Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000678 | FUW mCherry-GFP-LC3 | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The FUW mCherry-GFP-LC3 contains GFP and mCherry fluorescence. It can to observe free autophagosomes and autophagosomes fused to lysosomes (autolysosomes; mCherry fluorescence only because GFP is acid sensitive).
- Vector Name:
- FUW mCherry-GFP-LC3
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11138 bp
- Type:
- Lentiviral
- Replication origin:
- ori
- Promoter:
- UbC
- Cloning Method:
- TOPO Cloning
- 5' Primer:
- CGCGGATCCGGTCGCCACCATGGTGAGCAAGGGCGAG
- 3' Primer:
- CGCGGCGCGCCGCTGGGTCTAGATGCATGC
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
FUW mCherry-GFP-LC3 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
- Leeman DS, Hebestreit K, Ruetz T, Webb AE, McKay A, Pollina EA, Dulken BW, Zhao X, Yeo RW, Ho TT, Mahmoudi S, Devarajan K, Passegué E, Rando TA, Frydman J, Brunet A. Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging. Science. 2018 Mar 16;359(6381):1277-1283.
FUW mCherry-GFP-LC3 vector Sequence
LOCUS V000678 11138 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V000678
VERSION V000678
KEYWORDS FUW mCherry-GFP-LC3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 11138)
AUTHORS Leeman DS, Hebestreit K, Ruetz T, Webb AE, McKay A, Pollina EA,
Dulken BW, Zhao X, Yeo RW, Ho TT, Mahmoudi S, Devarajan K, Passegue
E, Rando TA, Frydman J, Brunet A
TITLE Lysosome activation clears aggregates and enhances quiescent neural
stem cell activation during aging.
JOURNAL Science. 2018 Mar 16;359(6381):1277-1283. doi:
10.1126/science.aag3048. Epub 2018 Mar 15.
PUBMED 29590078
REFERENCE 2 (bases 1 to 11138)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 11138)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1126/science.aag3048"; journalName: "Science"; date:
"2018-03-16- 16"; volume: "359"; issue: "6381"; pages: "1277-1283"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..11138
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 7..386
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 387..589
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 604..784
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 831..956
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1449..1682
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1867..1911
/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 2060..2101
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2209..2326
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2391..3602
/label="UbC promoter"
/note="human ubiquitin C promoter"
CDS 3647..4354
/label="mCherry"
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
protein_bind 4382..4406
/label="attB1"
/note="recombination site for the Gateway(R) BP reaction"
CDS 4421..5137
/label="EGFP"
/note="enhanced GFP"
CDS 5165..5539
/codon_start=1
/gene="MAP1LC3B"
/product="human LC3 protein"
/label="LC3B"
/note="Atg8 family member found on autophagosomes"
/translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
EDGFLYMVYASQETFGMKLSV"
misc_feature 5599..6187
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(6190..6206)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6191..6207)
/label="pBluescriptKS"
/note="For pBluescript vector"
LTR 6712..6892
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 6924..7148
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 7194..7622
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 7636..7965
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 8013..8060
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 8079..8450
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 8583..8716
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(8753..8769)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8753..8769)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8766..8788)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 8777..8793
/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(8801..8831)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(8846..8867)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8984..9001)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(9155..9743)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(9917..10774)
/label="AmpR"
/note="beta-lactamase"
promoter complement(10775..10879)
/label="AmpR promoter"
primer_bind complement(10954..10973)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"