| 出品公司: | Promega |
|---|---|
| 载体名称: | pGL4.32 ; pGL4.32[luc2P/NF-κB-RE/Hygro] |
| 质粒类型: | 信号通路报告载体；哺乳动物载体 |
| 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: | Minimal Promotor |
| 载体大小: | 6049 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: | pGL4.32[luc2P/NF-κB-RE/Hygro]载体含有NF-κB应答原件，驱动荧光素酶报告基因luc2P的表达。 |
| 产品目录号: | E849A |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
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pGL4.32[luc2P/NF-κB-RE/Hygro]载体质粒基本信息
pGL4.32[luc2P/NF-κB-RE/Hygro]质粒图谱载体图谱和pGL4.32[luc2P/NF-κB-RE/Hygro]载体序列质粒序列多克隆位点信息
pGL4.32[luc2P/NF-κB-RE/Hygro]质粒载体简介
实验材料 Dulbecco’s PBS (DPBS) 0.05% (w/v) trypsin in DPBS DMEM DMEM supplemented with 10% fetal bovine serum (DMEM/FBS) Tumor necrosis factor-α (Sigma T0157), 10μg/ml solution in PBS containing 1mg/ml BSA Bright-Glo Luciferase Assay System (Cat.# E2610) HEK 293 cells 实验流程 Day 1: Plate Cells 1. Grow HEK 293 cells in DMEM/FBS to approximately 75% confluency. 2. Harvest cells via trypsinization: Remove the DMEM/FBS, wash the cells with DPBS and add the trypsin/DPBS (1X volume). After 2 minutes, add a 4X volume of DMEM/FBS, collect the cell suspension and pellet the cells by centrifugation. Aspirate the supernatant and resuspend in DMEM/FBS. We have routinely used a concentration of 15,000 viable cells/100μl DMEM/FBS. 3. Dispense 100μl of the cell suspension into the wells of a 96-well plate. Plate enough wells to perform each test condition in triplicate. 4. Cover the plate and place it in a tissue culture incubator at 37°C overnight (or for 24 hours). Day 2: Transfect Cells 1. Transfect the cells using a high-efficiency transfection reagent. Each well of 96-well plate to be transfected requires 0.1μg pGL4.32[luc2P/NF-κB-RE/Hygro] plasmid DNA. Transfection conditions may require optimization. 2. Cover the plate and place it in a tissue culture incubator at 37°C overnight or as needed for cell recovery depending on the transfection method used. We have used 24 hours recovery time for lipid-mediated transfections. Day 3: Induce Transfected Cells 1. Prepare 1X induction and 1X control solutions. Calculate the volume of 1X induction and 1X control solution by multiplying the number of wells needed for each solution by 100μl and prepare 110% of this amount. 1X induction solution: Dilute 10μg/ml TNFα solution to 20ng/ml in DMEM/FBS. Final TNFα concentration will be 20ng/ml. 1X control solution: DMEM/FBS. 2. Remove media from wells that will be treated with either 1X induction solution or 1X control solution. 3. Add 100μl of 1X induction solution to the cells to be induced and 100μl of 1X control solution to the control noninduced cells. 4. Return the plate to the tissue culture incubator and induce for 5 hours. 5. Analyze luciferase activity using an appropriate luciferase detection assay. We have observed comparable results for fold induction of the vector using a variety of luciferase reagents, including: Bright-Glo Luciferase Assay System (Cat.# E2610, Technical Manual #TM052); ONE-Glo Luciferase Assay System (Cat.# E6110, Technical Manual #TM292); Dual-Luciferase Reporter Assay System (Cat.# E1910, Technical Manual #TM040); and Dual-Glo Luciferase Assay System (Cat.# E2920, Technical Manual #TM058). 6. Calculate the fold induction as follows: Fold Induction = Average relative light units of induced cells/Average relative light units of control cells
pGL4.32[luc2P/NF-κB-RE/Hygro]质粒序列载体序列
LOCUS EU581860 6049 bp DNA linear SYN 20-APR-2008
DEFINITION Firefly luciferase reporter vector pGL4.32[luc2P/NFkB-RE/Hygro],
complete sequence.
