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Plasmid Files

pGuide-it-tdTomato Vector (Linear)

Linearized CRISPR/Cas9 vector with a tdTomato marker, for cloning and expression of a single guide RNA (sgRNA).

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pGuide-it-tdTomato Vector (Linear).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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End (9038) Guide-it™ Sequencing Primer 1 (8959 .. 8982) EcoRV (8473) SalI (8465) BspDI - ClaI (8454) BspEI (8396) AgeI (8342) MreI - SgrAI (7934) KflI (7663) PmlI (7075) PciI (6747) BstAPI (5821) AarI (4704) KpnI (4323) Start (0) DrdI (196) PvuI (1351) SspI (1785) XbaI (2095) Bsu36I (2108) EcoRI (3563) Eco53kI (3598) SacI (3600) PaeR7I - PspXI - XhoI (3643) SnaBI (3988) BamHI (4244) MluI (4262) NheI (4268) BmtI (4272) NotI (4275) Acc65I (4319) pGuide-it-tdTomato Vector (Linear) 9038 bp
End  (9038)
0 sites
EcoRV  (8473)
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.
SalI  (8465)
1 site
G T C G A C C A G C T G
BspDI  (8454)
1 site
A T C G A T T A G C T A
ClaI  (8454)
1 site
A T C G A T T A G C T A
BspEI  (8396)
1 site
T C C G G A A G G C C T
AgeI  (8342)
1 site
A C C G G T T G G C C A
MreI  (7934)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (7934)
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.
KflI  (7663)
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  (7075)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
PciI  (6747)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstAPI  (5821)
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.
AarI  (4704)
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.
KpnI  (4323)
1 site
G G T A C C C C A T G G
Start  (0)
0 sites
DrdI  (196)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
PvuI  (1351)
1 site
C G A T C G G C T A G C
SspI  (1785)
1 site
A A T A T T T T A T A A
XbaI  (2095)
1 site
T C T A G A A G A T C T
Bsu36I  (2108)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
EcoRI  (3563)
1 site
G A A T T C C T T A A G
Eco53kI  (3598)
1 site
G A G C T C C T C G A G
SacI  (3600)
1 site
G A G C T C C T C G A G
PaeR7I  (3643)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (3643)
1 site
V C T C G A G B B G A G C T C V
XhoI  (3643)
1 site
C T C G A G G A G C T C
SnaBI  (3988)
1 site
T A C G T A A T G C A T
BamHI  (4244)
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.
MluI  (4262)
1 site
A C G C G T T G C G C A
NheI  (4268)
1 site
G C T A G C C G A T C G
BmtI  (4272)
1 site
G C T A G C C G A T C G
NotI  (4275)
1 site
G C G G C C G C C G C C G G C G
Acc65I  (4319)
1 site
G G T A C C C C A T G G
Guide-it™ Sequencing Primer 1
24-mer  /  38% GC
1 binding site
8959 .. 8982  =  24 annealed bases
Tm  =  56°C
Cas9
4286 .. 8389  =  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
4286 .. 8389  =  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
tdTomato
2126 .. 3556  =  1431 bp
476 amino acids  =  54.2 kDa
Product: tandem dimeric (pseudo-monomeric) derivative of DsRed (Shaner et al., 2004)
mammalian codon-optimized
tdTomato
2126 .. 3556  =  1431 bp
476 amino acids  =  54.2 kDa
Product: tandem dimeric (pseudo-monomeric) derivative of DsRed (Shaner et al., 2004)
mammalian codon-optimized
AmpR
908 .. 1768  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   908 .. 1699  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
908 .. 1768  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   1700 .. 1768  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
908 .. 1768  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
149 .. 737  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
149 .. 737  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
3709 .. 4012  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
3709 .. 4012  =  304 bp
human cytomegalovirus immediate early enhancer
U6 promoter
8792 .. 9032  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
8792 .. 9032  =  241 bp
RNA polymerase III promoter for human U6 snRNA
CMV promoter
4013 .. 4216  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
4013 .. 4216  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
AmpR promoter
1769 .. 1873  =  105 bp
AmpR promoter
1769 .. 1873  =  105 bp
gRNA scaffold
1 .. 86  =  86 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
gRNA scaffold
1 .. 86  =  86 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
SV40 poly(A) signal
1906 .. 1987  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1906 .. 1987  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
8590 .. 8671  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
8590 .. 8671  =  82 bp
SV40 polyadenylation signal
minimal CMV promoter
3597 .. 3634  =  38 bp
human cytomegalovirus (CMV) immediate early promoter
minimal CMV promoter
3597 .. 3634  =  38 bp
human cytomegalovirus (CMV) immediate early promoter
FLAG
8426 .. 8449  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
8426 .. 8449  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
SV40 NLS
8405 .. 8425  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
8405 .. 8425  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
polIII terminator
87 .. 92  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
87 .. 92  =  6 bp
RNA polymerase III transcription terminator
ORF:  2078 .. 3658  =  1581 bp
ORF:  526 amino acids  =  52.7 kDa
ORF:  4286 .. 8452  =  4167 bp
ORF:  1388 amino acids  =  160.7 kDa
ORF:  1038 .. 1304  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  5635 .. 5928  =  294 bp
ORF:  97 amino acids  =  10.9 kDa
ORF:  6250 .. 6648  =  399 bp
ORF:  132 amino acids  =  15.6 kDa
ORF:  2109 .. 3641  =  1533 bp
ORF:  510 amino acids  =  56.9 kDa
ORF:  908 .. 1768  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2126 .. 3556  =  1431 bp
ORF:  476 amino acids  =  54.2 kDa
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