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pCLIP-Cas9-Nickase-TRE3G-Puro

Lentiviral CRISPR vector encoding a Cas9 nickase mutant and a puromycin resistance marker expressed from a Tet-responsive promoter.

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pCLIP-Cas9(D10a)-Nickase-TRE3G-Puro Sequence and MappCLIP-Cas9(D10a)-Nickase-TRE3G-Puro.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SspI (10,676) PvuI (10,242) FspI (10,094) DrdI (9090) AflIII - PciI (8982) SmaI (8962) TspMI - XmaI (8960) AvrII (8939) PmeI (8614) PsiI (8564) KpnI (8229) Acc65I (8225) Bsu36I (8221) SalI (8101) EcoRI (7479) BstEII (7013) RsrII (6995) BsiWI (6935) PflFI - Tth111I (6921) BamHI (6816) FLAG BsaBI * (6138) PmlI (5429) SpeI (41) CMV enhancer NdeI (276) SnaBI (382) NheI (405) BmtI (409) HIV-1 Ψ NruI * (867) BspQI - SapI (1136) FseI (1184) MfeI (1223) BbvCI (1458) AleI (1611) AbsI - PspXI (2238) XbaI (2623) AfeI (2630) PspOMI (3995) ApaI (3999) pCLIP-Cas9-Nickase-TRE3G-Puro 10,794 bp
SspI  (10,676)
1 site
A A T A T T T T A T A A
PvuI  (10,242)
1 site
C G A T C G G C T A G C
FspI  (10,094)
1 site
T G C G C A A C G C G T
DrdI  (9090)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
AflIII  (8982)
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  (8982)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SmaI  (8962)
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  (8960)
1 site
C C C G G G G G G C C C
XmaI  (8960)
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  (8939)
1 site
C C T A G G G G A T C C
PmeI  (8614)
1 site
G T T T A A A C C A A A T T T G
PsiI  (8564)
1 site
T T A T A A A A T A T T
KpnI  (8229)
1 site
G G T A C C C C A T G G
Acc65I  (8225)
1 site
G G T A C C C C A T G G
Bsu36I  (8221)
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.
SalI  (8101)
1 site
G T C G A C C A G C T G
EcoRI  (7479)
1 site
G A A T T C C T T A A G
BstEII  (7013)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
RsrII  (6995)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BsiWI  (6935)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
PflFI  (6921)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (6921)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BamHI  (6816)
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.
BsaBI  (6138)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
PmlI  (5429)
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
NheI  (405)
1 site
G C T A G C C G A T C G
BmtI  (409)
1 site
G C T A G C C G A T C G
NruI  (867)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
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.
FseI  (1184)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
MfeI  (1223)
1 site
C A A T T G G T T A A C
BbvCI  (1458)
1 site
C C T C A G C G G A G T C G
AleI  (1611)
1 site
C A C N N N N G T G G T G N N N N C A C
AbsI  (2238)
1 site
C C T C G A G G G G A G C T C C
PspXI  (2238)
1 site
V C T C G A G B B G A G C T C V
XbaI  (2623)
1 site
T C T A G A A G A T C T
AfeI  (2630)
1 site
A G C G C T T C G C G A
PspOMI  (3995)
1 site
G G G C C C C C C G G G
ApaI  (3999)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
Cas9(D10A)
2640 .. 6743  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   2640 .. 2666  =  27 bp
   9 amino acids  =  1.1 kDa
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
2640 .. 6743  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   2667 .. 2669  =  3 bp
   1 amino acid  =  89.1 Da
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
2640 .. 6743  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 3:  
   2670 .. 6743  =  4074 bp
   1358 amino acids  =  157.3 kDa
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
2640 .. 6743  =  4104 bp
1368 amino acids  =  158.4 kDa
3 segments
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
AmpR
9799 .. 10,659  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9799 .. 10,590  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9799 .. 10,659  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,591 .. 10,659  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9799 .. 10,659  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
6882 .. 7478  =  597 bp
198 amino acids  =  21.4 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
6882 .. 7478  =  597 bp
198 amino acids  =  21.4 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
7503 .. 8091  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7503 .. 8091  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
9043 .. 9628  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9043 .. 9628  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
TRE3G promoter
2244 .. 2622  =  379 bp
3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On® 3G
TRE3G promoter
2244 .. 2622  =  379 bp
3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On® 3G
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
8625 .. 8954  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
8625 .. 8954  =  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
1337 .. 1570  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1337 .. 1570  =  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)
8296 .. 8529  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
8296 .. 8529  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
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
2062 .. 2177  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
cPPT/CTS
2062 .. 2177  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
AmpR promoter
10,660 .. 10,764  =  105 bp
AmpR promoter
10,660 .. 10,764  =  105 bp
P2A
6825 .. 6881  =  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
6825 .. 6881  =  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
6744 .. 6791  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
6744 .. 6791  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
FLAG
6792 .. 6815  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
6792 .. 6815  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
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