pGL4.33[luc2P/SRE/Hygro]

价格:2800元

联系方式:I47-825O-882O

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pGL4.33载体基本信息

出品公司: Promega
载体名称: pGL4.33 ; pGL4.33[luc2P/SRE/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: 6112 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: pGL4.33[luc2P]载体是信号通路报告载体;含SRE应答元件;含萤火 虫荧光素酶报告基因luc2P。
产品目录号: E134A
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.33载体质粒图谱和多克隆位点信息

pGL4.33载体图谱



pGL4.33 载体特征

pGL4.33载体简介

The pGL4.33[luc2P/SRE/Hygro] Vector(a–c) contains a Serum Response Element (SRE) that drives transcription of the luciferase reporter gene luc2P in response to activation of MAPK/ERK signaling pathway. luc2P is a synthetically derived luciferase sequence with humanized codon optimization. The luc2P gene also contains hPEST, a protein destabilization sequence. The protein encoded by luc2P responds more quickly to induction than the protein encoded by the luc2 gene. The vector backbone contains an ampicillin resistance gene to allow selection in E. coli and the mammalian selectable marker for hygromycin resistance.


Sample Protocol to Determine Induction of Luciferase by FBS + PMA in HEK293 Cells Transfected with the pGL4.33[luc2P/SRE/Hygro] Vector

实验材料
 Dulbecco's PBS (DPBS)
 0.05% (w/v) trypsin in DPBS
 DMEM
 DMEM supplemented with 0.5%, 10% and 40% fetal bovine serum (DMEM/FBS)
 Phorbol 12-myristate 13-acetate (PMA, Promega Cat.# V1171 or Sigma Cat.# P8139),
1mg/ml solution in DMSO
 ONE-Glo Luciferase Assay System (Cat.# E6110)
 HEK293 cells
 transfection reagent

Day 1: Plate Cells
1. Grow HEK293 cells in DMEM/FBS to approximately 75% confluency.
2. Harvest cells via trypsinization: Remove the DMEM/FBS, wash the cells with DPBS
and add the trypsin/DPBS (1X volume). After 2 minutes, add a 4X volume of
DMEM/FBS, collect the cell suspension and pellet the cells by centrifugation.
Aspirate the supernatant, and resuspend in DMEM/FBS. We have routinely used a
concentration of 10,000–15,000 viable cells/100μl DMEM/FBS.
3. Dispense 100μl of the cell suspension into the wells of a 96-well plate. Plate enough
wells to perform each test condition in triplicate.
4. Cover the plate, and place it in a tissue culture incubator at 37°C overnight (or for 24 hours).

Day 2: Transfect Cells
1. Transfect the cells using a high-efficiency transfection reagent. Each well of cells in a 96-well plate requires 0.1μg pGL4.33[luc2P/SRE/Hygro] Vector DNA. Transfection conditions may require optimization.
2. Cover the plate, and place it in a tissue culture incubator at 37°C.
3. After 4–6 hours, change the medium to DMEM/0.5%FBS (100μl per well) to start
serum starvation.

Day 3: Induce Transfected Cells
1. Prepare 2X induction and 2X control solutions. Calculate the volume of 2X induction
and 2X control solution by multiplying the number of wells needed for each solution
by 50μl, and prepare 110% of this amount.
 2X induction solution: 40%FBS plus 20ng/ml PMA in DMEM
 2X control solution: DMEM
2. Remove 50μl of medium from wells that will be treated with either 2X induction solution or 2X control solution.
3. Add 50μl of 2X induction solution to the cells to be induced and 50μl of 2X control
solution to the control noninduced cells.
4. Return the plate to the tissue culture incubator, and induce for 6 hours.
5. Analyze luciferase activity using an appropriate luciferase detection assay. We have
observed comparable results for fold induction of the vector using a variety of
luciferase reagents, including: Bright-Glo Luciferase Assay System (Cat.# E2610,
Technical Manual #TM052); ONE-Glo Luciferase Assay System (Cat.# E6110,
Technical Manual #TM292); Dual-Luciferase Reporter Assay System (Cat.# E1910,
Technical Manual #TM040); and Dual-Glo Luciferase Assay System (Cat.# E2920,
Technical Manual #TM058).
6. Calculate the fold induction as follows:
fold induction = average relative light units of induced cells
average relative light units of control cells

