| 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 |
买家导航
pGL4.49[luc2P/TCF-LEF RE/Hygro]载体pGL4.49质粒基本信息
pGL4.49[luc2P/TCF-LEF RE/Hygro]质粒图谱pGL4.49载体图谱和pGL4.49[luc2P/TCF-LEF RE/Hygro]载体序列pGL4.49质粒序列多克隆位点信息
pGL4.49[luc2P/TCF-LEF RE/Hygro]质粒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[luc2P/TCF-LEF RE/Hygro]质粒序列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
//
