pGL4.52[luc2P/STAT5RE/Hygro]

pGL4.52[luc2P/STAT5RE/Hygro]

pGL4.52载体基本信息

出品公司:	Ybscience
载体名称:	pGL4.52 ; pGL4.52[luc2P/STAT5RE/Hygro]
质粒类型:	信号通路报告载体；哺乳动物载体；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:	6079 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:	pGL4.52[luc2P/STAT5RE/Hygro]载体是信号通路报告载体；含STAT5应答元件；含荧光 素酶报告基因luc2P。
稳定性:	瞬表达或稳表达
组成型/诱导型:	组成型
病毒/非病毒:	非病毒

 

pGL4.52载体质粒图谱和多克隆位点信息

pGL4.52载体简介

 

pGL4.52载体简介
The pGL4.52[luc2P/STAT5 RE/Hygro] Vector contains five copies of a STAT5 response element (STAT5 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 pGL4.52[luc2P/STAT5 RE/Hygro] Vector is used to measure
activation of the STAT5 RE in Ba/F3 cells upon treatment with mIL-3. 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)
 Complete medium (RPMI Medium [Life Technologies Cat.# 22400]
+ 10% heat-inactivated FBS [Life Technologies Cat.# 10082-139]
+ 1 ng/ml mIL-3 [R&D Systems Cat.# 403-ML])
 Opti-MEM I (Life Technologies Cat.# 11058)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 mIL-3 (R&D Systems Cat.# 403-ML)
 BSA (Proliant Cat.# 68700)
 ONE-Glo Luciferase Assay System (Cat.# E6120)
 Ba/F3 cells

实验流程
Day 1: Reverse Transfection
Preparation of Cells
1. Grow Ba/F3 cells in suspension in complete medium [RPMI Medium + 10%
heat-inactivated FBS + 1ng/ml mIL-3].
2. Quantify cells and dilute them in complete medium to 1 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.52[luc2P/STAT5 RE/Hygro] Vector to 10ng total DNA/μl in Opti-MEM I.
2. Add FuGENE HD to a 6:1 lipid:DNA ratio and mix gently. Incubate at room
temperature for 15 minutes.
3. Dilute lipid:DNA mixture 20-fold with 1 × 105 cells/ml cell suspension and mix by
inversion.
4. Place cells in a flask and incubate for 18–24 hours in a 37°C, 5% CO2 incubator.

Day 2: Plating, Cell Treatment and Luminescence Measurement
Plating Cells
1. Pellet the cells by centrifugation at 200 × g for 5 minutes in a swinging-bucket rotor.
Wash once with DPBS and spin again.
2. Resuspend cells in Opti-MEM I to 1 × 106 cells/ml.
3. Plate 100μl per well to a solid, white 96-well plate (Corning Cat.# 3917).
4. Incubate for 4 hours in a 37°C, 5% CO2 incubator.
Cell Treatment
1. Resuspend mIL-3 to 0.1 mg/mL in DPBS + 0.1% BSA. Make serial dilutions into
Opti-MEM I to make 10X stocks.
2. Add 10μl of the 10X stocks of mIL-3 to each well and incubate for 4 hours in a 37°C,
5% CO2 incubator.
Luminescence Measurement
3. Remove plates from the 37°C, 5% CO2 incubator and allow them to cool to room
temperature for approximately 15 minutes.
4. Add 100μl of ONE-Glo Luciferase Assay System detection reagent to each well and
measure luminescence following the recommended protocol 

 

