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 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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