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Plasmid Files

phRG-B

Promoterless mammalian vector encoding humanized Renilla luciferase.

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phRG-B Sequence and MapphRG-B.dna
Map and Sequence File   
Sequence Author:  Promega
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 BsgI (4092) AarI - BfuAI - BspMI (4064) RVprimer3 (4043 .. 4062) poly(A) signal NotI (3934) BsaAI (3585) BtgZI (3580) AseI (2608) AhdI (2436) Acc65I (1) KpnI (5) Eco53kI (9) SacI (11) MluI (15) NheI (21) BmtI (25) TspMI - XmaI (26) SmaI - SrfI (28) PaeR7I - XhoI (32) BglII (36) HindIII (53) NcoI (86) BstAPI (276) NsiI (307) PfoI * (368) EcoRV (573) Bsu36I (674) NruI (749) ZraI (783) PflFI - Tth111I (784) AatII (785) PasI (862) EcoO109I (933) XbaI (1025) FseI (1044) HpaI (1185) MfeI (1194) BamHI (1287) SalI (1293) AccI (1294) PshAI (1358) RVprimer4 (1344 .. 1363) BbsI (1372) AfeI (1419) BspQI - SapI (1427) PciI (1543) NspI (1547) BseYI (1847) PspFI (1851) AlwNI (1959) phRG-B 4101 bp
BsgI  (4092)
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).
AarI  (4064)
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 AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
BfuAI  (4064)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (4064)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
NotI  (3934)
1 site
G C G G C C G C C G C C G G C G
BsaAI  (3585)
1 site
Y A C G T R R T G C A Y
BtgZI  (3580)
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.
AseI  (2608)
1 site
A T T A A T T A A T T A
AhdI  (2436)
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.
Acc65I  (1)
1 site
G G T A C C C C A T G G
KpnI  (5)
1 site
G G T A C C C C A T G G
Eco53kI  (9)
1 site
G A G C T C C T C G A G
SacI  (11)
1 site
G A G C T C C T C G A G
MluI  (15)
1 site
A C G C G T T G C G C A
NheI  (21)
1 site
G C T A G C C G A T C G
BmtI  (25)
1 site
G C T A G C C G A T C G
TspMI  (26)
1 site
C C C G G G G G G C C C
XmaI  (26)
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  (28)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (28)
1 site
G C C C G G G C C G G G C C C G
PaeR7I  (32)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (32)
1 site
C T C G A G G A G C T C
BglII  (36)
1 site
A G A T C T T C T A G A
HindIII  (53)
1 site
A A G C T T T T C G A A
NcoI  (86)
1 site
C C A T G G G G T A C C
BstAPI  (276)
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.
NsiI  (307)
1 site
A T G C A T T A C G T A
PfoI  (368)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
EcoRV  (573)
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.
Bsu36I  (674)
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.
NruI  (749)
1 site
T C G C G A A G C G C T
ZraI  (783)
1 site
G A C G T C C T G C A G
PflFI  (784)
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  (784)
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.
AatII  (785)
1 site
G A C G T C C T G C A G
PasI  (862)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
EcoO109I  (933)
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.
XbaI  (1025)
1 site
T C T A G A A G A T C T
FseI  (1044)
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.
HpaI  (1185)
1 site
G T T A A C C A A T T G
MfeI  (1194)
1 site
C A A T T G G T T A A C
BamHI  (1287)
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.
SalI  (1293)
1 site
G T C G A C C A G C T G
AccI  (1294)
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.
PshAI  (1358)
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.
BbsI  (1372)
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.
AfeI  (1419)
1 site
A G C G C T T C G C G A
BspQI  (1427)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (1427)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
PciI  (1543)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (1547)
1 site
R C A T G Y Y G T A C R
BseYI  (1847)
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.
PspFI  (1851)
1 site
C C C A G C G G G T C G
AlwNI  (1959)
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.
RVprimer3
20-mer  /  50% GC
1 binding site
4043 .. 4062  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
1344 .. 1363  =  20 annealed bases
Tm  =  61°C
hRluc
88 .. 1023  =  936 bp
311 amino acids  =  36.0 kDa
Product: Renilla luciferase
human codon-optimized
hRluc
88 .. 1023  =  936 bp
311 amino acids  =  36.0 kDa
Product: Renilla luciferase
human codon-optimized
AmpR
2363 .. 3223  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2363 .. 3154  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2363 .. 3223  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3155 .. 3223  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2363 .. 3223  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1604 .. 2192  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1604 .. 2192  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3355 .. 3810  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3355 .. 3810  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 poly(A) signal
1064 .. 1185  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1064 .. 1185  =  122 bp
SV40 polyadenylation signal
AmpR promoter
3224 .. 3328  =  105 bp
AmpR promoter
3224 .. 3328  =  105 bp
pause site
4003 .. 4094  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
4003 .. 4094  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
MCS
1 .. 58  =  58 bp
multiple cloning site
MCS
1 .. 58  =  58 bp
multiple cloning site
poly(A) signal
3941 .. 3989  =  49 bp
synthetic polyadenylation signal
poly(A) signal
3941 .. 3989  =  49 bp
synthetic polyadenylation signal
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