Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001621 | lentiSAMv2 | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The lentiSAMv2 features a lentiviral sgRNA cloning backbone with MS2 loops positioned at both the tetraloop and stemloop 2. It includes a dCas9-VP64 fusion protein and a blasticidin resistance marker for selection. Additionally, it is equipped with BsmBI restriction sites to facilitate the insertion of spacer sequences.
- Vector Name:
- lentiSAMv2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13500 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Blasticidin
- Copy Number:
- High Copy
- Promoter:
- U6 and EF1A
- Cloning Method:
- Restriction Enzyme
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
lentiSAMv2 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.
References
- Joung J, Konermann S, Gootenberg JS, et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening [published correction appears in Nat Protoc. 2019 Jul;14(7):2259. doi: 10.1038/s41596-018-0063-0]. Nat Protoc. 2017;12(4):828-863. doi:10.1038/nprot.2017.016
lentiSAMv2 vector Sequence
LOCUS V001621 13500 bp DNA circular SYN 21-DEC-2021
DEFINITION Exported.
ACCESSION V001621
VERSION V001621
KEYWORDS lentiSAMv2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 13500)
AUTHORS Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham
MD, Sanjana NE, Zhang F
TITLE Genome-scale CRISPR-Cas9 knockout and transcriptional activation
screening.
JOURNAL Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016.
Epub 2017 Mar 23.
PUBMED 28333914
REFERENCE 2 (bases 1 to 13500)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 13500)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nprot.2017.016"; journalName: "Nat Protoc"; date:
"2017-04"; volume: "12"; issue: "4"; pages: "828-863"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..13500
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 130..459
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 507..554
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 573..944
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 1077..1210
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(1247..1263)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(1271..1287)
/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)."
promoter complement(1295..1325)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(1340..1361)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1478..1495)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(1649..2237)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2411..3268)
/label="AmpR"
/note="beta-lactamase"
promoter complement(3269..3373)
/label="AmpR promoter"
primer_bind complement(3448..3467)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 3639..4018
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 4019..4221
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 4236..4416
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 4463..4588
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 5081..5314
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 5499..5543
/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 5692..5733
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 5841..5958
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 6008..6248
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 6294..6312
/label="MS2 stem loop"
/note="stem loop that binds the bacteriophage MS2 coat
protein"
misc_RNA 6364..6382
/label="MS2 stem loop"
/note="stem loop that binds the bacteriophage MS2 coat
protein"
promoter 6469..6680
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 6697..6744
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 6745..10845
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 10900..10920
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 10945..11094
/label="VP64"
/note="tetrameric repeat of the minimal activation domain
of herpes simplex virus VP16 (Beerli et al., 1998)"
CDS 11116..11169
/label="T2A"
/note="2A peptide from Thosea asigna virus capsid protein"
CDS 11170..11565
/label="BSD"
/note="blasticidin S deaminase"
misc_feature 11593..12181
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(12184..12200)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12185..12201)
/label="pBluescriptKS"
/note="For pBluescript vector"
LTR 12706..12886
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 12918..13142
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(13275..13294)
/label="F1ori-R"
/note="F1 origin, reverse primer"