| 出品公司: | Promega |
|---|---|
| 載體名稱: | pGL4.44 |
| 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: | 6052 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: | pGL4.44[luc2P/AP1 RE/Hygro]載體是信號通路報告載體；含AP1應答元件；含螢火蟲熒光素酶報告基因luc2P。 |
| 產品目錄號: | E4111 |
| 穩定性: | 穩表達 |
| 組成型/誘導型: | 組成型 |
| 病毒/非病毒: | 非病毒 |
pGL4.44[luc2P/AP1 RE/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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pGL4.44[luc2P/AP1 RE/Hygro]載體
pGL4.44載體基本信息
pGL4.44載體質粒圖譜和多克隆位點信息
pGL4.44載體簡介
pGL4.44載體簡介 The pGL4.44[luc2P/AP1 RE/Hygro] Vector contains six copies of an AP1 response element (AP1 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 pGL4.44[luc2P/AP1 RE/Hygro] Vector is used to measure activation of the AP1 RE in HEK293 cells upon treatment with PMA. 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. 實驗材料 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) PMA (Cat.# V1171) Dual-Glo Luciferase Assay System (Cat.# E2940) HEK293 cells pGL4.75[hRluc/CMV] Vector (Cat.# E6931) 實驗流程 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 pGL4.44[luc2P/AP1 RE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase control 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: Medium Replacement 1. Aspirate medium and replace with 75μl DMEM + 0.1% FBS. 2. Incubate for 17 hours in a 37°C, 5% CO2 incubator. Day 3: Cell Treatment and Luminescence Measurement 1. Dissolve PMA in DMSO to a final concentration of 10mM. Serially dilute this solution in DMSO to give a range of concentrated stock solutions (1,000X). Dilute each concentrated stock solution using Opti-MEM I to give a range of dilute stock solutions (16X). Add 5μl of dilute stock solution to the existing 75μl of medium per well, covering a final concentration range of PMA from 1pM to 1μM. 2. Incubate for 6 hours in a 37°C, 5% CO2 incubator. 3. Remove plates from the incubator and allow them 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).
pGL4.44載體序列
LOCUS JQ858516 6052 bp DNA circular SYN 02-JUL-2012
DEFINITION Reporter vector PGL4.44[luc2P/AP1/Hygro], complete sequence.
ACCESSION JQ858516
VERSION JQ858516.1 GI:392934094
KEYWORDS .
SOURCE Reporter vector PGL4.44[luc2P/AP1/Hygro]
ORGANISM Reporter vector PGL4.44[luc2P/AP1/Hygro]
other sequences; artificial sequences; vectors.
REFERENCE 1 (bases 1 to 6052)
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..6052
/organism="Reporter vector PGL4.44[luc2P/AP1/Hygro]"
/mol_type="other DNA"
/db_xref="taxon:1203010"
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..332
/note="AP1 response element"
regulatory 378..408
/regulatory_class="promoter"
/note="minP promoter"
gene 441..2216
/gene="luc2P"
/note="synthetic luciferase"
CDS 441..2216
/gene="luc2P"
/note="synthetic luciferase"
/codon_start=1
/transl_table=11
/product="luciferase"
/protein_id="AFM92236.1"
/db_xref="GI:392934096"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
LPMSCAQESGMDRHPAACASARINV"
regulatory 2256..2477
/regulatory_class="polyA_signal_sequence"
/note="SV40 late poly(A) signal"
regulatory 2525..2943
/regulatory_class="enhancer"
/note="SV40 enhancer and early promoter"
CDS 2968..4005
/note="hygromycin resistance"
/codon_start=1
/transl_table=11
/product="hygromycin phosphotransferase"
/protein_id="AFM92237.1"
/db_xref="GI:392934097"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
TRPRAKEVGRV"
regulatory 4029..4077
/regulatory_class="polyA_signal_sequence"
/note="synthetic poly(A) signal sequence"
