pGL4.38[luc2P/p53 RE/Hygro]

价格:1500元

联系方式:I47-825O-882O

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pGL4.38载体基本信息

出品公司: Promega
载体名称: pGL4.38; pGL4.38[luc2P/p53 RE/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: Minimal Promotor
载体大小: 6062 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: pGL4.38[luc2P/p53 RE/Hygro]载体是信号通路报告载体;含p53应答元件;含萤火虫荧光素酶报告基因luc2P。
产品目录号: E3651
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.38载体质粒图谱和多克隆位点信息

pGL4.38载体图谱



pGL4.38 载体特征

pGL4.38载体简介

The pGL4.38[luc2P/p53 RE/Hygro] Vector contains two copies of a p53 response element (p53 RE) 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 pGL4.38[luc2P/p53 RE/Hygro] Vector is used to measure
activation of the p53 RE in U2OS cells upon treatment with doxorubicin, etoposide, nutlin-
3, or mitomycin c. 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
 Complete medium [McCoy’s 5A (Life Technologies Cat.# 16600) + 10% FBS (Life
Technologies Cat.# 16000) + 1X NEAA (Life Technologies Cat.# 11140) + 1X
sodium pyruvate (Life Technologies Cat.# 11360)]
 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)
 Doxorubicin (Sigma Cat.# D1515)
 Etoposide (Calbiochem Cat.# 341205)
 Mitomycin c (Sigma Cat.# 705436)
 Nutlin-3 (Sigma Cat.# N6287)
 DMSO (Sigma Cat.# D2650)
 Dual-Glo Luciferase Assay System (Cat.# E2940)
 U2OS cells
 pGL4.75[hRluc/CMV] Vector (Cat.# E6931)

实验流程
Day 1: Plate Cells
1. Grow U2OS cells in complete medium (McCoy’s 5A + 10% FBS + 1X NEAA + 1X
sodium pyruvate). Wash with DPBS and treat with one volume of 0.05% trypsin-
EDTA. Resuspend the cells in four volumes of complete medium.
2. Quantify the cells and dilute to 1 × 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 pGL4.38[luc2P/p53 RE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
control 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 24 hours
in a 37°C, 5% CO2 incubator.

Day 3: Medium Replacement and Cell Treatment
1. Resuspend doxorubicin to 50mM in water. Resuspend etoposide to 50mM in DMSO.
Resuspend mitomycin c to 1mM in water. Resuspend nutlin-3 to 10mM in DMSO.
Make serial dilutions in either water or DMSO and then dilute into Opti-MEM I to
make 10X stocks.
2. Remove existing medium from cells and replace with 72μl of McCoy’s 5A + 0.5%
charcoal-stripped FBS per well.
3. Add 8μl of the 10X compound dilutions and incubate for 18 or 40 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).

