pRham N-His SUMO Kan

Vector for Expresso® cloning to add N-terminal 6xHis and SUMO tags, with rhamnose-inducible expression.

Sequence Author: Lucigen

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SwaI (2538) tonB terminator BsaAI (2469) AvaI - BsoBI - PaeR7I - PspXI - XhoI (2354) EarI (2140) TsoI (2117) PasI (2081) EcoNI (2043) SspI (2031) BsrFI (1998) AsiSI - PvuI (1958) Bpu10I - BsmBI - Esp3I (1936) AseI (1755) TaqII (1704) PflMI (1696) XmnI (1453) PvuII (1420) ApaI (1376) PspOMI (1372) pRham Forward (101 .. 124) BsaI (116) RBS NdeI (181) ATG 6xHis KflI - PpuMI (205) PstI - SbfI (214) DrdI (222) BglII (310) AclI (399) BclI * (431) BsrBI (511) EaeI - EagI - NotI (512) StyI (546) pETite Reverse (553 .. 572) T3Te terminator AlwNI (965) ApaLI (1060) BsiHKAI (1064) BseYI (1070) PspFI (1074) BciVI (1176) BssSI - BssSαI (1201) pRham N-His SUMO Kan 2575 bp
SwaI  (2538)
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.
BsaAI  (2469)
1 site
Y A C G T R R T G C A Y
AvaI  (2354)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (2354)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
PaeR7I  (2354)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2354)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2354)
1 site
C T C G A G G A G C T C
EarI  (2140)
1 site
C T C T T C N G A G A A G N N N N

Cleavage may be enhanced when more than one copy of the EarI recognition sequence is present.
Sticky ends from different EarI sites may not be compatible.
TsoI  (2117)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PasI  (2081)
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.
EcoNI  (2043)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
SspI  (2031)
1 site
A A T A T T T T A T A A
BsrFI  (1998)
1 site
R C C G G Y Y G G C C R

Cleavage may be enhanced when more than one copy of the BsrFI recognition sequence is present.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
AsiSI  (1958)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1958)
1 site
C G A T C G G C T A G C
Bpu10I  (1936)
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.
BsmBI  (1936)
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.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (1936)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
AseI  (1755)
1 site
A T T A A T T A A T T A
TaqII  (1704)
1 site
G A C C G A ( N ) 9 N N C T G G C T ( N ) 9

Sticky ends from different TaqII sites may not be compatible.
PflMI  (1696)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
XmnI  (1453)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuII  (1420)
1 site
C A G C T G G T C G A C
ApaI  (1376)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (1372)
1 site
G G G C C C C C C G G G
BsaI  (116)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
NdeI  (181)
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.
KflI  (205)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (205)
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.
PstI  (214)
1 site
C T G C A G G A C G T C
SbfI  (214)
1 site
C C T G C A G G G G A C G T C C
DrdI  (222)
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.
BglII  (310)
1 site
A G A T C T T C T A G A
AclI  (399)
1 site
A A C G T T T T G C A A
BclI  (431)
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.
BsrBI  (511)
1 site
C C G C T C G G C G A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BsrBI will not always regenerate a BsrBI site.
BsrBI is typically used at 37°C, but can be used at temperatures up to 50°C.
EaeI  (512)
1 site
Y G G C C R R C C G G Y
EagI  (512)
1 site
C G G C C G G C C G G C
NotI  (512)
1 site
G C G G C C G C C G C C G G C G
StyI  (546)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
AlwNI  (965)
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.
ApaLI  (1060)
1 site
G T G C A C C A C G T G
BsiHKAI  (1064)
1 site
G W G C W C C W C G W G

Sticky ends from different BsiHKAI sites may not be compatible.
BseYI  (1070)
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  (1074)
1 site
C C C A G C G G G T C G
BciVI  (1176)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
BssSI  (1201)
1 site
C A C G A G G T G C T C
BssSαI  (1201)
1 site
C A C G A G G T G C T C
pRham Forward
24-mer  /  50% GC
1 binding site
101 .. 124  =  24 annealed bases
Tm  =  58°C
pETite Reverse
20-mer  /  55% GC
1 binding site
553 .. 572  =  20 annealed bases
Tm  =  56°C
KanR
1573 .. 2388  =  816 bp
271 amino acids  =  30.9 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
1573 .. 2388  =  816 bp
271 amino acids  =  30.9 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
731 .. 1318  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
731 .. 1318  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ATG
183 .. 185  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
183 .. 185  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
186 .. 203  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
186 .. 203  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
SUMO
216 .. 503  =  288 bp
96 amino acids  =  11.0 kDa
Product: ubiquitin-like protein tag
modified by Lucigen to prevent cleavage by eukaryotic SUMO proteases
SUMO
216 .. 503  =  288 bp
96 amino acids  =  11.0 kDa
Product: ubiquitin-like protein tag
modified by Lucigen to prevent cleavage by eukaryotic SUMO proteases
rop
1381 .. 1572  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
1381 .. 1572  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rhaB promoter
1 .. 119  =  119 bp
promoter of the E. coli rhaBAD operon, conferring tight induction with L-rhamnose and repression with D-glucose in the presence of RhaR and RhaS (Giacalone et al., 2006)
rhaB promoter
1 .. 119  =  119 bp
promoter of the E. coli rhaBAD operon, conferring tight induction with L-rhamnose and repression with D-glucose in the presence of RhaR and RhaS (Giacalone et al., 2006)
cat promoter
2389 .. 2479  =  91 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
cat promoter
2389 .. 2479  =  91 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
T7 terminator
535 .. 582  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
535 .. 582  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
tonB terminator
2480 .. 2511  =  32 bp
bidirectional E. coli tonB-P14 transcription terminator
tonB terminator
2480 .. 2511  =  32 bp
bidirectional E. coli tonB-P14 transcription terminator
T3Te terminator
680 .. 709  =  30 bp
phage T3 early transcription terminator
T3Te terminator
680 .. 709  =  30 bp
phage T3 early transcription terminator
T7Te terminator
1330 .. 1357  =  28 bp
phage T7 early transcription terminator
T7Te terminator
1330 .. 1357  =  28 bp
phage T7 early transcription terminator
RBS
153 .. 175  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
RBS
153 .. 175  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
ORF:  1288 .. 1515  =  228 bp
ORF:  75 amino acids  =  8.6 kDa
ORF:  183 .. 506  =  324 bp
ORF:  107 amino acids  =  12.3 kDa
ORF:  1573 .. 2388  =  816 bp
ORF:  271 amino acids  =  30.9 kDa
ORF:  993 .. 1478  =  486 bp
ORF:  161 amino acids  =  18.0 kDa
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