| 出品公司: | Promega |
|---|---|
| 载体名称: | pGL4.42 ; pGL4.42[luc2P/HRE/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: | 6080 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: | pGL4.42[luc2P/HRE/Hygro]载体是信号通路报告载体；含氧缺乏应答元件HRE；含萤火 虫荧光素酶报告基因luc2P。 |
| 产品目录号: | E4001 |
| 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
pGL4.42[luc2P/HRE/Hygro]载体pGL4.42质粒基本信息
pGL4.42[luc2P/HRE/Hygro]质粒图谱pGL4.42载体图谱和pGL4.42[luc2P/HRE/Hygro]载体序列pGL4.42质粒序列多克隆位点信息
pGL4.42[luc2P/HRE/Hygro]质粒pGL4.42载体简介
pGL4.42载体介绍 The pGL4.42[luc2P/HRE/Hygro] Vector contains four copies of a hypoxia response element (HRE) 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 pGL4.42[luc2P/HRE/Hygro] Vector is used to measure activation of the HRE in HEK293 cells upon treatment with 1,10-phenanthroline. 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 Dulbecco’s PBS (DPBS; Life Technologies Cat.# 14190) 0.05% Tryspin-EDTA (Life Technologies Cat.# 25300) 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) Opti-MEM I (Life Technologies Cat.# 31985) FuGENE HD Transfection Reagent (Cat.# E2311) DMSO (Sigma Cat.# D2650) 1,10-phenanthroline (Sigma Cat.# 131377)) Dual-Glo Luciferase Assay System (Cat.# E2940) HEK293 cells pGL4.75[hRluc/CMV] Vector (Cat.# E6931) 实验流程 Day 1: Reverse Transfection Preparation of Cells 1. Grow HEK293 cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash with DPBS and treat with one volume of 0.05% trypsin-EDTA. Resuspend cells in four volumes of complete medium. 2. Pellet the cells by centrifugation at 233 x g for 5 minutes in a swinging-bucket rotor. Resuspend in complete medium at a concentration of 1 × 105 cells/ml. Preparation of Lipid:DNA Mixture 1. Dilute pGL4.42[luc2P/HRE/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 3:1 lipid:DNA ratio. Mix by pipetting. Incubate at room temperature for 30 minutes. 3. Dilute lipid:DNA mixture 20-fold with 1 × 105 cells/ml cell suspension. Mix by pipetting. 4. Plate 100μl per well into a solid, white 96-well plate (Corning Cat.# 3917). 5. Incubate for 24 hours in a 37°C, 5% CO2 incubator. Day 2: Cell Treatment and Luminescence Measurement 1. Dissolve 1,10-phenanthroline to a final concentration of 50mM in DMSO. Serially dilute this solution using DMSO to give a range of concentrated stock solutions (500X). Dilute each concentrated stock solution using Opti-MEM I to give a range of dilute stock solutions (10X). Add 10μl of the 10X stocks to each well. 2. Incubate for 5 hours in a 37°C, 5% CO2 incubator. 3. Remove plates from the 37°C, 5% CO2 incubator. Allow plates to cool to room temperature for approximately 15 minutes. 4. Add 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).
pGL4.42[luc2P/HRE/Hygro]质粒序列pGL4.42载体序列
LOCUS JQ858518 6080 bp DNA circular SYN 02-JUL-2012
DEFINITION Reporter vector PGL4.42 [luc2P/HRE/Hygro], complete sequence.
ACCESSION JQ858518
VERSION JQ858518.1 GI:392934102
KEYWORDS .
SOURCE Reporter vector PGL4.42 [luc2P/HRE/Hygro]
ORGANISM Reporter vector PGL4.42 [luc2P/HRE/Hygro]
other sequences; artificial sequences; vectors.
