| 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: | 无 |
| 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 |
买家导航
pGL4.37[luc2P/ARE/Hygro]载体pGL4.37质粒基本信息
pGL4.37[luc2P/ARE/Hygro]质粒图谱pGL4.37载体图谱和pGL4.37[luc2P/ARE/Hygro]载体序列pGL4.37质粒序列多克隆位点信息
pGL4.37[luc2P/ARE/Hygro]质粒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[luc2P/ARE/Hygro]质粒序列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
//
