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Plasmid Files

pCMV-MIR

Vector for expressing microRNA (miRNA) precursors using the CMV promoter, with a GFP reporter for transfection and a neomycin resistance marker.

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pCMV-MIR Sequence and MappCMV-MIR.dna
Map and Sequence File   
Sequence Author:  OriGene
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 AanI - PsiI (6136) SexAI * (5530) SfiI (5352) StuI (5303) BspDI * - ClaI * (5282) PflFI - Tth111I (5005) AclI (4418) KpnI (4002) Acc65I (3998) BmgBI (3900) PstI (3283) BsrGI (301) SpeI (357) AseI (365) CMV enhancer NdeI (592) SnaBI (698) VP1.5 (forward primer) (839 .. 856) Eco53kI (924) SacI (926) SalI (985) AccI (986) BamHI (992) AsiSI - SgfI (1023) MreI - SgrAI (1025) AscI - BssHII (1029) NheI (1054) BmtI (1058) RsrII (1060) MluI (1066) BsiWI (1069) NotI (1075) PaeR7I - PspXI - XhoI (1081) EcoRV (1125) PmeI (1159) SacII (1173) TspMI - XmaI (1199) SmaI (1201) AleI (1259) XL39 (reverse primer) (1265 .. 1284) AhdI (1315) BbvCI - Bpu10I (1485) BsmBI (1549) AgeI (1747) pCMV-MIR 6219 bp
AanI  (6136)
1 site
T T A T A A A A T A T T
PsiI  (6136)
1 site
T T A T A A A A T A T T
SexAI  (5530)
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.
SfiI  (5352)
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.
StuI  (5303)
1 site
A G G C C T T C C G G A
BspDI  (5282)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (5282)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PflFI  (5005)
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  (5005)
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  (4418)
1 site
A A C G T T T T G C A A
KpnI  (4002)
1 site
G G T A C C C C A T G G
Acc65I  (3998)
1 site
G G T A C C C C A T G G
BmgBI  (3900)
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.
PstI  (3283)
1 site
C T G C A G G A C G T C
BsrGI  (301)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
SpeI  (357)
1 site
A C T A G T T G A T C A
AseI  (365)
1 site
A T T A A T T A A T T A
NdeI  (592)
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  (698)
1 site
T A C G T A A T G C A T
Eco53kI  (924)
1 site
G A G C T C C T C G A G
SacI  (926)
1 site
G A G C T C C T C G A G
SalI  (985)
1 site
G T C G A C C A G C T G
AccI  (986)
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.
BamHI  (992)
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.
AsiSI  (1023)
1 site
G C G A T C G C C G C T A G C G
SgfI  (1023)
1 site
G C G A T C G C C G C T A G C G
MreI  (1025)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (1025)
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.
AscI  (1029)
1 site
G G C G C G C C C C G C G C G G
BssHII  (1029)
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.
NheI  (1054)
1 site
G C T A G C C G A T C G
BmtI  (1058)
1 site
G C T A G C C G A T C G
RsrII  (1060)
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.
MluI  (1066)
1 site
A C G C G T T G C G C A
BsiWI  (1069)
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.
NotI  (1075)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (1081)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1081)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1081)
1 site
C T C G A G G A G C T C
EcoRV  (1125)
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  (1159)
1 site
G T T T A A A C C A A A T T T G
SacII  (1173)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
TspMI  (1199)
1 site
C C C G G G G G G C C C
XmaI  (1199)
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.
SmaI  (1201)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AleI  (1259)
1 site
C A C N N N N G T G G T G N N N N C A C
AhdI  (1315)
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.
BbvCI  (1485)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1485)
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.
BsmBI  (1549)
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.
AgeI  (1747)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
VP1.5 (forward primer)
18-mer  /  44% GC
1 binding site
839 .. 856  =  18 annealed bases
Tm  =  51°C
XL39 (reverse primer)
20-mer  /  55% GC
1 binding site
1265 .. 1284  =  20 annealed bases
Tm  =  59°C
NeoR/KanR
4459 .. 5253  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
4459 .. 5253  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
TurboGFP
3159 .. 3857  =  699 bp
232 amino acids  =  25.7 kDa
Product: improved green fluorescent protein from
Pontellina plumata (Evdokimov et al., 2006)
mammalian codon-optimized
TurboGFP
3159 .. 3857  =  699 bp
232 amino acids  =  25.7 kDa
Product: improved green fluorescent protein from
Pontellina plumata (Evdokimov et al., 2006)
mammalian codon-optimized
hGH poly(A) signal
1202 .. 1824  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1202 .. 1824  =  623 bp
human growth hormone polyadenylation signal
ori
1973 .. 2561  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1973 .. 2561  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
3869 .. 4421  =  553 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
3869 .. 4421  =  553 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
f1 ori
5778 .. 6206  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5778 .. 6206  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
343 .. 722  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
343 .. 722  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
5288 .. 5645  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
5288 .. 5645  =  358 bp
SV40 enhancer and early promoter
CMV promoter
723 .. 926  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
723 .. 926  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
AmpR promoter
5647 .. 5751  =  105 bp
AmpR promoter
5647 .. 5751  =  105 bp
HSV TK poly(A) signal
2890 .. 2937  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
2890 .. 2937  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
Myc
1084 .. 1113  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
1084 .. 1113  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
FLAG
1132 .. 1155  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an
enterokinase cleavage site
FLAG
1132 .. 1155  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an
enterokinase cleavage site
T7 promoter
952 .. 970  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
952 .. 970  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
5302 .. 5437  =  136 bp
SV40 origin of replication
SV40 ori
5302 .. 5437  =  136 bp
SV40 origin of replication
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