pCMV6-Entry

Mammalian expression and dual tagging vector that serves as an entry vector to transfer an ORF into a destination vector using the PrecisionShuttle™ system.

Sequence Author: OriGene

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BsrGI (301) AanI - PsiI (4847) DraIII (4722) SexAI * (4241) SfiI (4063) StuI (4014) BspDI * - ClaI * (3993) PluTI (3836) SfoI (3834) NarI (3833) KasI (3832) PflFI - Tth111I (3716) BstBI (3151) ApaLI (2227) SpeI (357) AseI (365) CMV enhancer NdeI (592) SnaBI (698) VP1.5 (forward primer) (839 .. 856) Eco53kI (924) SacI (926) T7 promoter EcoRI (979) SalI (985) AccI (986) BamHI (992) Acc65I (998) KpnI (1002) AsiSI - SgfI (1024) MreI - SgrAI (1026) AscI - BssHII (1030) HindIII (1044) NheI (1055) BmtI (1059) RsrII (1061) MluI (1067) BsiWI (1070) NotI (1076) PaeR7I - PspXI - XhoI (1082) EcoRV (1126) PmeI (1160) FseI (1170) SacII (1174) TspMI - XmaI (1200) SmaI (1202) AleI (1260) XL39 (reverse primer) (1266 .. 1285) AhdI (1316) BbsI (1383) BsgI (1444) BbvCI - Bpu10I (1486) BlpI (1530) BsmBI (1550) BstXI (1637) AgeI (1748) PflMI (1751) PciI (1913) pCMV6-Entry 4919 bp
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.
AanI  (4847)
1 site
T T A T A A A A T A T T
PsiI  (4847)
1 site
T T A T A A A A T A T T
DraIII  (4722)
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.
SexAI  (4241)
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  (4063)
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  (4014)
1 site
A G G C C T T C C G G A
BspDI  (3993)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (3993)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PluTI  (3836)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SfoI  (3834)
1 site
G G C G C C C C G C G G
NarI  (3833)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
KasI  (3832)
1 site
G G C G C C C C G C G G
PflFI  (3716)
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  (3716)
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.
BstBI  (3151)
1 site
T T C G A A A A G C T T
ApaLI  (2227)
1 site
G T G C A C C A C G T G
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
EcoRI  (979)
1 site
G A A T T C C T T A 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.
Acc65I  (998)
1 site
G G T A C C C C A T G G
KpnI  (1002)
1 site
G G T A C C C C A T G G
AsiSI  (1024)
1 site
G C G A T C G C C G C T A G C G
SgfI  (1024)
1 site
G C G A T C G C C G C T A G C G
MreI  (1026)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (1026)
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  (1030)
1 site
G G C G C G C C C C G C G C G G
BssHII  (1030)
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.
HindIII  (1044)
1 site
A A G C T T T T C G A A
NheI  (1055)
1 site
G C T A G C C G A T C G
BmtI  (1059)
1 site
G C T A G C C G A T C G
RsrII  (1061)
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  (1067)
1 site
A C G C G T T G C G C A
BsiWI  (1070)
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  (1076)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (1082)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1082)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1082)
1 site
C T C G A G G A G C T C
EcoRV  (1126)
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  (1160)
1 site
G T T T A A A C C A A A T T T G
FseI  (1170)
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.
SacII  (1174)
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  (1200)
1 site
C C C G G G G G G C C C
XmaI  (1200)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (1202)
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  (1260)
1 site
C A C N N N N G T G G T G N N N N C A C
AhdI  (1316)
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.
BbsI  (1383)
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.
BsgI  (1444)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BbvCI  (1486)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1486)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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.
BlpI  (1530)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
BsmBI  (1550)
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.
BsmBI-v2 is an improved version of BsmBI.
BstXI  (1637)
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.
AgeI  (1748)
1 site
A C C G G T T G G C C A
PflMI  (1751)
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.
PciI  (1913)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
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
1266 .. 1285  =  20 annealed bases
Tm  =  59°C
NeoR/KanR
3170 .. 3964  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
3170 .. 3964  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
hGH poly(A) signal
1203 .. 1825  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1203 .. 1825  =  623 bp
human growth hormone polyadenylation signal
ori
1974 .. 2562  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1974 .. 2562  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4489 .. 25  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4489 .. 25  =  456 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
3999 .. 4356  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
3999 .. 4356  =  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
MCS
979 .. 1087  =  109 bp
multiple cloning site
MCS
979 .. 1087  =  109 bp
multiple cloning site
AmpR promoter
4358 .. 4462  =  105 bp
AmpR promoter
4358 .. 4462  =  105 bp
HSV TK poly(A) signal
2891 .. 2938  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
2891 .. 2938  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
T7 promoter
952 .. 970  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
952 .. 970  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  2711 .. 2944  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
ORF:  3119 .. 3655  =  537 bp
ORF:  178 amino acids  =  19.9 kDa
ORF:  2700 .. 3149  =  450 bp
ORF:  149 amino acids  =  16.3 kDa
ORF:  3170 .. 3964  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  1567 .. 1791  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  3406 .. 3792  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
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