ACCESSION EU581860
VERSION EU581860.1 GI:183181581
KEYWORDS .
SOURCE Firefly luciferase reporter vector pGL4.32[luc2P/NFkB-RE/Hygro]
ORGANISM Firefly luciferase reporter vector pGL4.32[luc2P/NFkB-RE/Hygro]
other sequences; artificial sequences; vectors.
REFERENCE 1 (bases 1 to 6049)
AUTHORS Brouette,C., TenHarmsel,A. and Garvin,D.
TITLE pGL4 Luciferase Reporter Vectors Technical Manual, TM259, Promega
Corporation
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 6049)
AUTHORS Brouette,C., TenHarmsel,A. and Garvin,D.
TITLE Direct Submission
JOURNAL Submitted (20-MAR-2008) Scientific Communications, Promega
Corporation, 5500 East Cheryl Parkway, Suite 110, Madison, WI
53711, USA
FEATURES Location/Qualifiers
source 1..6049
/organism="Firefly luciferase reporter vector
pGL4.32[luc2P/NFkB-RE/Hygro]"
/mol_type="other DNA"
/db_xref="taxon:517435"
regulatory 33..84
/regulatory_class="promoter"
/note="NFkB response element protein promoter; NFkB-RE;
response element"
regulatory 117..147
/regulatory_class="promoter"
/note="minimal promoter; minP"
gene 180..1955
/gene="luc2"
CDS 180..1955
/gene="luc2"
/codon_start=1
/transl_table=11
/product="firefly luciferase"
/protein_id="ACC44846.1"
/db_xref="GI:183181582"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
LPMSCAQESGMDRHPAACASARINV"
regulatory 1995..2216
/regulatory_class="polyA_signal_sequence"
/note="SV40 late poly(A)"
regulatory 2264..2682
/regulatory_class="promoter"
/note="SV40 early enhancer/promoter"
gene 2707..3744
/gene="hygR"
CDS 2707..3744
/gene="hygR"
/codon_start=1
/transl_table=11
/product="hygromycin resistance protein"
/protein_id="ACC44847.1"
/db_xref="GI:183181583"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
TRPRAKEVGRV"
regulatory 3768..3816
/regulatory_class="polyA_signal_sequence"
/note="synthetic poly(A) signal"
primer_bind 3883..3902
/note="RVprimer4 binding region"
rep_origin 4140
/note="ColE1-derived plasmid replication origin"
gene complement(4931..5791)
/gene="ampR"
CDS complement(4931..5791)
/gene="ampR"
/note="ampicillin resistance"
/codon_start=1
/transl_table=11
/product="synthetic beta-lactamase resistance protein"
/protein_id="ACC44848.1"
/db_xref="GI:183181584"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW"
misc_feature 5896..6049
/note="synthetic poly(A) signal/transcriptional pause"
primer_bind 5998..6017
/note="RVprimer3 binding region"
ORIGIN
1 ggcctaactg gccggtacct gagctcgcta gcgggaattt ccggggactt tccgggaatt
61 tccggggact ttccgggaat ttccagatct ggcctcggcg gccaagctta gacactagag
121 ggtatataat ggaagctcga cttccagctt ggcaatccgg tactgttggt aaagccacca
181 tggaagatgc caaaaacatt aagaagggcc cagcgccatt ctacccactc gaagacggga
241 ccgccggcga gcagctgcac aaagccatga agcgctacgc cctggtgccc ggcaccatcg
301 cctttaccga cgcacatatc gaggtggaca ttacctacgc cgagtacttc gagatgagcg
361 ttcggctggc agaagctatg aagcgctatg ggctgaatac aaaccatcgg atcgtggtgt
421 gcagcgagaa tagcttgcag ttcttcatgc ccgtgttggg tgccctgttc atcggtgtgg
481 ctgtggcccc agctaacgac atctacaacg agcgcgagct gctgaacagc atgggcatca
541 gccagcccac cgtcgtattc gtgagcaaga aagggctgca aaagatcctc aacgtgcaaa
601 agaagctacc gatcatacaa aagatcatca tcatggatag caagaccgac taccagggct
