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Plasmid Files

gRNA_GFP-T2

Plasmid for guide RNA (gRNA) targeting of an integrated GFP gene in human cells using the GFP T2 target sequence.

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gRNA_GFP-T2 Sequence and MapgRNA_GFP-T2.dna
Map and Sequence File   
Sequence Author:  Church Lab / Addgene #41820
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 PciI (3848) DraIII (3045) FseI (2968) SexAI * (2855) SgrAI (2806) SmaI (2782) TspMI - XmaI (2780) AatII (2738) ZraI (2736) BssHII - MauBI (2728) SfiI (2692) TsoI (2349) RsrII (2336) BsrDI (2053) HindIII (276) Eco53kI (290) SacI (292) SpeI (300) XmnI (365) SalI (372) AccI (373) NdeI (584) GFP T2 polIII terminator PstI (807) EcoRV (810) NotI (825) PaeR7I - PspXI - XhoI (831) PspOMI (849) ApaI (853) PvuI (1004) BsiWI - SnaBI (1034) BsaI (1259) XcmI (1636) BclI * (1661) PflFI - Tth111I (1938) gRNA_GFP-T2 3973 bp
PciI  (3848)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DraIII  (3045)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
FseI  (2968)
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  (2855)
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.
SgrAI  (2806)
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.
SmaI  (2782)
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  (2780)
1 site
C C C G G G G G G C C C
XmaI  (2780)
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.
AatII  (2738)
1 site
G A C G T C C T G C A G
ZraI  (2736)
1 site
G A C G T C C T G C A G
BssHII  (2728)
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.
MauBI  (2728)
1 site
C G C G C G C G G C G C G C G C
SfiI  (2692)
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.
TsoI  (2349)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
RsrII  (2336)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BsrDI  (2053)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
HindIII  (276)
1 site
A A G C T T T T C G A A
Eco53kI  (290)
1 site
G A G C T C C T C G A G
SacI  (292)
1 site
G A G C T C C T C G A G
SpeI  (300)
1 site
A C T A G T T G A T C A
XmnI  (365)
1 site
G A A N N N N T T C C T T N N N N A A G
SalI  (372)
1 site
G T C G A C C A G C T G
AccI  (373)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
NdeI  (584)
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.
PstI  (807)
1 site
C T G C A G G A C G T C
EcoRV  (810)
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.
NotI  (825)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (831)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (831)
1 site
V C T C G A G B B G A G C T C V
XhoI  (831)
1 site
C T C G A G G A G C T C
PspOMI  (849)
1 site
G G G C C C C C C G G G
ApaI  (853)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PvuI  (1004)
1 site
C G A T C G G C T A G C
BsiWI  (1034)
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.
SnaBI  (1034)
1 site
T A C G T A A T G C A T
BsaI  (1259)
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.
XcmI  (1636)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
BclI  (1661)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
PflFI  (1938)
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  (1938)
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.
NeoR/KanR
1692 .. 2486  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
1692 .. 2486  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
3204 .. 3792  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3204 .. 3792  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
BleoR
2692 .. 3066  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
2692 .. 3066  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
U6 promoter
406 .. 646  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
406 .. 646  =  241 bp
RNA polymerase III promoter for human U6 snRNA
gRNA scaffold
675 .. 750  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
675 .. 750  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
GFP T2
655 .. 674  =  20 bp
GFP gRNA Target 2 sequence
GFP T2
655 .. 674  =  20 bp
GFP gRNA Target 2 sequence
polIII terminator
751 .. 756  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
751 .. 756  =  6 bp
RNA polymerase III transcription terminator
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