出品公司: Promega
载体名称: pGL4.43 ; pGL4.43[luc2P/XRE/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: 6067 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: pGL4.43[luc2P/XRE/Hygro]载体是信号通路报告载体;含异己物应答元件XRE;含萤火虫荧光素酶报告基因luc2P。
产品目录号: E3751
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒



pGL4.43 载体特征


The pGL4.43[luc2P/XRE/Hygro] Vector contains three copies of a xenobiotic response element (XRE) 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 pGL4.43[luc2P/XRE/Hygro] Vector is used to measure
activation of the XRE in HepG2 cells upon treatment with TCDD. The pGL4.75 Vector
(encoding Renilla luciferase) is used as a normalization control. 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.

 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])
 Dulbecco’s PBS (DPBS; Life Technologies Cat. # 14190)
 0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
 Charcoal-stripped FBS (Life Technologies Cat.# 126776-011)
 Opti-MEM I (Life Technologies Cat.# 31985)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin; AccuStandard Cat.# D-404N)
 DMSO (Sigma Cat.# D2650)
 Dual-Glo Luciferase Assay System (Cat.# E2940)
 HepG2 cells
 pGL4.75[hRluc/CMV] Vector (Cat.# E6931)

Day 1: Plate Cells
1. Grow HepG2 cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash
twice with DPBS and treat with one volume of 0.05% trypsin-EDTA, followed by
four volumes of complete medium.
2. Vigorously resuspend the cells by pipetting and allow cell clumps to settle. Remove
the cell suspension from any cell clumps, quantify the cells and dilute in complete
medium to 1 × 105 cells/ml.
3. Plate 100μl per well to a solid, white 96-well plate (Corning Cat.# 3917).
4. Incubate for 24 hours in a 37°C, 5% CO2 incubator.

.Day 2: Transfection
1. Dilute pGL4.43[luc2P/XRE/hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
vector constructs in a 10:1 mass ratio, respectively, to 12.5ng total DNA/μl in
Opti-MEM I.
2. Add FuGENE HD to a 4.5:1 lipid:DNA ratio. Mix by pipetting. Incubate at room
temperature for 20 minutes.
3. Add 8μl transfection complex per well (100ng DNA/well) and incubate for 18 hours
in a 37°C, 5% CO2 incubator.

Day 3: Medium Replacement and Cell Treatment
1. Resuspend TCDD to 31.6μM in DMSO. Serially dilute by half logs into DMSO to
give concentrated stock solutions (316X). Dilute these 31.6-fold into Opti-MEM I
to give 10X stocks.
2. Remove existing medium from cells and replace with 72μl of DMEM + 0.5%
charcoal-stripped FBS per well.
3. Add 8μl of the 10X TCDD dilutions and incubate for 24 hours in a 37°C, 5% CO2

Day 4: Luminescence Measurement
1. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room
temperature for approximately 15 minutes.
2. Add 80μl of the Dual-Glo Luciferase Assay System detection reagents and measure
luminescence following the recommended protocol (Refer to the Dual-Glo
Luciferase Assay System Technical Manual, #TM058 for details).


