Basic Vector Information
- Vector Name:
- pLX_311-Cas9
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12494 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Blasticidin
- Promoter:
- EF-1α
pLX_311-Cas9 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_311-Cas9 vector Sequence
LOCUS 40924_29501 12494 bp DNA circular SYN 13-MAY-2021
DEFINITION for Cas9 knockout, lentiviral expression of Cas9. Alternate plasmid
name: pXR111.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12494)
AUTHORS Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I,
Sullender M, Ebert BL, Xavier RJ, Root DE
TITLE Rational design of highly active sgRNAs for CRISPR-Cas9-mediated
gene inactivation.
JOURNAL Nat Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026.
PUBMED 25184501
REFERENCE 2 (bases 1 to 12494)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 12494)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..12494
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(30..47)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(201..789)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(963..1820)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(1821..1925)
/label=AmpR promoter
rep_origin complement(1951..2406)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 2533..2555
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 2547..2564
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 2548..2564
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 2574..2592
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin complement(2613..2748)
/direction=LEFT
/label=SV40 ori
/note="SV40 origin of replication"
polyA_signal complement(2775..2909)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
LTR complement(2987..3220)
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
misc_feature complement(3292..3880)
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(3931..3972)
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"
protein_bind 3981..4001
/label=attB2
/note="core recombination site for the Gateway(R) BP
reaction"
CDS complement(4007..4054)
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS complement(4055..8155)
/codon_start=1
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS complement(8180..8200)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS complement(8207..8272)
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
protein_bind complement(8275..8299)
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
promoter complement(8319..9497)
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
misc_feature complement(9635..9752)
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
CDS complement(9826..10221)
/codon_start=1
/label=BSD
/note="blasticidin S deaminase"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
promoter complement(10251..10461)
/label=SV40 promoter
/note="SV40 early promoter"
CDS complement(10692..10736)
/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"
misc_feature complement(10921..11154)
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature complement(11647..11772)
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
LTR complement(11819..11999)
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
promoter complement(12000..12226)
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
promoter complement(12254..12272)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(12293..12309)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12293..12309)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12306..12328)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12317..12333
/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(12341..12371)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(12386..12407)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
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