| 出品公司: | Promega |
|---|---|
| 载体名称: | pGL4.40 |
| 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: | 6079 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: | pGL4.40[luc2P/MRE/Hygro]载体是信号通路报告载体；含金属应答元件MRE；含萤火虫荧光素酶报告基因luc2P。 |
| 产品目录号: | E4131 |
| 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
pGL4.40载体基本信息
pGL4.40载体图谱质粒图谱和多克隆位点信息
pGL4.40载体简介
The pGL4.40[luc2P/MRE/Hygro] Vector contains five copies of a metal response element (MRE) 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 pGL4.40[luc2P/MRE/Hygro] vector is used to measure activation of the MRE in HepG2 cells upon treatment with Zinc Sulfate. 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) ZnSO4 (Sigma Cat.# Z4750) 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 pGL4.40[luc2P/MRE/hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase 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 4.5:1 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 18 hours in a 37°C, 5% CO2 incubator. Day 3: Medium Replacement 1. Remove existing medium from cells and replace with 72μl of DMEM + 0.5% charcoal-stripped FBS per well. 2. Incubate for 24 hours in a 37°C, 5% CO2 incubator. Day 4: Cell Treatment and Luminescence Measurement 1. Serially dilute a 10mM aqueous stock of ZnSO4 into water to give 10X stocks. 2. Add 8μl of the 10X dilutions of ZnSO4 and incubate for 6 hours in a 37°C, 5% CO2 incubator. 3. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room temperature for approximately 15 minutes. 4. 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).
pGL4.40载体序列
LOCUS JQ858515 6079 bp DNA circular SYN 02-JUL-2012
DEFINITION Reporter vector PGL4.40[luc2P/MRE/Hygro], complete sequence.
ACCESSION JQ858515
VERSION JQ858515.1 GI:392934090
KEYWORDS .
SOURCE Reporter vector PGL4.40[luc2P/MRE/Hygro]
ORGANISM Reporter vector PGL4.40[luc2P/MRE/Hygro]
other sequences; artificial sequences; vectors.
REFERENCE 1 (bases 1 to 6079)
AUTHORS McNamara,B.
TITLE Direct Submission
JOURNAL Submitted (28-MAR-2012) Scientific Communications, Promega
Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA
FEATURES Location/Qualifiers
source 1..6079
/organism="Reporter vector PGL4.40[luc2P/MRE/Hygro]"
/mol_type="other DNA"
/db_xref="taxon:1203009"
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..359
/note="MRE response element"
regulatory 405..435
/regulatory_class="promoter"
/note="minP promoter"
gene 468..2243
/gene="luc2P"
/note="synthetic luciferase"
CDS 468..2243
/gene="luc2P"
/note="synthetic luciferase"
/codon_start=1
/transl_table=11
/product="luciferase"
/protein_id="AFM92233.1"
/db_xref="GI:392934092"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
LPMSCAQESGMDRHPAACASARINV"
regulatory 2283..2504
/regulatory_class="polyA_signal_sequence"
/note="SV40 late poly(A) signal"
regulatory 2552..2970
/regulatory_class="enhancer"
/note="SV40 enhancer and early promoter"
CDS 2995..4032
/note="hygromycin resistance"
/codon_start=1
/transl_table=11
/product="hygromycin phosphotransferase"
/protein_id="AFM92234.1"
/db_xref="GI:392934093"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
TRPRAKEVGRV"
regulatory 4056..4104
/regulatory_class="polyA_signal_sequence"
/note="synthetic poly(A) signal sequence"
primer_bind complement(4171..4190)
/note="RVprimer4 binding site"
rep_origin 4428..4464
gene complement(5219..6079)
/gene="bla"
/note="AmpR"
CDS complement(5219..6079)
/gene="bla"
/note="AmpR"
/codon_start=1
/transl_table=11
/product="beta-lactamase"
/protein_id="AFM92232.1"
/db_xref="GI:392934091"
/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 gctcgtctgc acacggcccg
301 tctgcacacg gcccgtctgc acacggcccg tctgcacacg gcccgtctgc acacggcccc
361 tcgaggatat caagatctgg cctcggcggc caagcttaga cactagaggg tatataatgg
421 aagctcgact tccagcttgg caatccggta ctgttggtaa agccaccatg gaagatgcca
481 aaaacattaa gaagggccca gcgccattct acccactcga agacgggacc gccggcgagc
541 agctgcacaa agccatgaag cgctacgccc tggtgcccgg caccatcgcc tttaccgacg
601 cacatatcga ggtggacatt acctacgccg agtacttcga gatgagcgtt cggctggcag
661 aagctatgaa gcgctatggg ctgaatacaa accatcggat cgtggtgtgc agcgagaata
721 gcttgcagtt cttcatgccc gtgttgggtg ccctgttcat cggtgtggct gtggccccag
781 ctaacgacat ctacaacgag cgcgagctgc tgaacagcat gggcatcagc cagcccaccg
841 tcgtattcgt gagcaagaaa gggctgcaaa agatcctcaa cgtgcaaaag aagctaccga
901 tcatacaaaa gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt
961 acaccttcgt gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga
1021 gcttcgaccg ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc
1081 ccaagggcgt agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc
1141 ccatcttcgg caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc
1201 acggcttcgg catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca
1261 tgtaccgctt cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg
1321 ccctgctggt gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg
1381 acctaagcaa cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg
1441 aggccgtggc caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa
1501 caaccagcgc cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg
1561 tggtgccctt cttcgaggct aaggtggtgg acttggacac cggtaagaca ctgggtgtga
1621 accagcgcgg cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc
1681 ccgaggctac aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct
1741 actgggacga ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca
1801 agggctacca ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct
1861 tcgacgccgg ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg
1921 tcgtgctgga acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc
1981 aggttacaac cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag
2041 gactgaccgg caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg
2101 gcggcaagat cgccgtgaat tctcacggct tccctcccga ggtggaggag caggccgccg
2161 gcaccctgcc catgagctgc gcccaggaga gcggcatgga tagacaccct gctgcttgcg
2221 ccagcgccag gatcaacgtc taaggccgcg actctagagt cggggcggcc ggccgcttcg
2281 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
2341 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
2401 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
2461 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc
2521 gtttgcgtat tgggcgctct tccgctgatc tgcgcagcac catggcctga aataacctct
2581 gaaagaggaa cttggttagc taccttctga ggcggaaaga accagctgtg gaatgtgtgt
2641 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
2701 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
2761 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
2821 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
2881 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
2941 tttggaggcc taggcttttg caaaaagctc gattcttctg acactagcgc caccatgaag
3001 aagcccgaac tcaccgctac cagcgttgaa aaatttctca tcgagaagtt cgacagtgtg
3061 agcgacctga tgcagttgtc ggagggcgaa gagagccgag ccttcagctt cgatgtcggc
3121 ggacgcggct atgtactgcg ggtgaatagc tgcgctgatg gcttctacaa agaccgctac
3181 gtgtaccgcc acttcgccag cgctgcacta cccatccccg aagtgttgga catcggcgag
3241 ttcagcgaga gcctgacata ctgcatcagt agacgcgccc aaggcgttac tctccaagac
3301 ctccccgaaa cagagctgcc tgctgtgtta cagcctgtcg ccgaagctat ggatgctatt
3361 gccgccgccg acctcagtca aaccagcggc ttcggcccat tcgggcccca aggcatcggc
3421 cagtacacaa cctggcggga tttcatttgc gccattgctg atccccatgt ctaccactgg
3481 cagaccgtga tggacgacac cgtgtccgcc agcgtagctc aagccctgga cgaactgatg
3541 ctgtgggccg aagactgtcc cgaggtgcgc cacctcgtcc atgccgactt cggcagcaac
3601 aacgtcctga ccgacaacgg ccgcatcacc gccgtaatcg actggtccga agctatgttc
3661 ggggacagtc agtacgaggt ggccaacatc ttcttctggc ggccctggct ggcttgcatg
3721 gagcagcaga ctcgctactt cgagcgccgg catcccgagc tggccggcag ccctcgtctg
3781 cgagcctaca tgctgcgcat cggcctggat cagctctacc agagcctcgt ggacggcaac
3841 ttcgacgatg ctgcctgggc tcaaggccgc tgcgatgcca tcgtccgcag cggggccggc
3901 accgtcggtc gcacacaaat cgctcgccgg agcgcagccg tatggaccga cggctgcgtc
3961 gaggtgctgg ccgacagcgg caaccgccgg cccagtacac gaccgcgcgc taaggaggta
4021 ggtcgagttt aaactctaga accggtcatg gccgcaataa aatatcttta ttttcattac
4081 atctgtgtgt tggttttttg tgtgttcgaa ctagatgctg tcgaccgatg cccttgagag
4141 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
4201 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc
4261 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
4321 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
4381 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
4441 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
4501 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
4561 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
4621 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
4681 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
4741 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
4801 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
4861 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
4921 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
4981 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
5041 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
5101 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
5161 agtatatatg agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt
5221 accagtgctt gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag
5281 tggcctgact ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca
5341 gcgcagcaat gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc
5401 agccagcagg gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt
5461 ctatgagctg ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag
5521 ttgtggccat tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca
5581 actctggttc ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag
5641 tcagctcctt agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca
5701 tggtaatggc agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg
5761 tgaccggcga gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct
5821 cttgcccggc gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca
5881 tcatcgggaa tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca
5941 gttcgatata gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg
6001 tttcggggtg tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac
6061 gaaaatgttg gatgctcat
//
