pGL4.49[luc2P/TCF-LEF RE/Hygro]

价格:1500元

联系方式:I47-825O-882O

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pGL4.49载体基本信息

出品公司: Promega
载体名称: pGL4.49; pGL4.49[luc2P/TCF-LEF RE/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: 6124 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: pGL4.49[luc2P/TCF-LEF RE/Hygro]载体是信号通路报告载体;含TCF-LEF应答元件;含萤火 虫荧光素酶报告基因luc2P。
产品目录号: E461A
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.49载体质粒图谱和多克隆位点信息

pGL4.49载体图谱



pGL4.49 载体特征

pGL4.49载体简介

pGL4.49载体简介
The pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector(a–c) contains eight copies of a TCF-LEF response element (TCF-LEF 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 pGL4.49[luc2P/TCF-LEF RE/Hygro] vector is used to
measure activation of the TCF-LEF RE in HEK293 cells upon treatment with mWnt3a or
LiCl. 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
 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])
 Charcoal-stripped FBS (Life Technologies Cat.# 126776-011)
 Opti-MEM I (Life Technologies Cat.# 31985)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 mWnt3a (R&D Systems Cat.# 1324-WN)
 BSA (Proliant cat# 68700)
 LiCl (Sigma Cat.# 40113)
 ONE-Glo Luciferase Assay System (Cat.# E6110)
 HEK293 cells

实验流程
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. Quantify cells and dilute in complete medium to 1.5 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector 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 1.5 × 105 cells/ml cell suspension. Mix by
inversion.
4. Plate 100μl per well into a solid, white 96-well plate (Corning Cat.# 3917).
5. Incubate for 18 hours in a 37°C, 5% CO2 incubator.

Day 2: Medium Replacement and Cell Treatment
1. Resuspend mWnt3a to 100μg/ml in DPBS + 1 mg/ml BSA. Dilute mWnt3a to 3μg/ml
and then serially dilute into PBS/BSA to give a range of 10X stock solutions. Serially
dilute 1M LiCl into water to give a range of 10X stock solutions.
2. Remove existing medium from cells and replace with 90μl of DMEM + 0.5%
charcoal-stripped FBS per well.
3. Add 10μl of the 10X dilutions of mWnt3a or LiCl.
4. Incubate for 8 hours in a 37°C, 5% CO2 incubator.

Day 3: Luminescence Measurement
1. Remove plates from the incubator and allow them to cool to room temperature for
approximately 15 minutes.
4. Add ONE-Glo Luciferase Assay System detection reagent, and measure
luminescence following the recommended protocol (refer to the ONE-Glo
Luciferase Assay System Technical Manual, #TM292 for details). 

