pGL4.32[luc2P/NF-κB-RE/Hygro]

价格:1500元

联系方式:I47-825O-882O

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载体基本信息

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

载体质粒图谱和多克隆位点信息

pGL4.32载体图谱



pGL4.32 载体特征

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

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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