pMCSG76 (linearized)

Linearized bacterial vector with a CloDF13 origin for ligation-independent cloning (LIC), with a 6xHis-TEV leader, a second LIC site, and tRNA genes for rare Arg and Ile codons.

Sequence Author: Midwest Center for Structural Genomics

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EcoNI (4029) PfoI (3993) PflMI (3986) AclI (3901) BstAPI (3885) MluI (3561) ApaI (3358) PspOMI (3354) HpaI (3059) BsmBI - Esp3I (2946) PciI (1941) BmtI (1845) NheI (1841) BspEI * (4037) T7 terminator PaeR7I - PspXI - XhoI (4193) EagI - NotI (4201) HindIII (4208) SalI (4214) AccI (4215) Eco53kI (4223) SacI (4225) EcoRI (4227) TspMI - XmaI (4267) SmaI (4269) RBS End (4295) Start (0) TEV site Acc65I (25) KpnI (29) BglII (32) 6xHis ATG NdeI (68) RBS T7 promoter BspDI * - ClaI * (175) AgeI (217) XmnI (286) AscI (434) PstI - SbfI (445) BstBI (450) MreI (582) AfeI (668) NcoI (852) PsiI (867) PflFI - Tth111I (901) BsaAI (990) BglI (1118) PvuI (1368) pMCSG76 4295 bp
EcoNI  (4029)
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.
PfoI  (3993)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
PflMI  (3986)
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.
AclI  (3901)
1 site
A A C G T T T T G C A A
BstAPI  (3885)
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  (3561)
1 site
A C G C G T T G C G C A
ApaI  (3358)
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  (3354)
1 site
G G G C C C C C C G G G
HpaI  (3059)
1 site
G T T A A C C A A T T G
BsmBI  (2946)
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  (2946)
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.
PciI  (1941)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BmtI  (1845)
1 site
G C T A G C C G A T C G
NheI  (1841)
1 site
G C T A G C C G A T C G
BspEI  (4037)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
PaeR7I  (4193)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4193)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4193)
1 site
C T C G A G G A G C T C
EagI  (4201)
1 site
C G G C C G G C C G G C
NotI  (4201)
1 site
G C G G C C G C C G C C G G C G
HindIII  (4208)
1 site
A A G C T T T T C G A A
SalI  (4214)
1 site
G T C G A C C A G C T G
AccI  (4215)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
Eco53kI  (4223)
1 site
G A G C T C C T C G A G
SacI  (4225)
1 site
G A G C T C C T C G A G
EcoRI  (4227)
1 site
G A A T T C C T T A A G
TspMI  (4267)
1 site
C C C G G G G G G C C C
XmaI  (4267)
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  (4269)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
End  (4295)
0 sites
Start  (0)
0 sites
Acc65I  (25)
1 site
G G T A C C C C A T G G
KpnI  (29)
1 site
G G T A C C C C A T G G
BglII  (32)
1 site
A G A T C T T C T A G A
NdeI  (68)
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.
BspDI  (175)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (175)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (217)
1 site
A C C G G T T G G C C A
XmnI  (286)
1 site
G A A N N N N T T C C T T N N N N A A G
AscI  (434)
1 site
G G C G C G C C C C G C G C G G
PstI  (445)
1 site
C T G C A G G A C G T C
SbfI  (445)
1 site
C C T G C A G G G G A C G T C C
BstBI  (450)
1 site
T T C G A A A A G C T T
MreI  (582)
1 site
C G C C G G C G G C G G C C G C
AfeI  (668)
1 site
A G C G C T T C G C G A
NcoI  (852)
1 site
C C A T G G G G T A C C
PsiI  (867)
1 site
T T A T A A A A T A T T
PflFI  (901)
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  (901)
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.
BsaAI  (990)
1 site
Y A C G T R R T G C A Y
BglI  (1118)
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.
PvuI  (1368)
1 site
C G A T C G G C T A G C
lacI
2834 .. 3916  =  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
2834 .. 3916  =  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).
SmR
955 .. 1746  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase (Murphy, 1985)
confers resistance to spectinomycin and streptomycin
SmR
955 .. 1746  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase (Murphy, 1985)
confers resistance to spectinomycin and streptomycin
CloDF13 ori
1886 .. 2624  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins.
CloDF13 ori
1886 .. 2624  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins.
AmpR promoter
1747 .. 1838  =  92 bp
AmpR promoter
1747 .. 1838  =  92 bp
lacI promoter
3917 .. 3994  =  78 bp
lacI promoter
3917 .. 3994  =  78 bp
argU
432 .. 508  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
432 .. 508  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
778 .. 853  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
778 .. 853  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
6xHis
49 .. 66  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
49 .. 66  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
67 .. 69  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
67 .. 69  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
T7 terminator
4061 .. 4108  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
4061 .. 4108  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
114 .. 138  =  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
114 .. 138  =  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
139 .. 157  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
139 .. 157  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
4175 .. 4192  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4175 .. 4192  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
78 .. 83  =  6 bp
ribosome binding site
RBS
78 .. 83  =  6 bp
ribosome binding site
RBS
4278 .. 4283  =  6 bp
ribosome binding site
RBS
4278 .. 4283  =  6 bp
ribosome binding site
ORF:  1 .. 282  =  282 bp
ORF:  93 amino acids  =  10.4 kDa  (no start codon)
ORF:  1324 .. 1707  =  384 bp
ORF:  127 amino acids  =  14.6 kDa
ORF:  2800 .. 3051  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  1169 .. 1447  =  279 bp
ORF:  92 amino acids  =  9.4 kDa
ORF:  1457 .. 1714  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
ORF:  567 .. 821  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  2814 .. 3077  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  3666 .. 4031  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  2834 .. 3793  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  955 .. 1746  =  792 bp
ORF:  263 amino acids  =  29.3 kDa
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