pGL4.49载体序列

LOCUS       JX099537                6124 bp    DNA     circular SYN 08-JUL-2012
DEFINITION  Reporter vector pGL4.49[luc2P/TCF-LEF RE/Hygro], complete sequence.
ACCESSION   JX099537
VERSION     JX099537.1  GI:393693183
KEYWORDS    .
SOURCE      Reporter vector pGL4.49[luc2P/TCF-LEF RE/Hygro]
  ORGANISM  Reporter vector pGL4.49[luc2P/TCF-LEF RE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6124)
  AUTHORS   McNamara,B.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-MAY-2012) Scientific Communications, Promega
            Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA
FEATURES             Location/Qualifiers
     source          1..6124
                     /organism="Reporter vector pGL4.49[luc2P/TCF-LEF
                     RE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203536"
     regulatory      105..258
                     /regulatory_class="other"
                     /note="poly(A) signal/transcriptional pause site"
     primer_bind     207..226
                     /note="RVprimer3 binding region"
     misc_feature    285..404
                     /note="TCF-LEF response element"
     regulatory      450..480
                     /regulatory_class="promoter"
                     /note="minimal promoter"
     gene            513..2288
                     /gene="luc2P"
     CDS             513..2288
                     /gene="luc2P"
                     /note="dervied from firefly"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFN11858.1"
                     /db_xref="GI:393693184"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2328..2549
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     CDS             3040..4077
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin resistance"
                     /protein_id="AFN11860.1"
                     /db_xref="GI:393693186"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     primer_bind     4216..4235
                     /note="RVprimer4 binding site"
     rep_origin      4473
     gene            complement(5264..6124)
                     /gene="bla"
     CDS             complement(5264..6124)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFN11859.1"
                     /db_xref="GI:393693185"
                     /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 gctcagatca aagggtttaa
      301 gatcaaaggg cttaagatca aagggtataa gatcaaaggg cctaagatca aagggactaa
      361 gatcaaaggg tttaagatca aagggcttaa gatcaaaggg cctactcgag gatatcaaga
      421 tctggcctcg gcggccaagc ttagacacta gagggtatat aatggaagct cgacttccag
      481 cttggcaatc cggtactgtt ggtaaagcca ccatggaaga tgccaaaaac attaagaagg
      541 gcccagcgcc attctaccca ctcgaagacg ggaccgccgg cgagcagctg cacaaagcca
      601 tgaagcgcta cgccctggtg cccggcacca tcgcctttac cgacgcacat atcgaggtgg
      661 acattaccta cgccgagtac ttcgagatga gcgttcggct ggcagaagct atgaagcgct
      721 atgggctgaa tacaaaccat cggatcgtgg tgtgcagcga gaatagcttg cagttcttca
      781 tgcccgtgtt gggtgccctg ttcatcggtg tggctgtggc cccagctaac gacatctaca
      841 acgagcgcga gctgctgaac agcatgggca tcagccagcc caccgtcgta ttcgtgagca
      901 agaaagggct gcaaaagatc ctcaacgtgc aaaagaagct accgatcata caaaagatca
      961 tcatcatgga tagcaagacc gactaccagg gcttccaaag catgtacacc ttcgtgactt
     1021 cccatttgcc acccggcttc aacgagtacg acttcgtgcc cgagagcttc gaccgggaca
     1081 aaaccatcgc cctgatcatg aacagtagtg gcagtaccgg attgcccaag ggcgtagccc
     1141 taccgcaccg caccgcttgt gtccgattca gtcatgcccg cgaccccatc ttcggcaacc
     1201 agatcatccc cgacaccgct atcctcagcg tggtgccatt tcaccacggc ttcggcatgt
     1261 tcaccacgct gggctacttg atctgcggct ttcgggtcgt gctcatgtac cgcttcgagg
     1321 aggagctatt cttgcgcagc ttgcaagact ataagattca atctgccctg ctggtgccca
     1381 cactatttag cttcttcgct aagagcactc tcatcgacaa gtacgaccta agcaacttgc
     1441 acgagatcgc cagcggcggg gcgccgctca gcaaggaggt aggtgaggcc gtggccaaac
     1501 gcttccacct accaggcatc cgccagggct acggcctgac agaaacaacc agcgccattc
     1561 tgatcacccc cgaaggggac gacaagcctg gcgcagtagg caaggtggtg cccttcttcg
     1621 aggctaaggt ggtggacttg gacaccggta agacactggg tgtgaaccag cgcggcgagc
     1681 tgtgcgtccg tggccccatg atcatgagcg gctacgttaa caaccccgag gctacaaacg
     1741 ctctcatcga caaggacggc tggctgcaca gcggcgacat cgcctactgg gacgaggacg
     1801 agcacttctt catcgtggac cggctgaaga gcctgatcaa atacaagggc taccaggtag
     1861 ccccagccga actggagagc atcctgctgc aacaccccaa catcttcgac gccggggtcg
     1921 ccggcctgcc cgacgacgat gccggcgagc tgcccgccgc agtcgtcgtg ctggaacacg
     1981 gtaaaaccat gaccgagaag gagatcgtgg actatgtggc cagccaggtt acaaccgcca
     2041 agaagctgcg cggtggtgtt gtgttcgtgg acgaggtgcc taaaggactg accggcaagt
     2101 tggacgcccg caagatccgc gagattctca ttaaggccaa gaagggcggc aagatcgccg
