| 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 |
买家导航
pGL4.41[luc2P/HSE/Hygro]载体pGL4.41质粒基本信息
pGL4.41[luc2P/HSE/Hygro]质粒图谱pGL4.41载体图谱和pGL4.41[luc2P/HSE/Hygro]载体序列pGL4.41质粒序列多克隆位点信息
pGL4.41[luc2P/HSE/Hygro]质粒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[luc2P/HSE/Hygro]质粒序列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
//
