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pMirTarget

Reporter construct with TurboRFP and neomycin resistance markers, for cloning 3' UTR sequences downstream of firefly luciferase to validate miRNA targets.

To see this sequence with restriction sites, features, and translations, please download
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pMirTarget.dna
Map and Sequence File:    Download    Open   
Sequence Author:  OriGene
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SpeI (7507) AseI (7501) NdeI (7274) SnaBI (7170) AccI (6881) SalI (6880) BamHI (6873) BglII (6857) PshAI (6214) PaeR7I - PspXI - XhoI (6136) EcoRV (6096) PmeI (6062) FseI (6056) SmaI (6020) TspMI - XmaI (6018) AhdI (5907) BsmBI (5668) BstXI (5589) BstBI (4065) XbaI (4058) NotI (4047) MluI (4038) RsrII (4030) AscI - BssHII (4022) AsiSI - SgfI (4015) AanI - PsiI (72) StuI (884) BspDI * - ClaI * (903) PflFI - Tth111I (1181) AclI (1766) PspOMI (1856) ApaI (1860) XmnI (1955) HindIII (1967) PmlI (2059) AarI (2082) Acc65I (2184) KpnI (2188) BmgBI (2286) MreI - SgrAI (2397) HpaI (3539) EcoRI (4003) pMirTarget 7868 bp
SpeI  (7507)
1 site
A C T A G T T G A T C A
AseI  (7501)
1 site
A T T A A T T A A T T A
NdeI  (7274)
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.
SnaBI  (7170)
1 site
T A C G T A A T G C A T
AccI  (6881)
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.
SalI  (6880)
1 site
G T C G A C C A G C T G
BamHI  (6873)
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.
BglII  (6857)
1 site
A G A T C T T C T A G A
PshAI  (6214)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
PaeR7I  (6136)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (6136)
1 site
V C T C G A G B B G A G C T C V
XhoI  (6136)
1 site
C T C G A G G A G C T C
EcoRV  (6096)
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.
PmeI  (6062)
1 site
G T T T A A A C C A A A T T T G
FseI  (6056)
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.
SmaI  (6020)
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  (6018)
1 site
C C C G G G G G G C C C
XmaI  (6018)
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.
AhdI  (5907)
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.
BsmBI  (5668)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BstXI  (5589)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BstBI  (4065)
1 site
T T C G A A A A G C T T
XbaI  (4058)
1 site
T C T A G A A G A T C T
NotI  (4047)
1 site
G C G G C C G C C G C C G G C G
MluI  (4038)
1 site
A C G C G T T G C G C A
RsrII  (4030)
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.
AscI  (4022)
1 site
G G C G C G C C C C G C G C G G
BssHII  (4022)
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.
AsiSI  (4015)
1 site
G C G A T C G C C G C T A G C G
SgfI  (4015)
1 site
G C G A T C G C C G C T A G C G
AanI  (72)
1 site
T T A T A A A A T A T T
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PsiI  (72)
1 site
T T A T A A A A T A T T