661 tccaaagcat gtacaccttc gtgacttccc atttgccacc cggcttcaac gagtacgact
721 tcgtgcccga gagcttcgac cgggacaaaa ccatcgccct gatcatgaac agtagtggca
781 gtaccggatt gcccaagggc gtagccctac cgcaccgcac cgcttgtgtc cgattcagtc
841 atgcccgcga ccccatcttc ggcaaccaga tcatccccga caccgctatc ctcagcgtgg
901 tgccatttca ccacggcttc ggcatgttca ccacgctggg ctacttgatc tgcggctttc
961 gggtcgtgct catgtaccgc ttcgaggagg agctattctt gcgcagcttg caagactata
1021 agattcaatc tgccctgctg gtgcccacac tatttagctt cttcgctaag agcactctca
1081 tcgacaagta cgacctaagc aacttgcacg agatcgccag cggcggggcg ccgctcagca
1141 aggaggtagg tgaggccgtg gccaaacgct tccacctacc aggcatccgc cagggctacg
1201 gcctgacaga aacaaccagc gccattctga tcacccccga aggggacgac aagcctggcg
1261 cagtaggcaa ggtggtgccc ttcttcgagg ctaaggtggt ggacttggac accggtaaga
1321 cactgggtgt gaaccagcgc ggcgagctgt gcgtccgtgg ccccatgatc atgagcggct
1381 acgttaacaa ccccgaggct acaaacgctc tcatcgacaa ggacggctgg ctgcacagcg
1441 gcgacatcgc ctactgggac gaggacgagc acttcttcat cgtggaccgg ctgaagagcc
1501 tgatcaaata caagggctac caggtagccc cagccgaact ggagagcatc ctgctgcaac
1561 accccaacat cttcgacgcc ggggtcgccg gcctgcccga cgacgatgcc ggcgagctgc
1621 ccgccgcagt cgtcgtgctg gaacacggta aaaccatgac cgagaaggag atcgtggact
1681 atgtggccag ccaggttaca accgccaaga agctgcgcgg tggtgttgtg ttcgtggacg
1741 aggtgcctaa aggactgacc ggcaagttgg acgcccgcaa gatccgcgag attctcatta
1801 aggccaagaa gggcggcaag atcgccgtga attctcacgg cttccctccc gaggtggagg
1861 agcaggccgc cggcaccctg cccatgagct gcgcccagga gagcggcatg gatagacacc
1921 ctgctgcttg cgccagcgcc aggatcaacg tctaaggccg cgactctaga gtcggggcgg
1981 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
2041 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
2101 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
2161 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
2221 cgataaggat ccgtttgcgt attgggcgct cttccgctga tctgcgcagc accatggcct
2281 gaaataacct ctgaaagagg aacttggtta gctaccttct gaggcggaaa gaaccagctg
2341 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
2401 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
2461 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
2521 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
2581 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
2641 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcgattcttc tgacactagc
2701 gccaccatga agaagcccga actcaccgct accagcgttg aaaaatttct catcgagaag
2761 ttcgacagtg tgagcgacct gatgcagttg tcggagggcg aagagagccg agccttcagc
2821 ttcgatgtcg gcggacgcgg ctatgtactg cgggtgaata gctgcgctga tggcttctac
2881 aaagaccgct acgtgtaccg ccacttcgcc agcgctgcac tacccatccc cgaagtgttg
2941 gacatcggcg agttcagcga gagcctgaca tactgcatca gtagacgcgc ccaaggcgtt
3001 actctccaag acctccccga aacagagctg cctgctgtgt tacagcctgt cgccgaagct
3061 atggatgcta ttgccgccgc cgacctcagt caaaccagcg gcttcggccc attcgggccc
3121 caaggcatcg gccagtacac aacctggcgg gatttcattt gcgccattgc tgatccccat
3181 gtctaccact ggcagaccgt gatggacgac accgtgtccg ccagcgtagc tcaagccctg
3241 gacgaactga tgctgtgggc cgaagactgt cccgaggtgc gccacctcgt ccatgccgac
3301 ttcggcagca acaacgtcct gaccgacaac ggccgcatca ccgccgtaat cgactggtcc