primer_bind complement(4144..4163)
/note="RVprimer4 binding site"
rep_origin 4401..4437
gene complement(5192..6052)
/gene="bla"
/note="AmpR"
CDS complement(5192..6052)
/gene="bla"
/note="AmpR"
/codon_start=1
/transl_table=11
/product="beta-lactamase"
/protein_id="AFM92235.1"
/db_xref="GI:392934095"
/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 gctctgagtc agtgactcag
301 tgagtcagtg actcagtgag tcagtgactc agctcgagga tatcaagatc tggcctcggc
361 ggccaagctt agacactaga gggtatataa tggaagctcg acttccagct tggcaatccg
421 gtactgttgg taaagccacc atggaagatg ccaaaaacat taagaagggc ccagcgccat
481 tctacccact cgaagacggg accgccggcg agcagctgca caaagccatg aagcgctacg
541 ccctggtgcc cggcaccatc gcctttaccg acgcacatat cgaggtggac attacctacg
601 ccgagtactt cgagatgagc gttcggctgg cagaagctat gaagcgctat gggctgaata
661 caaaccatcg gatcgtggtg tgcagcgaga atagcttgca gttcttcatg cccgtgttgg
721 gtgccctgtt catcggtgtg gctgtggccc cagctaacga catctacaac gagcgcgagc
781 tgctgaacag catgggcatc agccagccca ccgtcgtatt cgtgagcaag aaagggctgc
841 aaaagatcct caacgtgcaa aagaagctac cgatcataca aaagatcatc atcatggata
901 gcaagaccga ctaccagggc ttccaaagca tgtacacctt cgtgacttcc catttgccac
961 ccggcttcaa cgagtacgac ttcgtgcccg agagcttcga ccgggacaaa accatcgccc
1021 tgatcatgaa cagtagtggc agtaccggat tgcccaaggg cgtagcccta ccgcaccgca
1081 ccgcttgtgt ccgattcagt catgcccgcg accccatctt cggcaaccag atcatccccg
1141 acaccgctat cctcagcgtg gtgccatttc accacggctt cggcatgttc accacgctgg
1201 gctacttgat ctgcggcttt cgggtcgtgc tcatgtaccg cttcgaggag gagctattct
1261 tgcgcagctt gcaagactat aagattcaat ctgccctgct ggtgcccaca ctatttagct
1321 tcttcgctaa gagcactctc atcgacaagt acgacctaag caacttgcac gagatcgcca
1381 gcggcggggc gccgctcagc aaggaggtag gtgaggccgt ggccaaacgc ttccacctac
1441 caggcatccg ccagggctac ggcctgacag aaacaaccag cgccattctg atcacccccg
1501 aaggggacga caagcctggc gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg
1561 tggacttgga caccggtaag acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg
1621 gccccatgat catgagcggc tacgttaaca accccgaggc tacaaacgct ctcatcgaca
1681 aggacggctg gctgcacagc ggcgacatcg cctactggga cgaggacgag cacttcttca
1741 tcgtggaccg gctgaagagc ctgatcaaat acaagggcta ccaggtagcc ccagccgaac
1801 tggagagcat cctgctgcaa caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg
1861 acgacgatgc cggcgagctg cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga
1921 ccgagaagga gatcgtggac tatgtggcca gccaggttac aaccgccaag aagctgcgcg
1981 gtggtgttgt gttcgtggac gaggtgccta aaggactgac cggcaagttg gacgcccgca
2041 agatccgcga gattctcatt aaggccaaga agggcggcaa gatcgccgtg aattctcacg
2101 gcttccctcc cgaggtggag gagcaggccg ccggcaccct gcccatgagc tgcgcccagg
2161 agagcggcat ggatagacac cctgctgctt gcgccagcgc caggatcaac gtctaaggcc
2221 gcgactctag agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga
2281 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
2341 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
2401 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
2461 cctctacaaa tgtggtaaaa tcgataagga tccgtttgcg tattgggcgc tcttccgctg
2521 atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt agctaccttc
2581 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
2641 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
2701 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
2761 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
2821 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
2881 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
2941 ctcgattctt ctgacactag cgccaccatg aagaagcccg aactcaccgc taccagcgtt
