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Plasmid Files

pET28a/Cas9-Cys

Bacterial plasmid for regulated expression of S. pyogenes Cas9 with a C-terminal cysteine.

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pET28a Cas9-Cys Sequence and MappET28a Cas9-Cys.dna
Map and Sequence File   
Sequence Author:  Kim Lab / Addgene #53261
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 NruI (8566) BspDI - ClaI (8530) SmaI (8349) TspMI - XmaI (8347) AsiSI - PvuI (8225) PsiI (7651) PaeR7I - PspXI - XhoI (7119) Cys HA SV40 NLS AgeI (6993) MreI (6585) KflI (6314) PmlI (5726) PflFI - Tth111I (130) FspAI (895) PshAI (1132) HpaI (1471) EcoRV (1527) BssHII (1562) MluI (1973) T7 promoter XbaI (2761) RBS NcoI (2800) ATG 6xHis thrombin site NdeI (2860) NheI (2865) BmtI (2869) BamHI (2898) EcoRI (2904) Eco53kI (2912) SacI (2914) SalI (2917) Acc65I (2970) KpnI (2974) SbfI (3184) AarI (3355) BsrGI (4495) pET28a/Cas9-Cys 9546 bp
NruI  (8566)
1 site
T C G C G A A G C G C T
BspDI  (8530)
1 site
A T C G A T T A G C T A
ClaI  (8530)
1 site
A T C G A T T A G C T A
SmaI  (8349)
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  (8347)
1 site
C C C G G G G G G C C C
XmaI  (8347)
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.
AsiSI  (8225)
1 site
G C G A T C G C C G C T A G C G
PvuI  (8225)
1 site
C G A T C G G C T A G C
PsiI  (7651)
1 site
T T A T A A A A T A T T
PaeR7I  (7119)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (7119)
1 site
V C T C G A G B B G A G C T C V
XhoI  (7119)
1 site
C T C G A G G A G C T C
AgeI  (6993)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
MreI  (6585)
1 site
C G C C G G C G G C G G C C G C
KflI  (6314)
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.
PmlI  (5726)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
PflFI  (130)
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  (130)
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.
FspAI  (895)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (1132)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
HpaI  (1471)
1 site
G T T A A C C A A T T G
EcoRV  (1527)
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.
BssHII  (1562)
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.
MluI  (1973)
1 site
A C G C G T T G C G C A
XbaI  (2761)
1 site
T C T A G A A G A T C T
NcoI  (2800)
1 site
C C A T G G G G T A C C
NdeI  (2860)
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.
NheI  (2865)
1 site
G C T A G C C G A T C G
BmtI  (2869)
1 site
G C T A G C C G A T C G
BamHI  (2898)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
EcoRI  (2904)
1 site
G A A T T C C T T A A G
Eco53kI  (2912)
1 site
G A G C T C C T C G A G
SacI  (2914)
1 site
G A G C T C C T C G A G
SalI  (2917)
1 site
G T C G A C C A G C T G
Acc65I  (2970)
1 site
G G T A C C C C A T G G
KpnI  (2974)
1 site
G G T A C C C C A T G G
SbfI  (3184)
1 site
C C T G C A G G G G A C G T C C
AarI  (3355)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
BsrGI  (4495)
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.
Cas9
2937 .. 7040  =  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
2937 .. 7040  =  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
lacI
1246 .. 2328  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
1246 .. 2328  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
KanR
7840 .. 8655  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
7840 .. 8655  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
8777 .. 9365  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8777 .. 9365  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
7293 .. 7748  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
7293 .. 7748  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
246 .. 437  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
246 .. 437  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
2329 .. 2406  =  78 bp
lacI promoter
2329 .. 2406  =  78 bp
T7 terminator
7209 .. 7256  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
7209 .. 7256  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 tag (gene 10 leader)
2862 .. 2894  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene
10
promotes efficient translation in E. coli
T7 tag (gene 10 leader)
2862 .. 2894  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene
10
promotes efficient translation in E. coli
HA
7077 .. 7103  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
7077 .. 7103  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
lac operator
2734 .. 2758  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
2734 .. 2758  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
RBS
2773 .. 2795  =  23 bp
efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)
RBS
2773 .. 2795  =  23 bp
efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)
SV40 NLS
7056 .. 7076  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7056 .. 7076  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
2715 .. 2733  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2715 .. 2733  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
2814 .. 2831  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2814 .. 2831  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
thrombin site
2841 .. 2858  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
thrombin site
2841 .. 2858  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
ATG
2802 .. 2804  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
2802 .. 2804  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
Cys
7104 .. 7106  =  3 bp
1 amino acid  =  121.2 Da
Cys
7104 .. 7106  =  3 bp
1 amino acid  =  121.2 Da
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