3361 gaagctatgt tcggggacag tcagtacgag gtggccaaca tcttcttctg gcggccctgg
3421 ctggcttgca tggagcagca gactcgctac ttcgagcgcc ggcatcccga gctggccggc
3481 agccctcgtc tgcgagccta catgctgcgc atcggcctgg atcagctcta ccagagcctc
3541 gtggacggca acttcgacga tgctgcctgg gctcaaggcc gctgcgatgc catcgtccgc
3601 agcggggccg gcaccgtcgg tcgcacacaa atcgctcgcc ggagcgcagc cgtatggacc
3661 gacggctgcg tcgaggtgct ggccgacagc ggcaaccgcc ggcccagtac acgaccgcgc
3721 gctaaggagg taggtcgagt ttaaactcta gaaccggtca tggccgcaat aaaatatctt
3781 tattttcatt acatctgtgt gttggttttt tgtgtgttcg aactagatgc tgtcgaccga
3841 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
3901 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
3961 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
4021 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
4081 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
4141 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
4201 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
4261 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
4321 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
4381 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
4441 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
4501 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
4561 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
4621 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
4681 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
4741 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
4801 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
4861 aaatcaatct aaagtatata tgagtaaact tggtctgaca gcggccgcaa atgctaaacc
4921 actgcagtgg ttaccagtgc ttgatcagtg aggcaccgat ctcagcgatc tgcctatttc
4981 gttcgtccat agtggcctga ctccccgtcg tgtagatcac tacgattcgt gagggcttac
5041 catcaggccc cagcgcagca atgatgccgc gagagccgcg ttcaccggcc cccgatttgt
5101 cagcaatgaa ccagccagca gggagggccg agcgaagaag tggtcctgct actttgtccg
5161 cctccatcca gtctatgagc tgctgtcgtg atgctagagt aagaagttcg ccagtgagta
5221 gtttccgaag agttgtggcc attgctactg gcatcgtggt atcacgctcg tcgttcggta
5281 tggcttcgtt caactctggt tcccagcggt caagccgggt cacatgatca cccatattat
5341 gaagaaatgc agtcagctcc ttagggcctc cgatcgttgt cagaagtaag ttggccgcgg
5401 tgttgtcgct catggtaatg gcagcactac acaattctct taccgtcatg ccatccgtaa
5461 gatgcttttc cgtgaccggc gagtactcaa ccaagtcgtt ttgtgagtag tgtatacggc
5521 gaccaagctg ctcttgcccg gcgtctatac gggacaacac cgcgccacat agcagtactt
5581 tgaaagtgct catcatcggg aatcgttctt cggggcggaa agactcaagg atcttgccgc
5641 tattgagatc cagttcgata tagcccactc ttgcacccag ttgatcttca gcatctttta
5701 ctttcaccag cgtttcgggg tgtgcaaaaa caggcaagca aaatgccgca aagaagggaa
5761 tgagtgcgac acgaaaatgt tggatgctca tactcgtcct ttttcaatat tattgaagca
5821 tttatcaggg ttactagtac gtctctcaag gataagtaag taatattaag gtacgggagg
5881 tattggacag gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg
5941 tgtgaatcga tagtactaac atacgctctc catcaaaaca aaacgaaaca aaacaaacta
6001 gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa catttctct
//
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pGL4.32[luc2P/NF-κB-RE/Hygro]质粒载体应用举例
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