pGL4.37[luc2P/ARE/Hygro]

价格:1500元

联系方式:I47-825O-882O

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pGL4.37载体基本信息

出品公司: Promega
载体名称: pGL4.37 ; pGL4.37[luc2P/ARE/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:
载体大小: 6086 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
备注:
pGL4.37[luc2P]载体是信号通路报告载体;含ARE应答元件;含萤火虫荧光素酶报告基因luc2P。
产品目录号: E3641
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.37载体质粒图谱和多克隆位点信息

pGL4.37载体图谱



pGL4.37 载体特征

pGL4.37载体简介

The pGL4.37[luc2P/ARE/Hygro] Vector contains four copies of an antioxidant response element (ARE) 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.


In this example protocol, the pGL4.37[luc2P/ARE/Hygro] Vector is used to measure activation of the ARE in HEK293 cells upon treatment with tert-Butylhydroquinone or D,LSulforaphane.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
[Life Technologies Cat.# 11995] and 1X NEAA [Life Technologies Cat.# 11140])
 Charcoal-stripped FBS (Life Technologies Cat.# 126776-011)
 DMSO (Sigma Cat.# D2650)
 Opti-MEM I (Life Technologies Cat.# 31985)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 tert-Butylhydroquinone (Sigma Cat.# 112941)
or D,L-Sulforaphane (Sigma Cat.# S4441)
 Dual-Glo Luciferase Assay System (Cat.# E2940)
 HEK293 cells
 pGL4.75[hRluc/CMV] Vector (Cat.# E6931)


实验流程

Day 1: Plate 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. Quantify the cells and dilute to 1.5 × 105 cells/ml in complete medium.
3. Plate 100μl per well to a solid, white 96-well plate (Corning Cat.# 3917).
4. Incubate for 24 hours in a 37°C, 5% CO2 incubator.

Day 2: Transfection

1. Dilute pGL4.37[luc2P/ARE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase vector
constructs in a 10:1 mass ratio, respectively, to 12.5ng 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 20 minutes.
3. Add 8μl transfection complex per well (100ng DNA/well) and incubate for 18 hours
in a 37°C, 5% CO2 incubator.

Day 3: Medium Replacement and Cell Treatment

1. Remove medium from cells and replace with 72μl of DMEM + 0.5% charcoalstripped
FBS per well.
2. Incubate for 6 hours in a 37°C, 5% CO2 incubator.
3. Resuspend tert-Butylhydroquinone (tBHQ) to 500mM in ethanol. Serially dilute into
ethanol to give 1,000X stocks. Resuspend D,L-Sulforaphane to 200mM in DMSO.
Serially dilute into DMSO to give 1,000X stocks. Dilute the 1,000X stocks into Opti-
MEM I to give 10X stocks.
4. Add 8 μl of the 10X stocks of tBHQ or D,L-Sulforaphane to each well and incubate
for 18 hours in a 37°C, 5% CO2 incubator.

Day 4: Luciferase Measurement

1. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room temperature
for approximately 15 minutes.
2. Add 80μl of the 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).

