Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V017137 | pSIN-PAmCherry-KFERQ-NE | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pSIN-PAmCherry-KFERQ-NE plasmid is a lentiviral vector designed for monitoring chaperone-mediated autophagy (CMA) activity. Key features include: CMA substrate: Contains the "KFERQ" pentapeptide motif recognized by HSPA8/HSC70 for lysosomal targeting; Photoactivatable reporter: PAmCherry fluorescence enables pulse-chase degradation assays; NE epitope tag: Allows antibody-based detection; Puromycin resistance: For stable cell line selection.
- Vector Name:
- pSIN-PAmCherry-KFERQ-NE
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8388 bp
- Type:
- Constitutive expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- EF-1α
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pSIN-PAmCherry-KFERQ-NE 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
- Ho PW, Leung CT, Liu H, Pang SY, Lam CS, Xian J, Li L, Kung MH, Ramsden DB, Ho SL. Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA). Autophagy. 2020 Feb;16(2):347-370. doi: 10.1080/15548627.2019.1603545. Epub 2019 Apr 14. PMID: 30983487; PMCID: PMC6984454.
pSIN-PAmCherry-KFERQ-NE vector Sequence
LOCUS Exported 8388 bp DNA circular SYN 20-JUL-2025
DEFINITION Exported.
ACCESSION V017137
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8388)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8388)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8388
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 2096..2115
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..1179
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 1193..1900
/label=PAmCherry1
/note="photoactivatable monomeric derivative of DsRed
fluorescent protein (Subach et al., 2009)"
misc_feature 2083..2681
/label=IRES2
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 2701..3297
/label=PuroR
/note="puromycin N-acetyltransferase"
LTR 3503..3736
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 3893..4117
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 4213..4317
/label=AmpR promoter
CDS 4318..5175
/label=AmpR
/note="beta-lactamase"
rep_origin 5349..5937
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
LTR 6096..6729
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 6776..6901
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7394..7627
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7812..7856
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 8005..8046
/label=Protein Tat
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
misc_feature 8143..8260
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 8388
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"