pGL4.33载体序列

LOCUS       FJ773212                6112 bp    DNA     circular SYN 21-MAR-2009
DEFINITION  Reporter vector pGL4.33[luc2P/SRE/Hygro], complete sequence.
ACCESSION   FJ773212
VERSION     FJ773212.1  GI:224998143
KEYWORDS    .
SOURCE      Reporter vector pGL4.33[luc2P/SRE/Hygro]
  ORGANISM  Reporter vector pGL4.33[luc2P/SRE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6112)
  AUTHORS   Kopish,K., Paguio,A. and Hung,L.
  TITLE     pGL4 Luciferase Reporter Vectors Technical Manual, TM259
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6112)
  AUTHORS   Kopish,K., Paguio,A. and Hung,L.
  TITLE     Direct Submission
  JOURNAL   Submitted (20-FEB-2009) Scientific Communications, Promega
            Corporation, 5500 E. Cheryl Parkway, Suite 110, Madison, WI 53711,
            USA
FEATURES             Location/Qualifiers
     source          1..6112
                     /organism="Reporter vector pGL4.33[luc2P/SRE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:624118"
     regulatory      33..147
                     /regulatory_class="promoter"
                     /note="SRE; serum response element"
     regulatory      180..210
                     /regulatory_class="promoter"
                     /note="minP; minimal promoter"
     gene            243..2018
                     /gene="luc2P"
     CDS             243..2018
                     /gene="luc2P"
                     /note="luminescent reporter protein"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase reporter"
                     /protein_id="ACN77840.1"
                     /db_xref="GI:224998145"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     misc_feature    252..941
                     /gene="luc2P"
                     /note="GloSensor(TM) 10F; C-terminal domain"
     misc_feature    2058..2279
                     /note="SV40 late"
     regulatory      2327..2745
                     /regulatory_class="promoter"
                     /note="SV40 early enhancer/promoter"
     regulatory      2400..2636
                     /regulatory_class="enhancer"
                     /note="SV40 enhancer"
     gene            2770..3807
                     /gene="hygR"
     CDS             2770..3807
                     /gene="hygR"
                     /note="Hygro(r)"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin-resistance protein"
                     /protein_id="ACN77839.1"
                     /db_xref="GI:224998144"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     primer_bind     3946..3965
                     /note="reporter vector primer 4; RVprimer4"
     rep_origin      4203..4239
                     /note="ColEI-derived"
     gene            complement(4994..5854)
                     /gene="ampR"
     CDS             complement(4994..5854)
                     /gene="ampR"
                     /function="resistance to ampicillin"
                     /note="Amp(r)"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase resistance protein"
                     /protein_id="ACN77841.1"
                     /db_xref="GI:224998146"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
     regulatory      5959..6112
                     /regulatory_class="terminator"
                     /note="poly(A) signal/transcriptional pause site for
                     background reduction"
     primer_bind     6061..6080
                     /note="reporter vector primer 3; RVprimer3"
ORIGIN      
        1 ggcctaactg gccggtacct gagctcgcta gcaggatgtc catattagga catctaggat
       61 gtccatatta ggacatctag gatgtccata ttaggacatc taggatgtcc atattaggac
