Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V002360 | pXPR_011 | 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:
- pXPR_011
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8395 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- hPGK
- 5' Primer:
- LKO.1 5'
pXPR_011 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.
pXPR_011 vector Sequence
LOCUS 40924_47263 8395 bp DNA circular SYN 13-MAY-2021
DEFINITION This lentiviral vector can be used to assay Cas9 activity..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8395)
AUTHORS Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I,
Sullender M, Ebert BL, Xavier RJ, Root DE
TITLE Rational design of highly active sgRNAs for CRISPR-Cas9-mediated
gene inactivation.
JOURNAL Nat Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026.
PUBMED 25184501
REFERENCE 2 (bases 1 to 8395)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8395)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8395
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 7..507
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
primer_bind complement(519..538)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 1015..1035
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
CDS 1155..1220
/codon_start=1
/product="2A peptide from foot-and-mouth disease virus
polyprotein"
/label=F2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="VKQTLNFDLLKLAGDVESNPGP"
CDS 1227..1943
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
misc_feature 1964..2552
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 2624..2857
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 2935..3069
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 3096..3231
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(3252..3270)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3280..3296)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3438..3893
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3919..4023
/label=AmpR promoter
CDS 4024..4881
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5055..5643
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5797..5814
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 5931..5952
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5967..5997
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6005..6021
/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 6029..6045
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 6066..6084
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 6112..6338
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 6339..6519
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6566..6691
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7184..7417
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7602..7646
/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"
promoter 7824..8064
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 8226..8343
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 8395
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"