Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007333 | pCW57-CMV-ssRFP-GFP-KDEL | 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:
- pCW57-CMV-ssRFP-GFP-KDEL
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9213 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- Low Copy
- Promoter:
- hPGK
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- CCATCCACGCTGTTTTGACCTC
pCW57-CMV-ssRFP-GFP-KDEL 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.
pCW57-CMV-ssRFP-GFP-KDEL vector Sequence
LOCUS 40924_13885 9213 bp DNA circular SYN 13-MAY-2021
DEFINITION Fluorescence reporter to monitor ER-phagy activity.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9213)
AUTHORS Chino H, Hatta T, Natsume T, Mizushima N
TITLE Intrinsically Disordered Protein TEX264 Mediates ER-phagy.
JOURNAL Mol Cell. 2019 Apr 11. pii: S1097-2765(19)30257-6. doi:
10.1016/j.molcel.2019.03.033.
PUBMED 31006538
REFERENCE 2 (bases 1 to 9213)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9213)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell.
2019 Apr 11. pii: S1097-2765(19)30257-6. doi:
10.1016/j.molcel.2019.03.033."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9213
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..227
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 228..408
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 455..580
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1073..1306
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1491..1535
/codon_start=1
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
/translation="KNEQELLELDKWASL"
misc_feature 1724..1841
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind 1909..2179
/label=tetracycline response element
/note="contains seven copies of the tetracycline operator
tetO"
promoter 2212..2250
/label=minimal CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 2247..2271
/label=LNCX
/note="Human CMV promoter, forward primer"
sig_peptide 2350..2403
/product="Drosophila BiP signal sequence"
/label=BiP signal sequence
CDS 2404..3078
/codon_start=1
/label=mRFP1
/note="monomeric derivative of DsRed (Campbell et al.,
2002)"
/translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
TGA"
CDS 3124..3840
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
promoter 3862..4372
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 4382..4978
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 4979..5032
/codon_start=1
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
/translation="EGRGSLLTCGDVEENPGP"
CDS 5033..5776
/codon_start=1
/label=rtTA-Advanced
/note="improved tetracycline-controlled transactivator"
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
primer_bind complement(5819..5839)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(6237..6248)
/codon_start=1
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
/translation="IEGR"
LTR 6453..6686
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 6758..6892
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 6919..7054
/label=SV40 ori
/note="SV40 origin of replication"
promoter 7055..7125
/label=AmpR promoter
CDS 7126..7983
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 8157..8745
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8899..8916
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 9033..9054
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9069..9099
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9107..9123
/label=lac operator
/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)."
primer_bind 9131..9147
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 9168..9186
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"