Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017271 | pcDNA3.1 iCasper T2A HO1 | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
Infrared fluorescent proteins (IFPs) were derived and engineered from bacterial phytochrome photoreceptors (BphPs). The photosensory module of BphPs consists of the PAS, GAF and PHY domains. Light irradiation activates the photosensory module, which then transmits the signal to the effector domain to initiate a molecular signaling pathway. As a chromophore, BphPs use biliverdin (BV), which is located in a pocket of the GAF domain and is covalently bound to the Cys in the PAS domain. IFPs are composed of the N-terminal PAS and GAF domains of BphPs, and autocatalytically incorporate the chromophore biliverdin (BV). BV is a catabolic metabolite of heme by heme oxygenase and is nonfluorescent by itself. BV binds to the GAF domain noncovalently and forms a thioether bond with a conserved cysteine near N terminus of IFPs. iCasper consists of spGFP-N, spGFP-C and caspase3 cleavage site-separated IFPs PAS and GAF domains. Cells expressing iCasper were green but not infrared fluorescent. Upon addition of staurosporine, which is a protein kinase inhibitor and induces apoptosis by activating caspase 3, iCasper became infrared fluorescent as the cells underwent apoptosis.
- Vector Name:
- pcDNA3.1 iCasper T2A HO1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7922 bp
- Type:
- Gene expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Neo/G418
- Promoter:
- CMV
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
pcDNA3.1 iCasper T2A HO1 vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- To TL, Piggott BJ, Makhijani K, Yu D, Jan YN, Shu X. Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3338-43. doi: 10.1073/pnas.1502857112. Epub 2015 Mar 2. PMID: 25733847; PMCID: PMC4371907.
pcDNA3.1 iCasper T2A HO1 vector Sequence
LOCUS Exported 7922 bp DNA circular SYN 14-AUG-2024
DEFINITION Expresses infrared fluorescent executioner caspase reporter
(iCasper) plus human heme oxygenase 1.
ACCESSION .
VERSION .
KEYWORDS pcDNA3.1 iCasper T2A HO1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7922)
AUTHORS To TL, Piggott BJ, Makhijani K, Yu D, Jan YN, Shu X
TITLE Rationally designed fluorogenic protease reporter visualizes
spatiotemporal dynamics of apoptosis in vivo.
JOURNAL Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3338-43. doi:
10.1073/pnas.1502857112. Epub 2015 Mar 2.
PUBMED 25733847
REFERENCE 2 (bases 1 to 7922)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1073/pnas.1502857112"; journalName: "Proc Natl Acad Sci U S A";
date: "2015-03-17- 17"; volume: "112"; issue: "11"; pages: "3338-43"
FEATURES Location/Qualifiers
source 1..7922
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(44..63)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 769..789
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 863..882
/label=T7
/note="T7 promoter, forward primer"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 923..1564
/codon_start=1
/product="fragment of superfolder GFP consisting of the
first 10 beta-strands"
/label=GFP(1-10)
/note="associates with GFP11, the 11th beta-strand, to
reconstitute fluorescent GFP (Cabantous et al., 2005;
Kamiyama et al., 2016)"
/translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
CDS 1580..2146
/codon_start=1
/product="cGMP phosphodiesterase/adenylate cyclase/FhlA
transcriptional activator"
/label=GAF
/translation="TTNIAPALDGAFHRITSSSSLMGLCDETATIIREITGYDRVMVVR
FDEEGNAEILSERRRADLEAFLGNRYPASTIPQIARRLYEHNRVRLLVDVNYTPVPLQP
RISPLNGRDLDMSLSCLRSMSPVHQKYMQDMGVGATLVCSLMVSGRLWGLIACHHYEPR
FVPFHIRAAGEALAETCANRIATLES"
CDS 2150..2164
/codon_start=1
/label=Caspase 3/7 cleavage site
/translation="DEVDG"
CDS 2171..2491
/codon_start=1
/product="Per-ARNT-Sim repeats"
/label=PAS
/translation="CEREQIHLAGSIQPHGILLAVKEPDNVVIQASINAAEFLNTNSVV
GRPLRDLGGDLPLQILPHLNGPLHLAPVTLRCTVGSPPRRVDCTIHRPSNGGLIVELEP
ATK"
CDS 2507..2554
/codon_start=1
/product="11th beta-strand of superfolder GFP"
/label=GFP11
/note="associates with GFP(1-10), consisting of the first
10 beta-strands, to reconstitute fluorescent GFP (Cabantous
et al., 2005; Kamiyama et al., 2016)"
/translation="RDHMVLHEYVNAAGIT"
CDS 2564..2617
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 2618..3484
/codon_start=1
/product="heme oxygenase 1"
/label=HO1
/translation="MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRD
GFKLVMASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPRWQEV
IPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEG
LAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLT
HDTKDQSPSRAPGLRQRASNKVQDSAPVETPRGKPPLNTRSQAPLLRWVLTLSFLVATV
AVGLYAM"
primer_bind complement(3516..3533)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3522..3746
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 3792..4220
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3879..3898)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4089..4110
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4229..4249)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 4234..4563
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 4414..4549
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 4476..4495
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 4630..5424
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(4684..4703)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 5294..5313
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 5598..5719
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5635..5654)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 5689..5708
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(5768..5784)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5768..5784)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5781..5803)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5792..5808
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(5816..5846)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5861..5882
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5999..6016)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6170..6755)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6250..6269)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6926..7786)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 7549..7568
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7787..7891)
/gene="bla"
/label=AmpR promoter