Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012618 | pLKO5.sgRNA.EFS.tRFP | 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:
- pLKO5.sgRNA.EFS.tRFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7408 bp
- Type:
- Lentiviral, CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- U6
pLKO5.sgRNA.EFS.tRFP 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.
pLKO5.sgRNA.EFS.tRFP vector Sequence
LOCUS 40924_28371 7408 bp DNA circular SYN 13-MAY-2021
DEFINITION 3rd generation Lentiviral Vector for Sp sgRNA delivery without
SpCas9, tagRFP, EFS Promoter driven.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7408)
AUTHORS Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey
ME, Thielke A, Aster JC, Regev A, Ebert BL
TITLE Generation of mouse models of myeloid malignancy with combinatorial
genetic lesions using CRISPR-Cas9 genome editing.
JOURNAL Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
PUBMED 24952903
REFERENCE 2 (bases 1 to 7408)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7408)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7408
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 98..115
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 232..253
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 268..298
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 306..322
/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 330..346
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 367..385
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 413..639
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 640..820
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 867..992
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1485..1718
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1903..1947
/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 2125..2365
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 2392..2467
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 2515..2632
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2701..2912
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2938..3648
/codon_start=1
/label=TagRFP
/note="monomeric derivative of red fluorescent protein from
Entacmaea quadricolor (Merzlyak et al., 2007)"
/translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
ARYCDLPSKLGHKLN"
misc_feature 3673..4261
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 4333..4566
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 4644..4778
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 4805..4940
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(4961..4979)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4989..5005)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 5147..5602
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 5628..5732
/label=AmpR promoter
CDS 5733..6590
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 6764..7352
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"