LOCUS       JQ858513                6067 bp    DNA     circular SYN 02-JUL-2012
DEFINITION  Reporter vector PGL4.43[luc2P/XRE/Hygro], complete sequence.
VERSION     JQ858513.1  GI:392934082
SOURCE      Reporter vector PGL4.43[luc2P/XRE/Hygro]
  ORGANISM  Reporter vector PGL4.43[luc2P/XRE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6067)
  AUTHORS   McNamara,B.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-MAR-2012) Scientific Communications, Promega
            Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA
FEATURES             Location/Qualifiers
     source          1..6067
                     /organism="Reporter vector PGL4.43[luc2P/XRE/Hygro]"
                     /mol_type="other DNA"
     regulatory      105..258
                     /note="transcriptional pause site"
     regulatory      105..153
                     /note="synthetic poly(A) sequence"
     primer_bind     207..226
                     /note="RVprimer3 binding site"
     misc_feature    285..347
                     /note="XRE response element"
     regulatory      393..423
                     /note="minP promoter"
     gene            456..2231
     CDS             456..2231
     regulatory      2271..2492
                     /note="SV40 late poly(A) signal"
     regulatory      2540..2958
                     /note="SV40 enhancer and early promoter"
     CDS             2983..4020
                     /note="hygromycin resistance"
                     /product="hygromycin phosphotransferase"
     regulatory      4044..4092
                     /note="synthetic poly(A) sequence"
     primer_bind     complement(4159..4178)
                     /note="RVprimer4 binding site"
     rep_origin      4416..4452
     gene            complement(5207..6067)
     CDS             complement(5207..6067)
        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 gctctgagtt ctcacgctag
      301 cagattgagt tctcacgcta gcagattgag ttctcacgct agcagatctc gaggatatca
      361 agatctggcc tcggcggcca agcttagaca ctagagggta tataatggaa gctcgacttc
      421 cagcttggca atccggtact gttggtaaag ccaccatgga agatgccaaa aacattaaga
      481 agggcccagc gccattctac ccactcgaag acgggaccgc cggcgagcag ctgcacaaag
      541 ccatgaagcg ctacgccctg gtgcccggca ccatcgcctt taccgacgca catatcgagg
      601 tggacattac ctacgccgag tacttcgaga tgagcgttcg gctggcagaa gctatgaagc
      661 gctatgggct gaatacaaac catcggatcg tggtgtgcag cgagaatagc ttgcagttct
      721 tcatgcccgt gttgggtgcc ctgttcatcg gtgtggctgt ggccccagct aacgacatct
      781 acaacgagcg cgagctgctg aacagcatgg gcatcagcca gcccaccgtc gtattcgtga
      841 gcaagaaagg gctgcaaaag atcctcaacg tgcaaaagaa gctaccgatc atacaaaaga
      901 tcatcatcat ggatagcaag accgactacc agggcttcca aagcatgtac accttcgtga
      961 cttcccattt gccacccggc ttcaacgagt acgacttcgt gcccgagagc ttcgaccggg
     1021 acaaaaccat cgccctgatc atgaacagta gtggcagtac cggattgccc aagggcgtag
     1081 ccctaccgca ccgcaccgct tgtgtccgat tcagtcatgc ccgcgacccc atcttcggca
     1141 accagatcat ccccgacacc gctatcctca gcgtggtgcc atttcaccac ggcttcggca
     1201 tgttcaccac gctgggctac ttgatctgcg gctttcgggt cgtgctcatg taccgcttcg
     1261 aggaggagct attcttgcgc agcttgcaag actataagat tcaatctgcc ctgctggtgc
     1321 ccacactatt tagcttcttc gctaagagca ctctcatcga caagtacgac ctaagcaact
     1381 tgcacgagat cgccagcggc ggggcgccgc tcagcaagga ggtaggtgag gccgtggcca
     1441 aacgcttcca cctaccaggc atccgccagg gctacggcct gacagaaaca accagcgcca
     1501 ttctgatcac ccccgaaggg gacgacaagc ctggcgcagt aggcaaggtg gtgcccttct
     1561 tcgaggctaa ggtggtggac ttggacaccg gtaagacact gggtgtgaac cagcgcggcg
     1621 agctgtgcgt ccgtggcccc atgatcatga gcggctacgt taacaacccc gaggctacaa
     1681 acgctctcat cgacaaggac ggctggctgc acagcggcga catcgcctac tgggacgagg
     1741 acgagcactt cttcatcgtg gaccggctga agagcctgat caaatacaag ggctaccagg
     1801 tagccccagc cgaactggag agcatcctgc tgcaacaccc caacatcttc gacgccgggg
     1861 tcgccggcct gcccgacgac gatgccggcg agctgcccgc cgcagtcgtc gtgctggaac
     1921 acggtaaaac catgaccgag aaggagatcg tggactatgt ggccagccag gttacaaccg
     1981 ccaagaagct gcgcggtggt gttgtgttcg tggacgaggt gcctaaagga ctgaccggca
     2041 agttggacgc ccgcaagatc cgcgagattc tcattaaggc caagaagggc ggcaagatcg
     2101 ccgtgaattc tcacggcttc cctcccgagg tggaggagca ggccgccggc accctgccca
     2161 tgagctgcgc ccaggagagc ggcatggata gacaccctgc tgcttgcgcc agcgccagga
     2221 tcaacgtcta aggccgcgac tctagagtcg gggcggccgg ccgcttcgag cagacatgat
