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Plasmid Files

pMB66

C. elegans plasmid for expression of nematode codon-optimized Cas9 fused to monomeric EGFP and nuclear localization signals.

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pMB66 Sequence and MappMB66.dna
Map and Sequence File   
Sequence Author:  Boxem Lab / Addgene #47946
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 PsiI (102) AhdI (8088) AlwNI (7611) SpeI (6759) NdeI (6726) AfeI (6630) EcoRI (6378) PacI (5745) BsgI (5550) BfuAI - BspMI (5426) KflI (4692) BsaAI - DraIII (230) NgoMIV (331) NaeI (333) Acc65I (653) KpnI (657) NsiI (913) ATG SV40 NLS BstEII (2886) BbsI (4609) pMB66 9000 bp
PsiI  (102)
1 site
T T A T A A A A T A T T
AhdI  (8088)
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  (7611)
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.
SpeI  (6759)
1 site
A C T A G T T G A T C A
NdeI  (6726)
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.
AfeI  (6630)
1 site
A G C G C T T C G C G A
EcoRI  (6378)
1 site
G A A T T C C T T A A G
PacI  (5745)
1 site
T T A A T T A A A A T T A A T T
BsgI  (5550)
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).
BfuAI  (5426)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (5426)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
KflI  (4692)
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.
BsaAI  (230)
1 site
Y A C G T R R T G C A Y
DraIII  (230)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
NgoMIV  (331)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (333)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
Acc65I  (653)
1 site
G G T A C C C C A T G G
KpnI  (657)
1 site
G G T A C C C C A T G G
NsiI  (913)
1 site
A T G C A T T A C G T A
BstEII  (2886)
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.
BbsI  (4609)
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.
Cas9
1165 .. 5418  =  4254 bp
1367 amino acids  =  158.3 kDa
4 segments
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with synthetic introns
Cas9
1165 .. 5418  =  4254 bp
1367 amino acids  =  158.3 kDa
4 segments
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with synthetic introns
Cas9
1165 .. 5418  =  4254 bp
1367 amino acids  =  158.3 kDa
4 segments
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with synthetic introns
Cas9
1165 .. 5418  =  4254 bp
1367 amino acids  =  158.3 kDa
   Segment 1:  
   1165 .. 2205  =  1041 bp
   347 amino acids  =  39.7 kDa
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with synthetic introns
Cas9
1165 .. 5418  =  4254 bp
1367 amino acids  =  158.3 kDa
   Segment 2:  
   2257 .. 3243  =  987 bp
   329 amino acids  =  38.9 kDa
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with synthetic introns
Cas9
1165 .. 5418  =  4254 bp
1367 amino acids  =  158.3 kDa
   Segment 3:  
   3295 .. 4356  =  1062 bp
   354 amino acids  =  41.2 kDa
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with synthetic introns
Cas9
1165 .. 5418  =  4254 bp
1367 amino acids  =  158.3 kDa
   Segment 4:  
   4408 .. 5418  =  1011 bp
   337 amino acids  =  38.5 kDa
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with synthetic introns
Cas9
1165 .. 5418  =  4254 bp
1367 amino acids  =  158.3 kDa
4 segments
Product: S. pyogenes Cas9, codon optimized for C.
elegans
with synthetic introns
mEGFP
5425 .. 6293  =  869 bp
238 amino acids  =  26.9 kDa
4 segments
Product: monomeric enhanced GFP, optimized with
synthetic introns for expression in C. elegans
mEGFP
5425 .. 6293  =  869 bp
238 amino acids  =  26.9 kDa
4 segments
Product: monomeric enhanced GFP, optimized with
synthetic introns for expression in C. elegans
mEGFP
5425 .. 6293  =  869 bp
238 amino acids  =  26.9 kDa
4 segments
Product: monomeric enhanced GFP, optimized with
synthetic introns for expression in C. elegans
mEGFP
5425 .. 6293  =  869 bp
238 amino acids  =  26.9 kDa
   Segment 1:  
   5425 .. 5727  =  303 bp
   101 amino acids  =  11.4 kDa
Product: monomeric enhanced GFP, optimized with
synthetic introns for expression in C. elegans
mEGFP
5425 .. 6293  =  869 bp
238 amino acids  =  26.9 kDa
   Segment 2:  
   5781 .. 5855  =  75 bp
   25 amino acids  =  2.9 kDa
Product: monomeric enhanced GFP, optimized with
synthetic introns for expression in C. elegans
mEGFP
5425 .. 6293  =  869 bp
238 amino acids  =  26.9 kDa
   Segment 3:  
   5907 .. 6026  =  120 bp
   40 amino acids  =  4.7 kDa
Product: monomeric enhanced GFP, optimized with
synthetic introns for expression in C. elegans
mEGFP
5425 .. 6293  =  869 bp
238 amino acids  =  26.9 kDa
   Segment 4:  
   6078 .. 6293  =  216 bp
   72 amino acids  =  8.1 kDa
Product: monomeric enhanced GFP, optimized with
synthetic introns for expression in C. elegans
mEGFP
5425 .. 6293  =  869 bp
238 amino acids  =  26.9 kDa
4 segments
Product: monomeric enhanced GFP, optimized with
synthetic introns for expression in C. elegans
AmpR
8015 .. 8875  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8015 .. 8806  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8015 .. 8875  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8807 .. 8875  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8015 .. 8875  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
7256 .. 7844  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7256 .. 7844  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6 .. 461  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6 .. 461  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
tbb-2 terminator
6384 .. 6707  =  324 bp
transcription terminator for the C. elegans tubulin
beta-2 gene
tbb-2 terminator
6384 .. 6707  =  324 bp
transcription terminator for the C. elegans tubulin
beta-2 gene
hsp16-48 promoter
730 .. 1034  =  305 bp
promoter for the C. elegans heat shock protein
16-48 gene (Russnak and Candido, 1985)
hsp16-48 promoter
730 .. 1034  =  305 bp
promoter for the C. elegans heat shock protein
16-48 gene (Russnak and Candido, 1985)
AmpR promoter
8876 .. 8980  =  105 bp
AmpR promoter
8876 .. 8980  =  105 bp
EGL-13 NLS
6300 .. 6374  =  75 bp
25 amino acids  =  2.9 kDa
Product: nuclear localization signal from the C.
elegans
EGL-13 transcription factor (Lyssenko et al.,
2007)
EGL-13 NLS
6300 .. 6374  =  75 bp
25 amino acids  =  2.9 kDa
Product: nuclear localization signal from the C.
elegans
EGL-13 transcription factor (Lyssenko et al.,
2007)
3xFLAG
1048 .. 1113  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
1048 .. 1113  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
SV40 NLS
1120 .. 1140  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1120 .. 1140  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
1045 .. 1047  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1045 .. 1047  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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