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Plasmid Files

pCLIP-Cas9-Nuclease-EFS-Blast

Lentiviral CRISPR vector encoding Cas9 and a blasticidin resistance marker expressed from the EF-1α core promoter.

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pCLIP-Cas9-Nuclease-EFS-Blast Sequence and MappCLIP-Cas9-Nuclease-EFS-Blast.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SspI (10,377) FspI (9795) AflIII - PciI (8683) SmaI (8663) TspMI - XmaI (8661) AvrII (8640) SexAI * (8407) PmeI (8315) PsiI (8265) KpnI (7930) Acc65I (7926) Bsu36I (7922) PaeR7I - XhoI (7808) SalI (7802) BspDI - ClaI (7794) SacII (7707) EcoRI (7180) BamHI (6715) FLAG PmlI (5328) SpeI (41) CMV enhancer NdeI (276) SnaBI (382) KasI (671) NarI (672) SfoI (673) PluTI (675) BssHII (745) HIV-1 Ψ BspQI - SapI (1136) NotI (1178) BbvCI (1454) AleI (1607) AgeI (2516) XbaI (2522) AfeI (2529) PspOMI (3894) ApaI (3898) PasI (4308) pCLIP-Cas9-Nuclease-EFS-Blast 10,495 bp
SspI  (10,377)
1 site
A A T A T T T T A T A A
FspI  (9795)
1 site
T G C G C A A C G C G T
AflIII  (8683)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (8683)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SmaI  (8663)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (8661)
1 site
C C C G G G G G G C C C
XmaI  (8661)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
AvrII  (8640)
1 site
C C T A G G G G A T C C
SexAI  (8407)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
PmeI  (8315)
1 site
G T T T A A A C C A A A T T T G
PsiI  (8265)
1 site
T T A T A A A A T A T T
KpnI  (7930)
1 site
G G T A C C C C A T G G
Acc65I  (7926)
1 site
G G T A C C C C A T G G
Bsu36I  (7922)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
PaeR7I  (7808)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (7808)
1 site
C T C G A G G A G C T C
SalI  (7802)
1 site
G T C G A C C A G C T G
BspDI  (7794)
1 site
A T C G A T T A G C T A
ClaI  (7794)
1 site
A T C G A T T A G C T A
SacII  (7707)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
EcoRI  (7180)
1 site
G A A T T C C T T A A G
BamHI  (6715)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
PmlI  (5328)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
SpeI  (41)
1 site
A C T A G T T G A T C A
NdeI  (276)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
SnaBI  (382)
1 site
T A C G T A A T G C A T
KasI  (671)
1 site
G G C G C C C C G C G G
NarI  (672)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (673)
1 site
G G C G C C C C G C G G
PluTI  (675)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
BssHII  (745)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BspQI  (1136)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (1136)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
NotI  (1178)
1 site
G C G G C C G C C G C C G G C G
BbvCI  (1454)
1 site
C C T C A G C G G A G T C G
AleI  (1607)
1 site
C A C N N N N G T G G T G N N N N C A C
AgeI  (2516)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
XbaI  (2522)
1 site
T C T A G A A G A T C T
AfeI  (2529)
1 site
A G C G C T T C G C G A
PspOMI  (3894)
1 site
G G G C C C C C C G G G
ApaI  (3898)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PasI  (4308)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
Cas9
2539 .. 6642  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
2539 .. 6642  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
9500 .. 10,360  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9500 .. 10,291  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9500 .. 10,360  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,292 .. 10,360  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9500 .. 10,360  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
WPRE
7204 .. 7792  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7204 .. 7792  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
8744 .. 9329  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8744 .. 9329  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
BSD
6781 .. 7179  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
6781 .. 7179  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
8326 .. 8655  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
8326 .. 8655  =  330 bp
SV40 enhancer and early promoter
5' LTR (truncated)
411 .. 668  =  258 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
411 .. 668  =  258 bp
truncated 5' long terminal repeat (LTR) from HIV-1
RRE
1333 .. 1566  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1333 .. 1566  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
3' LTR (ΔU3)
7997 .. 8230  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
7997 .. 8230  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
EF-1α core promoter
2303 .. 2514  =  212 bp
core promoter for human elongation factor EF-1α
EF-1α core promoter
2303 .. 2514  =  212 bp
core promoter for human elongation factor EF-1α
HIV-1 Ψ
715 .. 840  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
715 .. 840  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
2093 .. 2210  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2093 .. 2210  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
10,361 .. 10,465  =  105 bp
AmpR promoter
10,361 .. 10,465  =  105 bp
P2A
6724 .. 6780  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1
polyprotein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
P2A
6724 .. 6780  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1
polyprotein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
nucleoplasmin NLS
6643 .. 6690  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
6643 .. 6690  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
FLAG
6691 .. 6714  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
6691 .. 6714  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
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