pCLIP-All-Nickase(D10A)-hCMV-tRFP

Lentiviral CRISPR vector encoding a guide RNA (gRNA), plus a Cas9 nickase mutant and the red fluorescent protein TurboRFP expressed from the human CMV promoter.

Sequence Author: transOMIC

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FLAG CMV enhancer HIV-1 Ψ BsmBI (2505) BsmBI (4390) polIII terminator pCLIP-All-Nickase(D10A)-hCMV-tRFP 13,367 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-v2 is an improved version of BsmBI.
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.
BsmBI-v2 is an improved version of BsmBI.
Cas9(D10A)
5123 .. 9226  =  4104 bp
1368 amino acids  =  158.4 kDa
3 segments
   Segment 1:  
   5123 .. 5149  =  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)
5123 .. 9226  =  4104 bp
1368 amino acids  =  158.4 kDa
3 segments
   Segment 2:  
   5150 .. 5152  =  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)
5123 .. 9226  =  4104 bp
1368 amino acids  =  158.4 kDa
3 segments
   Segment 3:  
   5153 .. 9226  =  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)
5123 .. 9226  =  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
nucleoplasmin NLS
9227 .. 9274  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
nucleoplasmin NLS
9227 .. 9274  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
FLAG
9275 .. 9298  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
9275 .. 9298  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
P2A
9308 .. 9364  =  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
9308 .. 9364  =  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.
TurboRFP
9365 .. 10,060  =  696 bp
231 amino acids  =  26.1 kDa
Product: red fluorescent protein from Entacmaea quadricolor
mammalian codon-optimized
TurboRFP
9365 .. 10,060  =  696 bp
231 amino acids  =  26.1 kDa
Product: red fluorescent protein from Entacmaea quadricolor
mammalian codon-optimized
Stuffer
2506 .. 4390  =  1885 bp
Stuffer
2506 .. 4390  =  1885 bp
AmpR
12,372 .. 13,232  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   12,372 .. 13,163  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
12,372 .. 13,232  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   13,164 .. 13,232  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
12,372 .. 13,232  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
WPRE
10,073 .. 10,661  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
WPRE
10,073 .. 10,661  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
ori
11,616 .. 12,201  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
11,616 .. 12,201  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
4479 .. 4858  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4479 .. 4858  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
11,198 .. 11,527  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
11,198 .. 11,527  =  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)
10,869 .. 11,102  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from HIV-1
3' LTR (ΔU3)
10,869 .. 11,102  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from HIV-1
CMV promoter
4859 .. 5062  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
4859 .. 5062  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
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
13,233 .. 13,337  =  105 bp
AmpR promoter
13,233 .. 13,337  =  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
polIII terminator
4467 .. 4472  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
4467 .. 4472  =  6 bp
RNA polymerase III transcription terminator
ORF:  12,502 .. 12,768  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1211 .. 2098  =  888 bp
ORF:  295 amino acids  =  33.8 kDa
ORF:  5123 .. 10,060  =  4938 bp
ORF:  1645 amino acids  =  189.4 kDa
ORF:  10,163 .. 10,690  =  528 bp
ORF:  175 amino acids  =  19.0 kDa
ORF:  10,176 .. 10,472  =  297 bp
ORF:  98 amino acids  =  10.2 kDa
ORF:  12,372 .. 13,232  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  13,129 .. 31  =  270 bp
ORF:  89 amino acids  =  10.4 kDa
ORF:  2684 .. 3079  =  396 bp
ORF:  131 amino acids  =  14.0 kDa
ORF:  5069 .. 7642  =  2574 bp
ORF:  857 amino acids  =  89.9 kDa
ORF:  1126 .. 1506  =  381 bp
ORF:  126 amino acids  =  13.3 kDa
ORF:  6472 .. 6765  =  294 bp
ORF:  97 amino acids  =  10.8 kDa
ORF:  7036 .. 7485  =  450 bp
ORF:  149 amino acids  =  17.3 kDa
ORF:  9466 .. 9711  =  246 bp
ORF:  81 amino acids  =  9.1 kDa
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