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Plasmid Files

pDD162 (Peft-3::Cas9 + Empty sgRNA)

C. elegans plasmid for expressing nematode codon-optimized Cas9 together with a single guide RNA (sgRNA).

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pDD162 (Peft-3_Cas9 + Empty sgRNA).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Goldstein Lab / Addgene #47549
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BsrBI (7861) PfoI (7664) BstAPI (7533) NdeI (7529) PluTI (7480) SfoI (7478) NarI (7477) KasI (7476) SphI (7123) BsrGI (6999) AfeI (6821) SpeI (6564) KflI (5771) BbsI (5688) BlpI (5050) AflII (4242) BsrFI (505) BpmI (521) AhdI (590) AlwNI (1069) BsgI (1851) XbaI (2231) BstXI (2507) MscI (2511) BspQI - SapI (2796) NcoI (3714) BstEII (3965) pDD162 (Peft-3::Cas9 + Empty sgRNA) 8113 bp
BsrBI  (7861)
1 site
C C G C T C G G C G A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BsrBI will not always regenerate a BsrBI site.
BsrBI is typically used at 37°C, but can be used at temperatures up to 50°C.
PfoI  (7664)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
BstAPI  (7533)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
NdeI  (7529)
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.
PluTI  (7480)
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.
SfoI  (7478)
1 site
G G C G C C C C G C G G
NarI  (7477)
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.
KasI  (7476)
1 site
G G C G C C C C G C G G
SphI  (7123)
1 site
G C A T G C C G T A C G
BsrGI  (6999)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
AfeI  (6821)
1 site
A G C G C T T C G C G A
SpeI  (6564)
1 site
A C T A G T T G A T C A
KflI  (5771)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
BbsI  (5688)
1 site
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.
BlpI  (5050)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
AflII  (4242)
1 site
C T T A A G G A A T T C
BsrFI  (505)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
BpmI  (521)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
AhdI  (590)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
AlwNI  (1069)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
BsgI  (1851)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
XbaI  (2231)
1 site
T C T A G A A G A T C T
BstXI  (2507)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
MscI  (2511)
1 site
T G G C C A A C C G G T
BspQI  (2796)
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  (2796)
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.
NcoI  (3714)
1 site
C C A T G G G G T A C C
BstEII  (3965)
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.
Cas9
2241 .. 6497  =  4257 bp
1368 amino acids  =  158.4 kDa
4 segments
Product: S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns
Cas9
2241 .. 6497  =  4257 bp
1368 amino acids  =  158.4 kDa
4 segments
Product: S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns
Cas9
2241 .. 6497  =  4257 bp
1368 amino acids  =  158.4 kDa
4 segments
Product: S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns
Cas9
2241 .. 6497  =  4257 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   2241 .. 3284  =  1044 bp
   348 amino acids  =  39.8 kDa
Product: S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns
Cas9
2241 .. 6497  =  4257 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   3336 .. 4406  =  1071 bp
   357 amino acids  =  42.2 kDa
Product: S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns
Cas9
2241 .. 6497  =  4257 bp
1368 amino acids  =  158.4 kDa
   Segment 3:  
   4458 .. 5483  =  1026 bp
   342 amino acids  =  39.9 kDa
Product: S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns
Cas9
2241 .. 6497  =  4257 bp
1368 amino acids  =  158.4 kDa
   Segment 4:  
   5535 .. 6497  =  963 bp
   321 amino acids  =  36.6 kDa
Product: S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns
Cas9
2241 .. 6497  =  4257 bp
1368 amino acids  =  158.4 kDa
4 segments
Product: S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns
AmpR
7916 .. 663  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7916 .. 7984  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7916 .. 663  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7985 .. 663  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7916 .. 663  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
eft-3 promoter
1625 .. 2224  =  600 bp
promoter for the C. elegans elongation factor 1α gene (Mitrovich and Anderson, 2000)
eft-3 promoter
1625 .. 2224  =  600 bp
promoter for the C. elegans elongation factor 1α gene (Mitrovich and Anderson, 2000)
ori
834 .. 1422  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
834 .. 1422  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CeU6 promoter
6967 .. 7316  =  350 bp
promoter for the C. elegans R07E5.16 U6 snRNA gene (Dickinson et al., 2013)
CeU6 promoter
6967 .. 7316  =  350 bp
promoter for the C. elegans R07E5.16 U6 snRNA gene (Dickinson et al., 2013)
tbb-2 terminator
6575 .. 6898  =  324 bp
transcription terminator for the C. elegans tubulin beta-2 gene
tbb-2 terminator
6575 .. 6898  =  324 bp
transcription terminator for the C. elegans tubulin beta-2 gene
AmpR promoter
7811 .. 7915  =  105 bp
AmpR promoter
7811 .. 7915  =  105 bp
gRNA scaffold
7318 .. 7393  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
gRNA scaffold
7318 .. 7393  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
HA
6534 .. 6560  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin) epitope tag
HA
6534 .. 6560  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin) epitope tag
attB4
1604 .. 1624  =  21 bp
core recombination site for the Gateway® BP reaction
attB4
1604 .. 1624  =  21 bp
core recombination site for the Gateway® BP reaction
SV40 NLS
6513 .. 6533  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
6513 .. 6533  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
attB3
6902 .. 6922  =  21 bp
core recombination site for the Gateway® BP reaction
attB3
6902 .. 6922  =  21 bp
core recombination site for the Gateway® BP reaction
ORF:  7916 .. 663  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2241 .. 5516  =  3276 bp
ORF:  1091 amino acids  =  126.9 kDa
ORF:  5658 .. 6563  =  906 bp
ORF:  301 amino acids  =  34.3 kDa
ORF:  267 .. 533  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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