pVP65K

Bacterial Flexi® Vector with a kanamycin resistance marker and the barnase gene, encoding an N-terminal MBP-TVMV-8xHis cassette plus TVMV protease.

Sequence Author: Center for Eukaryotic Structural Genomics

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HomePlasmidsStructural Genomics VectorspVP65K
FspI (6775) NaeI (6375) NgoMIV (6373) RsrII (6358) AvrII (6206) BspHI (6105) PciI (5385) NdeI (5208) BstZ17I (5158) BstEII (4352) ApaI (4331) PspOMI (4327) EcoRV (4088) HpaI (4032) HindIII (3589) BstBI (3586) BspDI - ClaI (3575) BsiWI (1) SacII (267) SpeI (380) BglI (397) DraIII (456) AgeI (617) PaeR7I - XhoI (947) AanI (995) MfeI (1005) RBS NcoI (1059) BsaI (1127) AanI (1416) BmgBI (1606) AfeI (1974) PacI (2169) TVMV site NsiI (2236) AsiSI - SgfI (2244) NotI (2247) Bpu10I (2698) PasI (3157) SspI (3237) ScaI (3342) PmeI (3376) Eco53kI (3389) SacI (3391) SbfI (3424) BsrGI (3530) AscI (3547) SgrDI (3562) pVP65K 7174 bp
FspI  (6775)
1 site		 T G C G C A A C G C G T
NaeI  (6375)
1 site		 G C C G G C C G G C C G
Efficient cleavage requires at least two copies of the NaeI recognition sequence.
NgoMIV  (6373)
1 site		 G C C G G C C G G C C G
Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
RsrII  (6358)
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.
AvrII  (6206)
1 site		 C C T A G G G G A T C C
BspHI  (6105)
1 site		 T C A T G A A G T A C T
PciI  (5385)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
NdeI  (5208)
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.
BstZ17I  (5158)
1 site		 G T A T A C C A T A T G
BstEII  (4352)
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.
ApaI  (4331)
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  (4327)
1 site		 G G G C C C C C C G G G
EcoRV  (4088)
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.
HpaI  (4032)
1 site		 G T T A A C C A A T T G
HindIII  (3589)
1 site		 A A G C T T T T C G A A
BstBI  (3586)
1 site		 T T C G A A A A G C T T
BspDI  (3575)
1 site		 A T C G A T T A G C T A
ClaI  (3575)
1 site		 A T C G A T T A G C T A
BsiWI  (1)
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.
SacII  (267)
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.
SpeI  (380)
1 site		 A C T A G T T G A T C A
BglI  (397)
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.
DraIII  (456)
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.
AgeI  (617)
1 site		 A C C G G T T G G C C A
PaeR7I  (947)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (947)
1 site		 C T C G A G G A G C T C
AanI  (995)
2 sites		 T T A T A A A A T A T T
MfeI  (1005)
1 site		 C A A T T G G T T A A C
NcoI  (1059)
1 site		 C C A T G G G G T A C C
BsaI  (1127)
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.
AanI  (1416)
2 sites		 T T A T A A A A T A T T
BmgBI  (1606)
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.
AfeI  (1974)
1 site		 A G C G C T T C G C G A
PacI  (2169)
1 site		 T T A A T T A A A A T T A A T T
NsiI  (2236)
1 site		 A T G C A T T A C G T A
AsiSI  (2244)
1 site		 G C G A T C G C C G C T A G C G
SgfI  (2244)
1 site		 G C G A T C G C C G C T A G C G
NotI  (2247)
1 site		 G C G G C C G C C G C C G G C G
Bpu10I  (2698)
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.
PasI  (3157)
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.
SspI  (3237)
1 site		 A A T A T T T T A T A A
ScaI  (3342)
1 site		 A G T A C T T C A T G A
PmeI  (3376)
1 site		 G T T T A A A C C A A A T T T G
Eco53kI  (3389)
1 site		 G A G C T C C T C G A G
SacI  (3391)
1 site		 G A G C T C C T C G A G
SbfI  (3424)
1 site		 C C T G C A G G G G A C G T C C
BsrGI  (3530)
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.
AscI  (3547)
1 site		 G G C G C G C C C C G C G C G G
SgrDI  (3562)
1 site		 C G T C G A C G G C A G C T G C
MBP
1061 .. 2164  =  1104 bp
368 amino acids  =  40.4 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
MBP
1061 .. 2164  =  1104 bp
368 amino acids  =  40.4 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
TVMV site
2189 .. 2209  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
2189 .. 2209  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
8xHis
2210 .. 2233  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
2210 .. 2233  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
lacI
3807 .. 4889  =  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
3807 .. 4889  =  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).
NeoR/KanR
6212 .. 7006  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
6212 .. 7006  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
TVMV protease
139 .. 849  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
TVMV protease
139 .. 849  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
CmR
2712 .. 3371  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2712 .. 3371  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5446 .. 6034  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5446 .. 6034  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
barnase
2277 .. 2612  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
barnase
