psiCHECK-2
Catalog No. PVT10837
Packing 2ug
psiCHECK-2 Information
Function Mammal reporter plasmid
Promoters: HSV TK, T7, SV40
Replicator: pUC ori, SV40 ori
Terminator: SV40 poly (A) signal
Plasmid classification: mammalian cells, signal pathway reporting vectors
Plasmid size: 6273bp
Prokaryotic resistance: Amp
Cloned strain: DH5 alpha
Culture conditions: 37 centigrade, aerobic LB
Expression host: mammalian cells
Induction mode: no induction, instantaneous expression
5'sequencing primers: T7:TAATACGACTCACTATAGGG
3'sequencing primers: primers designed according to sequence
psiCHECK-2 Description
psiCHEC-2 Vector is designed to provide a quantitative and rapid approach for optimization of RNA interference (RNAi). The vectors enable the monitoring of changes in expression of a target gene fused to the reporter gene. In both vectors, Renilla luciferase is used as a primary reporter gene, and the gene of interest can be cloned into the multiple cloning region located downstream of the Renilla luciferase translational stop codon. Initiation of the RNAi process toward a gene of interest results in cleavage and subsequent degradation of fusion mRNA. Measurement of decreased Renilla luciferase activity is a convenient indicator of RNAi effect . RNAi is a phenomenon by which double-stranded RNA complementary to a target mRNA can specifically inactivate gene function by stimulating the degradation of the target mRNA . Because of the ability to inactivate genes, RNAi has emerged as a powerful tool for analyzing gene function. In mammalian systems, including cultured mammalian cells, chemically synthesized double-stranded short interfering RNA molecules (<30 nucleotides; siRNA) result in dsRNA duplexes <30 base pairs in length that induce RNAi .RNAi duplexes >30bp induce the interferon response and nonspecific degradation of mRNA and cannot be used as tools for specific gene silencing .
psiCHECK-2 Multiple site
psiCHECK-2 Sequence
LOCUS Exported 6273 bp ds-DNA circular SYN 07-JUL-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS psiCHECK-2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6273)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Thursday, July 7, 2016 from SnapGene Viewer 3.1.4
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6273
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 62..419
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 270..405
/note="SV40 ori"
/note="SV40 origin of replication"
intron 489..621
/note="chimeric intron"
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
promoter 666..684
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 694..1629
/codon_start=1
/product="Renilla luciferase"
/note="hRluc"
/note="human codon-optimized"
/translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
polyA_signal 1688..1736
/note="synthetic polyadenylation signal"
promoter 1745..2496
/note="HSV TK promoter"
/note="herpes simplex virus thymidine kinase promoter"
CDS 2532..4184
/codon_start=1
/note="hluc+"
/translation="MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
polyA_signal 4228..4349
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter 4482..4586
/gene="bla"
/note="AmpR promoter"
CDS 4587..5447
/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"
rep_origin 5618..6206
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
61 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
241 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
301 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
361 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
421 agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
481 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
541 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
601 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
661 gtacttaata cgactcacta taggctagcc accatggctt ccaaggtgta cgaccccgag
721 caacgcaaac gcatgatcac tgggcctcag tggtgggctc gctgcaagca aatgaacgtg
781 ctggactcct tcatcaacta ctatgattcc gagaagcacg ccgagaacgc cgtgattttt
841 ctgcatggta acgctgcctc cagctacctg tggaggcacg tcgtgcctca catcgagccc
901 gtggctagat gcatcatccc tgatctgatc ggaatgggta agtccggcaa gagcgggaat
961 ggctcatatc gcctcctgga tcactacaag tacctcaccg cttggttcga gctgctgaac
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