pGL4.41[luc2P/HSE/Hygro]

价格:1500元

联系方式:I47-825O-882O

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pGL4.41载体基本信息

出品公司: Promega
载体名称: pGL4.41
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: 6045 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: pGL4.41[luc2P/HSE/Hygro]载体是信号通路报告载体;含热激应答元件HSE;含萤火 虫荧光素酶报告基因luc2P。
产品目录号: E3751
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.41载体质粒图谱和多克隆位点信息

pGL4.41载体图谱



pGL4.41 载体特征

pGL4.41载体简介

The pGL4.41[luc2P/HSE/Hygro] Vector contains four copies of a heat shock response element (HSE) 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 pGL4.41[luc2P/HSE/Hygro] Vector is used to measure activation of the HSE in HepG2 cells upon treatment with 17-AAG or CdCl2. 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
 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])
 Dulbecco’s PBS (DPBS; Life Technologies Cat.# 14190)
 0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
 Charcoal-stripped FBS (Life Technologies Cat.# 126776-011)
 Opti-MEM I (Life Technologies Cat.# 31985)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin; Calbiochem Cat.# 100068)
 CdCl2 (Sigma Cat.# 202908)
 DMSO (Sigma Cat.# D2650)
 Dual-Glo Luciferase Assay System (Cat.# E2940)
 HepG2 cells
 pGL4.75[hRluc/CMV] Vector (Cat.# E6931)

实验流程

Day 1: Plate Cells
1. Grow HepG2 cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash
twice with DPBS and treat with one volume of 0.05% trypsin-EDTA, followed by four
volumes of complete medium.
2. Vigorously resuspend the cells by pipetting and allow cell clumps to settle. Remove
the cell suspension from any cell clumps, quantify the cells and dilute in complete
medium to 1 × 105 cells/ml.
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 pGL4.41[luc2P/HSE/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 4.5:1 lipid:DNA ratio. Mix by pipetting. Incubate at room
temperature for 20 minutes.
3. Add 10μl 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. Resuspend 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) to 1mM in
DMSO. Serially dilute into DMSO to give concentrated stock solutions (1,000X).
Serially dilute a 1mM aqueous stock of CdCl2 into water to give concentrated stock
solutions (1,000X). Dilute the 1,000X stocks of 17-AAG and CdCl2 into DMEM to
give 10X stocks.
2. Remove existing medium from cells and replace with 72μl of DMEM + 0.5%
charcoal-stripped FBS per well.
3. Add 8μl of the 10X dilutions of 17-AAG or CdCl2 and incubate for 6 hours in a 37°C,
5% CO2 incubator.

Day 4: Luminescence 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).

