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Plasmid Files

pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6

Mammalian vector for expressing a single guide RNA (sgRNA) together with S. pyogenes Cas9 plus mCherry and the Ad4 E4orf6 protein.

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pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6 Sequence and MappU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6.dna
Map and Sequence File   
Sequence Author:  Kuhn Lab / Addgene #64222
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 BbsI (251) BbsI (6651) BbsI (6499) BbsI (273) polIII terminator hybrid intron ATG 3xFLAG SV40 NLS pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6 10,249 bp
BbsI  (251)
4 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BbsI  (6651)
4 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BbsI  (6499)
4 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BbsI  (273)
4 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
Cas9
1377 .. 5477  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
1377 .. 5477  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
E4orf6
6366 .. 7265  =  900 bp
299 amino acids  =  34.7 kDa
Product: adenovirus 4 protein that mediates the
ubiquitination and proteasomal degradation of DNA
ligase IV (Baker et al., 2007)
E4orf6
6366 .. 7265  =  900 bp
299 amino acids  =  34.7 kDa
Product: adenovirus 4 protein that mediates the
ubiquitination and proteasomal degradation of DNA
ligase IV (Baker et al., 2007)
AmpR
8569 .. 9429  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8569 .. 8637  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8569 .. 9429  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8638 .. 9429  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8569 .. 9429  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
mCherry
5589 .. 6296  =  708 bp
236 amino acids  =  26.7 kDa
Product: monomeric derivative of DsRed fluorescent
protein (Shaner et al., 2004)
mammalian codon-optimized
mCherry
5589 .. 6296  =  708 bp
236 amino acids  =  26.7 kDa
Product: monomeric derivative of DsRed fluorescent
protein (Shaner et al., 2004)
mammalian codon-optimized
ori
9600 .. 10,188  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9600 .. 10,188  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
7727 .. 8182  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
7727 .. 8182  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
446 .. 731  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
CMV enhancer
446 .. 731  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
chicken β-actin promoter
733 .. 1010  =  278 bp
chicken β-actin promoter
733 .. 1010  =  278 bp
U6 promoter
7 .. 247  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
7 .. 247  =  241 bp
RNA polymerase III promoter for human U6 snRNA
hybrid intron
1011 .. 1239  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
1011 .. 1239  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
7296 .. 7503  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
7296 .. 7503  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
7512 .. 7652  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
7512 .. 7652  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
8464 .. 8568  =  105 bp
AmpR promoter
8464 .. 8568  =  105 bp
gRNA scaffold
274 .. 349  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
274 .. 349  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
3xFLAG
1260 .. 1325  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
1260 .. 1325  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
P2A
6303 .. 6359  =  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
6303 .. 6359  =  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.
T2A
5529 .. 5582  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
5529 .. 5582  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
nucleoplasmin NLS
5478 .. 5525  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
5478 .. 5525  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
1332 .. 1352  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1332 .. 1352  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
350 .. 355  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
350 .. 355  =  6 bp
RNA polymerase III transcription terminator
ATG
1257 .. 1259  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1257 .. 1259  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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