| 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 |
买家导航
pGL4.33[luc2P/SRE/Hygro]载体pGL4.33质粒基本信息
pGL4.33[luc2P/SRE/Hygro]质粒图谱pGL4.33载体图谱和pGL4.33[luc2P/SRE/Hygro]载体序列pGL4.33质粒序列多克隆位点信息
pGL4.33[luc2P/SRE/Hygro]质粒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[luc2P/SRE/Hygro]质粒序列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
//
