pTandem-1

Mammalian vector for the co-expression of two genes from a bicistronic mRNA using the CMV promoter and an IRES.

Sequence Author: MilliporeSigma (Novagen)

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Bpu10I (3785) SspI (3752) ScaI (3428) PvuI (3318) FspI (3170) NmeAIII (3096) BpmI (3018) AhdI (2948) PspFI (2521) BseYI (2517) DrdI (2321) BstAPI (2041) BspHI (1880) BamHI (3806) BtgZI (329) SnaBI (335) BsgI (669) SacII (726) RsrII (901) RBS NcoI (991) ATG 6xHis XcmI (1017) TspMI - XmaI (1093) SmaI (1095) enterokinase site PshAI (1135) MfeI (1140) EcoRV (1147) AscI - BssHII (1152) NsiI (1163) PacI (1168) SwaI (1174) BsiWI (1183) SfiI (1186) BstEII (1192) AgeI (1196) NotI (1204) PvuII (1214) AccI (1218) BstZ17I (1219) PaeR7I - XhoI (1266) Bsu36I (1308) SpeI (1314) AvrII (1397) HindIII (1470) PaqCI (1585) Acc65I (1687) KpnI (1691) BmgBI (1788) PmeI (1809) NheI (1814) BmtI (1818) AfeI (1824) BstXI (1826) HpaI (1832) BlpI (1837) BspDI * - ClaI * (1844) pTandem™-1 3813 bp
Bpu10I  (3785)
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.
SspI  (3752)
1 site
A A T A T T T T A T A A
ScaI  (3428)
1 site
A G T A C T T C A T G A
PvuI  (3318)
1 site
C G A T C G G C T A G C
FspI  (3170)
1 site
T G C G C A A C G C G T
NmeAIII  (3096)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BpmI  (3018)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
AhdI  (2948)
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.
PspFI  (2521)
1 site
C C C A G C G G G T C G
BseYI  (2517)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
DrdI  (2321)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BstAPI  (2041)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
BspHI  (1880)
1 site
T C A T G A A G T A C T
BamHI  (3806)
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.
BtgZI  (329)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
SnaBI  (335)
1 site
T A C G T A A T G C A T
BsgI  (669)
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).
SacII  (726)
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.
RsrII  (901)
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.
NcoI  (991)
1 site
C C A T G G G G T A C C
XcmI  (1017)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
TspMI  (1093)
1 site
C C C G G G G G G C C C
XmaI  (1093)
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  (1095)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
PshAI  (1135)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
MfeI  (1140)
1 site
C A A T T G G T T A A C
EcoRV  (1147)
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.
AscI  (1152)
1 site
G G C G C G C C C C G C G C G G
BssHII  (1152)
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.
NsiI  (1163)
1 site
A T G C A T T A C G T A
PacI  (1168)
1 site
T T A A T T A A A A T T A A T T
SwaI  (1174)
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.
BsiWI  (1183)
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.
SfiI  (1186)
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.
BstEII  (1192)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
AgeI  (1196)
1 site
A C C G G T T G G C C A
NotI  (1204)
1 site
G C G G C C G C C G C C G G C G
PvuII  (1214)
1 site
C A G C T G G T C G A C
AccI  (1218)
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.
BstZ17I  (1219)
1 site
G T A T A C C A T A T G
PaeR7I  (1266)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1266)
1 site
C T C G A G G A G C T C
Bsu36I  (1308)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
SpeI  (1314)
1 site
A C T A G T T G A T C A
AvrII  (1397)
1 site
C C T A G G G G A T C C
HindIII  (1470)
1 site
A A G C T T T T C G A A
PaqCI  (1585)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
Acc65I  (1687)
1 site
G G T A C C C C A T G G
KpnI  (1691)
1 site
G G T A C C C C A T G G
BmgBI  (1788)
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.
PmeI  (1809)
1 site
G T T T A A A C C A A A T T T G
NheI  (1814)
1 site
G C T A G C C G A T C G
BmtI  (1818)
1 site
G C T A G C C G A T C G
AfeI  (1824)
1 site
A G C G C T T C G C G A
BstXI  (1826)
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.
HpaI  (1832)
1 site
G T T A A C C A A T T G
BlpI  (1837)
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.
BspDI  (1844)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1844)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
bla(M)
2875 .. 3663  =  789 bp
262 amino acids  =  28.8 kDa
Product: β-lactamase lacking the signal sequence
allows cytosolic expression of β-lactamase
bla(M)
2875 .. 3663  =  789 bp
262 amino acids  =  28.8 kDa
Product: β-lactamase lacking the signal sequence
allows cytosolic expression of β-lactamase
ori
2275 .. 2862  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2275 .. 2862  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
1321 .. 1805  =  485 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
1321 .. 1805  =  485 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
CMV enhancer
56 .. 359  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
56 .. 359  =  304 bp
human cytomegalovirus immediate early enhancer
ATG
993 .. 995  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
993 .. 995  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
999 .. 1016  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
999 .. 1016  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
S-Tag
1026 .. 1070  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
1026 .. 1070  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
thrombin site
1086 .. 1103  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
1086 .. 1103  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
enterokinase site
1122 .. 1136  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
1122 .. 1136  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
HSV tag
1233 .. 1265  =  33 bp
11 amino acids  =  1.2 kDa
Product: HSV (herpes simplex virus) epitope tag
HSV tag
1233 .. 1265  =  33 bp
11 amino acids  =  1.2 kDa
Product: HSV (herpes simplex virus) epitope tag
8xHis
1272 .. 1295  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
1272 .. 1295  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
β-globin terminator
1846 .. 2055  =  210 bp
rabbit β-globin terminator
β-globin terminator
1846 .. 2055  =  210 bp
rabbit β-globin terminator
CMV promoter
361 .. 560  =  200 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
361 .. 560  =  200 bp
human cytomegalovirus (CMV) immediate early promoter
intron
679 .. 842  =  164 bp
intron
679 .. 842  =  164 bp
AmpR promoter
3736 .. 3807  =  72 bp
AmpR promoter
3736 .. 3807  =  72 bp
T7 promoter
911 .. 929  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
911 .. 929  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
977 .. 982  =  6 bp
ribosome binding site
RBS
977 .. 982  =  6 bp
ribosome binding site
MCS 1
991 .. 1271  =  281 bp
multiple cloning site 1
MCS 1
991 .. 1271  =  281 bp
multiple cloning site 1
β-globin poly(A) signal
1913 .. 1968  =  56 bp
rabbit β-globin polyadenylation signal
β-globin poly(A) signal
1913 .. 1968  =  56 bp
rabbit β-globin polyadenylation signal
MCS 2
1806 .. 1848  =  43 bp
multiple cloning site 2
MCS 2
1806 .. 1848  =  43 bp
multiple cloning site 2
ORF:  3005 .. 3271  =  267 bp
ORF:  88 amino acids  =  9.3 kDa
ORF:  993 .. 1298  =  306 bp
ORF:  101 amino acids  =  11.1 kDa
ORF:  910 .. 1164  =  255 bp
ORF:  84 amino acids  =  9.4 kDa
ORF:  2875 .. 3735  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  567 .. 821  =  255 bp
ORF:  84 amino acids  =  9.0 kDa
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