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Plasmid Files

pDream2.1/MCS

Vector for expression of N-terminally FLAG-tagged proteins in bacterial, insect, and mammalian cells.

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pDream2.1 MCS Sequence and MappDream2.1 MCS.dna
Map and Sequence File   
Sequence Author:  GenScript
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 SalI (243) ScaI (6790) AlwNI (5991) PciI (5576) AsiSI (5432) BmgBI (5390) SwaI (4976) AfeI (4844) BspHI (4486) BsmI (4434) BstBI (4196) RsrII (4030) BssHII (3911) PflFI - Tth111I (3632) PstI (3567) AccI (244) HincII (245) MluI (399) FseI (1017) PaeR7I - PspXI - XhoI (1020) CMV enhancer T7 primer (1615 .. 1634) T7 promoter PpuMI (1660) PacI (1732) attB1 ATG FLAG BamHI (1837) BglII (1844) EcoRI (1851) Acc65I (1857) KpnI (1861) Eco53kI (1865) SacI (1867) HindIII (1869) NotI (1876) SP6 primer (1883 .. 1904) attB2 AleI (1988) hGH poly(A) signal BsgI (2172) BlpI (2258) XbaI (2456) DraIII (2781) SexAI * (3071) BseRI (3300) StuI (3303) AvrII (3304) TspMI - XmaI (3325) SmaI (3327) BclI * (3355) BsaBI * (3373) KasI (3513) NarI (3514) SfoI (3515) PluTI (3517) pDream2.1/MCS 7159 bp
SalI  (243)
1 site
G T C G A C C A G C T G
ScaI  (6790)
1 site
A G T A C T T C A T G A
AlwNI  (5991)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
PciI  (5576)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AsiSI  (5432)
1 site
G C G A T C G C C G C T A G C G
BmgBI  (5390)
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.
SwaI  (4976)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
AfeI  (4844)
1 site
A G C G C T T C G C G A
BspHI  (4486)
1 site
T C A T G A A G T A C T
BsmI  (4434)
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.
BstBI  (4196)
1 site
T T C G A A A A G C T T
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.
BssHII  (3911)
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  (3632)
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  (3632)
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.
PstI  (3567)
1 site
C T G C A G G A C G T C
AccI  (244)
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.
HincII  (245)
1 site
G T Y R A C C A R Y T G
MluI  (399)
1 site
A C G C G T T G C G C A
FseI  (1017)
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.
PaeR7I  (1020)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1020)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1020)
1 site
C T C G A G G A G C T C
PpuMI  (1660)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
PacI  (1732)
1 site
T T A A T T A A A A T T A A T T
BamHI  (1837)
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  (1844)
1 site
A G A T C T T C T A G A
EcoRI  (1851)
1 site
G A A T T C C T T A A G
Acc65I  (1857)
1 site
G G T A C C C C A T G G
KpnI  (1861)
1 site
G G T A C C C C A T G G
Eco53kI  (1865)
1 site
G A G C T C C T C G A G
SacI  (1867)
1 site
G A G C T C C T C G A G
HindIII  (1869)
1 site
A A G C T T T T C G A A
NotI  (1876)
1 site
G C G G C C G C C G C C G G C G
AleI  (1988)
1 site
C A C N N N N G T G G T G N N N N C A C
BsgI  (2172)
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).
BlpI  (2258)
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.
XbaI  (2456)
1 site
T C T A G A A G A T C T
DraIII  (2781)
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  (3071)
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  (3300)
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  (3303)
1 site
A G G C C T T C C G G A
AvrII  (3304)
1 site
C C T A G G G G A T C C
TspMI  (3325)
1 site
C C C G G G G G G C C C
XmaI  (3325)
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  (3327)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BclI  (3355)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BsaBI  (3373)
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  (3513)
1 site
G G C G C C C C G C G G
NarI  (3514)
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  (3515)
1 site
G G C G C C C C G C G G
PluTI  (3517)
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.
T7 primer
20-mer  /  40% GC
1 binding site
1615 .. 1634  =  20 annealed bases
Tm  =  49°C
SP6 primer
22-mer  /  36% GC
1 binding site
1883 .. 1904  =  22 annealed bases
Tm  =  50°C
AmpR
6237 .. 7097  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6237 .. 7028  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6237 .. 7097  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7029 .. 7097  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6237 .. 7097  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
baculovirus recombination region (lef2/ORF603)
181 .. 1008  =  828 bp
contains ORF603 and part of lef2
baculovirus recombination region (lef2/ORF603)
181 .. 1008  =  828 bp
contains ORF603 and part of lef2
NeoR/KanR
3386 .. 4180  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
3386 .. 4180  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
baculovirus recombination region (ORF1629)
4722 .. 5427  =  706 bp
contains part of ORF1629
baculovirus recombination region (ORF1629)
4722 .. 5427  =  706 bp
contains part of ORF1629
ori
5637 .. 6224  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5637 .. 6224  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hGH poly(A) signal
1931 .. 2407  =  477 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1931 .. 2407  =  477 bp
human growth hormone polyadenylation signal
f1 ori
2548 .. 2976  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2548 .. 2976  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
2990 .. 3319  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
2990 .. 3319  =  330 bp
SV40 enhancer and early promoter
CMV enhancer
1086 .. 1389  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
1086 .. 1389  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
1390 .. 1593  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
1390 .. 1593  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
4354 .. 4475  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4354 .. 4475  =  122 bp
SV40 polyadenylation signal
p10 promoter
1661 .. 1770  =  110 bp
baculovirus promoter for expression in insect cells
p10 promoter
1661 .. 1770  =  110 bp
baculovirus promoter for expression in insect cells
β-globin poly(A) signal
4519 .. 4574  =  56 bp
rabbit β-globin polyadenylation signal
β-globin poly(A) signal
4519 .. 4574  =  56 bp
rabbit β-globin polyadenylation signal
T7 terminator
2408 .. 2455  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
2408 .. 2455  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
MCS
1837 .. 1882  =  46 bp
multiple cloning site
MCS
1837 .. 1882  =  46 bp
multiple cloning site
lac operator
1634 .. 1658  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
1634 .. 1658  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
attB1
1772 .. 1796  =  25 bp
recombination site for the Gateway® BP reaction
attB1
1772 .. 1796  =  25 bp
recombination site for the Gateway® BP reaction
attB2
1905 .. 1929  =  25 bp
recombination site for the Gateway® BP reaction
attB2
1905 .. 1929  =  25 bp
recombination site for the Gateway® BP reaction
FLAG
1813 .. 1836  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an
enterokinase cleavage site
FLAG
1813 .. 1836  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an
enterokinase cleavage site
T7 promoter
1615 .. 1633  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1615 .. 1633  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ATG
1810 .. 1812  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1810 .. 1812  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF603
343 .. 948  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
ORF603
343 .. 948  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
ORF1629
4839 .. 5427  =  589 bp
195 amino acids  =  22.3 kDa
Product: baculovirus capsid-associated protein
required for viral replication
ORF1629
4839 .. 5427  =  589 bp
195 amino acids  =  22.3 kDa
Product: baculovirus capsid-associated protein
required for viral replication
SV40 ori
3170 .. 3305  =  136 bp
SV40 origin of replication
SV40 ori
3170 .. 3305  =  136 bp
SV40 origin of replication
lef2
181 .. 305  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
lef2
181 .. 305  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
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