| 出品公司: | Promega |
|---|---|
| 載體名稱: | pGL4.47; pGL4.47[luc2P/SIE/Hygro] |
| 質粒類型: | 信號通路報告載體；哺乳動物載體；螢火蟲熒光素酶報告載體 |
| 高拷貝/低拷貝: | -- |
| 克隆方法: | 限制性內切酶，多克隆位點 |
| 啟動子: | Minimal Promotor |
| 載體大小: | 6129 bp |
| 5' 測序引物及序列: | -- |
| 3' 測序引物及序列: | -- |
| 載體標簽: | luc2P |
| 載體抗性: | 氨芐青霉素 |
| 篩選標記: | 潮霉素(Hygromycin) |
| 克隆菌株: | TOP10等常規菌株 |
| 宿主細胞（系）: | HEK293等 |
| 備注: | pGL4.47[luc2P/SIE/Hygro]載體是信號通路報告載體；含SIS-inducible element(SIE)元件；含螢火蟲熒光素酶報告基因luc2P。 |
| 產品目錄號: | E4041 |
| 穩定性: | 穩表達 |
| 組成型/誘導型: | 組成型 |
| 病毒/非病毒: | 非病毒 |
pGL4.47[luc2P/SIE/Hygro]信號通路報告載體；哺乳動物載體；螢火蟲熒光素酶報告載體-BioVector Inc
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pGL4.47[luc2P/SIE/Hygro]載體
pGL4.47載體基本信息
pGL4.47載體質粒圖譜和多克隆位點信息
pGL4.47載體簡介
The pGL4.47[luc2P/SIE/Hygro] Vector contains five copies of the SIS-inducible element (SIE) 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 pGL4.47[luc2P/SIE/Hygro] vector is used to measure activation of the SIE in HEK293 cells upon treatment with interleukin 6. 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. 實驗材料 DMEM (Life Technologies Cat.# 11995) FBS (HyClone Cat.# SH30070.03) Dulbecco’s PBS (DPBS; Life Technologies Cat. # 14190) 0.05% Tryspin-EDTA (Life Technologies Cat.# 25300) Opti-MEM I (Life Technologies Cat.# 31985) FuGENE HD Transfection Reagent (Cat.# E2311) Human recombinant interleukin 6 (IL-6, Life Technologies Cat.# PHC0061) DMSO (Sigma Cat.# D2650) ONE-Glo Luciferase Assay System (Cat.# E6120) HEK293 cells 實驗流程 Day 1: Plate Cells 1. Plate 10ml of HEK293 cells at 2 × 105 cells/ml in a 10cm dish in complete medium (DMEM + 10% FBS). 2 Incubate for 24 hours in a 37°C, 5% CO2 incubator. Day 2: Transfection 1. Dilute 10μg pGL4.47[luc2P/SIE/Hygro] Vector DNA in 500μl Opti-MEM I. 2. Add 30μl FuGENE HD to a 3:1 lipid:DNA ratio and mix. Incubate at room temperature for 15 minutes. 3. Add DNA-lipid complex to cells and mix gently to ensure even distribution. 4 Incubate for 18 hours in a 37°C, 5% CO2 incubator. Day 3: Medium Replacement and Cell Treatment 1. Wash cells with DPBS and treat with one volume of 0.05% trypsin-EDTA. Resuspend cells in four volumes of complete medium. 2. Quantify the cells and dilute to 2 × 105 cells/ml in complete medium. 3. Plate 50μl per well into a solid, white 96-well plate (Corning Cat.# 3917). 4. Serially dilute human recombinant interleukin 6 into complete medium to give 2X stock solutions. 5. Add 50μl of the 2X dilutions of IL-6 to each well. 6. Incubate for 24 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 100μl ONE-Glo detection reagent to each well and measure luminescence following the recommended protocol. (Refer to the ONE-Glo Luciferase Assay System Technical Manual, #TM292 for details).
pGL4.47載體序列
LOCUS JQ858512 6129 bp DNA circular SYN 02-JUL-2012
DEFINITION Reporter vector PGL4.47[luc2P/SIE/Hygro], complete sequence.
