Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012182 | pBABE-puro mCherry-EGFP-LC3B | 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
- Vector Name:
- pBABE-puro mCherry-EGFP-LC3B
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6952 bp
- Type:
- Mammalian Expression, Retroviral
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Cloning Method:
- Restriction Enzyme
pBABE-puro mCherry-EGFP-LC3B 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.
pBABE-puro mCherry-EGFP-LC3B vector Sequence
LOCUS 40924_5424 6952 bp DNA circular SYN 13-MAY-2021
DEFINITION retroviral expression of mCherry, EGFP-tagged LC3.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6952)
AUTHORS N'Diaye EN, Kajihara KK, Hsieh I, Morisaki H, Debnath J, Brown EJ
TITLE PLIC proteins or ubiquilins regulate autophagy-dependent cell
survival during nutrient starvation.
JOURNAL EMBO Rep. 2009 Feb;10(2):173-9. doi: 10.1038/embor.2008.238. Epub
2009 Jan 16.
PUBMED 19148225
REFERENCE 2 (bases 1 to 6952)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6952)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/embor.2008.238"; journalName: "EMBO Rep"; date: "2009-02";
volume: "10"; issue: "2"; pages: "173-9"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6952
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..470
/label=LTR
/note="long terminal repeat from Moloney murine leukemia
virus"
misc_feature 535..734
/label=MMLV Psi
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 935..1351
/codon_start=1
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
/translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
CDS 1368..2075
/codon_start=1
/label=mCherry
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
protein_bind 2103..2127
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 2142..2858
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 2886..3260
/codon_start=1
/gene="MAP1LC3B"
/product="human LC3 protein"
/label=LC3B
/note="Atg8 family member found on autophagosomes"
/translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
EDGFLYMVYASQETFGMKLSV"
promoter 3280..3609
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 3619..4215
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 4317..4908
/label=LTR
/note="long terminal repeat from Moloney murine leukemia
virus"
primer_bind complement(5038..5055)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5209..5797)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5960..6817)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6818..6922)
/label=AmpR promoter