Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007469 | pLentiCRISPR-E | 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:
- pLentiCRISPR-E
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14849 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- SV40
- Cloning Method:
- Restriction Enzyme
pLentiCRISPR-E 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.
pLentiCRISPR-E vector Sequence
LOCUS V007469 14849 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V007469
VERSION V007469
KEYWORDS pLentiCRISPR-E
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14849)
AUTHORS Sikder RK, Ellithi M, Uzzo RN, Weader D, Metz AL, Behbahani A,
McKenzie ER, El-Deiry WS, Abbosh PH
TITLE Differential effects of clinically-relevant N- vs C-terminal
truncating CDKN1A mutations on cisplatin sensitivity in bladder
cancer.
JOURNAL Mol Cancer Res. 2020 Dec 3. pii: 1541-7786.MCR-19-1200. doi:
10.1158/1541-7786.MCR-19-1200.
PUBMED 33272936
REFERENCE 2 (bases 1 to 14849)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14849)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cancer
Res. 2020 Dec 3. pii: 1541-7786.MCR-19-1200. doi:
10.1158/1541-7786.MCR-19-1200."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14849
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(47..66)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 238..617
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 618..820
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2098..2142
/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 2291..2332
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2440..2557
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2608..2848
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 4738..4813
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 4875..5086
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 5111..9214
/label="eSpCas9(1.1)"
/note="Cas9 endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system, mutated to improve targeting
specificity (Slaymaker et al., 2016)"
CDS 9215..9262
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 9272..9328
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label="P2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 9329..9925
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label="PuroR"
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind 9822..9842
/label="Puro-F"
/note="Puromycin resistance gene, forward primer"
primer_bind complement(9994..10014)
/label="WPRE-R"
/note="WPRE, reverse primer"
CDS complement(10412..10423)
/label="Factor Xa site"
/note="Factor Xa recognition and cleavage site"
LTR 10601..10834
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 10866..11090
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 11136..11564
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 11578..11907
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 11955..12002
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 12021..12392
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 12525..12658
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(12695..12711)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12695..12711)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12708..12730)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 12719..12735
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/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(12743..12773)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(12788..12809)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12926..12943)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(13097..13685)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13859..14716)
/label="AmpR"
/note="beta-lactamase"
promoter complement(14717..14821)
/label="AmpR promoter"