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Plasmid Files

pX600

Zhang lab plasmid for expression of human codon-optimized Staphylococcus aureus Cas9. Also known as pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA.

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pX600 Sequence and MappX600.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #61592
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 AAV2 ITR EcoRI (6711) AflII (6706) BamHI (6621) FseI (6601) SexAI * (5347) BstAPI (4997) BspEI (4939) HindIII (4769) PpuMI * (4053) BspDI * - ClaI * (3499) PciI (1) SacII (960) BpmI (964) FspI (1116) PvuI (1264) PsiI (2164) EagI - NotI (2740) AbsI - PaeR7I - PspXI - XhoI (2747) HincII (2757) SpeI (2772) CMV enhancer NdeI (3007) SnaBI (3113) AgeI (3354) ATG pX600 7101 bp
EcoRI  (6711)
1 site
G A A T T C C T T A A G
AflII  (6706)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BamHI  (6621)
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.
FseI  (6601)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
SexAI  (5347)
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.
BstAPI  (4997)
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.
BspEI  (4939)
1 site
T C C G G A A G G C C T
HindIII  (4769)
1 site
A A G C T T T T C G A A
PpuMI  (4053)
1 site
R G G W C C Y Y C C W G G R
* Blocked by Dcm methylation.
Sticky ends from different PpuMI sites may not be compatible.
BspDI  (3499)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (3499)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PciI  (1)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SacII  (960)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BpmI  (964)
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.
FspI  (1116)
1 site
T G C G C A A C G C G T
PvuI  (1264)
1 site
C G A T C G G C T A G C
PsiI  (2164)
1 site
T T A T A A A A T A T T
EagI  (2740)
1 site
C G G C C G G C C G G C
NotI  (2740)
1 site
G C G G C C G C C G C C G G C G
AbsI  (2747)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (2747)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2747)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2747)
1 site
C T C G A G G A G C T C
HincII  (2757)
1 site
G T Y R A C C A R Y T G
SpeI  (2772)
1 site
A C T A G T T G A T C A
NdeI  (3007)
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  (3113)
1 site
T A C G T A A T G C A T
AgeI  (3354)
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.
SaCas9
3417 .. 6572  =  3156 bp
1052 amino acids  =  123.8 kDa
Product: Cas9 (Csn1) endonuclease from the
Staphylococcus aureus Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
SaCas9
3417 .. 6572  =  3156 bp
1052 amino acids  =  123.8 kDa
Product: Cas9 (Csn1) endonuclease from the
Staphylococcus aureus Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
821 .. 1681  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   821 .. 1612  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
821 .. 1681  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1613 .. 1681  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
821 .. 1681  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
62 .. 650  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
62 .. 650  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
2068 .. 2523  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2068 .. 2523  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
2758 .. 3137  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2758 .. 3137  =  380 bp
human cytomegalovirus immediate early enhancer
bGH poly(A) signal
6741 .. 6948  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
6741 .. 6948  =  208 bp
bovine growth hormone polyadenylation signal
CMV promoter
3138 .. 3341  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
3138 .. 3341  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
AAV2 ITR
2598 .. 2738  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
2598 .. 2738  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
6961 .. 7101  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
6961 .. 7101  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
1682 .. 1786  =  105 bp
AmpR promoter
1682 .. 1786  =  105 bp
3xHA
6627 .. 6707  =  81 bp
27 amino acids  =  3.3 kDa
Product: three tandem HA epitope tags
3xHA
6627 .. 6707  =  81 bp
27 amino acids  =  3.3 kDa
Product: three tandem HA epitope tags
nucleoplasmin NLS
6573 .. 6620  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
6573 .. 6620  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
3372 .. 3392  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
3372 .. 3392  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
3366 .. 3368  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3366 .. 3368  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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