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Plasmid Files

pGuide-it-ZsGreen1 Vector (Linear)

CRISPR/Cas9 vector with a ZsGreen1 marker, for cloning and expression of a single guide RNA (sgRNA).

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pGuide-it-ZsGreen1 Vector (Linear) Sequence and MappGuide-it-ZsGreen1 Vector (Linear).dna
Map and Sequence File   
Sequence Author:  Clontech
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 End (8303) Guide-it™ Sequencing Primer 1 (8224 .. 8247) EcoRV (7738) BspDI - ClaI (7719) FLAG SV40 NLS BspEI (7661) AgeI (7607) MreI (7199) KflI (6928) PmlI (6340) PciI (6012) BstEII (5224) BstAPI (5086) AarI (3969) Start (0) polIII terminator DrdI (196) PvuI (1351) SspI (1785) XbaI (2095) PflFI - Tth111I (2585) EcoRI (2828) Eco53kI (2863) SacI (2865) StuI (2878) PaeR7I - PspXI - XhoI (2908) SnaBI (3253) BamHI (3509) MluI (3527) NheI (3533) BmtI (3537) NotI (3540) Acc65I (3584) KpnI (3588) SbfI (3798) pGuide-it-ZsGreen1 Vector (Linear) 8303 bp
End  (8303)
0 sites
EcoRV  (7738)
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.
BspDI  (7719)
1 site
A T C G A T T A G C T A
ClaI  (7719)
1 site
A T C G A T T A G C T A
BspEI  (7661)
1 site
T C C G G A A G G C C T
AgeI  (7607)
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  (7199)
1 site
C G C C G G C G G C G G C C G C
KflI  (6928)
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  (6340)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
PciI  (6012)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstEII  (5224)
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.
BstAPI  (5086)
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  (3969)
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.
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
PflFI  (2585)
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  (2585)
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.
EcoRI  (2828)
1 site
G A A T T C C T T A A G
Eco53kI  (2863)
1 site
G A G C T C C T C G A G
SacI  (2865)
1 site
G A G C T C C T C G A G
StuI  (2878)
1 site
A G G C C T T C C G G A
PaeR7I  (2908)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2908)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2908)
1 site
C T C G A G G A G C T C
SnaBI  (3253)
1 site
T A C G T A A T G C A T
BamHI  (3509)
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  (3527)
1 site
A C G C G T T G C G C A
NheI  (3533)
1 site
G C T A G C C G A T C G
BmtI  (3537)
1 site
G C T A G C C G A T C G
NotI  (3540)
1 site
G C G G C C G C C G C C G G C G
Acc65I  (3584)
1 site
G G T A C C C C A T G G
KpnI  (3588)
1 site
G G T A C C C C A T G G
SbfI  (3798)
1 site
C C T G C A G G G G A C G T C C
Guide-it™ Sequencing Primer 1
24-mer  /  38% GC
1 binding site
8224 .. 8247  =  24 annealed bases
Tm  =  56°C
Cas9
3551 .. 7654  =  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
3551 .. 7654  =  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
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
ZsGreen1
2126 .. 2821  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
2126 .. 2821  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
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
2974 .. 3277  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2974 .. 3277  =  304 bp
human cytomegalovirus immediate early enhancer
U6 promoter
8057 .. 8297  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
8057 .. 8297  =  241 bp
RNA polymerase III promoter for human U6 snRNA
CMV promoter
3278 .. 3481  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
3278 .. 3481  =  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
7855 .. 7936  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
7855 .. 7936  =  82 bp
SV40 polyadenylation signal
minimal CMV promoter
2862 .. 2899  =  38 bp
human cytomegalovirus (CMV) immediate early
promoter
minimal CMV promoter
2862 .. 2899  =  38 bp
human cytomegalovirus (CMV) immediate early
promoter
FLAG
7691 .. 7714  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
7691 .. 7714  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 NLS
7670 .. 7690  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7670 .. 7690  =  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
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