Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010419 | pLVX-TetOne-Puro | 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
In the Tet-Off expression system, a tetracycline-controlled transactivator protein (tTA), which is composed of the Tet repressor DNA binding protein (TetR) from the Tc resistance operon of Escherichia coli transposon Tn10 fused to the strong transactivating domain(sequence: PTDALDDFDLDML) of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE is made up of Tet operator (tetO) sequence concatemers fused to a minimal promoter. In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive. The Tet-On system is based on a reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change(E19G, A56P, D148E, H179R) in the tetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of the Dox effector. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.
- Vector Name:
- pLVX-TetOne-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9181 bp
- Type:
- Viral Expression & Packaging Vectors
- Replication origin:
- ori
- Source/Author:
- Clontech
- Copy Number:
- High copy number
- Promoter:
- hPGK
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pLVX-TetOne-Puro 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
- Moonmuang S, Saoin S, Chupradit K, Sakkhachornphop S, Israsena N, Rungsiwiwut R, Tayapiwatana C. Modulated expression of the HIV-1 2LTR zinc finger efficiently interferes with the HIV integration process. Biosci Rep. 2018 Sep 7;38(5):BSR20181109. doi: 10.1042/BSR20181109. PMID: 30068696; PMCID: PMC6127673.
pLVX-TetOne-Puro vector Sequence
LOCUS Exported 9181 bp DNA circular SYN 02-SEP-2024
DEFINITION Exported.
ACCESSION V010419
VERSION .
KEYWORDS pLVX-TetOne-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9181)
AUTHORS Clontech
TITLE Direct Submission
REFERENCE 2 (bases 1 to 9181)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9181)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9181
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..634
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/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 1914..1955
/label=Protein Tat
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
misc_feature 2027..2144
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
polyA_signal 2187..2321
/label=SV40 poly(A) signal
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
misc_feature 2496..2527
/label=MCS
/note="MCS"
/note="multiple cloning site"
promoter complement(2528..2895)
/label=TRE3GS promoter
/note="3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On(R) 3G, modified to eliminate
binding sites for endogenous mammalian transcription
factors"
promoter 2912..3422
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 3441..4184
/label=Tet-On(R) 3G
/note="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
promoter 4198..4527
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 4536..5132
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 5149..5737
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5944..6577
/label=5' LTR
/note="5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(6706..6722)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6730..6746)
/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)."
promoter complement(6754..6784)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(6799..6820)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(7108..7696)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7870..8727)
/label=AmpR
/note="beta-lactamase"
promoter complement(8728..8832)
/label=AmpR promoter
polyA_signal 8880..9014
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"