Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010813 | pgRNA-humanized | 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:
- pgRNA-humanized
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8297 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- CMV
- Cloning Method:
- Restriction Enzyme
pgRNA-humanized 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.
pgRNA-humanized vector Sequence
LOCUS V010813 8297 bp DNA circular SYN 16-AUG-2021
DEFINITION Exported.
ACCESSION V010813
VERSION V010813
KEYWORDS pgRNA-humanized
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8297)
AUTHORS Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim
WA
TITLE Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific
Control of Gene Expression.
JOURNAL Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.
PUBMED 23452860
REFERENCE 2 (bases 1 to 8297)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8297)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.cell.2013.02"; journalName: "Cell"; date: "2013-02-28-
28"; volume: "152"; issue: "5"; pages: "1173-83"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8297
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 205..238
/label="loxP"
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind 242..262
/label="mU6-F"
/note="Mouse U6 promoter, forward primer"
misc_RNA 345..420
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 545..848
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 849..1052
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 1086..1682
/label="PuroR"
/note="puromycin N-acetyltransferase"
CDS 1692..1745
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 1758..2465
/label="mCherry"
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
protein_bind complement(2488..2521)
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_feature 2577..3165
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3168..3184)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
LTR 3694..3874
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
rep_origin complement(3936..4524)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4698..5555)
/label="AmpR"
/note="beta-lactamase"
promoter complement(5556..5660)
/label="AmpR promoter"
primer_bind complement(5735..5754)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 5926..6305
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 6307..6505
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 6523..6703
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6750..6875
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7372..7605
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7790..7834
/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 7983..8024
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 8132..8249
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"