pcDNA3.1 NT-GFP

Vector for expressing N-terminally GFP-tagged proteins in mammalian cells.

Sequence Author: Thermo Fisher (Invitrogen)

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SgrDI (6158) SspI (6041) ScaI (5717) PvuI (5607) AhdI (5237) PciI (4344) BsmI (3913) PfoI (3768) BssHII (3390) PflFI - Tth111I (3111) PluTI (2996) BglII (12) Bpu10I (180) NruI (208) CMV enhancer SnaBI (590) HindIII (1028) BsrGI (1183) AgeI (1633) NheI (1639) BmtI (1643) Acc65I (1645) KpnI (1649) EcoRI (1680) EcoRV (1692) NotI (1707) XbaI (1719) EcoO109I - PspOMI (1725) ApaI (1729) PmeI (1734) BbsI (1945) DraIII (2259) CsiI - SexAI * (2550) BseRI (2779) StuI (2782) AvrII (2783) TspMI - XmaI (2804) SmaI (2806) BsaBI * (2852) KasI (2992) NarI (2993) SfoI (2994) pcDNA™3.1/NT-GFP 6160 bp
SgrDI  (6158)
1 site
C G T C G A C G G C A G C T G C
SspI  (6041)
1 site
A A T A T T T T A T A A
ScaI  (5717)
1 site
A G T A C T T C A T G A
PvuI  (5607)
1 site
C G A T C G G C T A G C
AhdI  (5237)
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.
PciI  (4344)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BsmI  (3913)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
PfoI  (3768)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
BssHII  (3390)
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.
PflFI  (3111)
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  (3111)
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.
PluTI  (2996)
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.
BglII  (12)
1 site
A G A T C T T C T A G A
Bpu10I  (180)
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.
NruI  (208)
1 site
T C G C G A A G C G C T
SnaBI  (590)
1 site
T A C G T A A T G C A T
HindIII  (1028)
1 site
A A G C T T T T C G A A
BsrGI  (1183)
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.
AgeI  (1633)
1 site
A C C G G T T G G C C A
NheI  (1639)
1 site
G C T A G C C G A T C G
BmtI  (1643)
1 site
G C T A G C C G A T C G
Acc65I  (1645)
1 site
G G T A C C C C A T G G
KpnI  (1649)
1 site
G G T A C C C C A T G G
EcoRI  (1680)
1 site
G A A T T C C T T A A G
EcoRV  (1692)
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.
NotI  (1707)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1719)
1 site
T C T A G A A G A T C T
EcoO109I  (1725)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
PspOMI  (1725)
1 site
G G G C C C C C C G G G
ApaI  (1729)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PmeI  (1734)
1 site
G T T T A A A C C A A A T T T G
BbsI  (1945)
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.
DraIII  (2259)
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.
CsiI  (2550)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (2550)
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.
BseRI  (2779)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
StuI  (2782)
1 site
A G G C C T T C C G G A
AvrII  (2783)
1 site
C C T A G G G G A T C C
TspMI  (2804)
1 site
C C C G G G G G G C C C
XmaI  (2804)
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  (2806)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BsaBI  (2852)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
KasI  (2992)
1 site
G G C G C C C C G C G G
NarI  (2993)
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.
SfoI  (2994)
1 site
G G C G C C C C G C G G
AmpR
5164 .. 6024  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   5164 .. 5955  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5164 .. 6024  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   5956 .. 6024  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5164 .. 6024  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
2865 .. 3659  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
2865 .. 3659  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
Cycle 3 GFP
905 .. 1621  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 1:  
   905 .. 907  =  3 bp
   1 amino acid  =  149.2 Da
Product: Cycle 3 GFP (Crameri et al., 1996)
mammalian codon-optimized
Cycle 3 GFP
905 .. 1621  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 2:  1a  
   908 .. 910  =  3 bp
   1 amino acid  =  89.1 Da
Product: Cycle 3 GFP (Crameri et al., 1996)
mammalian codon-optimized
Cycle 3 GFP
905 .. 1621  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 3:  
   911 .. 1621  =  711 bp
   237 amino acids  =  26.7 kDa
Product: Cycle 3 GFP (Crameri et al., 1996)
mammalian codon-optimized
Cycle 3 GFP
905 .. 1621  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
Product: Cycle 3 GFP (Crameri et al., 1996)
mammalian codon-optimized
ori
4405 .. 4993  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4405 .. 4993  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2026 .. 2454  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2026 .. 2454  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
2468 .. 2798  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
2468 .. 2798  =  331 bp
SV40 enhancer and early promoter
bGH poly(A) signal
1756 .. 1980  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
1756 .. 1980  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
3833 .. 3954  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3833 .. 3954  =  122 bp
SV40 polyadenylation signal
MCS
1633 .. 1738  =  106 bp
multiple cloning site
MCS
1633 .. 1738  =  106 bp
multiple cloning site
AmpR promoter
6025 .. 6129  =  105 bp
AmpR promoter
6025 .. 6129  =  105 bp
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
2649 .. 2784  =  136 bp
SV40 origin of replication
SV40 ori
2649 .. 2784  =  136 bp
SV40 origin of replication
ORF:  3037 .. 3423  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  905 .. 1723  =  819 bp
ORF:  272 amino acids  =  30.0 kDa
ORF:  5294 .. 5560  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1977 .. 2243  =  267 bp
ORF:  88 amino acids  =  9.3 kDa
ORF:  2865 .. 3659  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  5164 .. 6024  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3174 .. 3710  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
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