      121 atctaggatg tccatattag gacatctaga tctggcctcg gcggccaagc ttagacacta
      181 gagggtatat aatggaagct cgacttccag cttggcaatc cggtactgtt ggtaaagcca
      241 ccatggaaga tgccaaaaac attaagaagg gcccagcgcc attctaccca ctcgaagacg
      301 ggaccgccgg cgagcagctg cacaaagcca tgaagcgcta cgccctggtg cccggcacca
      361 tcgcctttac cgacgcacat atcgaggtgg acattaccta cgccgagtac ttcgagatga
      421 gcgttcggct ggcagaagct atgaagcgct atgggctgaa tacaaaccat cggatcgtgg
      481 tgtgcagcga gaatagcttg cagttcttca tgcccgtgtt gggtgccctg ttcatcggtg
      541 tggctgtggc cccagctaac gacatctaca acgagcgcga gctgctgaac agcatgggca
      601 tcagccagcc caccgtcgta ttcgtgagca agaaagggct gcaaaagatc ctcaacgtgc
      661 aaaagaagct accgatcata caaaagatca tcatcatgga tagcaagacc gactaccagg
      721 gcttccaaag catgtacacc ttcgtgactt cccatttgcc acccggcttc aacgagtacg
      781 acttcgtgcc cgagagcttc gaccgggaca aaaccatcgc cctgatcatg aacagtagtg
      841 gcagtaccgg attgcccaag ggcgtagccc taccgcaccg caccgcttgt gtccgattca
      901 gtcatgcccg cgaccccatc ttcggcaacc agatcatccc cgacaccgct atcctcagcg
      961 tggtgccatt tcaccacggc ttcggcatgt tcaccacgct gggctacttg atctgcggct
     1021 ttcgggtcgt gctcatgtac cgcttcgagg aggagctatt cttgcgcagc ttgcaagact
     1081 ataagattca atctgccctg ctggtgccca cactatttag cttcttcgct aagagcactc
     1141 tcatcgacaa gtacgaccta agcaacttgc acgagatcgc cagcggcggg gcgccgctca
     1201 gcaaggaggt aggtgaggcc gtggccaaac gcttccacct accaggcatc cgccagggct
     1261 acggcctgac agaaacaacc agcgccattc tgatcacccc cgaaggggac gacaagcctg
     1321 gcgcagtagg caaggtggtg cccttcttcg aggctaaggt ggtggacttg gacaccggta
     1381 agacactggg tgtgaaccag cgcggcgagc tgtgcgtccg tggccccatg atcatgagcg
     1441 gctacgttaa caaccccgag gctacaaacg ctctcatcga caaggacggc tggctgcaca
     1501 gcggcgacat cgcctactgg gacgaggacg agcacttctt catcgtggac cggctgaaga
     1561 gcctgatcaa atacaagggc taccaggtag ccccagccga actggagagc atcctgctgc
     1621 aacaccccaa catcttcgac gccggggtcg ccggcctgcc cgacgacgat gccggcgagc
     1681 tgcccgccgc agtcgtcgtg ctggaacacg gtaaaaccat gaccgagaag gagatcgtgg
     1741 actatgtggc cagccaggtt acaaccgcca agaagctgcg cggtggtgtt gtgttcgtgg
     1801 acgaggtgcc taaaggactg accggcaagt tggacgcccg caagatccgc gagattctca
     1861 ttaaggccaa gaagggcggc aagatcgccg tgaattctca cggcttccct cccgaggtgg
     1921 aggagcaggc cgccggcacc ctgcccatga gctgcgccca ggagagcggc atggatagac
     1981 accctgctgc ttgcgccagc gccaggatca acgtctaagg ccgcgactct agagtcgggg
     2041 cggccggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     2101 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     2161 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     2221 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa
     2281 aatcgataag gatccgtttg cgtattgggc gctcttccgc tgatctgcgc agcaccatgg
     2341 cctgaaataa cctctgaaag aggaacttgg ttagctacct tctgaggcgg aaagaaccag
     2401 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2461 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2521 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2581 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2641 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     2701 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcgattc ttctgacact
     2761 agcgccacca tgaagaagcc cgaactcacc gctaccagcg ttgaaaaatt tctcatcgag
     2821 aagttcgaca gtgtgagcga cctgatgcag ttgtcggagg gcgaagagag ccgagccttc
     2881 agcttcgatg tcggcggacg cggctatgta ctgcgggtga atagctgcgc tgatggcttc
     2941 tacaaagacc gctacgtgta ccgccacttc gccagcgctg cactacccat ccccgaagtg
     3001 ttggacatcg gcgagttcag cgagagcctg acatactgca tcagtagacg cgcccaaggc
