pMCSG71 (linearized)

Linearized compact bacterial vector for ligation-independent cloning (LIC), encoding N-terminal MBP-TVMV and C-terminal TEV-6xHis plus tRNA genes for rare Arg and Ile codons.

Sequence Author: Midwest Center for Structural Genomics

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HomePlasmidsStructural Genomics VectorspMCSG71 (linearized)
6xHis ScaI (4990) FspI (4732) BglI (4630) AhdI (4510) DrdI (3725) PciI (3617) BspQI - SapI (3501) BstZ17I (3388) PshAI (3362) BsmBI - Esp3I (3132) HpaI (3023) EcoRV (2967) ApaI (2728) PspOMI (2724) BstEII (2698) AvaI - BsoBI - PaeR7I - PspXI - XhoI (5843) EagI - NotI (5851) HindIII (5858) SalI (5864) Eco53kI (5873) SacI (5875) EcoRI (5877) BamHI (5883) 6xHis TEV site End (5930) Start (0) TVMV site BsrGI (43) BsmI (546) BmgBI (622) BglII (801) BsiWI (870) NdeI (1164) RBS XbaI (1202) T7 promoter BspDI * - ClaI * (1271) AgeI (1313) AscI (1530) SbfI (1541) BstBI (1546) MreI (1678) EcoNI (2052) PflMI (2099) MluI (2517) pMCSG71 5930 bp
ScaI  (4990)
1 site		 A G T A C T T C A T G A
FspI  (4732)
1 site		 T G C G C A A C G C G T
BglI  (4630)
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  (4510)
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.
DrdI  (3725)
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.
PciI  (3617)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
BspQI  (3501)
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  (3501)
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.
BstZ17I  (3388)
1 site		 G T A T A C C A T A T G
PshAI  (3362)
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.
BsmBI  (3132)
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  (3132)
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.
HpaI  (3023)
1 site		 G T T A A C C A A T T G
EcoRV  (2967)
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.
ApaI  (2728)
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  (2724)
1 site		 G G G C C C C C C G G G
BstEII  (2698)
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.
AvaI  (5843)
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  (5843)
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  (5843)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5843)
1 site		 V C T C G A G B B G A G C T C V
XhoI  (5843)
1 site		 C T C G A G G A G C T C
EagI  (5851)
1 site		 C G G C C G G C C G G C
NotI  (5851)
1 site		 G C G G C C G C C G C C G G C G
HindIII  (5858)
1 site		 A A G C T T T T C G A A
SalI  (5864)
1 site		 G T C G A C C A G C T G
Eco53kI  (5873)
1 site		 G A G C T C C T C G A G
SacI  (5875)
1 site		 G A G C T C C T C G A G
EcoRI  (5877)
1 site		 G A A T T C C T T A A G
BamHI  (5883)
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.
End  (5930)
0 sites
Start  (0)
0 sites
BsrGI  (43)
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.
BsmI  (546)
1 site		 G A A T G C N C T T A C G N
Sticky ends from different BsmI sites may not be compatible.
BmgBI  (622)
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.
BglII  (801)
1 site		 A G A T C T T C T A G A
BsiWI  (870)
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.
NdeI  (1164)
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.
XbaI  (1202)
1 site		 T C T A G A A G A T C T
BspDI  (1271)
1 site		 A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1271)
1 site		 A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (1313)
1 site		 A C C G G T T G G C C A
AscI  (1530)
1 site		 G G C G C G C C C C G C G C G G
SbfI  (1541)
1 site		 C C T G C A G G G G A C G T C C
BstBI  (1546)
1 site		 T T C G A A A A G C T T
MreI  (1678)
1 site		 C G C C G G C G G C G G C C G C
EcoNI  (2052)
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.
PflMI  (2099)
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.
MluI  (2517)
1 site		 A C G C G T T G C G C A
TVMV site
8 .. 28  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
8 .. 28  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
MBP
65 .. 1165  =  1101 bp
367 amino acids  =  40.3 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
65 .. 1165  =  1101 bp
367 amino acids  =  40.3 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.
lacI
2167 .. 3249  =  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
2167 .. 3249  =  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
4437 .. 5297  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   4437 .. 5228  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4437 .. 5297  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   5229 .. 5297  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4437 .. 5297  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3678 .. 4266  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3678 .. 4266  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
5298 .. 5401  =  104 bp
AmpR promoter
5298 .. 5401  =  104 bp
lacI promoter
2089 .. 2166  =  78 bp
lacI promoter
2089 .. 2166  =  78 bp
argU
1528 .. 1604  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
1528 .. 1604  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
1874 .. 1949  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
1874 .. 1949  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
T7 terminator
5711 .. 5758  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
5711 .. 5758  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
6xHis
5893 .. 5910  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5893 .. 5910  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TEV site
5917 .. 5937  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
5917 .. 5937  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
lac operator
1210 .. 1234  =  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
1210 .. 1234  =  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
1235 .. 1253  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1235 .. 1253  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
5825 .. 5842  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5825 .. 5842  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
1174 .. 1179  =  6 bp
ribosome binding site
RBS
1174 .. 1179  =  6 bp
ribosome binding site
ORF:  1663 .. 1917  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  2290 .. 3249  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  4567 .. 4833  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2 .. 826  =  825 bp
ORF:  274 amino acids  =  31.8 kDa  (no start codon)
ORF:  1046 .. 1378  =  333 bp
ORF:  110 amino acids  =  12.3 kDa
ORF:  1950 .. 2417  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  3006 .. 3269  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4437 .. 5297  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2 .. 1165  =  1164 bp
ORF:  388 amino acids  =  42.6 kDa
ORF:  3032 .. 3283  =  252 bp
ORF:  83 amino acids  =  9.1 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

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