| 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 |
买家导航
pGL4.38载体基本信息
pGL4.38载体图谱质粒图谱和多克隆位点信息
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
//
