pNIC28-Bsa4

Bacterial vector encoding an N-terminal 6xHis-TEV cassette and kanamycin resistance plus a SacB negative selection marker, for purification of recombinant proteins.

Sequence Author: Structural Genomics Consortium

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HomePlasmidsStructural Genomics VectorspNIC28-Bsa4
SgrAI (7224) SphI (7076) BstAPI (6867) MluI (6543) BclI * (6529) BstEII (6361) NmeAIII (6343) ApaI (6340) PspOMI (6336) BssHII (6132) BglI (5486) FspI - FspAI (5465) PpuMI (5437) PflFI - Tth111I (4700) BspQI - SapI (4559) BssSI (4269) AlwNI (4033) T7 promoter XbaI (47) RBS NdeI (87) ATG 6xHis TEV site BsaI (152) NcoI (153) ZraI (166) AatII (168) BfuAI - BspMI (186) StuI * (857) MscI (879) AanI (1256) BsrGI (1296) SnaBI (1365) SacII (1695) BsaI (2083) BamHI (2099) EcoRI (2105) Eco53kI (2113) SacI (2115) SalI (2118) HindIII (2124) EagI - NotI (2131) PaeR7I - PspXI - XhoI (2139) BlpI (2218) DraIII (2546) AanI (2671) AsiSI - PvuI (3246) TspMI - XmaI (3368) SmaI (3370) NruI (3587) pNIC28-Bsa4 7284 bp
SgrAI  (7224)
1 site		 C R C C G G Y G G Y G G C C R C
Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
SphI  (7076)
1 site		 G C A T G C C G T A C G
BstAPI  (6867)
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.
MluI  (6543)
1 site		 A C G C G T T G C G C A
BclI  (6529)
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.
BstEII  (6361)
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.
NmeAIII  (6343)
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).
ApaI  (6340)
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  (6336)
1 site		 G G G C C C C C C G G G
BssHII  (6132)
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.
BglI  (5486)
1 site		 G C C N N N N N G G C C G G N N N N N C C G
Sticky ends from different BglI sites may not be compatible.
FspI  (5465)
1 site		 T G C G C A A C G C G T
FspAI  (5465)
1 site		 R T G C G C A Y Y A C G C G T R
PpuMI  (5437)
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.
PflFI  (4700)
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  (4700)
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.
BspQI  (4559)
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  (4559)
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.
BssSI  (4269)
1 site		 C A C G A G G T G C T C
AlwNI  (4033)
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.
XbaI  (47)
1 site		 T C T A G A A G A T C T
NdeI  (87)
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.
BsaI  (152)
2 sites		 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.
NcoI  (153)
1 site		 C C A T G G G G T A C C
ZraI  (166)
1 site		 G A C G T C C T G C A G
AatII  (168)
1 site		 G A C G T C C T G C A G
BfuAI  (186)
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  (186)
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.
StuI  (857)
1 site		 A G G C C T T C C G G A
* Blocked by Dcm methylation.
MscI  (879)
1 site		 T G G C C A A C C G G T
AanI  (1256)
2 sites		 T T A T A A A A T A T T
BsrGI  (1296)
1 site		 T G T A C A A C A T G T
BsrGI is typically used at 37°C, but is even more active at 60°C.
SnaBI  (1365)
1 site		 T A C G T A A T G C A T
SacII  (1695)
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.
BsaI  (2083)
2 sites		 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.
BamHI  (2099)
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.
EcoRI  (2105)
1 site		 G A A T T C C T T A A G
Eco53kI  (2113)
1 site		 G A G C T C C T C G A G
SacI  (2115)
1 site		 G A G C T C C T C G A G
SalI  (2118)
1 site		 G T C G A C C A G C T G
HindIII  (2124)
1 site		 A A G C T T T T C G A A
EagI  (2131)
1 site		 C G G C C G G C C G G C
NotI  (2131)
1 site		 G C G G C C G C C G C C G G C G
PaeR7I  (2139)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2139)
1 site		 V C T C G A G B B G A G C T C V
XhoI  (2139)
1 site		 C T C G A G G A G C T C
BlpI  (2218)
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.
DraIII  (2546)
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.
AanI  (2671)
2 sites		 T T A T A A A A T A T T
AsiSI  (3246)
1 site		 G C G A T C G C C G C T A G C G
PvuI  (3246)
1 site		 C G A T C G G C T A G C
TspMI  (3368)
1 site		 C C C G G G G G G C C C
XmaI  (3368)
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  (3370)
1 site		 C C C G G G G G G C C C
SmaI can be used at 37°C for brief incubations.
NruI  (3587)
1 site		 T C G C G A A G C G C T
SacB
616 .. 2037  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 1:  signal peptide  
   616 .. 702  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
616 .. 2037  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 2:  
   703 .. 2037  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
616 .. 2037  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
lacI
5816 .. 6898  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
lacI
5816 .. 6898  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
KanR
2861 .. 3676  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
2861 .. 3676  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
3798 .. 4386  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3798 .. 4386  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2313 .. 2768  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2313 .. 2768  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
sacB promoter
170 .. 615  =  446 bp
sacB promoter and control region
sacB promoter
170 .. 615  =  446 bp
sacB promoter and control region
rop
4816 .. 5007  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4816 .. 5007  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
lacI promoter
6899 .. 6976  =  78 bp
lacI promoter
6899 .. 6976  =  78 bp
ATG
89 .. 91  =  3 bp
1 amino acid  =  149.2 Da
ATG
89 .. 91  =  3 bp
1 amino acid  =  149.2 Da
6xHis
92 .. 109  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
92 .. 109  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TEV site
134 .. 154  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
134 .. 154  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 terminator
2229 .. 2276  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
2229 .. 2276  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
20 .. 44  =  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
20 .. 44  =  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).
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
2145 .. 2162  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2145 .. 2162  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
75 .. 80  =  6 bp
ribosome binding site
RBS
75 .. 80  =  6 bp
ribosome binding site
ORF:  616 .. 2037  =  1422 bp
ORF:  473 amino acids  =  53.0 kDa
ORF:  5782 .. 6033  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  6937 .. 7200  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  5039 .. 5407  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  7074 .. 29  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  4816 .. 5040  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  5404 .. 5760  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  5796 .. 6059  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  6648 .. 7148  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
ORF:  7145 .. 109  =  249 bp
ORF:  82 amino acids  =  8.8 kDa
ORF:  2861 .. 3676  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  5816 .. 6775  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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Individual Sequences & Maps