2277 .. 2612  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
rrnB T1 terminator
20 .. 106  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
20 .. 106  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
3628 .. 3714  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
3628 .. 3714  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacI promoter
4890 .. 4967  =  78 bp
lacI promoter
4890 .. 4967  =  78 bp
PLtetO-1 promoter
873 .. 946  =  74 bp
5 segments
   Segment 5:  
   873 .. 898  =  26 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
5 segments
   Segment 4:  -10  
   899 .. 904  =  6 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
5 segments
   Segment 3:  
   905 .. 921  =  17 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
5 segments
   Segment 2:  -35  
   922 .. 927  =  6 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
5 segments
   Segment 1:  
   928 .. 946  =  19 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
5 segments
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
T5 promoter
956 .. 1000  =  45 bp
4 segments
   Segment 1:  
   956 .. 970  =  15 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
956 .. 1000  =  45 bp
4 segments
   Segment 2:  -35  
   971 .. 976  =  6 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
956 .. 1000  =  45 bp
4 segments
   Segment 3:  
   977 .. 993  =  17 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
956 .. 1000  =  45 bp
4 segments
   Segment 4:  -10  
   994 .. 1000  =  7 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
956 .. 1000  =  45 bp
4 segments
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
lac UV5 promoter
2628 .. 2658  =  31 bp
3 segments
   Segment 1:  -35  
   2628 .. 2633  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2628 .. 2658  =  31 bp
3 segments
   Segment 2:  
   2634 .. 2651  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2628 .. 2658  =  31 bp
3 segments
   Segment 3:  -10  
   2652 .. 2658  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2628 .. 2658  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
lac operator
1008 .. 1024  =  17 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
1008 .. 1024  =  17 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).
RBS
1047 .. 1052  =  6 bp
ribosome binding site
RBS
1047 .. 1052  =  6 bp
ribosome binding site
tet operator
903 .. 921  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
903 .. 921  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
928 .. 946  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
928 .. 946  =  19 bp
bacterial operator O2 for the tetR and tetA genes
lac operator
976 .. 992  =  17 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
976 .. 992  =  17 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).
ORF:  448 .. 864  =  417 bp
ORF:  138 amino acids  =  16.0 kDa
ORF:  1061 .. 2263  =  1203 bp
ORF:  400 amino acids  =  43.8 kDa
ORF:  3773 .. 4024  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  6443 .. 6697  =  255 bp
ORF:  84 amino acids  =  9.8 kDa
ORF:  2277 .. 2612  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  2712 .. 3371  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  6212 .. 7006  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  139 .. 849  =  711 bp
ORF:  236 amino acids  =  26.6 kDa
ORF:  3787 .. 4050  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4639 .. 4956  =  318 bp
ORF:  105 amino acids  =  11.2 kDa
ORF:  6448 .. 6834  =  387 bp
ORF:  128 amino acids  =  14.7 kDa
ORF:  27 .. 272  =  246 bp
ORF:  81 amino acids  =  9.2 kDa
ORF:  3807 .. 4766  =  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
pMCSG34 (linearized)
pMCSG60
pNIC-Zb (linearized)
pMCSG10 (linearized)
pMCSG34B
pMCSG60 (linearized)
pNIC28-Bsa4
pMCSG11
pMCSG34B (linearized)
pMCSG61
pNIC28-Bsa4 (linearized)
pMCSG11 (linearized)
pMCSG35
pMCSG61 (linearized)
pNYCOMPS-LIC-FH10T+ (N term) - linearized
pMCSG12
pMCSG35 (linearized)
pMCSG62
pNYCOMPS-LIC-FH10T+ (N term)
pMCSG12 (linearized)
pMCSG35B
pMCSG62 (linearized)
pSpeedET
pMCSG13
pMCSG35B (linearized)
pMCSG63
pVP13
pMCSG13 (linearized)
pMCSG37
pMCSG63 (linearized)
pVP16
pMCSG14
pMCSG37 (linearized)
pMCSG64
pVP33A
pMCSG14 (linearized)
pMCSG38
pMCSG64 (linearized)
pVP33K
pMCSG17
pMCSG38 (linearized)
pMCSG65
pVP56A
pMCSG17 (linearized)
pMCSG38C
pMCSG65 (linearized)
pVP56K
pMCSG18
pMCSG38C (linearized)
pMCSG66
pVP65K
pMCSG18 (linearized)
pMCSG39
pMCSG66 (linearized)
pVP68K
pMCSG19
pMCSG39 (linearized)
pMCSG67
pVP81K
pMCSG19 (linearized)
pMCSG40
pMCSG67 (linearized)
pVP91A
pMCSG19B
pMCSG40 (linearized)
pMCSG68

Plasmid Sets

Basic Cloning Vectors
I.M.A.G.E. Consortium Plasmids
pET & Duet Vectors (Novagen)
TA and GC Cloning Vectors
Coronavirus Resources
Insect Cell Vectors
pGEX Vectors (GE Healthcare)
TOPO Cloning Vectors
CRISPR Plasmids
Luciferase Vectors
Plant Vectors
Viral Expression & Packaging Vectors
Fluorescent Protein Genes & Plasmids
Lucigen Vectors
Qiagen Vectors
Yeast Plasmids
Gateway® Cloning Vectors
Mammalian Expression Vectors
Structural Genomics Vectors

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