Basic Vector Information
- Vector Name:
- pLX-sgRNA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7477 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Blasticidin
- Promoter:
- hPGK
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- cgggtttattacagggacagcag
- 3' Primer:
- taccagtcaatctttcacaaattttgt
pLX-sgRNA vector Vector Map
Plasmid Resuspension 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 ℃.
pLX-sgRNA vector Sequence
LOCUS 40924_29421 7477 bp DNA circular SYN 13-MAY-2021
DEFINITION Lentiviral expression of AAVS1-targeting sgRNA; insert can be
replaced with custom sgRNA (see protocol).
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7477)
AUTHORS Wang T, Wei JJ, Sabatini DM, Lander ES
TITLE Genetic screens in human cells using the CRISPR-Cas9 system.
JOURNAL Science. 2014 Jan 3;343(6166):80-4. doi: 10.1126/science.1246981.
Epub 2013 Dec 12.
PUBMED 24336569
REFERENCE 2 (bases 1 to 7477)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7477)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1126/science.1246981"; journalName: "Science"; date: "2014-01-3-
3"; volume: "343"; issue: "6166"; pages: "80-4"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7477
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 12..238
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 239..419
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 466..591
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1084..1317
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1502..1546
/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 1721..2221
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 2233..2628
/codon_start=1
/label=BSD
/note="blasticidin S deaminase"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 2688..2805
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2923..3163
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 3192..3267
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
protein_bind complement(3279..3303)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
misc_feature 3341..3929
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 4001..4234
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 4312..4446
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 4473..4608
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(4629..4647)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4657..4673)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 4815..5270
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 5296..5400
/label=AmpR promoter
CDS 5401..6258
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 6432..7020
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7174..7191
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 7308..7329
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 7344..7374
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7382..7398
/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 7406..7422
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 7443..7461
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
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