pGL4.41载体序列

LOCUS       JQ858520                6045 bp    DNA     circular SYN 02-JUL-2012
DEFINITION  Reporter vector pGL4.41[luc2P/HSE/Hygro], complete sequence.
ACCESSION   JQ858520
VERSION     JQ858520.1  GI:392934110
KEYWORDS    .
SOURCE      Reporter vector pGL4.41[luc2P/HSE/Hygro]
  ORGANISM  Reporter vector pGL4.41[luc2P/HSE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6045)
  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..6045
                     /organism="Reporter vector pGL4.41[luc2P/HSE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203014"
     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..325
                     /regulatory_class="enhancer"
                     /note="HSE response element"
     regulatory      371..401
                     /regulatory_class="promoter"
                     /note="minP promoter"
     gene            434..2209
                     /gene="luc2P"
                     /note="synthetic luciferase"
     CDS             434..2209
                     /gene="luc2P"
                     /note="synthetic luciferase"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFM92248.1"
                     /db_xref="GI:392934112"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2249..2470
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     regulatory      2518..2936
                     /regulatory_class="enhancer"
                     /note="SV40 enhancer and early promoter"
     CDS             2961..3998
                     /note="hygromycin resistance"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin phosphotransferase"
                     /protein_id="AFM92249.1"
                     /db_xref="GI:392934113"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      4022..4070
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     complement(4137..4156)
                     /note="RVprimer4 binding site"
     rep_origin      4394..4430
     gene            complement(5185..6045)
                     /gene="bla"
                     /note="AmpR"
     CDS             complement(5185..6045)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFM92247.1"
                     /db_xref="GI:392934111"
                     /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 gctcctggaa gattctagaa
      301 cgttctggaa gattctagaa cgttcctcga ggatatcaag atctggcctc ggcggccaag
      361 cttagacact agagggtata taatggaagc tcgacttcca gcttggcaat ccggtactgt
      421 tggtaaagcc accatggaag atgccaaaaa cattaagaag ggcccagcgc cattctaccc
      481 actcgaagac gggaccgccg gcgagcagct gcacaaagcc atgaagcgct acgccctggt
      541 gcccggcacc atcgccttta ccgacgcaca tatcgaggtg gacattacct acgccgagta
      601 cttcgagatg agcgttcggc tggcagaagc tatgaagcgc tatgggctga atacaaacca
      661 tcggatcgtg gtgtgcagcg agaatagctt gcagttcttc atgcccgtgt tgggtgccct
      721 gttcatcggt gtggctgtgg ccccagctaa cgacatctac aacgagcgcg agctgctgaa
      781 cagcatgggc atcagccagc ccaccgtcgt attcgtgagc aagaaagggc tgcaaaagat
      841 cctcaacgtg caaaagaagc taccgatcat acaaaagatc atcatcatgg atagcaagac
      901 cgactaccag ggcttccaaa gcatgtacac cttcgtgact tcccatttgc cacccggctt
      961 caacgagtac gacttcgtgc ccgagagctt cgaccgggac aaaaccatcg ccctgatcat
     1021 gaacagtagt ggcagtaccg gattgcccaa gggcgtagcc ctaccgcacc gcaccgcttg
     1081 tgtccgattc agtcatgccc gcgaccccat cttcggcaac cagatcatcc ccgacaccgc
     1141 tatcctcagc gtggtgccat ttcaccacgg cttcggcatg ttcaccacgc tgggctactt
     1201 gatctgcggc tttcgggtcg tgctcatgta ccgcttcgag gaggagctat tcttgcgcag
     1261 cttgcaagac tataagattc aatctgccct gctggtgccc acactattta gcttcttcgc
     1321 taagagcact ctcatcgaca agtacgacct aagcaacttg cacgagatcg ccagcggcgg
     1381 ggcgccgctc agcaaggagg taggtgaggc cgtggccaaa cgcttccacc taccaggcat
     1441 ccgccagggc tacggcctga cagaaacaac cagcgccatt ctgatcaccc ccgaagggga
     1501 cgacaagcct ggcgcagtag gcaaggtggt gcccttcttc gaggctaagg tggtggactt
     1561 ggacaccggt aagacactgg gtgtgaacca gcgcggcgag ctgtgcgtcc gtggccccat
     1621 gatcatgagc ggctacgtta acaaccccga ggctacaaac gctctcatcg acaaggacgg
     1681 ctggctgcac agcggcgaca tcgcctactg ggacgaggac gagcacttct tcatcgtgga
     1741 ccggctgaag agcctgatca aatacaaggg ctaccaggta gccccagccg aactggagag
     1801 catcctgctg caacacccca acatcttcga cgccggggtc gccggcctgc ccgacgacga
     1861 tgccggcgag ctgcccgccg cagtcgtcgt gctggaacac ggtaaaacca tgaccgagaa
     1921 ggagatcgtg gactatgtgg ccagccaggt tacaaccgcc aagaagctgc gcggtggtgt
     1981 tgtgttcgtg gacgaggtgc ctaaaggact gaccggcaag ttggacgccc gcaagatccg
     2041 cgagattctc attaaggcca agaagggcgg caagatcgcc gtgaattctc acggcttccc