pGL4.37载体序列

LOCUS       JQ858521                6086 bp    DNA     circular SYN 02-JUL-2012
DEFINITION  Reporter vector pGL4.37[luc2P/ARE/Hygro], complete sequence.
ACCESSION   JQ858521
VERSION     JQ858521.1  GI:392934114
KEYWORDS    .
SOURCE      Reporter vector pGL4.37[luc2P/ARE/Hygro]
  ORGANISM  Reporter vector pGL4.37[luc2P/ARE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6086)
  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..6086
                     /organism="Reporter vector pGL4.37[luc2P/ARE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203015"
     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"
     regulatory      285..366
                     /regulatory_class="enhancer"
                     /note="antioxidant response element (ARE)"
     regulatory      412..442
                     /regulatory_class="promoter"
                     /note="minP promoter"
     gene            475..2250
                     /gene="luc2P"
                     /note="synthetic luciferase"
     CDS             475..2250
                     /gene="luc2P"
                     /note="synthetic luciferase"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFM92251.1"
                     /db_xref="GI:392934116"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2290..2511
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     regulatory      2559..2977
                     /regulatory_class="enhancer"
                     /note="SV40 enhancer and early promoter"
     CDS             3002..4039
                     /note="hygromycin resistance"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin phosphotransferase"
                     /protein_id="AFM92252.1"
                     /db_xref="GI:392934117"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      4063..4111
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     complement(4178..4197)
                     /note="RVprimer4 binding site"
     rep_origin      4435..4471
     gene            complement(5226..6086)
                     /gene="bla"
                     /note="AmpR"
     CDS             complement(5226..6086)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFM92250.1"
                     /db_xref="GI:392934115"
                     /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 gctctagctt ggaaatgaca
      301 ttgctaatgg tgacaaagca acttttagct tggaaatgac attgctaatg gtgacaaagc
      361 aactttctcg aggatatcaa gatctggcct cggcggccaa gcttagacac tagagggtat
      421 ataatggaag ctcgacttcc agcttggcaa tccggtactg ttggtaaagc caccatggaa
      481 gatgccaaaa acattaagaa gggcccagcg ccattctacc cactcgaaga cgggaccgcc
      541 ggcgagcagc tgcacaaagc catgaagcgc tacgccctgg tgcccggcac catcgccttt
      601 accgacgcac atatcgaggt ggacattacc tacgccgagt acttcgagat gagcgttcgg
      661 ctggcagaag ctatgaagcg ctatgggctg aatacaaacc atcggatcgt ggtgtgcagc
      721 gagaatagct tgcagttctt catgcccgtg ttgggtgccc tgttcatcgg tgtggctgtg
      781 gccccagcta acgacatcta caacgagcgc gagctgctga acagcatggg catcagccag
      841 cccaccgtcg tattcgtgag caagaaaggg ctgcaaaaga tcctcaacgt gcaaaagaag
      901 ctaccgatca tacaaaagat catcatcatg gatagcaaga ccgactacca gggcttccaa
      961 agcatgtaca ccttcgtgac ttcccatttg ccacccggct tcaacgagta cgacttcgtg
     1021 cccgagagct tcgaccggga caaaaccatc gccctgatca tgaacagtag tggcagtacc
     1081 ggattgccca agggcgtagc cctaccgcac cgcaccgctt gtgtccgatt cagtcatgcc
     1141 cgcgacccca tcttcggcaa ccagatcatc cccgacaccg ctatcctcag cgtggtgcca
     1201 tttcaccacg gcttcggcat gttcaccacg ctgggctact tgatctgcgg ctttcgggtc
     1261 gtgctcatgt accgcttcga ggaggagcta ttcttgcgca gcttgcaaga ctataagatt
     1321 caatctgccc tgctggtgcc cacactattt agcttcttcg ctaagagcac tctcatcgac
     1381 aagtacgacc taagcaactt gcacgagatc gccagcggcg gggcgccgct cagcaaggag
     1441 gtaggtgagg ccgtggccaa acgcttccac ctaccaggca tccgccaggg ctacggcctg
     1501 acagaaacaa ccagcgccat tctgatcacc cccgaagggg acgacaagcc tggcgcagta
     1561 ggcaaggtgg tgcccttctt cgaggctaag gtggtggact tggacaccgg taagacactg
     1621 ggtgtgaacc agcgcggcga gctgtgcgtc cgtggcccca tgatcatgag cggctacgtt
     1681 aacaaccccg aggctacaaa cgctctcatc gacaaggacg gctggctgca cagcggcgac
     1741 atcgcctact gggacgagga cgagcacttc ttcatcgtgg accggctgaa gagcctgatc
     1801 aaatacaagg gctaccaggt agccccagcc gaactggaga gcatcctgct gcaacacccc
     1861 aacatcttcg acgccggggt cgccggcctg cccgacgacg atgccggcga gctgcccgcc
     1921 gcagtcgtcg tgctggaaca cggtaaaacc atgaccgaga aggagatcgt ggactatgtg
     1981 gccagccagg ttacaaccgc caagaagctg cgcggtggtg ttgtgttcgt ggacgaggtg
     2041 cctaaaggac tgaccggcaa gttggacgcc cgcaagatcc gcgagattct cattaaggcc