REFERENCE 1 (bases 1 to 6080)
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..6080
/organism="Reporter vector PGL4.42 [luc2P/HRE/Hygro]"
/mol_type="other DNA"
/db_xref="taxon:1203012"
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"
misc_feature 285..360
/note="HRE response element"
regulatory 406..436
/regulatory_class="promoter"
/note="minP promoter"
gene 469..2244
/gene="luc2P"
/note="synthetic luciferase"
CDS 469..2244
/gene="luc2P"
/note="synthetic luciferase"
/codon_start=1
/transl_table=11
/product="luciferase"
/protein_id="AFM92242.1"
/db_xref="GI:392934104"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
LPMSCAQESGMDRHPAACASARINV"
regulatory 2284..2505
/regulatory_class="polyA_signal_sequence"
/note="SV40 late poly(A) signal"
regulatory 2553..2971
/regulatory_class="enhancer"
/note="SV40 enhancer and early promoter"
CDS 2996..4033
/note="hygromycin resistance"
/codon_start=1
/transl_table=11
/product="hygromycin phosphotransferase"
/protein_id="AFM92243.1"
/db_xref="GI:392934105"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
TRPRAKEVGRV"
regulatory 4057..4105
/regulatory_class="polyA_signal_sequence"
/note="synthetic poly(A) signal sequence"
primer_bind complement(4172..4191)
/note="RVprimer4 binding site"
rep_origin 4429..4465
gene complement(5220..6080)
/gene="bla"
/note="AmpR"
CDS complement(5220..6080)
/gene="bla"
/note="AmpR"
/codon_start=1
/transl_table=11
/product="beta-lactamase"
/protein_id="AFM92241.1"
/db_xref="GI:392934103"
/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 gctcgtgact acgtgctgcc
301 taggtgacta cgtgctgcct aggtgactac gtgctgccta ggtgactacg tgctgcctag
361 ctcgaggata tcaagatctg gcctcggcgg ccaagcttag acactagagg gtatataatg
421 gaagctcgac ttccagcttg gcaatccggt actgttggta aagccaccat ggaagatgcc
481 aaaaacatta agaagggccc agcgccattc tacccactcg aagacgggac cgccggcgag
541 cagctgcaca aagccatgaa gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac
601 gcacatatcg aggtggacat tacctacgcc gagtacttcg agatgagcgt tcggctggca
661 gaagctatga agcgctatgg gctgaataca aaccatcgga tcgtggtgtg cagcgagaat
721 agcttgcagt tcttcatgcc cgtgttgggt gccctgttca tcggtgtggc tgtggcccca
781 gctaacgaca tctacaacga gcgcgagctg ctgaacagca tgggcatcag ccagcccacc
841 gtcgtattcg tgagcaagaa agggctgcaa aagatcctca acgtgcaaaa gaagctaccg
901 atcatacaaa agatcatcat catggatagc aagaccgact accagggctt ccaaagcatg
961 tacaccttcg tgacttccca tttgccaccc ggcttcaacg agtacgactt cgtgcccgag
1021 agcttcgacc gggacaaaac catcgccctg atcatgaaca gtagtggcag taccggattg
1081 cccaagggcg tagccctacc gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac
1141 cccatcttcg gcaaccagat catccccgac accgctatcc tcagcgtggt gccatttcac
1201 cacggcttcg gcatgttcac cacgctgggc tacttgatct gcggctttcg ggtcgtgctc
1261 atgtaccgct tcgaggagga gctattcttg cgcagcttgc aagactataa gattcaatct
1321 gccctgctgg tgcccacact atttagcttc ttcgctaaga gcactctcat cgacaagtac
1381 gacctaagca acttgcacga gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt
1441 gaggccgtgg ccaaacgctt ccacctacca ggcatccgcc agggctacgg cctgacagaa
1501 acaaccagcg ccattctgat cacccccgaa ggggacgaca agcctggcgc agtaggcaag
1561 gtggtgccct tcttcgaggc taaggtggtg gacttggaca ccggtaagac actgggtgtg
1621 aaccagcgcg gcgagctgtg cgtccgtggc cccatgatca tgagcggcta cgttaacaac
1681 cccgaggcta caaacgctct catcgacaag gacggctggc tgcacagcgg cgacatcgcc
1741 tactgggacg aggacgagca cttcttcatc gtggaccggc tgaagagcct gatcaaatac
1801 aagggctacc aggtagcccc agccgaactg gagagcatcc tgctgcaaca ccccaacatc
1861 ttcgacgccg gggtcgccgg cctgcccgac gacgatgccg gcgagctgcc cgccgcagtc
1921 gtcgtgctgg aacacggtaa aaccatgacc gagaaggaga tcgtggacta tgtggccagc
1981 caggttacaa ccgccaagaa gctgcgcggt ggtgttgtgt tcgtggacga ggtgcctaaa
2041 ggactgaccg gcaagttgga cgcccgcaag atccgcgaga ttctcattaa ggccaagaag
2101 ggcggcaaga tcgccgtgaa ttctcacggc ttccctcccg aggtggagga gcaggccgcc
2161 ggcaccctgc ccatgagctg cgcccaggag agcggcatgg atagacaccc tgctgcttgc
2221 gccagcgcca ggatcaacgt ctaaggccgc gactctagag tcggggcggc cggccgcttc
2281 gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
2341 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
2401 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
