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Plasmid Files

pCLIP-All-Nickase(D10A)-EFS-Puro

Lentiviral CRISPR vector encoding a guide RNA (gRNA), plus a Cas9 nickase mutant and a puromycin resistance marker expressed from the EF-1α core promoter.

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pCLIP-All-Nickase(D10A)-EFS-Puro Sequence and MappCLIP-All-Nickase(D10A)-EFS-Puro.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 FLAG CMV enhancer HIV-1 Ψ BsmBI (2505) BsmBI (4390) polIII terminator pCLIP-All-Nickase(D10A)-EFS-Puro 12,916 bp
BsmBI  (2505)
2 sites
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI  (4390)
2 sites
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
Cas9(D10A)
4770 .. 8873  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   4770 .. 4796  =  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)
4770 .. 8873  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   4797 .. 4799  =  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)
4770 .. 8873  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 3:  
   4800 .. 8873  =  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)
4770 .. 8873  =  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
Stuffer
2506 .. 4390  =  1885 bp
Stuffer
2506 .. 4390  =  1885 bp
AmpR
11,921 .. 12,781  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   11,921 .. 12,712  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
11,921 .. 12,781  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   12,713 .. 12,781  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
11,921 .. 12,781  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
9012 .. 9608  =  597 bp
198 amino acids  =  21.4 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
9012 .. 9608  =  597 bp
198 amino acids  =  21.4 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
9622 .. 10,210  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
9622 .. 10,210  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
11,165 .. 11,750  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
11,165 .. 11,750  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
10,747 .. 11,076  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
10,747 .. 11,076  =  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
U6 promoter
2261 .. 2501  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
2261 .. 2501  =  241 bp
RNA polymerase III promoter for human U6 snRNA
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)
10,418 .. 10,651  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
10,418 .. 10,651  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
EF-1α core promoter
4528 .. 4739  =  212 bp
core promoter for human elongation factor EF-1α
EF-1α core promoter
4528 .. 4739  =  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
12,782 .. 12,886  =  105 bp
AmpR promoter
12,782 .. 12,886  =  105 bp
gRNA scaffold
4391 .. 4466  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
4391 .. 4466  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
P2A
8955 .. 9011  =  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
8955 .. 9011  =  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
8874 .. 8921  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
8874 .. 8921  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
FLAG
8922 .. 8945  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
8922 .. 8945  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
polIII terminator
4467 .. 4472  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
4467 .. 4472  =  6 bp
RNA polymerase III transcription terminator
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