     2161 tgaattctca cggcttccct cccgaggtgg aggagcaggc cgccggcacc ctgcccatga
     2221 gctgcgccca ggagagcggc atggatagac accctgctgc ttgcgccagc gccaggatca
     2281 acgtctaagg ccgcgactct agagtcgggg cggccggccg cttcgagcag acatgataag
     2341 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     2401 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     2461 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     2521 aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatccgtttg cgtattgggc
     2581 gctcttccgc tgatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg
     2641 ttagctacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     2701 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2761 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2821 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2881 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2941 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3001 ttttgcaaaa agctcgattc ttctgacact agcgccacca tgaagaagcc cgaactcacc
     3061 gctaccagcg ttgaaaaatt tctcatcgag aagttcgaca gtgtgagcga cctgatgcag
     3121 ttgtcggagg gcgaagagag ccgagccttc agcttcgatg tcggcggacg cggctatgta
     3181 ctgcgggtga atagctgcgc tgatggcttc tacaaagacc gctacgtgta ccgccacttc
     3241 gccagcgctg cactacccat ccccgaagtg ttggacatcg gcgagttcag cgagagcctg
     3301 acatactgca tcagtagacg cgcccaaggc gttactctcc aagacctccc cgaaacagag
     3361 ctgcctgctg tgttacagcc tgtcgccgaa gctatggatg ctattgccgc cgccgacctc
     3421 agtcaaacca gcggcttcgg cccattcggg ccccaaggca tcggccagta cacaacctgg
     3481 cgggatttca tttgcgccat tgctgatccc catgtctacc actggcagac cgtgatggac
     3541 gacaccgtgt ccgccagcgt agctcaagcc ctggacgaac tgatgctgtg ggccgaagac
     3601 tgtcccgagg tgcgccacct cgtccatgcc gacttcggca gcaacaacgt cctgaccgac
     3661 aacggccgca tcaccgccgt aatcgactgg tccgaagcta tgttcgggga cagtcagtac
     3721 gaggtggcca acatcttctt ctggcggccc tggctggctt gcatggagca gcagactcgc
     3781 tacttcgagc gccggcatcc cgagctggcc ggcagccctc gtctgcgagc ctacatgctg
     3841 cgcatcggcc tggatcagct ctaccagagc ctcgtggacg gcaacttcga cgatgctgcc
     3901 tgggctcaag gccgctgcga tgccatcgtc cgcagcgggg ccggcaccgt cggtcgcaca
     3961 caaatcgctc gccggagcgc agccgtatgg accgacggct gcgtcgaggt gctggccgac
     4021 agcggcaacc gccggcccag tacacgaccg cgcgctaagg aggtaggtcg agtttaaact
     4081 ctagaaccgg tcatggccgc aataaaatat ctttattttc attacatctg tgtgttggtt
     4141 ttttgtgtgt tcgaactaga tgctgtcgac cgatgccctt gagagccttc aacccagtca
     4201 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     4261 tcatgcaact cgtaggacag gtgccggcag cgctcttccg cttcctcgct cactgactcg
     4321 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4381 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4441 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4501 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4561 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4621 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4681 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4741 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4801 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4861 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4921 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4981 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5041 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5101 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5161 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5221 acttggtctg acagcggccg caaatgctaa accactgcag tggttaccag tgcttgatca
     5281 gtgaggcacc gatctcagcg atctgcctat ttcgttcgtc catagtggcc tgactccccg
     5341 tcgtgtagat cactacgatt cgtgagggct taccatcagg ccccagcgca gcaatgatgc
     5401 cgcgagagcc gcgttcaccg gcccccgatt tgtcagcaat gaaccagcca gcagggaggg
     5461 ccgagcgaag aagtggtcct gctactttgt ccgcctccat ccagtctatg agctgctgtc
     5521 gtgatgctag agtaagaagt tcgccagtga gtagtttccg aagagttgtg gccattgcta
     5581 ctggcatcgt ggtatcacgc tcgtcgttcg gtatggcttc gttcaactct ggttcccagc
     5641 ggtcaagccg ggtcacatga tcacccatat tatgaagaaa tgcagtcagc tccttagggc
     5701 ctccgatcgt tgtcagaagt aagttggccg cggtgttgtc gctcatggta atggcagcac
     5761 tacacaattc tcttaccgtc atgccatccg taagatgctt ttccgtgacc ggcgagtact
     5821 caaccaagtc gttttgtgag tagtgtatac ggcgaccaag ctgctcttgc ccggcgtcta
     5881 tacgggacaa caccgcgcca catagcagta ctttgaaagt gctcatcatc gggaatcgtt
     5941 cttcggggcg gaaagactca aggatcttgc cgctattgag atccagttcg atatagccca
     6001 ctcttgcacc cagttgatct tcagcatctt ttactttcac cagcgtttcg gggtgtgcaa
     6061 aaacaggcaa gcaaaatgcc gcaaagaagg gaatgagtgc gacacgaaaa tgttggatgc
     6121 tcat
//

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