ACCESSION JQ858512
VERSION JQ858512.1 GI:392934078
KEYWORDS .
SOURCE Reporter vector PGL4.47[luc2P/SIE/Hygro]
ORGANISM Reporter vector PGL4.47[luc2P/SIE/Hygro]
other sequences; artificial sequences; vectors.
REFERENCE 1 (bases 1 to 6129)
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..6129
/organism="Reporter vector PGL4.47[luc2P/SIE/Hygro]"
/mol_type="other DNA"
/db_xref="taxon:1203006"
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..409
/regulatory_class="enhancer"
/note="SIE response element"
regulatory 455..485
/regulatory_class="promoter"
/note="minP"
gene 518..2293
/gene="luc2P"
CDS 518..2293
/gene="luc2P"
/codon_start=1
/transl_table=11
/product="luciferase"
/protein_id="AFM92223.1"
/db_xref="GI:392934079"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
LPMSCAQESGMDRHPAACASARINV"
regulatory 2333..2554
/regulatory_class="polyA_signal_sequence"
/note="SV40 late poly(A) signal"
regulatory 2602..3020
/regulatory_class="promoter"
/note="SV40 enhancer and early promoter"
CDS 3045..4082
/note="hygromycin phosphotransferase"
/codon_start=1
/transl_table=11
/product="hygromycin resistance"
/protein_id="AFM92224.1"
/db_xref="GI:392934080"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
TRPRAKEVGRV"
regulatory 4106..4154
/regulatory_class="polyA_signal_sequence"
/note="synthetic poly(A) signal sequence"
primer_bind complement(4221..4240)
/note="RVprimer4 binding site"
rep_origin 4478..4514
gene complement(5269..6129)
/gene="bla"
CDS complement(5269..6129)
/gene="bla"
/note="AmpR"
/codon_start=1
/transl_table=11
/product="beta-lactamase"
/protein_id="AFM92225.1"
/db_xref="GI:392934081"
/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 gctcagcttc atttcccgta
301 aatcgtcgaa gcttcatttc ccgtaaatcg tcgaagcttc atttcccgta aatcgtcgaa
361 gcttcatttc ccgtaaatcg tcgaagcttc atttcccgta aatcgtcgac tcgaggatat
421 caagatctgg cctcggcggc caagcttaga cactagaggg tatataatgg aagctcgact
481 tccagcttgg caatccggta ctgttggtaa agccaccatg gaagatgcca aaaacattaa
541 gaagggccca gcgccattct acccactcga agacgggacc gccggcgagc agctgcacaa
601 agccatgaag cgctacgccc tggtgcccgg caccatcgcc tttaccgacg cacatatcga
661 ggtggacatt acctacgccg agtacttcga gatgagcgtt cggctggcag aagctatgaa
721 gcgctatggg ctgaatacaa accatcggat cgtggtgtgc agcgagaata gcttgcagtt
781 cttcatgccc gtgttgggtg ccctgttcat cggtgtggct gtggccccag ctaacgacat
841 ctacaacgag cgcgagctgc tgaacagcat gggcatcagc cagcccaccg tcgtattcgt
901 gagcaagaaa gggctgcaaa agatcctcaa cgtgcaaaag aagctaccga tcatacaaaa
961 gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt acaccttcgt
1021 gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga gcttcgaccg
1081 ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc ccaagggcgt
1141 agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc ccatcttcgg
1201 caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc acggcttcgg
1261 catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca tgtaccgctt
1321 cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg ccctgctggt
1381 gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg acctaagcaa
1441 cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg aggccgtggc
1501 caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa caaccagcgc
1561 cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg tggtgccctt
1621 cttcgaggct aaggtggtgg acttggacac cggtaagaca ctgggtgtga accagcgcgg
1681 cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc ccgaggctac
1741 aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct actgggacga
1801 ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca agggctacca
1861 ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct tcgacgccgg
1921 ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg tcgtgctgga
1981 acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc aggttacaac
2041 cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag gactgaccgg
2101 caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg gcggcaagat
2161 cgccgtgaat tctcacggct tccctcccga ggtggaggag caggccgccg gcaccctgcc
2221 catgagctgc gcccaggaga gcggcatgga tagacaccct gctgcttgcg ccagcgccag
2281 gatcaacgtc taaggccgcg actctagagt cggggcggcc ggccgcttcg agcagacatg
2341 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
