pGL4.44[luc2P/AP1 RE/Hygro]

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聯系方式:I47-825O-882O

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pGL4.44載體基本信息

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載體質粒圖譜和多克隆位點信息

pGL4.44載體圖譜



pGL4.44 載體特征

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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