     2101 tcccgaggtg gaggagcagg ccgccggcac cctgcccatg agctgcgccc aggagagcgg
     2161 catggataga caccctgctg cttgcgccag cgccaggatc aacgtctaag gccgcgactc
     2221 tagagtcggg gcggccggcc gcttcgagca gacatgataa gatacattga tgagtttgga
     2281 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     2341 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     2401 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     2461 aaatgtggta aaatcgataa ggatccgttt gcgtattggg cgctcttccg ctgatctgcg
     2521 cagcaccatg gcctgaaata acctctgaaa gaggaacttg gttagctacc ttctgaggcg
     2581 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2641 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2701 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2761 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2821 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     2881 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcgatt
     2941 cttctgacac tagcgccacc atgaagaagc ccgaactcac cgctaccagc gttgaaaaat
     3001 ttctcatcga gaagttcgac agtgtgagcg acctgatgca gttgtcggag ggcgaagaga
     3061 gccgagcctt cagcttcgat gtcggcggac gcggctatgt actgcgggtg aatagctgcg
     3121 ctgatggctt ctacaaagac cgctacgtgt accgccactt cgccagcgct gcactaccca
     3181 tccccgaagt gttggacatc ggcgagttca gcgagagcct gacatactgc atcagtagac
     3241 gcgcccaagg cgttactctc caagacctcc ccgaaacaga gctgcctgct gtgttacagc
     3301 ctgtcgccga agctatggat gctattgccg ccgccgacct cagtcaaacc agcggcttcg
     3361 gcccattcgg gccccaaggc atcggccagt acacaacctg gcgggatttc atttgcgcca
     3421 ttgctgatcc ccatgtctac cactggcaga ccgtgatgga cgacaccgtg tccgccagcg
     3481 tagctcaagc cctggacgaa ctgatgctgt gggccgaaga ctgtcccgag gtgcgccacc
     3541 tcgtccatgc cgacttcggc agcaacaacg tcctgaccga caacggccgc atcaccgccg
     3601 taatcgactg gtccgaagct atgttcgggg acagtcagta cgaggtggcc aacatcttct
     3661 tctggcggcc ctggctggct tgcatggagc agcagactcg ctacttcgag cgccggcatc
     3721 ccgagctggc cggcagccct cgtctgcgag cctacatgct gcgcatcggc ctggatcagc
     3781 tctaccagag cctcgtggac ggcaacttcg acgatgctgc ctgggctcaa ggccgctgcg
     3841 atgccatcgt ccgcagcggg gccggcaccg tcggtcgcac acaaatcgct cgccggagcg
     3901 cagccgtatg gaccgacggc tgcgtcgagg tgctggccga cagcggcaac cgccggccca
     3961 gtacacgacc gcgcgctaag gaggtaggtc gagtttaaac tctagaaccg gtcatggccg
     4021 caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg ttcgaactag
     4081 atgctgtcga ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg
     4141 gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca
     4201 ggtgccggca gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4261 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4321 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4381 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4441 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4501 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4561 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4621 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4681 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4741 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4801 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4861 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4921 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4981 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5041 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5101 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagcggcc
     5161 gcaaatgcta aaccactgca gtggttacca gtgcttgatc agtgaggcac cgatctcagc
     5221 gatctgccta tttcgttcgt ccatagtggc ctgactcccc gtcgtgtaga tcactacgat
     5281 tcgtgagggc ttaccatcag gccccagcgc agcaatgatg ccgcgagagc cgcgttcacc
     5341 ggcccccgat ttgtcagcaa tgaaccagcc agcagggagg gccgagcgaa gaagtggtcc
     5401 tgctactttg tccgcctcca tccagtctat gagctgctgt cgtgatgcta gagtaagaag
     5461 ttcgccagtg agtagtttcc gaagagttgt ggccattgct actggcatcg tggtatcacg
     5521 ctcgtcgttc ggtatggctt cgttcaactc tggttcccag cggtcaagcc gggtcacatg
     5581 atcacccata ttatgaagaa atgcagtcag ctccttaggg cctccgatcg ttgtcagaag
     5641 taagttggcc gcggtgttgt cgctcatggt aatggcagca ctacacaatt ctcttaccgt
     5701 catgccatcc gtaagatgct tttccgtgac cggcgagtac tcaaccaagt cgttttgtga
     5761 gtagtgtata cggcgaccaa gctgctcttg cccggcgtct atacgggaca acaccgcgcc
     5821 acatagcagt actttgaaag tgctcatcat cgggaatcgt tcttcggggc ggaaagactc
     5881 aaggatcttg ccgctattga gatccagttc gatatagccc actcttgcac ccagttgatc
     5941 ttcagcatct tttactttca ccagcgtttc ggggtgtgca aaaacaggca agcaaaatgc
     6001 cgcaaagaag ggaatgagtg cgacacgaaa atgttggatg ctcat
//

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