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Plasmid Files

pSQT834

Plasmid for mammalian cell expression of the Csy4 CRISPR-associated endoribonuclease and wild-type S. pyogenes Cas9.

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pSQT834.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Joung Lab / Addgene #53371
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SexAI * (9650) ScaI (9216) PvuI (9106) BsaI (8797) AhdI (8736) AvrII (7510) SfiI (7463) HindIII (7139) BstXI (6652) NotI (6592) PaeR7I - XhoI (6514) BmtI (6030) NheI (6026) FspAI (5334) SpeI (18) BlpI (1154) MreI - SgrAI (1311) XbaI (1623) Acc65I (1735) KpnI (1739) TspMI - XmaI (1746) SmaI (1748) AgeI (1756) BsmBI (2240) BstZ17I (2598) BmgBI (2603) BstEII (3098) BsiWI (3192) BsrGI (3942) EcoRV (4225) PshAI (4366) PvuII (4468) pSQT834 9654 bp
SexAI  (9650)
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.
ScaI  (9216)
1 site
A G T A C T T C A T G A
PvuI  (9106)
1 site
C G A T C G G C T A G C
BsaI  (8797)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (8736)
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.
AvrII  (7510)
1 site
C C T A G G G G A T C C
SfiI  (7463)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
HindIII  (7139)
1 site
A A G C T T T T C G A A
BstXI  (6652)
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.
NotI  (6592)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (6514)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (6514)
1 site
C T C G A G G A G C T C
BmtI  (6030)
1 site
G C T A G C C G A T C G
NheI  (6026)
1 site
G C T A G C C G A T C G
FspAI  (5334)
1 site
R T G C G C A Y Y A C G C G T R
SpeI  (18)
1 site
A C T A G T T G A T C A
BlpI  (1154)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
MreI  (1311)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (1311)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
XbaI  (1623)
1 site
T C T A G A A G A T C T
Acc65I  (1735)
1 site
G G T A C C C C A T G G
KpnI  (1739)
1 site
G G T A C C C C A T G G
TspMI  (1746)
1 site
C C C G G G G G G C C C
XmaI  (1746)
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.
SmaI  (1748)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AgeI  (1756)
1 site
A C C G G T T G G C C A
BsmBI  (2240)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BstZ17I  (2598)
1 site
G T A T A C C A T A T G
BmgBI  (2603)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
BstEII  (3098)
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.
BsiWI  (3192)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BsrGI  (3942)
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.
EcoRV  (4225)
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.
PshAI  (4366)
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.
PvuII  (4468)
1 site
C A G C T G G T C G A C
Cas9
2384 .. 6487  =  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
2384 .. 6487  =  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
chimeric intron
662 .. 1670  =  1009 bp
chimera between introns from chicken β-actin and rabbit β-globin
chimeric intron
662 .. 1670  =  1009 bp
chimera between introns from chicken β-actin and rabbit β-globin
AmpR
8663 .. 9523  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8663 .. 9454  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8663 .. 9523  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9455 .. 9523  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8663 .. 9523  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
7904 .. 8492  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7904 .. 8492  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
Csy4
1769 .. 2329  =  561 bp
187 amino acids  =  21.4 kDa
Product: CRISPR-associated endoribonuclease responsible for pre-crRNA processing in Pseudomonas aeruginosa (Haurwitz et al., 2010)
Csy4
1769 .. 2329  =  561 bp
187 amino acids  =  21.4 kDa
Product: CRISPR-associated endoribonuclease responsible for pre-crRNA processing in Pseudomonas aeruginosa (Haurwitz et al., 2010)
CMV enhancer
4 .. 383  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4 .. 383  =  380 bp
human cytomegalovirus immediate early enhancer
chicken β-actin promoter
386 .. 661  =  276 bp
chicken β-actin promoter
386 .. 661  =  276 bp
SV40 promoter
7330 .. 7525  =  196 bp
SV40 early promoter
SV40 promoter
7330 .. 7525  =  196 bp
SV40 early promoter
SV40 poly(A) signal
7531 .. 7665  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
7531 .. 7665  =  135 bp
SV40 polyadenylation signal
AmpR promoter
9524 .. 9628  =  105 bp
AmpR promoter
9524 .. 9628  =  105 bp
3xFLAG
6521 .. 6586  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags
3xFLAG
6521 .. 6586  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags
β-globin poly(A) signal
6741 .. 6796  =  56 bp
rabbit β-globin polyadenylation signal
β-globin poly(A) signal
6741 .. 6796  =  56 bp
rabbit β-globin polyadenylation signal
T2A
2330 .. 2383  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid protein
Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides.
T2A
2330 .. 2383  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid protein
Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides.
SV40 NLS
6494 .. 6514  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
6494 .. 6514  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 ori
7376 .. 7511  =  136 bp
SV40 origin of replication
SV40 ori
7376 .. 7511  =  136 bp
SV40 origin of replication
ORF:  1642 .. 2139  =  498 bp
ORF:  165 amino acids  =  16.8 kDa
ORF:  1355 .. 1627  =  273 bp
ORF:  90 amino acids  =  9.3 kDa
ORF:  1769 .. 6589  =  4821 bp
ORF:  1606 amino acids  =  185.5 kDa
ORF:  255 .. 734  =  480 bp
ORF:  159 amino acids  =  17.1 kDa
ORF:  8793 .. 9059  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  802 .. 1533  =  732 bp
ORF:  243 amino acids  =  26.3 kDa
ORF:  4348 .. 4635  =  288 bp
ORF:  95 amino acids  =  10.5 kDa
ORF:  5497 .. 5742  =  246 bp
ORF:  81 amino acids  =  9.1 kDa
ORF:  9420 .. 8  =  243 bp
ORF:  80 amino acids  =  9.3 kDa
ORF:  843 .. 1346  =  504 bp
ORF:  167 amino acids  =  16.7 kDa
ORF:  1706 .. 2014  =  309 bp
ORF:  102 amino acids  =  11.2 kDa
ORF:  6470 .. 6727  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
ORF:  8663 .. 9523  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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