TopFlash

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  • Model: PVT10787
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TopFlash


Catalog No. PVT10787
Packing 2ug

 

TopFlash Information

Promoter: Mini-TK promoter

Replicon: pUC ori

Terminator: SV40 poly (A) signal

Plasmid classification: lactation serial plasmid; lactating reporter plasmid; luciferase plasmid

Plasmid size: 5539bp

Plasmid tagging: Luc

Prokaryotic resistance: Amp (100 g/ml)

Cloning strains: E. coli DH5 and E.

Culture conditions: 37 C, LB, aerobic.

Expression host: lactation cells

Induction mode: no need to induce, transient expression.

5'sequencing primers: M13F (TGTAAAACGACGGCCAGT)

3'sequencing primers: M13R (CAGGAAACAGCTATGACC)

Function Mammal reporter plasmid

 

TopFlash Description

Wnt / Wg family encoding involves cell growth regulation, secretory glycoprotein determination of cell fate, organogenesis and tumorigenesis. From the genetic analysis of wingless (wg) signals, it is inferred that in mammalian cells, Wnts are transmitted to Dishevell via receptor signals of the convoluted family. Reduce the activity of glycogen synthase kinase -3 (GSK-3). This results in the stabilization and accumulation of cytoplasmic-catenin, a GSK-3 substrate that, when phosphorylated by GSK-3, is targeted at ubiquitin-mediated protein hydrolysis. -catenin is then transferred to the nucleus, where complex transcription regulators with members of the Tcf/LEF family activate transcription of Tcf-responsive genes. The Tcf reporter plasmid and FOPFLASH can quantify Wnt / Wg signaling in cells transfected with these constructs.

Transfection grade T cell factor (Tcf) reporter plasmid containing three copies of the Tcf binding site (wt) upstream of the Thymidine Kinase (TK) minimal promoter and Luciferase open reading frame. FOPFLASH (Catalog # 21-169) containing mutated Tcf binding sites is also available as a negative control.

 

TopFlash Multiple cloning site

TopFlash-vector

 

TopFlash Sequence

LOCUS       Exported                5539 bp ds-DNA    circular SYN 13-JULY-2017
KEYWORDS    TopFlash
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5539)
  TITLE     Direct Submission
  JOURNAL   Exported 2017-7-13
FEATURES             Location/Qualifiers
     source          1..5539
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        600..662
                     /note="Mini-TK promoter"
                     /note="minimal herpes simplex virus (HSV) thymidine kinase 
                     promoter"
     CDS             691..2343
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /EC_number="
                     /note="luciferase"
                     /experiment="
                     /protein_id="
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     intron          2502..2567
                     /note="small t intron"
                     /note="simian virus 40 (SV40) small t antigen intron"
     CDS             2697..2717
                     /codon_start=1
                     /locus_tag="
                     "
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /note="
                     /protein_id="
                     /translation="PKKKRKV"
     polyA_signal    3142..3276
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(3720..4308)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4479..5339)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5340..5444)
                     /gene="bla"
                     /note="AmpR promoter"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gttaagatca aagggggtaa
      421 gakcaaaggg ggtaaaatca aagggggccc cctttgatct tacccccttt gatcttaccc
      481 cctttgatct tactgcatgc ctgcaggtcg actctagagg atccggcccc gcccagcgtc
      541 ttgtcattgg cgaattcgaa cacgcagatg cagtcggggc ggcgcggtcc gaggtccact
      601 tcgcatatta aggtgacgcg tgtggcctcg aacaccgagc gaccctgcag cgacccgctt
      661 aacagcgtca acagcgtgcc gcagatctcc atggaagacg ccaaaaacat aaagaaaggc
      721 ccggcgccat tctatcctct agaggatgga accgctggag agcaactgca taaggctatg
      781 aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtgaac
      841 atcacgtacg cggaatactt cgaaatgtcc gttcggttgg cagaagctat gaaacgatat
      901 gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca attctttatg
      961 ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga catttataat
     1021 gaacgtgaat tgctcaacag tatgaacatt tcgcagccta ccgtagtgtt tgtttccaaa
     1081 aaggggttgc aaaaaatttt gaacgtgcaa aaaaaattac caataatcca gaaaattatt
     1141 atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt cgtcacatct
     1201 catctacctc ccggttttaa tgaatacgat tttgtaccag agtcctttga tcgtgacaaa
     1261 acaattgcac tgataatgaa ttcctctgga tctactgggt tacctaaggg tgtggccctt
     1321 ccgcatagaa ctgcctgcgt cagattctcg catgccagag atcctatttt tggcaatcaa
     1381 atcattccgg atactgcgat tttaagtgtt gttccattcc atcacggttt tggaatgttt
     1441 actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag atttgaagaa
     1501 gagctgtttt tacgatccct tcaggattac aaaattcaaa gtgcgttgct agtaccaacc
     1561 ctattttcat tcttcgccaa aagcactctg attgacaaat acgatttatc taatttacac
     1621 gaaattgctt ctgggggcgc acctctttcg aaagaagtcg gggaagcggt tgcaaaacgc
     1681 ttccatcttc cagggatacg acaaggatat gggctcactg agactacatc agctattctg
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