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Plasmid Files

pGen2.1

CMV promoter-based mammalian expression vector encoding an N-terminal FLAG tag.

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pGen2.1 Sequence and MappGen2.1.dna
Map and Sequence File   
Sequence Author:  GenScript
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 SgrDI (5772) ScaI (5331) PvuI (5221) PciI (3961) BstZ17I (3582) BsmI (3530) PfoI (3385) BstBI (3292) RsrII (3126) BssHII (3007) BglII (12) MfeI (161) NruI (208) MluI (228) SpeI (249) NdeI (484) SnaBI (590) T7 primer (863 .. 882) NheI (895) BmtI (899) PmeI (904) AflII (908) HindIII (911) ATG FLAG NotI (976) XbaI (994) BamHI (1000) PspOMI (1031) ApaI (1035) SP6 promoter AleI (1136) Reverse sequencing primer (1173 .. 1192) BsgI (1320) BbvCI (1362) BlpI (1406) BsmBI (1426) BstXI (1513) BbsI (1563) DraIII (1877) SexAI * (2167) BseRI (2396) StuI (2399) AvrII (2400) TspMI - XmaI (2421) SmaI (2423) BsaBI * (2469) KasI (2609) NarI (2610) SfoI (2611) PluTI (2613) PstI (2663) PflFI - Tth111I (2728) pGen2.1 5774 bp
SgrDI  (5772)
1 site
C G T C G A C G G C A G C T G C
ScaI  (5331)
1 site
A G T A C T T C A T G A
PvuI  (5221)
1 site
C G A T C G G C T A G C
PciI  (3961)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (3582)
1 site
G T A T A C C A T A T G
BsmI  (3530)
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  (3385)
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.
BstBI  (3292)
1 site
T T C G A A A A G C T T
RsrII  (3126)
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.
BssHII  (3007)
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.
BglII  (12)
1 site
A G A T C T T C T A G A
MfeI  (161)
1 site
C A A T T G G T T A A C
NruI  (208)
1 site
T C G C G A A G C G C T
MluI  (228)
1 site
A C G C G T T G C G C A
SpeI  (249)
1 site
A C T A G T T G A T C A
NdeI  (484)
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  (590)
1 site
T A C G T A A T G C A T
NheI  (895)
1 site
G C T A G C C G A T C G
BmtI  (899)
1 site
G C T A G C C G A T C G
PmeI  (904)
1 site
G T T T A A A C C A A A T T T G
AflII  (908)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
HindIII  (911)
1 site
A A G C T T T T C G A A
NotI  (976)
1 site
G C G G C C G C C G C C G G C G
XbaI  (994)
1 site
T C T A G A A G A T C T
BamHI  (1000)
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.
PspOMI  (1031)
1 site
G G G C C C C C C G G G
ApaI  (1035)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AleI  (1136)
1 site
C A C N N N N G T G G T G N N N N C A C
BsgI  (1320)
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  (1362)
1 site
C C T C A G C G G A G T C G
BlpI  (1406)
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  (1426)
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  (1513)
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.
BbsI  (1563)
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  (1877)
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  (2167)
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  (2396)
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  (2399)
1 site
A G G C C T T C C G G A
AvrII  (2400)
1 site
C C T A G G G G A T C C
TspMI  (2421)
1 site
C C C G G G G G G C C C
XmaI  (2421)
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  (2423)
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  (2469)
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  (2609)
1 site
G G C G C C C C G C G G
NarI  (2610)
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  (2611)
1 site
G G C G C C C C G C G G
PluTI  (2613)
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.
PstI  (2663)
1 site
C T G C A G G A C G T C
PflFI  (2728)
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  (2728)
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.
T7 primer
20-mer  /  40% GC
1 binding site
863 .. 882  =  20 annealed bases
Tm  =  49°C
Reverse sequencing primer
20-mer  /  40% GC
1 binding site
1173 .. 1192  =  20 annealed bases
Tm  =  51°C
AmpR
4778 .. 5638  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4778 .. 5569  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4778 .. 5638  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5570 .. 5638  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4778 .. 5638  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
2482 .. 3276  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
2482 .. 3276  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
4022 .. 4607  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4022 .. 4607  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hGH poly(A) signal
1079 .. 1555  =  477 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1079 .. 1555  =  477 bp
human growth hormone polyadenylation signal
f1 ori
1644 .. 2072  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1644 .. 2072  =  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
2086 .. 2415  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
2086 .. 2415  =  330 bp
SV40 enhancer and early promoter
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
3450 .. 3571  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3450 .. 3571  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5639 .. 5743  =  105 bp
AmpR promoter
5639 .. 5743  =  105 bp
MCS
975 .. 1036  =  62 bp
multiple cloning site
MCS
975 .. 1036  =  62 bp
multiple cloning site
attB1
917 .. 941  =  25 bp
recombination site for the Gateway® BP reaction
attB1
917 .. 941  =  25 bp
recombination site for the Gateway® BP reaction
FLAG
951 .. 974  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an
enterokinase cleavage site
FLAG
951 .. 974  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an
enterokinase cleavage site
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
1040 .. 1058  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
1040 .. 1058  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
ATG
948 .. 950  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
948 .. 950  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
2266 .. 2401  =  136 bp
SV40 origin of replication
SV40 ori
2266 .. 2401  =  136 bp
SV40 origin of replication
attB2
1006 .. 1030  =  25 bp
recombination site for the Gateway® BP reaction
attB2
1006 .. 1030  =  25 bp
recombination site for the Gateway® BP reaction
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