     3061 gttactctcc aagacctccc cgaaacagag ctgcctgctg tgttacagcc tgtcgccgaa
     3121 gctatggatg ctattgccgc cgccgacctc agtcaaacca gcggcttcgg cccattcggg
     3181 ccccaaggca tcggccagta cacaacctgg cgggatttca tttgcgccat tgctgatccc
     3241 catgtctacc actggcagac cgtgatggac gacaccgtgt ccgccagcgt agctcaagcc
     3301 ctggacgaac tgatgctgtg ggccgaagac tgtcccgagg tgcgccacct cgtccatgcc
     3361 gacttcggca gcaacaacgt cctgaccgac aacggccgca tcaccgccgt aatcgactgg
     3421 tccgaagcta tgttcgggga cagtcagtac gaggtggcca acatcttctt ctggcggccc
     3481 tggctggctt gcatggagca gcagactcgc tacttcgagc gccggcatcc cgagctggcc
     3541 ggcagccctc gtctgcgagc ctacatgctg cgcatcggcc tggatcagct ctaccagagc
     3601 ctcgtggacg gcaacttcga cgatgctgcc tgggctcaag gccgctgcga tgccatcgtc
     3661 cgcagcgggg ccggcaccgt cggtcgcaca caaatcgctc gccggagcgc agccgtatgg
     3721 accgacggct gcgtcgaggt gctggccgac agcggcaacc gccggcccag tacacgaccg
     3781 cgcgctaagg aggtaggtcg agtttaaact ctagaaccgg tcatggccgc aataaaatat
     3841 ctttattttc attacatctg tgtgttggtt ttttgtgtgt tcgaactaga tgctgtcgac
     3901 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     3961 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     4021 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4081 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4141 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4201 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4261 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4321 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4381 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4441 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4501 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4561 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4621 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4681 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4741 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4801 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4861 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4921 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagcggccg caaatgctaa
     4981 accactgcag tggttaccag tgcttgatca gtgaggcacc gatctcagcg atctgcctat
     5041 ttcgttcgtc catagtggcc tgactccccg tcgtgtagat cactacgatt cgtgagggct
     5101 taccatcagg ccccagcgca gcaatgatgc cgcgagagcc gcgttcaccg gcccccgatt
     5161 tgtcagcaat gaaccagcca gcagggaggg ccgagcgaag aagtggtcct gctactttgt
     5221 ccgcctccat ccagtctatg agctgctgtc gtgatgctag agtaagaagt tcgccagtga
     5281 gtagtttccg aagagttgtg gccattgcta ctggcatcgt ggtatcacgc tcgtcgttcg
     5341 gtatggcttc gttcaactct ggttcccagc ggtcaagccg ggtcacatga tcacccatat
     5401 tatgaagaaa tgcagtcagc tccttagggc ctccgatcgt tgtcagaagt aagttggccg
     5461 cggtgttgtc gctcatggta atggcagcac tacacaattc tcttaccgtc atgccatccg
     5521 taagatgctt ttccgtgacc ggcgagtact caaccaagtc gttttgtgag tagtgtatac
     5581 ggcgaccaag ctgctcttgc ccggcgtcta tacgggacaa caccgcgcca catagcagta
     5641 ctttgaaagt gctcatcatc gggaatcgtt cttcggggcg gaaagactca aggatcttgc
     5701 cgctattgag atccagttcg atatagccca ctcttgcacc cagttgatct tcagcatctt
     5761 ttactttcac cagcgtttcg gggtgtgcaa aaacaggcaa gcaaaatgcc gcaaagaagg
     5821 gaatgagtgc gacacgaaaa tgttggatgc tcatactcgt cctttttcaa tattattgaa
     5881 gcatttatca gggttactag tacgtctctc aaggataagt aagtaatatt aaggtacggg
     5941 aggtattgga caggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt
     6001 ttgtgtgaat cgatagtact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa
     6061 ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ct
//

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