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Plasmid Files

pMirTarget

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

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pMirTarget Sequence and MappMirTarget.dna
Map and Sequence File   
Sequence Author:  OriGene
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 SpeI (7507) AseI (7501) CMV enhancer 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
PsiI  (72)
1 site
T T A T A A A A T A T T
StuI  (884)
1 site
A G G C C T T C C G G A
BspDI  (903)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (903)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PflFI  (1181)
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  (1181)
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.
AclI  (1766)
1 site
A A C G T T T T G C A A
PspOMI  (1856)
1 site
G G G C C C C C C G G G
ApaI  (1860)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
XmnI  (1955)
1 site
G A A N N N N T T C C T T N N N N A A G
HindIII  (1967)
1 site
A A G C T T T T C G A A
PmlI  (2059)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (2082)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
Acc65I  (2184)
1 site
G G T A C C C C A T G G
KpnI  (2188)
1 site
G G T A C C C C A T G G
BmgBI  (2286)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
MreI  (2397)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (2397)
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.
HpaI  (3539)
1 site
G T T A A C C A A T T G
EcoRI  (4003)
1 site
G A A T T C C T T A A G
luciferase
2334 .. 3986  =  1653 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic version of the luciferase gene
luciferase
2334 .. 3986  =  1653 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic version of the luciferase gene
NeoR/KanR
935 .. 1729  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
935 .. 1729  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
TurboRFP
6150 .. 6842  =  693 bp
231 amino acids  =  26.1 kDa
Product: red fluorescent protein from Entacmaea
quadricolor

mammalian codon-optimized
TurboRFP
6150 .. 6842  =  693 bp
231 amino acids  =  26.1 kDa
Product: red fluorescent protein from Entacmaea
quadricolor

mammalian codon-optimized
hGH poly(A) signal
5398 .. 6020  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
5398 .. 6020  =  623 bp
human growth hormone polyadenylation signal
ori
4661 .. 5249  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4661 .. 5249  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
1766 .. 2318  =  553 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
1766 .. 2318  =  553 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
f1 ori
7844 .. 431  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
7844 .. 431  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
7147 .. 7526  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
7147 .. 7526  =  380 bp
human cytomegalovirus immediate early enhancer
CMV promoter
6943 .. 7146  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
6943 .. 7146  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 ori
752 .. 886  =  135 bp
SV40 origin of replication
SV40 ori
752 .. 886  =  135 bp
SV40 origin of replication
AmpR promoter
458 .. 562  =  105 bp
AmpR promoter
458 .. 562  =  105 bp
MCS
4003 .. 4069  =  67 bp
multiple cloning site
MCS
4003 .. 4069  =  67 bp
multiple cloning site
HSV TK poly(A) signal
4285 .. 4332  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
4285 .. 4332  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
T7 promoter
6899 .. 6917  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
6899 .. 6917  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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