     2101 aagaagggcg gcaagatcgc cgtgaattct cacggcttcc ctcccgaggt ggaggagcag
     2161 gccgccggca ccctgcccat gagctgcgcc caggagagcg gcatggatag acaccctgct
     2221 gcttgcgcca gcgccaggat caacgtctaa ggccgcgact ctagagtcgg ggcggccggc
     2281 cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     2341 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     2401 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     2461 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata
     2521 aggatccgtt tgcgtattgg gcgctcttcc gctgatctgc gcagcaccat ggcctgaaat
     2581 aacctctgaa agaggaactt ggttagctac cttctgaggc ggaaagaacc agctgtggaa
     2641 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2701 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2761 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2821 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2881 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     2941 aggctttttt ggaggcctag gcttttgcaa aaagctcgat tcttctgaca ctagcgccac
     3001 catgaagaag cccgaactca ccgctaccag cgttgaaaaa tttctcatcg agaagttcga
     3061 cagtgtgagc gacctgatgc agttgtcgga gggcgaagag agccgagcct tcagcttcga
     3121 tgtcggcgga cgcggctatg tactgcgggt gaatagctgc gctgatggct tctacaaaga
     3181 ccgctacgtg taccgccact tcgccagcgc tgcactaccc atccccgaag tgttggacat
     3241 cggcgagttc agcgagagcc tgacatactg catcagtaga cgcgcccaag gcgttactct
     3301 ccaagacctc cccgaaacag agctgcctgc tgtgttacag cctgtcgccg aagctatgga
     3361 tgctattgcc gccgccgacc tcagtcaaac cagcggcttc ggcccattcg ggccccaagg
     3421 catcggccag tacacaacct ggcgggattt catttgcgcc attgctgatc cccatgtcta
     3481 ccactggcag accgtgatgg acgacaccgt gtccgccagc gtagctcaag ccctggacga
     3541 actgatgctg tgggccgaag actgtcccga ggtgcgccac ctcgtccatg ccgacttcgg
     3601 cagcaacaac gtcctgaccg acaacggccg catcaccgcc gtaatcgact ggtccgaagc
     3661 tatgttcggg gacagtcagt acgaggtggc caacatcttc ttctggcggc cctggctggc
     3721 ttgcatggag cagcagactc gctacttcga gcgccggcat cccgagctgg ccggcagccc
     3781 tcgtctgcga gcctacatgc tgcgcatcgg cctggatcag ctctaccaga gcctcgtgga
     3841 cggcaacttc gacgatgctg cctgggctca aggccgctgc gatgccatcg tccgcagcgg
     3901 ggccggcacc gtcggtcgca cacaaatcgc tcgccggagc gcagccgtat ggaccgacgg
     3961 ctgcgtcgag gtgctggccg acagcggcaa ccgccggccc agtacacgac cgcgcgctaa
     4021 ggaggtaggt cgagtttaaa ctctagaacc ggtcatggcc gcaataaaat atctttattt
     4081 tcattacatc tgtgtgttgg ttttttgtgt gttcgaacta gatgctgtcg accgatgccc
     4141 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     4201 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc
     4261 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4321 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4381 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4441 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4501 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4561 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4621 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4681 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4741 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4801 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4861 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4921 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4981 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5041 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5101 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5161 aatctaaagt atatatgagt aaacttggtc tgacagcggc cgcaaatgct aaaccactgc
     5221 agtggttacc agtgcttgat cagtgaggca ccgatctcag cgatctgcct atttcgttcg
     5281 tccatagtgg cctgactccc cgtcgtgtag atcactacga ttcgtgaggg cttaccatca
     5341 ggccccagcg cagcaatgat gccgcgagag ccgcgttcac cggcccccga tttgtcagca
     5401 atgaaccagc cagcagggag ggccgagcga agaagtggtc ctgctacttt gtccgcctcc
     5461 atccagtcta tgagctgctg tcgtgatgct agagtaagaa gttcgccagt gagtagtttc
     5521 cgaagagttg tggccattgc tactggcatc gtggtatcac gctcgtcgtt cggtatggct
     5581 tcgttcaact ctggttccca gcggtcaagc cgggtcacat gatcacccat attatgaaga
     5641 aatgcagtca gctccttagg gcctccgatc gttgtcagaa gtaagttggc cgcggtgttg
     5701 tcgctcatgg taatggcagc actacacaat tctcttaccg tcatgccatc cgtaagatgc
     5761 ttttccgtga ccggcgagta ctcaaccaag tcgttttgtg agtagtgtat acggcgacca
     5821 agctgctctt gcccggcgtc tatacgggac aacaccgcgc cacatagcag tactttgaaa
     5881 gtgctcatca tcgggaatcg ttcttcgggg cggaaagact caaggatctt gccgctattg
     5941 agatccagtt cgatatagcc cactcttgca cccagttgat cttcagcatc ttttactttc
     6001 accagcgttt cggggtgtgc aaaaacaggc aagcaaaatg ccgcaaagaa gggaatgagt
     6061 gcgacacgaa aatgttggat gctcat
//

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