Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017360 | pLX-TRE-dCas9-KRAB-MeCP2-BSD | 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:
- pLX-TRE-dCas9-KRAB-MeCP2-BSD
- Antibiotic Resistance:
- Ampicillin
- Length:
- 16734 bp
- Type:
- Genome editing
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Blast
- Promoter:
- TRE3G
- Growth Temperature:
- 30℃
pLX-TRE-dCas9-KRAB-MeCP2-BSD vector 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.
pLX-TRE-dCas9-KRAB-MeCP2-BSD vector Sequence
LOCUS V017360 16734 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V017360
VERSION V017360
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 16734)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..16734
/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
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2050..2167
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2929..3307
/label="TRE3G promoter"
/note="3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On(R) 3G"
CDS 3340..7443
/label="Cas9m4"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 7579..7764
/label="KRAB"
/note="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
CDS 7798..7818
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 7828..8685
/label="MeCP2"
/note="transcriptional repression domain from rat
methyl-CpG-binding protein 2 (Nan et al., 1997)"
polyA_signal 8784..8832
/label="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
promoter 9063..9274
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 9320..10063
/label="Tet-On(R) 3G"
/note="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
CDS 10064..10117
/label="T2A"
/note="2A peptide from Thosea asigna virus capsid protein"
CDS 10139..10534
/label="BSD"
/note="blasticidin S deaminase"
misc_feature 10598..11186
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 11296..11529
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
promoter complement(11554..11572)
/label="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
promoter 12360..12464
/label="AmpR promoter"
CDS 12465..13322
/label="AmpR"
/note="beta-lactamase"
rep_origin 13496..14084
/direction=RIGHT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 14330..14659
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
intron 15793..15858
/label="small t intron"
/note="SV40 (simian virus 40) small t antigen intron"
CDS 15988..16008
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
polyA_signal 16433..16567
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"