pGL4.38载体序列

LOCUS       JQ858522                6062 bp    DNA     circular SYN 02-JUL-2012
DEFINITION  Reporter vector pGL4.38[luc2P/p53 RE/Hygro], complete sequence.
ACCESSION   JQ858522
VERSION     JQ858522.1  GI:392934118
KEYWORDS    .
SOURCE      Reporter vector pGL4.38[luc2P/p53 RE/Hygro]
  ORGANISM  Reporter vector pGL4.38[luc2P/p53 RE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6062)
  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..6062
                     /organism="Reporter vector pGL4.38[luc2P/p53 RE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203016"
     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..342
                     /regulatory_class="enhancer"
                     /note="p53 response element"
     regulatory      388..418
                     /regulatory_class="promoter"
                     /note="minP promoter"
     gene            451..2226
                     /gene="luc2P"
                     /note="luciferase"
     CDS             451..2226
                     /gene="luc2P"
                     /note="synthetic luciferase"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFM92254.1"
                     /db_xref="GI:392934120"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2266..2487
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     regulatory      2535..2953
                     /regulatory_class="enhancer"
                     /note="SV40 enhancer and early promoter"
     CDS             2978..4015
                     /note="hygromycin resistance"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin phosphotransferase"
                     /protein_id="AFM92255.1"
                     /db_xref="GI:392934121"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      4039..4087
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     complement(4154..4173)
                     /note="RVprimer4 binding site"
     rep_origin      4411..4447
     gene            complement(5202..6062)
                     /gene="bla"
                     /note="AmpR"
     CDS             complement(5202..6062)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFM92253.1"
                     /db_xref="GI:392934119"
                     /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 gctctacaga acatgtctaa
      301 gcatgctgtg ccttgcctgg acttgcctgg ccttgccttg ggctcgagga tatcaagatc
      361 tggcctcggc ggccaagctt agacactaga gggtatataa tggaagctcg acttccagct
      421 tggcaatccg gtactgttgg taaagccacc atggaagatg ccaaaaacat taagaagggc
      481 ccagcgccat tctacccact cgaagacggg accgccggcg agcagctgca caaagccatg
      541 aagcgctacg ccctggtgcc cggcaccatc gcctttaccg acgcacatat cgaggtggac
      601 attacctacg ccgagtactt cgagatgagc gttcggctgg cagaagctat gaagcgctat
      661 gggctgaata caaaccatcg gatcgtggtg tgcagcgaga atagcttgca gttcttcatg
      721 cccgtgttgg gtgccctgtt catcggtgtg gctgtggccc cagctaacga catctacaac
      781 gagcgcgagc tgctgaacag catgggcatc agccagccca ccgtcgtatt cgtgagcaag
      841 aaagggctgc aaaagatcct caacgtgcaa aagaagctac cgatcataca aaagatcatc
      901 atcatggata gcaagaccga ctaccagggc ttccaaagca tgtacacctt cgtgacttcc
      961 catttgccac ccggcttcaa cgagtacgac ttcgtgcccg agagcttcga ccgggacaaa
     1021 accatcgccc tgatcatgaa cagtagtggc agtaccggat tgcccaaggg cgtagcccta
     1081 ccgcaccgca ccgcttgtgt ccgattcagt catgcccgcg accccatctt cggcaaccag
     1141 atcatccccg acaccgctat cctcagcgtg gtgccatttc accacggctt cggcatgttc
     1201 accacgctgg gctacttgat ctgcggcttt cgggtcgtgc tcatgtaccg cttcgaggag
     1261 gagctattct tgcgcagctt gcaagactat aagattcaat ctgccctgct ggtgcccaca
     1321 ctatttagct tcttcgctaa gagcactctc atcgacaagt acgacctaag caacttgcac
     1381 gagatcgcca gcggcggggc gccgctcagc aaggaggtag gtgaggccgt ggccaaacgc
     1441 ttccacctac caggcatccg ccagggctac ggcctgacag aaacaaccag cgccattctg
     1501 atcacccccg aaggggacga caagcctggc gcagtaggca aggtggtgcc cttcttcgag
     1561 gctaaggtgg tggacttgga caccggtaag acactgggtg tgaaccagcg cggcgagctg
     1621 tgcgtccgtg gccccatgat catgagcggc tacgttaaca accccgaggc tacaaacgct
     1681 ctcatcgaca aggacggctg gctgcacagc ggcgacatcg cctactggga cgaggacgag
     1741 cacttcttca tcgtggaccg gctgaagagc ctgatcaaat acaagggcta ccaggtagcc
     1801 ccagccgaac tggagagcat cctgctgcaa caccccaaca tcttcgacgc cggggtcgcc
     1861 ggcctgcccg acgacgatgc cggcgagctg cccgccgcag tcgtcgtgct ggaacacggt
     1921 aaaaccatga ccgagaagga gatcgtggac tatgtggcca gccaggttac aaccgccaag
     1981 aagctgcgcg gtggtgttgt gttcgtggac gaggtgccta aaggactgac cggcaagttg
     2041 gacgcccgca agatccgcga gattctcatt aaggccaaga agggcggcaa gatcgccgtg
     2101 aattctcacg gcttccctcc cgaggtggag gagcaggccg ccggcaccct gcccatgagc
