttccgaaga
     5521 gttgtggcca ttgctactgg catcgtggta tcacgctcgt cgttcggtat ggcttcgttc
     5581 aactctggtt cccagcggtc aagccgggtc acatgatcac ccatattatg aagaaatgca
     5641 gtcagctcct tagggcctcc gatcgttgtc agaagtaagt tggccgcggt gttgtcgctc
     5701 atggtaatgg cagcactaca caattctctt accgtcatgc catccgtaag atgcttttcc
     5761 gtgaccggcg agtactcaac caagtcgttt tgtgagtagt gtatacggcg accaagctgc
     5821 tcttgcccgg cgtctatacg ggacaacacc gcgccacata gcagtacttt gaaagtgctc
     5881 atcatcggga atcgttcttc ggggcggaaa gactcaagga tcttgccgct attgagatcc
     5941 agttcgatat agcccactct tgcacccagt tgatcttcag catcttttac tttcaccagc
     6001 gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa agaagggaat gagtgcgaca
     6061 cgaaaatgtt ggatgctcat
//

pGL4.42载体质粒图谱pdf版和相关资料下载

pGL4.42载体应用举例

分享到:

全部载体分类

相关载体

相关文章

相关问答

Copyright © 20012-2013 BIOFENG. 生物风 版权所有 Powered by Biofeng