     2281 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     2341 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     2401 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     2461 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatccgt ttgcgtattg
     2521 ggcgctcttc cgctgatctg cgcagcacca tggcctgaaa taacctctga aagaggaact
     2581 tggttagcta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     2641 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2701 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2761 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2821 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2881 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2941 ggcttttgca aaaagctcga ttcttctgac actagcgcca ccatgaagaa gcccgaactc
     3001 accgctacca gcgttgaaaa atttctcatc gagaagttcg acagtgtgag cgacctgatg
     3061 cagttgtcgg agggcgaaga gagccgagcc ttcagcttcg atgtcggcgg acgcggctat
     3121 gtactgcggg tgaatagctg cgctgatggc ttctacaaag accgctacgt gtaccgccac
     3181 ttcgccagcg ctgcactacc catccccgaa gtgttggaca tcggcgagtt cagcgagagc
     3241 ctgacatact gcatcagtag acgcgcccaa ggcgttactc tccaagacct ccccgaaaca
     3301 gagctgcctg ctgtgttaca gcctgtcgcc gaagctatgg atgctattgc cgccgccgac
     3361 ctcagtcaaa ccagcggctt cggcccattc gggccccaag gcatcggcca gtacacaacc
     3421 tggcgggatt tcatttgcgc cattgctgat ccccatgtct accactggca gaccgtgatg
     3481 gacgacaccg tgtccgccag cgtagctcaa gccctggacg aactgatgct gtgggccgaa
     3541 gactgtcccg aggtgcgcca cctcgtccat gccgacttcg gcagcaacaa cgtcctgacc
     3601 gacaacggcc gcatcaccgc cgtaatcgac tggtccgaag ctatgttcgg ggacagtcag
     3661 tacgaggtgg ccaacatctt cttctggcgg ccctggctgg cttgcatgga gcagcagact
     3721 cgctacttcg agcgccggca tcccgagctg gccggcagcc ctcgtctgcg agcctacatg
     3781 ctgcgcatcg gcctggatca gctctaccag agcctcgtgg acggcaactt cgacgatgct
     3841 gcctgggctc aaggccgctg cgatgccatc gtccgcagcg gggccggcac cgtcggtcgc
     3901 acacaaatcg ctcgccggag cgcagccgta tggaccgacg gctgcgtcga ggtgctggcc
     3961 gacagcggca accgccggcc cagtacacga ccgcgcgcta aggaggtagg tcgagtttaa
     4021 actctagaac cggtcatggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttg
     4081 gttttttgtg tgttcgaact agatgctgtc gaccgatgcc cttgagagcc ttcaacccag
     4141 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     4201 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac
     4261 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4321 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4381 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4441 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4501 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4561 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4621 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4681 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4741 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4801 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4861 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4921 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4981 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5041 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5101 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5161 taaacttggt ctgacagcgg ccgcaaatgc taaaccactg cagtggttac cagtgcttga
     5221 tcagtgaggc accgatctca gcgatctgcc tatttcgttc gtccatagtg gcctgactcc
     5281 ccgtcgtgta gatcactacg attcgtgagg gcttaccatc aggccccagc gcagcaatga
     5341 tgccgcgaga gccgcgttca ccggcccccg atttgtcagc aatgaaccag ccagcaggga
     5401 gggccgagcg aagaagtggt cctgctactt tgtccgcctc catccagtct atgagctgct
     5461 gtcgtgatgc tagagtaaga agttcgccag tgagtagttt ccgaagagtt gtggccattg
     5521 ctactggcat cgtggtatca cgctcgtcgt tcggtatggc ttcgttcaac tctggttccc
     5581 agcggtcaag ccgggtcaca tgatcaccca tattatgaag aaatgcagtc agctccttag
     5641 ggcctccgat cgttgtcaga agtaagttgg ccgcggtgtt gtcgctcatg gtaatggcag
     5701 cactacacaa ttctcttacc gtcatgccat ccgtaagatg cttttccgtg accggcgagt
     5761 actcaaccaa gtcgttttgt gagtagtgta tacggcgacc aagctgctct tgcccggcgt
     5821 ctatacggga caacaccgcg ccacatagca gtactttgaa agtgctcatc atcgggaatc
     5881 gttcttcggg gcggaaagac tcaaggatct tgccgctatt gagatccagt tcgatatagc
     5941 ccactcttgc acccagttga tcttcagcat cttttacttt caccagcgtt tcggggtgtg
     6001 caaaaacagg caagcaaaat gccgcaaaga agggaatgag tgcgacacga aaatgttgga
     6061 tgctcat








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