pGL4.52载体序列

LOCUS       JX206457                6079 bp    DNA     circular SYN 31-JUL-2012
DEFINITION  Reporter vector pGL4.52[luc2P/STAT5 RE/Hygro], complete sequence.
ACCESSION   JX206457
VERSION     JX206457.1  GI:393885976
KEYWORDS    .
SOURCE      Reporter vector pGL4.52[luc2P/STAT5 RE/Hygro]
  ORGANISM  Reporter vector pGL4.52[luc2P/STAT5 RE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6079)
  AUTHORS   McNamara,B.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-JUN-2012) Scientific Communications, Promega
            Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA
FEATURES             Location/Qualifiers
     source          1..6079
                     /organism="Reporter vector pGL4.52[luc2P/STAT5 RE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203538"
     regulatory      105..258
                     /regulatory_class="other"
                     /note="poly(A) signal/transcriptional pause site"
     primer_bind     207..226
                     /note="reporter vector primer (RV primer3) binding site"
     misc_feature    285..359
                     /note="STAT5 response element"
     gene            405..435
                     /gene="minP"
     regulatory      405..435
                     /regulatory_class="promoter"
                     /gene="minP"
     gene            468..2243
                     /gene="luc2P"
     CDS             468..2243
                     /gene="luc2P"
                     /note="derived from firefly"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFN26006.1"
                     /db_xref="GI:393885978"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2283..2504
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     regulatory      2552..2970
                     /regulatory_class="enhancer"
                     /note="SV40 early enhancer/promoter"
     gene            2995..4032
                     /gene="hygR"
     CDS             2995..4032
                     /gene="hygR"
                     /note="synthetic"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin resistance"
                     /protein_id="AFN26007.1"
                     /db_xref="GI:393885979"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      4056..4104
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     complement(4171..4190)
                     /note="reporter vector primer 4 (RV primer4) binding
                     region"
     rep_origin      4428..4464
                     /note="ColE1-derived plasmid replication origin"
     gene            complement(5219..6079)
                     /gene="bla"
     CDS             complement(5219..6079)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFN26005.1"
                     /db_xref="GI:393885977"
                     /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 gctcagttct gagaaaagta
      301 gttctgagaa aagtagttct gagaaaagta gttctgagaa aagtagttct gagaaaagtc
      361 tcgaggatat caagatctgg cctcggcggc caagcttaga cactagaggg tatataatgg
      421 aagctcgact tccagcttgg caatccggta ctgttggtaa agccaccatg gaagatgcca
      481 aaaacattaa gaagggccca gcgccattct acccactcga agacgggacc gccggcgagc
      541 agctgcacaa agccatgaag cgctacgccc tggtgcccgg caccatcgcc tttaccgacg
      601 cacatatcga ggtggacatt acctacgccg agtacttcga gatgagcgtt cggctggcag
      661 aagctatgaa gcgctatggg ctgaatacaa accatcggat cgtggtgtgc agcgagaata
      721 gcttgcagtt cttcatgccc gtgttgggtg ccctgttcat cggtgtggct gtggccccag
      781 ctaacgacat ctacaacgag cgcgagctgc tgaacagcat gggcatcagc cagcccaccg
      841 tcgtattcgt gagcaagaaa gggctgcaaa agatcctcaa cgtgcaaaag aagctaccga
      901 tcatacaaaa gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt
      961 acaccttcgt gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga
     1021 gcttcgaccg ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc
     1081 ccaagggcgt agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc
     1141 ccatcttcgg caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc
     1201 acggcttcgg catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca
     1261 tgtaccgctt cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg
     1321 ccctgctggt gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg
     1381 acctaagcaa cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg
     1441 aggccgtggc caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa
     1501 caaccagcgc cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg
     1561 tggtgccctt cttcgaggct aaggtggtgg acttggacac cggtaagaca ctgggtgtga
     1621 accagcgcgg cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc
     1681 ccgaggctac aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct
     1741 actgggacga ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca
     1801 agggctacca ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct
     1861 tcgacgccgg ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg
     1921 tcgtgctgga acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc
     1981 aggttacaac cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag
     2041 gactgaccgg caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg
     2101 gcggcaagat cgccgtgaat tctcacggct tccctcccga ggtggaggag caggccgccg
     2161 gcaccctgcc catgagctgc gcccaggaga gcggcatgga tagacaccct gctgcttgcg
     2221 ccagcgccag gatcaacgtc taaggccgcg actctagagt cggggcggcc ggccgcttcg
     2281 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     2341 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     2401 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     2461 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc
     2521 gtttgcgtat tgggcgctct tccgctgatc tgcgcagcac catggcctga aataacctct
     2581 gaaagaggaa cttggttagc taccttctga ggcggaaaga accagctgtg gaatgtgtgt
     2641 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2701 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2761 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2821 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2881 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     2941 tttggaggcc taggcttttg caaaaagctc gattcttctg acactagcgc caccatgaag
     3001 aagcccgaac tcaccgctac cagcgttgaa aaatttctca tcgagaagtt cgacagtgtg
     3061 agcgacctga tgcagttgtc ggagggcgaa gagagccgag ccttcagctt cgatgtcggc
     3121 ggacgcggct atgtactgcg ggtgaatagc tgcgctgatg gcttctacaa agaccgctac
     3181 gtgtaccgcc acttcgccag cgctgcacta cccatccccg aagtgttgga catcggcgag
     3241 ttcagcgaga gcctgacata ctgcatcagt agacgcgccc aaggcgttac tctccaagac
     3301 ctccccgaaa cagagctgcc tgctgtgtta cagcctgtcg ccgaagctat ggatgctatt
     3361 gccgccgccg acctcagtca aaccagcggc ttcggcccat tcgggcccca aggcatcggc
     3421 cagtacacaa cctggcggga tttcatttgc gccattgctg atccccatgt ctaccactgg
     3481 cagaccgtga tggacgacac cgtgtccgcc agcgtagctc aagccctgga cgaactgatg
     3541 ctgtgggccg aagactgtcc cgaggtgcgc cacctcgtcc atgccgactt cggcagcaac
     3601 aacgtcctga ccgacaacgg ccgcatcacc gccgtaatcg actggtccga agctatgttc
     3661 ggggacagtc agtacgaggt ggccaacatc ttcttctggc ggccctggct ggcttgcatg
     3721 gagcagcaga ctcgctactt cgagcgccgg catcccgagc tggccggcag ccctcgtctg
     3781 cgagcctaca tgctgcgcat cggcctggat cagctctacc agagcctcgt ggacggcaac
     3841 ttcgacgatg ctgcctgggc tcaaggccgc tgcgatgcca tcgtccgcag cggggccggc
     3901 accgtcggtc gcacacaaat cgctcgccgg agcgcagccg tatggaccga cggctgcgtc
     3961 gaggtgctgg ccgacagcgg caaccgccgg cccagtacac gaccgcgcgc taaggaggta
     4021 ggtcgagttt aaactctaga accggtcatg gccgcaataa aatatcttta ttttcattac
     4081 atctgtgtgt tggttttttg tgtgttcgaa ctagatgctg tcgaccgatg cccttgagag
     4141 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     4201 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc
     4261 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4321 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4381 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4441 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4501 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4561 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4621 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4681 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4741 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4801 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4861 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4921 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4981 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5041 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5101 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5161 agtatatatg agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt
     5221 accagtgctt gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag
     5281 tggcctgact ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca
     5341 gcgcagcaat gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc
     5401 agccagcagg gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt
     5461 ctatgagctg ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag
     5521 ttgtggccat tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca
     5581 actctggttc ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag
     5641 tcagctcctt agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca
     5701 tggtaatggc agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg
     5761 tgaccggcga gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct
     5821 cttgcccggc gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca
     5881 tcatcgggaa tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca
     5941 gttcgatata gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg
     6001 tttcggggtg tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac
     6061 gaaaatgttg gatgctcat