2461 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc
2521 cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca ccatggcctg aaataacctc
2581 tgaaagagga acttggttag ctaccttctg aggcggaaag aaccagctgt ggaatgtgtg
2641 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
2701 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
2761 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
2821 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
2881 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
2941 ttttggaggc ctaggctttt gcaaaaagct cgattcttct gacactagcg ccaccatgaa
3001 gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc atcgagaagt tcgacagtgt
3061 gagcgacctg atgcagttgt cggagggcga agagagccga gccttcagct tcgatgtcgg
3121 cggacgcggc tatgtactgc gggtgaatag ctgcgctgat ggcttctaca aagaccgcta
3181 cgtgtaccgc cacttcgcca gcgctgcact acccatcccc gaagtgttgg acatcggcga
3241 gttcagcgag agcctgacat actgcatcag tagacgcgcc caaggcgtta ctctccaaga
3301 cctccccgaa acagagctgc ctgctgtgtt acagcctgtc gccgaagcta tggatgctat
3361 tgccgccgcc gacctcagtc aaaccagcgg cttcggccca ttcgggcccc aaggcatcgg
3421 ccagtacaca acctggcggg atttcatttg cgccattgct gatccccatg tctaccactg
3481 gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct caagccctgg acgaactgat
3541 gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc catgccgact tcggcagcaa
3601 caacgtcctg accgacaacg gccgcatcac cgccgtaatc gactggtccg aagctatgtt
3661 cggggacagt cagtacgagg tggccaacat cttcttctgg cggccctggc tggcttgcat
3721 ggagcagcag actcgctact tcgagcgccg gcatcccgag ctggccggca gccctcgtct
3781 gcgagcctac atgctgcgca tcggcctgga tcagctctac cagagcctcg tggacggcaa
3841 cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc atcgtccgca gcggggccgg
3901 caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc gtatggaccg acggctgcgt
3961 cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca cgaccgcgcg ctaaggaggt
4021 aggtcgagtt taaactctag aaccggtcat ggccgcaata aaatatcttt attttcatta
4081 catctgtgtg ttggtttttt gtgtgttcga actagatgct gtcgaccgat gcccttgaga
4141 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
4201 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc
4261 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
4321 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
4381 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
4441 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
4501 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
4561 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
4621 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
4681 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
4741 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
4801 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
4861 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
4921 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
4981 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
5041 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
5101 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
5161 aagtatatat gagtaaactt ggtctgacag cggccgcaaa tgctaaacca ctgcagtggt
5221 taccagtgct tgatcagtga ggcaccgatc tcagcgatct gcctatttcg ttcgtccata
5281 gtggcctgac tccccgtcgt gtagatcact acgattcgtg agggcttacc atcaggcccc
5341 agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc ccgatttgtc agcaatgaac
5401 cagccagcag ggagggccga gcgaagaagt ggtcctgcta ctttgtccgc ctccatccag
5461 tctatgagct gctgtcgtga tgctagagta agaagttcgc cagtgagtag tttccgaaga
5521 gttgtggcca ttgctactgg catcgtggta tcacgctcgt cgttcggtat ggcttcgttc
5581 aactctggtt cccagcggtc aagccgggtc acatgatcac ccatattatg aagaaatgca
5641 gtcagctcct tagggcctcc gatcgttgtc agaagtaagt tggccgcggt gttgtcgctc
5701 atggtaatgg cagcactaca caattctctt accgtcatgc catccgtaag atgcttttcc
5761 gtgaccggcg agtactcaac caagtcgttt tgtgagtagt gtatacggcg accaagctgc
5821 tcttgcccgg cgtctatacg ggacaacacc gcgccacata gcagtacttt gaaagtgctc
5881 atcatcggga atcgttcttc ggggcggaaa gactcaagga tcttgccgct attgagatcc
5941 agttcgatat agcccactct tgcacccagt tgatcttcag catcttttac tttcaccagc
6001 gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa agaagggaat gagtgcgaca
6061 cgaaaatgtt ggatgctcat
//
