Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012092 | pCW57-MCS1-P2A-MCS2 (Neo) | 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-MCS1-P2A-MCS2 (Neo)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7943 bp
- Type:
- Mammalian Expression, Lentiviral ; Doxycycline ind
- Replication origin:
- ori
- Selection Marker:
- Neomycin (select with G418)
- Promoter:
- hPGK
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- pCW57.MCS_Seq_F CGTATGTCGAGGTAGGCGTG
pCW57-MCS1-P2A-MCS2 (Neo) 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-MCS1-P2A-MCS2 (Neo) vector Sequence
LOCUS 40924_13890 7943 bp DNA circular SYN 13-MAY-2021
DEFINITION All-in-one doxycycline inducible lentiviral vector for expression of
one or two genes using the P2A self-cleaving peptide..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7943)
AUTHORS -
TITLE Karpf Lab Plasmids
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 7943)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7943)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7943
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 32..346
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
CDS 387..443
/codon_start=1
/label=P2A
/note="2A peptide from porcine teschovirus-1 polyprotein"
/translation="ATNFSLLKQAGDVEENPGP"
promoter 471..981
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 1005..1748
/codon_start=1
/label=rtTA-Advanced
/note="improved tetracycline-controlled transactivator"
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
CDS 1764..1817
/codon_start=1
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
/translation="EGRGSLLTCGDVEENPGP"
CDS 1827..2618
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(2662..2682)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3080..3091)
/codon_start=1
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
/translation="IEGR"
LTR 3296..3529
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 3601..3735
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 3762..3897
/label=SV40 ori
/note="SV40 origin of replication"
promoter 3898..3968
/label=AmpR promoter
CDS 3969..4826
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5000..5588
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5742..5759
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 5876..5897
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5912..5942
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5950..5966
/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 5974..5990
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 6011..6029
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 6057..6283
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 6284..6464
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6511..6636
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7129..7362
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7547..7591
/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 7804..7921
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"