     2161 tgcgcccagg agagcggcat ggatagacac cctgctgctt gcgccagcgc caggatcaac
     2221 gtctaaggcc gcgactctag agtcggggcg gccggccgct tcgagcagac atgataagat
     2281 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2341 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     2401 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     2461 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtttgcg tattgggcgc
     2521 tcttccgctg atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt
     2581 agctaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     2641 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2701 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2761 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     2821 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2881 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2941 ttgcaaaaag ctcgattctt ctgacactag cgccaccatg aagaagcccg aactcaccgc
     3001 taccagcgtt gaaaaatttc tcatcgagaa gttcgacagt gtgagcgacc tgatgcagtt
     3061 gtcggagggc gaagagagcc gagccttcag cttcgatgtc ggcggacgcg gctatgtact
     3121 gcgggtgaat agctgcgctg atggcttcta caaagaccgc tacgtgtacc gccacttcgc
     3181 cagcgctgca ctacccatcc ccgaagtgtt ggacatcggc gagttcagcg agagcctgac
     3241 atactgcatc agtagacgcg cccaaggcgt tactctccaa gacctccccg aaacagagct
     3301 gcctgctgtg ttacagcctg tcgccgaagc tatggatgct attgccgccg ccgacctcag
     3361 tcaaaccagc ggcttcggcc cattcgggcc ccaaggcatc ggccagtaca caacctggcg
     3421 ggatttcatt tgcgccattg ctgatcccca tgtctaccac tggcagaccg tgatggacga
     3481 caccgtgtcc gccagcgtag ctcaagccct ggacgaactg atgctgtggg ccgaagactg
     3541 tcccgaggtg cgccacctcg tccatgccga cttcggcagc aacaacgtcc tgaccgacaa
     3601 cggccgcatc accgccgtaa tcgactggtc cgaagctatg ttcggggaca gtcagtacga
     3661 ggtggccaac atcttcttct ggcggccctg gctggcttgc atggagcagc agactcgcta
     3721 cttcgagcgc cggcatcccg agctggccgg cagccctcgt ctgcgagcct acatgctgcg
     3781 catcggcctg gatcagctct accagagcct cgtggacggc aacttcgacg atgctgcctg
     3841 ggctcaaggc cgctgcgatg ccatcgtccg cagcggggcc ggcaccgtcg gtcgcacaca
     3901 aatcgctcgc cggagcgcag ccgtatggac cgacggctgc gtcgaggtgc tggccgacag
     3961 cggcaaccgc cggcccagta cacgaccgcg cgctaaggag gtaggtcgag tttaaactct
     4021 agaaccggtc atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt
     4081 ttgtgtgttc gaactagatg ctgtcgaccg atgcccttga gagccttcaa cccagtcagc
     4141 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc
     4201 atgcaactcg taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct
     4261 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4321 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4381 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4441 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4501 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4561 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4621 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4681 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4741 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4801 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4861 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4921 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4981 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5041 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5101 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5161 ttggtctgac agcggccgca aatgctaaac cactgcagtg gttaccagtg cttgatcagt
     5221 gaggcaccga tctcagcgat ctgcctattt cgttcgtcca tagtggcctg actccccgtc
     5281 gtgtagatca ctacgattcg tgagggctta ccatcaggcc ccagcgcagc aatgatgccg
     5341 cgagagccgc gttcaccggc ccccgatttg tcagcaatga accagccagc agggagggcc
     5401 gagcgaagaa gtggtcctgc tactttgtcc gcctccatcc agtctatgag ctgctgtcgt
     5461 gatgctagag taagaagttc gccagtgagt agtttccgaa gagttgtggc cattgctact
     5521 ggcatcgtgg tatcacgctc gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg
     5581 tcaagccggg tcacatgatc acccatatta tgaagaaatg cagtcagctc cttagggcct
     5641 ccgatcgttg tcagaagtaa gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta
     5701 cacaattctc ttaccgtcat gccatccgta agatgctttt ccgtgaccgg cgagtactca
     5761 accaagtcgt tttgtgagta gtgtatacgg cgaccaagct gctcttgccc ggcgtctata
     5821 cgggacaaca ccgcgccaca tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct
     5881 tcggggcgga aagactcaag gatcttgccg ctattgagat ccagttcgat atagcccact
     5941 cttgcaccca gttgatcttc agcatctttt actttcacca gcgtttcggg gtgtgcaaaa
     6001 acaggcaagc aaaatgccgc aaagaaggga atgagtgcga cacgaaaatg ttggatgctc
     6061 at
//

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