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Plasmid Files

Peft-3::cas9-SV40_NLS::tbb-2 3'UTR

C. elegans plasmid for expressing nematode codon-optimized Cas9 with a synthetic intron. Use together with PU6::klp‑12_sgRNA.

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Peft-3_cas9-SV40_NLS_tbb-2 3Peft-3_cas9-SV40_NLS_tbb-2 3'UTR.dna
Map and Sequence File   
Sequence Author:  Calarco Lab / Addgene #46168
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 NdeI (184) TsoI (7211) BsrFI (6892) AlwNI (6335) HindIII (5560) AfeI (5479) KflI (4465) BbsI (4382) BstAPI (185) KasI (235) NarI (236) SfoI (237) PluTI (239) EcoRI (396) BsgI (628) PsiI (764) BsrGI (1017) BstXI (1303) MscI (1307) NcoI (2459) BstEII (2710) PmeI (3120) Peft-3::cas9-SV40_NLS::tbb-2 3'UTR 7799 bp
NdeI  (184)
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.
TsoI  (7211)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
BsrFI  (6892)
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.
AlwNI  (6335)
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.
HindIII  (5560)
1 site
A A G C T T T T C G A A
AfeI  (5479)
1 site
A G C G C T T C G C G A
KflI  (4465)
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  (4382)
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.
BstAPI  (185)
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.
KasI  (235)
1 site
G G C G C C C C G C G G
NarI  (236)
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.
SfoI  (237)
1 site
G G C G C C C C G C G G
PluTI  (239)
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.
EcoRI  (396)
1 site
G A A T T C C T T A A G
BsgI  (628)
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).
PsiI  (764)
1 site
T T A T A A A A T A T T
BsrGI  (1017)
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.
BstXI  (1303)
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  (1307)
1 site
T G G C C A A C C G G T
NcoI  (2459)
1 site
C C A T G G G G T A C C
BstEII  (2710)
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.
PmeI  (3120)
1 site
G T T T A A A C C A A A T T T G
Cas9
1037 .. 5191  =  4155 bp
1368 amino acids  =  158.4 kDa
2 segments
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with a synthetic intron
Cas9
1037 .. 5191  =  4155 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   1037 .. 3112  =  2076 bp
   692 amino acids  =  80.5 kDa
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with a synthetic intron
Cas9
1037 .. 5191  =  4155 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   3164 .. 5191  =  2028 bp
   676 amino acids  =  78.0 kDa
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with a synthetic intron
Cas9
1037 .. 5191  =  4155 bp
1368 amino acids  =  158.4 kDa
2 segments
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with a synthetic intron
AmpR
6739 .. 7599  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6739 .. 7530  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6739 .. 7599  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7531 .. 7599  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6739 .. 7599  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
eft-3 promoter
402 .. 1001  =  600 bp
promoter for the C. elegans elongation factor 1α
gene (Mitrovich and Anderson, 2000)
eft-3 promoter
402 .. 1001  =  600 bp
promoter for the C. elegans elongation factor 1α
gene (Mitrovich and Anderson, 2000)
ori
5980 .. 6568  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5980 .. 6568  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
tbb-2 terminator
5233 .. 5556  =  324 bp
transcription terminator for the C. elegans tubulin
beta-2 gene
tbb-2 terminator
5233 .. 5556  =  324 bp
transcription terminator for the C. elegans tubulin
beta-2 gene
AmpR promoter
7600 .. 7704  =  105 bp
AmpR promoter
7600 .. 7704  =  105 bp
attB1
1010 .. 1034  =  25 bp
recombination site for the Gateway® BP reaction
attB1
1010 .. 1034  =  25 bp
recombination site for the Gateway® BP reaction
SV40 NLS
5204 .. 5224  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5204 .. 5224  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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