Resources
Plasmid Files

gRNA_GFP-T1

Plasmid for guide RNA (gRNA) targeting of an integrated GFP gene in human cells using the GFP T1 target sequence.

To see this sequence with restriction sites, features, and translations, please download
 SnapGene or the free  SnapGene Viewer.

gRNA_GFP-T1.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Church Lab / Addgene #41819
Download Free Trial Get SnapGene Viewer


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) NdeI (541) SalI (751) AccI (752) XmnI (762) 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-T1 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
NdeI  (541)
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.
SalI  (751)
1 site
G T C G A C C A G C T G
AccI  (752)
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.
XmnI  (762)
1 site
G A A N N N N T T C C T T N N N N A A G
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
482 .. 722  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
482 .. 722  =  241 bp
RNA polymerase III promoter for human U6 snRNA
gRNA scaffold
378 .. 453  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
gRNA scaffold
378 .. 453  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
GFP T1
454 .. 473  =  20 bp
GFP gRNA Target 1 sequence
GFP T1
454 .. 473  =  20 bp
GFP gRNA Target 1 sequence
polIII terminator
372 .. 377  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
372 .. 377  =  6 bp
RNA polymerase III transcription terminator
ORF:  838 .. 1137  =  300 bp
ORF:  99 amino acids  =  11.1 kDa
ORF:  1864 .. 2250  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  2692 .. 3066  =  375 bp
ORF:  124 amino acids  =  13.8 kDa
ORF:  1692 .. 2486  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  2511 .. 3095  =  585 bp
ORF:  194 amino acids  =  21.4 kDa
ORF:  2001 .. 2534  =  534 bp
ORF:  177 amino acids  =  19.9 kDa
ORF:  2664 .. 2972  =  309 bp
ORF:  102 amino acids  =  10.9 kDa
Try SnapGene and create your own beautiful maps

Individual Sequences & Maps

SnapGene offers the fastest and easiest way to plan, visualize, and document your molecular biology procedures.

Priced accessibly so that everyone in your lab can have a license.

Learn More...

SnapGene Viewer is a versatile tool for creating and sharing richly annotated sequence files. It opens many common file formats.

Free! Because there should be no barriers to seeing your data.

Learn More...

The map, notes, and annotations on this page and in the sequence/map file are copyrighted material. This material may be used without restriction by academic, nonprofit, and governmental entities, except that the source must be cited as "www.snapgene.com/resources". Commercial entities must contact GSL Biotech LLC for permission and terms of use.

Copyright © 2018 GSL Biotech LLC | Site Map | Privacy | Legal Disclaimers   Subscribe to Our Newsletter