Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V018104 | LentiGuide-zeo | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
LentiGuide-zeo is a lentiviral CRISPR vector expressing a S. pyogenes chimeric RNA with a U6 promoter-driven customizable sgRNA for genome targeting and a zeocin resistance marker for mammalian cell selection. It enables precise Cas9-mediated editing and stable cell population enrichment via zeocin-based antibiotic selection.
- Vector Name:
- LentiGuide-zeo
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9964 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Selection Marker:
- Zeocin
- Copy Number:
- High Copy
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
LentiGuide-zeo 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.
LentiGuide-zeo vector Sequence
LOCUS Exported 9964 bp ds-DNA circular SYN 11-JUN-2020
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM recombinant plasmid
REFERENCE 1 (bases 1 to 9964)
AUTHORS Sascha
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9964
/organism="recombinant plasmid"
/mol_type="other DNA"
protein_bind 107..128
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 143..173
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 181..197
/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)."
primer_bind 205..221
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 242..260
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 286..514
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 515..695
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 742..867
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1360..1593
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter 2000..2240
/label=U6 Promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 4130..4205
/label=gRNA scaffold
/note="guide RNA scaffold for the CRISPR/Cas9 system"
misc_feature 4261..4378
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 4517..5695
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 4748..5686
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind complement(5695..5713)
/label=Vector.REV
primer_bind 5699..5734
/label=Fragment.FOR (2)
CDS 5714..6088
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
primer_bind complement(6071..6103)
/label=Fragment.REV (2)
primer_bind 6089..6112
/label=Vector.FOR
misc_feature 6104..6692
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(6575..6586)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 6764..6997
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 7075..7196
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 7236..7371
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(7392..7410)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(7420..7436)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 7578..8033
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 8059..8163
/gene="bla"
/label=AmpR promoter
CDS 8164..9024
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 9195..9783
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"