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pY016 (pcDNA3.1-hLbCpf1)

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

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pY016 (pcDNA3.1-hLbCpf1).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Zhang Lab / Addgene #69988
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SgrDI (9225) SspI (9108) PciI (7414) BstBI (6745) RsrII (6579) PflFI - Tth111I (6181) SmaI (5876) TspMI - XmaI (5874) AvrII (5853) SexAI * (5620) BbsI (5016) ApaI (4800) PspOMI (4796) XbaI (4790) PaeR7I - PspXI - XhoI (4784) NotI (4778) EcoRI (4751) BamHI (4661) MfeI (161) Bpu10I (180) NruI (208) MluI (228) SpeI (249) SnaBI (590) Eco53kI (816) SacI (818) NheI (895) BmtI (899) AflII (908) HindIII (911) Acc65I (917) KpnI (921) Bsu36I (977) BstEII (2056) PflMI (2191) AscI (3169) AleI (4364) FseI (4641) pY016 (pcDNA3.1-hLbCpf1) 9227 bp
SgrDI  (9225)
1 site
C G T C G A C G G C A G C T G C
SspI  (9108)
1 site
A A T A T T T T A T A A
PciI  (7414)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstBI  (6745)
1 site
T T C G A A A A G C T T
RsrII  (6579)
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.
PflFI  (6181)
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  (6181)
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  (5876)
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  (5874)
1 site
C C C G G G G G G C C C
XmaI  (5874)
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  (5853)
1 site
C C T A G G G G A T C C
SexAI  (5620)
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.
BbsI  (5016)
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.
ApaI  (4800)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (4796)
1 site
G G G C C C C C C G G G
XbaI  (4790)
1 site
T C T A G A A G A T C T
PaeR7I  (4784)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4784)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4784)
1 site
C T C G A G G A G C T C
NotI  (4778)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (4751)
1 site
G A A T T C C T T A A G
BamHI  (4661)
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
Bpu10I  (180)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
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 G G
Bsu36I  (977)
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.
BstEII  (2056)
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.
PflMI  (2191)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
AscI  (3169)
1 site
G G C G C G C C C C G C G C G G
AleI  (4364)
1 site