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Plasmid Files

pMJ920

Doudna lab plasmid for mammalian cell expression of human codon-optimized Cas9 together with GFP.

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pMJ920.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Doudna Lab / Addgene #42234
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MfeI (161) SgrDI (10,355) PvuI (9804) PciI (8541) BstZ17I (8162) BstBI (7872) RsrII (7706) BssHII (7587) PflFI - Tth111I (7308) SmaI (7003) TspMI - XmaI (7001) AvrII (6980) StuI (6979) SexAI * (6747) BbsI (6142) XbaI (5894) NruI (208) MluI (228) SpeI (249) NdeI (484) SnaBI (590) HindIII (889) Acc65I (895) KpnI (899) PasI (2704) EcoRV (2788) PmlI (3724) KflI (4312) pMJ920 10,357 bp
MfeI  (161)
1 site
C A A T T G G T T A A C
SgrDI  (10,355)
1 site
C G T C G A C G G C A G C T G C
PvuI  (9804)
1 site
C G A T C G G C T A G C
PciI  (8541)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (8162)
1 site
G T A T A C C A T A T G
BstBI  (7872)
1 site
T T C G A A A A G C T T
RsrII  (7706)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BssHII  (7587)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
PflFI  (7308)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (7308)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
SmaI  (7003)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (7001)
1 site
C C C G G G G G G C C C
XmaI  (7001)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI recognition sequence.
Full cleavage with XmaI may require a long incubation.
AvrII  (6980)
1 site
C C T A G G G G A T C C
StuI  (6979)
1 site
A G G C C T T C C G G A
SexAI  (6747)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BbsI  (6142)
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.
XbaI  (5894)
1 site
T C T A G A A G A T C T
NruI  (208)
1 site
T C G C G A A G C G C T
MluI  (228)
1 site
A C G C G T T G C G C A
SpeI  (249)
1 site
A C T A G T T G A T C A
NdeI  (484)
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.
SnaBI  (590)
1 site
T A C G T A A T G C A T
HindIII  (889)
1 site
A A G C T T T T C G A A
Acc65I  (895)
1 site
G G T A C C C C A T G G
KpnI  (899)
1 site
G G T A C C C C A T G G
PasI  (2704)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
EcoRV  (2788)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
PmlI  (3724)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
KflI  (4312)
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.
Cas9
935 .. 5038  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
935 .. 5038  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
9361 .. 10,221  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9361 .. 10,152  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
9361 .. 10,221  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,153 .. 10,221  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
9361 .. 10,221  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
7062 .. 7856  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
7062 .. 7856  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
mEGFP
5174 .. 5890  =  717 bp
238 amino acids  =  26.7 kDa
   Segment 1:  1a  
   5174 .. 5176  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)
mammalian codon-optimized
mEGFP
5174 .. 5890  =  717 bp
238 amino acids  =  26.7 kDa
   Segment 2:  
   5177 .. 5890  =  714 bp
   237 amino acids  =  26.6 kDa
Product: enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)
mammalian codon-optimized
mEGFP
5174 .. 5890  =  717 bp
238 amino acids  =  26.7 kDa
2 segments
Product: enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)
mammalian codon-optimized
ori
8602 .. 9190  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
8602 .. 9190  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
6223 .. 6651  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
6223 .. 6651  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
6665 .. 6995  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
6665 .. 6995  =  331 bp
SV40 enhancer and early promoter
bGH poly(A) signal
5953 .. 6177  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5953 .. 6177  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
8030 .. 8151  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
8030 .. 8151  =  122 bp
SV40 polyadenylation signal
AmpR promoter
10,222 .. 10,326  =  105 bp
AmpR promoter
10,222 .. 10,326  =  105 bp
HA
5042 .. 5068  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin) epitope tag
HA
5042 .. 5068  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin) epitope tag
SV40 NLS
5087 .. 5107  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5087 .. 5107  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5114 .. 5134  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5114 .. 5134  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
6846 .. 6981  =  136 bp
SV40 origin of replication
SV40 ori
6846 .. 6981  =  136 bp
SV40 origin of replication
ORF:  7234 .. 7620  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  935 .. 5890  =  4956 bp
ORF:  1651 amino acids  =  189.8 kDa
ORF:  9491 .. 9757  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  6174 .. 6440  =  267 bp
ORF:  88 amino acids  =  9.3 kDa
ORF:  7062 .. 7856  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  875 .. 3454  =  2580 bp
ORF:  859 amino acids  =  90.3 kDa
ORF:  2284 .. 2577  =  294 bp
ORF:  97 amino acids  =  10.7 kDa
ORF:  2848 .. 3297  =  450 bp
ORF:  149 amino acids  =  17.3 kDa
ORF:  5062 .. 5313  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  9361 .. 10,221  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  7371 .. 7907  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
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