3001 gaaaaatttc tcatcgagaa gttcgacagt gtgagcgacc tgatgcagtt gtcggagggc
3061 gaagagagcc gagccttcag cttcgatgtc ggcggacgcg gctatgtact gcgggtgaat
3121 agctgcgctg atggcttcta caaagaccgc tacgtgtacc gccacttcgc cagcgctgca
3181 ctacccatcc ccgaagtgtt ggacatcggc gagttcagcg agagcctgac atactgcatc
3241 agtagacgcg cccaaggcgt tactctccaa gacctccccg aaacagagct gcctgctgtg
3301 ttacagcctg tcgccgaagc tatggatgct attgccgccg ccgacctcag tcaaaccagc
3361 ggcttcggcc cattcgggcc ccaaggcatc ggccagtaca caacctggcg ggatttcatt
3421 tgcgccattg ctgatcccca tgtctaccac tggcagaccg tgatggacga caccgtgtcc
3481 gccagcgtag ctcaagccct ggacgaactg atgctgtggg ccgaagactg tcccgaggtg
3541 cgccacctcg tccatgccga cttcggcagc aacaacgtcc tgaccgacaa cggccgcatc
3601 accgccgtaa tcgactggtc cgaagctatg ttcggggaca gtcagtacga ggtggccaac
3661 atcttcttct ggcggccctg gctggcttgc atggagcagc agactcgcta cttcgagcgc
3721 cggcatcccg agctggccgg cagccctcgt ctgcgagcct acatgctgcg catcggcctg
3781 gatcagctct accagagcct cgtggacggc aacttcgacg atgctgcctg ggctcaaggc
3841 cgctgcgatg ccatcgtccg cagcggggcc ggcaccgtcg gtcgcacaca aatcgctcgc
3901 cggagcgcag ccgtatggac cgacggctgc gtcgaggtgc tggccgacag cggcaaccgc
3961 cggcccagta cacgaccgcg cgctaaggag gtaggtcgag tttaaactct agaaccggtc
4021 atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgttc
4081 gaactagatg ctgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
4141 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
4201 taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
4261 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
4321 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
4381 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
4441 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
4501 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
4561 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
4621 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
4681 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
4741 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
4801 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
4861 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
4921 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
4981 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
5041 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
5101 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
5161 agcggccgca aatgctaaac cactgcagtg gttaccagtg cttgatcagt gaggcaccga
5221 tctcagcgat ctgcctattt cgttcgtcca tagtggcctg actccccgtc gtgtagatca
5281 ctacgattcg tgagggctta ccatcaggcc ccagcgcagc aatgatgccg cgagagccgc
5341 gttcaccggc ccccgatttg tcagcaatga accagccagc agggagggcc gagcgaagaa
5401 gtggtcctgc tactttgtcc gcctccatcc agtctatgag ctgctgtcgt gatgctagag
5461 taagaagttc gccagtgagt agtttccgaa gagttgtggc cattgctact ggcatcgtgg
5521 tatcacgctc gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg tcaagccggg
5581 tcacatgatc acccatatta tgaagaaatg cagtcagctc cttagggcct ccgatcgttg
5641 tcagaagtaa gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta cacaattctc
5701 ttaccgtcat gccatccgta agatgctttt ccgtgaccgg cgagtactca accaagtcgt
5761 tttgtgagta gtgtatacgg cgaccaagct gctcttgccc ggcgtctata cgggacaaca
5821 ccgcgccaca tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct tcggggcgga
5881 aagactcaag gatcttgccg ctattgagat ccagttcgat atagcccact cttgcaccca
5941 gttgatcttc agcatctttt actttcacca gcgtttcggg gtgtgcaaaa acaggcaagc
6001 aaaatgccgc aaagaaggga atgagtgcga cacgaaaatg ttggatgctc at
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