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pY010 (pcDNA3.1-hAsCpf1)

Zhang lab mammalian expression vector encoding a humanized version of the Acidaminococcus CRISPR-associated protein Cpf1.

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pY010 (pcDNA3.1-hAsCpf1).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Zhang Lab / Addgene #69982
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SgrDI (9462) SspI (9345) AhdI (8541) PciI (7651) BstBI (6982) RsrII (6816) BssHII (6697) PflFI - Tth111I (6418) SmaI (6113) TspMI - XmaI (6111) AvrII (6090) SexAI * (5857) DraIII (5567) BbsI (5253) XbaI (5027) PaeR7I - PspXI - XhoI (5021) NotI (5015) EcoRI (4988) BamHI (4898) MfeI (161) NruI (208) MluI (228) SpeI (249) SnaBI (590) Eco53kI (816) SacI (818) NheI (895) BmtI (899) AflII (908) HindIII (911) Acc65I (917) KpnI (921) SbfI (2190) BlpI (2219) AarI (2373) PmlI (3544) XcmI (4567) FseI (4878) pY010 (pcDNA3.1-hAsCpf1) 9464 bp
SgrDI  (9462)
1 site
C G T C G A C G G C A G C T G C
SspI  (9345)
1 site
A A T A T T T T A T A A
AhdI  (8541)
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.
PciI  (7651)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstBI  (6982)
1 site
T T C G A A A A G C T T
RsrII  (6816)
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.
BssHII  (6697)
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.
PflFI  (6418)
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  (6418)
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.
SmaI  (6113)
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  (6111)
1 site
C C C G G G G G G C C C
XmaI  (6111)
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.
AvrII  (6090)
1 site
C C T A G G G G A T C C
SexAI  (5857)
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.
DraIII  (5567)
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.
BbsI  (5253)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
XbaI  (5027)
1 site
T C T A G A A G A T C T
PaeR7I  (5021)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5021)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5021)
1 site
C T C G A G G A G C T C
NotI  (5015)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (4988)
1 site
G A A T T C C T T A A G
BamHI  (4898)
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.
MfeI  (161)
1 site
C A A T T G G T T A A C
NruI  (208)
1 site
T C G C G A A G C G C T
MluI  (228)
1 site
A C G C G T T G C G C A
SpeI  (249)
1 site
A C T A G T T G A T C A
SnaBI  (590)
1 site
T A C G T A A T G C A T
Eco53kI  (816)
1 site
G A G C T C C T C G A G
SacI  (818)
1 site
G A G C T C C T C G A G
NheI  (895)
1 site
G C T A G C C G A T C G
BmtI  (899)
1 site
G C T A G C C G A T C G
AflII  (908)
1 site
C T T A A G G A A T T C
HindIII  (911)
1 site
A A G C T T T T C G A A
Acc65I  (917)
1 site
G G T A C C C C A T G G
KpnI  (921)
1 site
G G T A C C C C A T