2401 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
2461 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
2521 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc gtttgcgtat
2581 tgggcgctct tccgctgatc tgcgcagcac catggcctga aataacctct gaaagaggaa
2641 cttggttagc taccttctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
2701 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
2761 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
2821 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
2881 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
2941 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
3001 taggcttttg caaaaagctc gattcttctg acactagcgc caccatgaag aagcccgaac
3061 tcaccgctac cagcgttgaa aaatttctca tcgagaagtt cgacagtgtg agcgacctga
3121 tgcagttgtc ggagggcgaa gagagccgag ccttcagctt cgatgtcggc ggacgcggct
3181 atgtactgcg ggtgaatagc tgcgctgatg gcttctacaa agaccgctac gtgtaccgcc
3241 acttcgccag cgctgcacta cccatccccg aagtgttgga catcggcgag ttcagcgaga
3301 gcctgacata ctgcatcagt agacgcgccc aaggcgttac tctccaagac ctccccgaaa
3361 cagagctgcc tgctgtgtta cagcctgtcg ccgaagctat ggatgctatt gccgccgccg
3421 acctcagtca aaccagcggc ttcggcccat tcgggcccca aggcatcggc cagtacacaa
3481 cctggcggga tttcatttgc gccattgctg atccccatgt ctaccactgg cagaccgtga
3541 tggacgacac cgtgtccgcc agcgtagctc aagccctgga cgaactgatg ctgtgggccg
3601 aagactgtcc cgaggtgcgc cacctcgtcc atgccgactt cggcagcaac aacgtcctga
3661 ccgacaacgg ccgcatcacc gccgtaatcg actggtccga agctatgttc ggggacagtc
3721 agtacgaggt ggccaacatc ttcttctggc ggccctggct ggcttgcatg gagcagcaga
3781 ctcgctactt cgagcgccgg catcccgagc tggccggcag ccctcgtctg cgagcctaca
3841 tgctgcgcat cggcctggat cagctctacc agagcctcgt ggacggcaac ttcgacgatg
3901 ctgcctgggc tcaaggccgc tgcgatgcca tcgtccgcag cggggccggc accgtcggtc
3961 gcacacaaat cgctcgccgg agcgcagccg tatggaccga cggctgcgtc gaggtgctgg
4021 ccgacagcgg caaccgccgg cccagtacac gaccgcgcgc taaggaggta ggtcgagttt
4081 aaactctaga accggtcatg gccgcaataa aatatcttta ttttcattac atctgtgtgt
4141 tggttttttg tgtgttcgaa ctagatgctg tcgaccgatg cccttgagag ccttcaaccc
4201 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
4261 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg
4321 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
4381 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
4441 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
4501 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
4561 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
4621 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
4681 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
4741 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
4801 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
4861 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
4921 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
4981 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
5041 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
5101 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
5161 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
5221 agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt accagtgctt
5281 gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag tggcctgact
5341 ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca gcgcagcaat
5401 gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc agccagcagg
5461 gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt ctatgagctg
5521 ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag ttgtggccat
5581 tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca actctggttc
5641 ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag tcagctcctt
5701 agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca tggtaatggc
5761 agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg tgaccggcga
5821 gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct cttgcccggc
5881 gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca tcatcgggaa
5941 tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca gttcgatata
6001 gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg tttcggggtg
6061 tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac gaaaatgttg
6121 gatgctcat
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