p11
pMCSG19B (linearized)
pMCSG41
pMCSG68 (linearized)
p15Tv-L
pMCSG19C
pMCSG41 (linearized)
pMCSG69
p283
pMCSG19C (linearized)
pMCSG42
pMCSG69 (linearized)
pBEN1-SGC
pMCSG20
pMCSG42 (linearized)
pMCSG7
pBEN1-SGC (linearized)
pMCSG20 (linearized)
pMCSG43
pMCSG7 (linearized)
pDB-HisGST
pMCSG21
pMCSG43 (linearized)
pMCSG70
pDB.GST
pMCSG21 (linearized)
pMCSG46
pMCSG70 (linearized)
pDB.His.MBP
pMCSG22
pMCSG46 (linearized)
pMCSG71
pDB.His.NusA
pMCSG22 (linearized)
pMCSG48
pMCSG71 (linearized)
pDB.His.TRX
pMCSG23
pMCSG48 (linearized)
pMCSG73
pDB.TRX
pMCSG23 (linearized)
pMCSG49
pMCSG73 (linearized)
pEU-GST-FV
pMCSG24
pMCSG49 (linearized)
pMCSG75
pEU-His-FV
pMCSG24 (linearized)
pMCSG50
pMCSG75 (linearized)
pEU-HSBC
pMCSG25
pMCSG50 (linearized)
pMCSG76
pFB-LIC-Bse
pMCSG25 (linearized)
pMCSG51
pMCSG76 (linearized)
pFB-LIC-Bse (linearized)
pMCSG26
pMCSG51 (linearized)
pMCSG77
pH3c-LIC
pMCSG26 (linearized)
pMCSG52
pMCSG77 (linearized)
pH3c-LIC (linearized)
pMCSG27
pMCSG52 (linearized)
pMCSG79
pHM3C-LIC
pMCSG27 (linearized)
pMCSG53
pMCSG79 (linearized)
pHM3C-LIC (linearized)
pMCSG28
pMCSG53 (linearized)
pMCSG8
pLIC-CH
pMCSG28 (linearized)
pMCSG54
pMCSG8 (linearized)
pLIC-CH (linearized)
pMCSG29
pMCSG54 (linearized)
pMCSG81
pLIC-EGFP
pMCSG29 (linearized)
pMCSG55
pMCSG81 (linearized)
pLIC-EGFP (linearized)
pMCSG30
pMCSG55 (linearized)
pMCSG9
pLIC-SGC1
pMCSG30 (linearized)
pMCSG56
pMCSG9 (linearized)
pLIC-SGC1 (linearized)
pMCSG31
pMCSG56 (linearized)
pNH-TrxT
pMCentr1
pMCSG31 (linearized)
pMCSG57
pNH-TrxT (linearized)
pMCentr1 (linearized)
pMCSG32
pMCSG57 (linearized)
pNIC-CH
pMCentr2
pMCSG32 (linearized)
pMCSG58
pNIC-CH (linearized)
pMCentr2 (linearized)
pMCSG33
pMCSG58 (linearized)
pNIC-CTHF
pMCentr3
pMCSG33 (linearized)
pMCSG59
pNIC-CTHF (linearized)
pMCentr3 (linearized)
pMCSG34
pMCSG59 (linearized)
pNIC-Zb
pMCSG10
pMCSG