pVP33A

Bacterial Flexi® Vector with an ampicillin resistance marker, for appending an N-terminal 8xHis-MBP-HRV 3C-tetracysteine-TEV cassette to an expressed protein.

Sequence Author: Center for Eukaryotic Structural Genomics

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HomePlasmidsStructural Genomics VectorspVP33A
PaeR7I - XhoI (37) XmnI (6871) BglI (6392) AhdI (6272) PciI (5379) BspQI - SapI (5263) NdeI (5202) BstZ17I (5152) PflFI - Tth111I (5126) FspAI (4959) PpuMI * (4942) BstEII (4307) EcoRV (4043) HpaI (3987) PluTI (3854) SfoI (3852) NarI * (3851) T5 promoter AanI (85) MfeI (95) lac operator RBS BseRI (151) ATG NsiI (183) BsiWI (474) AanI (536) BglII (543) BmgBI (726) BsaBI (859) AfeI (1094) PacI (1289) tetracysteine tag AsiSI - SgfI (1358) PmeI (2484) Eco53kI (2497) SacI (2499) PstI - SbfI (2532) SphI (2538) BsrGI (2641) AscI (2658) SgrDI (2673) BspDI - ClaI (2686) BstBI (2697) HindIII (2700) NheI (2818) BmtI (2822) KasI (3850) pVP33A 7228 bp
PaeR7I  (37)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (37)
1 site		 C T C G A G G A G C T C
XmnI  (6871)
1 site		 G A A N N N N T T C C T T N N N N A A G
BglI  (6392)
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.
AhdI  (6272)
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.
PciI  (5379)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
BspQI  (5263)
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  (5263)
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.
NdeI  (5202)
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  (5152)
1 site		 G T A T A C C A T A T G
PflFI  (5126)
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  (5126)
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.
FspAI  (4959)
1 site		 R T G C G C A Y Y A C G C G T R
PpuMI  (4942)
1 site		 R G G W C C Y Y C C W G G R
* Blocked by Dcm methylation.
Sticky ends from different PpuMI sites may not be compatible.
BstEII  (4307)
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.
EcoRV  (4043)
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  (3987)
1 site		 G T T A A C C A A T T G
PluTI  (3854)
1 site		 G G C G C C C C G C G G
Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SfoI  (3852)
1 site		 G G C G C C C C G C G G
NarI  (3851)
1 site		 G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
AanI  (85)
2 sites		 T T A T A A A A T A T T
MfeI  (95)
1 site		 C A A T T G G T T A A C
BseRI  (151)
1 site		 G A G G A G ( N ) 8 N N C T C C T C ( N ) 8
Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
NsiI  (183)
1 site		 A T G C A T T A C G T A
BsiWI  (474)
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.
AanI  (536)
2 sites		 T T A T A A A A T A T T
BglII  (543)
1 site		 A G A T C T T C T A G A
BmgBI  (726)
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.
BsaBI  (859)
1 site		 G A T N N N N A T C C T A N N N N T A G
AfeI  (1094)
1 site		 A G C G C T T C G C G A
PacI  (1289)
1 site		 T T A A T T A A A A T T A A T T
AsiSI  (1358)
1 site		 G C G A T C G C C G C T A G C G
SgfI  (1358)
1 site		 G C G A T C G C C G C T A G C G
PmeI  (2484)
1 site		 G T T T A A A C C A A A T T T G
Eco53kI  (2497)
1 site		 G A G C T C C T C G A G
SacI  (2499)
1 site		 G A G C T C C T C G A G
PstI  (2532)
1 site		 C T G C A G G A C G T C
SbfI  (2532)
1 site		 C C T G C A G G G G A C G T C C
SphI  (2538)
1 site		 G C A T G C C G T A C G
BsrGI  (2641)
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  (2658)
1 site		 G G C G C G C C C C G C G C G G
SgrDI  (2673)
1 site		 C G T C G A C G G C A G C T G C
BspDI  (2686)
1 site		 A T C G A T T A G C T A
ClaI  (2686)
1 site		 A T C G A T T A G C T A
BstBI  (2697)
1 site		 T T C G A A A A G C T T
HindIII  (2700)
1 site		 A A G C T T T T C G A A
NheI  (2818)
1 site		 G C T A G C C G A T C G
BmtI  (2822)
1 site		 G C T A G C C G A T C G
KasI  (3850)
1 site		 G G C G C C C C G C G G
ATG
151 .. 153  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
151 .. 153  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
8xHis
157 .. 180  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
157 .. 180  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
MBP
187 .. 1284  =  1098 bp
366 amino acids  =  40.2 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
187 .. 1284  =  1098 bp
366 amino acids  =  40.2 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.
HRV 3C site
1294 .. 1317  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
HRV 3C site
1294 .. 1317  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
tetracysteine tag
1318 .. 1335  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical labeling reagents
tetracysteine tag
1318 .. 1335  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical labeling reagents
TEV site
1336 .. 1356  =  21 bp
7 amino acids  =  884.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
1336 .. 1356  =  21 bp
7 amino acids  =  884.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
lacI
3762 .. 4844  =  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
3762 .. 4844  =  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).
AmpR
6199 .. 7059  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   6199 .. 6990  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6199 .. 7059  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   6991 .. 7059  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6199 .. 7059  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
1820 .. 2479  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1820 .. 2479  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5440 .. 6028  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5440 .. 6028  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
barnase
1385 .. 1720  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
barnase
1385 .. 1720  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
AmpR promoter
7060 .. 7164  =  105 bp
AmpR promoter
7060 .. 7164  =  105 bp
lambda t0 terminator
2719 .. 2813  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
2719 .. 2813  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
3583 .. 3669  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
3583 .. 3669  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacIq promoter
4845 .. 4922  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
lacIq promoter
4845 .. 4922  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
T5 promoter
46 .. 90  =  45 bp
4 segments
   Segment 1:  
   46 .. 60  =  15 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
4 segments
   Segment 2:  -35  
   61 .. 66  =  6 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
4 segments
   Segment 3:  
   67 .. 83  =  17 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
4 segments
   Segment 4:  -10  
   84 .. 90  =  7 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
4 segments
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
lac UV5 promoter
1736 .. 1766  =  31 bp
3 segments
   Segment 1:  -35  
   1736 .. 1741  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1736 .. 1766  =  31 bp
3 segments
   Segment 2:  
   1742 .. 1759  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1736 .. 1766  =  31 bp
3 segments
   Segment 3:  -10  
   1760 .. 1766  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1736 .. 1766  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
lac operator
98 .. 114  =  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
98 .. 114  =  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
137 .. 142  =  6 bp
ribosome binding site
RBS
137 .. 142  =  6 bp
ribosome binding site
lac operator
66 .. 82  =  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
66 .. 82  =  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:  151 .. 1371  =  1221 bp
ORF:  406 amino acids  =  44.8 kDa
ORF:  2857 .. 3099  =  243 bp
ORF:  80 amino acids  =  9.5 kDa
ORF:  1385 .. 1720  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  1820 .. 2479  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  3728 .. 3979  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  6329 .. 6595  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3282 .. 3518  =  237 bp
ORF:  78 amino acids  =  8.9 kDa
ORF:  3742 .. 4005  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4594 .. 4911  =  318 bp
ORF:  105 amino acids  =  11.2 kDa
ORF:  6199 .. 7059  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3762 .. 4721  =  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

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Fluorescent Protein Genes & Plasmids
Lucigen Vectors
Qiagen Vectors
Yeast Plasmids
Gateway® Cloning Vectors
Mammalian Expression Vectors
Structural Genomics Vectors

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