pCold TF

Vector for cold shock-induced expression in E. coli of a protein fused to the Trigger Factor chaperone.

Sequence Author: TaKaRa

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HomePlasmidsBasic Cloning VectorspCold TF
lac operator pCold-F (208 .. 226) TEE 6xHis thrombin site Factor Xa site NdeI (1664) Eco53kI (1671) SacI (1673) Acc65I (1675) KpnI (1679) AbsI - PaeR7I - PspXI - XhoI (1681) BamHI (1687) EcoRI (1693) HindIII (1699) SalI (1705) PstI (1715) XbaI (1717) BfuAI - BspMI (1718) pCold-R (1746 .. 1765) pCold™ TF 5769 bp
NdeI  (1664)
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.
Eco53kI  (1671)
1 site		 G A G C T C C T C G A G
SacI  (1673)
1 site		 G A G C T C C T C G A G
Acc65I  (1675)
1 site		 G G T A C C C C A T G G
KpnI  (1679)
1 site		 G G T A C C C C A T G G
AbsI  (1681)
1 site		 C C T C G A G G G G A G C T C C
PaeR7I  (1681)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1681)
1 site		 V C T C G A G B B G A G C T C V
XhoI  (1681)
1 site		 C T C G A G G A G C T C
BamHI  (1687)
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  (1693)
1 site		 G A A T T C C T T A A G
HindIII  (1699)
1 site		 A A G C T T T T C G A A
SalI  (1705)
1 site		 G T C G A C C A G C T G
PstI  (1715)
1 site		 C T G C A G G A C G T C
XbaI  (1717)
1 site		 T C T A G A A G A T C T
BfuAI  (1718)
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  (1718)
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.
pCold-F
19-mer  /  58% GC
1 binding site
208 .. 226  =  19 annealed bases
Tm  =  60°C
pCold-R
20-mer  /  50% GC
1 binding site
1746 .. 1765  =  20 annealed bases
Tm  =  56°C
TEE
256 .. 270  =  15 bp
5 amino acids  =  627.8 Da
Product: translation enhancing element for E. coli (Qing et al., 2004)
TEE
256 .. 270  =  15 bp
5 amino acids  =  627.8 Da
Product: translation enhancing element for E. coli (Qing et al., 2004)
6xHis
271 .. 288  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
271 .. 288  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
Trigger Factor
289 .. 1584  =  1296 bp
432 amino acids  =  48.2 kDa
Product: ribosome-associated chaperone from E. coli (Hoffmann et al., 2010)
Trigger Factor
289 .. 1584  =  1296 bp
432 amino acids  =  48.2 kDa
Product: ribosome-associated chaperone from E. coli (Hoffmann et al., 2010)
HRV 3C site
1594 .. 1617  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
HRV 3C site
1594 .. 1617  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
thrombin site
1627 .. 1644  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
1627 .. 1644  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
Factor Xa site
1651 .. 1662  =  12 bp
4 amino acids  =  473.5 Da
Product: Factor Xa recognition and cleavage site
Factor Xa site
1651 .. 1662  =  12 bp
4 amino acids  =  473.5 Da
Product: Factor Xa recognition and cleavage site
lacI
4524 .. 5606  =  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
4524 .. 5606  =  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
2733 .. 3593  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   2733 .. 2801  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2733 .. 3593  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   2802 .. 3593  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2733 .. 3593  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3764 .. 4352  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3764 .. 4352  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2073 .. 2528  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2073 .. 2528  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
cspA 3'UTR
1730 .. 1874  =  145 bp
3'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA 3'UTR
1730 .. 1874  =  145 bp
3'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA 5'UTR
122 .. 255  =  134 bp
5'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA 5'UTR
122 .. 255  =  134 bp
5'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
AmpR promoter
2628 .. 2732  =  105 bp
AmpR promoter
2628 .. 2732  =  105 bp
lacI promoter
5607 .. 5684  =  78 bp
lacI promoter
5607 .. 5684  =  78 bp
cspA promoter
15 .. 81  =  67 bp
promoter of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA promoter
15 .. 81  =  67 bp
promoter of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
MCS
1663 .. 1722  =  60 bp
multiple cloning site
MCS
1663 .. 1722  =  60 bp
multiple cloning site
lac operator
84 .. 100  =  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
84 .. 100  =  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:  256 .. 1725  =  1470 bp
ORF:  489 amino acids  =  54.4 kDa
ORF:  4322 .. 4741  =  420 bp
ORF:  139 amino acids  =  15.3 kDa
ORF:  2733 .. 3593  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  298 .. 1767  =  1470 bp
ORF:  489 amino acids  =  56.9 kDa
ORF:  4504 .. 4767  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5356 .. 5721  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  621 .. 1133  =  513 bp
ORF:  170 amino acids  =  18.0 kDa
ORF:  4524 .. 5483  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3197 .. 3463  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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Individual Sequences & Maps

BlueScribe
pEASY-T1 (linearized)
pJET1.2
pSMART LCAmp
CAG promoter
pEASY-T1 Simple (linearized)
pJET1.2 blunt
pSMART LCAmp (linearized)
chicken beta-actin promoter
pEASY-T3 (linearized)
pJW168
pSMART LCKan
CLIP-tag
pEASY-T5 Zero (linearized)
pKF 18k-2
pSMART LCKan (linearized)
CMV promoter
pEX-1
pKF 19k-2
pSP64 Poly(A)
GAL1 promoter
pEX-A
pKOV
pSP72
GST
pEX-C-GST
pLATE11 (linearized)
pSP73
HaloTag
pEX-C-His
pLATE31 (linearized)
pSpark Done (linearized)
HSV TK promoter
pEX-K
pLATE51 (linearized)
pSpark I (linearized)
lac operator
pEX-N-GST
pLATE52 (linearized)
pSpark II (linearized)
lac promoter
pEX-N-His
pLEXX-AK
pSpark III (linearized)
lac UV5 promoter
pEX-N-His-GST
pLPS-B (linearized)
pSpark IV (linearized)
lacZ
pEX1
pMAL-c5E
pSpark V (linearized)
lambda
pEX2
pMAL-c5G
pSpot1
LITMUS28
pEX3
pMAL-c5X
pSpot2
LITMUS28i
pEZSeq-Amp
pMAL-c5X-His
pSpot3
LITMUS29
pEZSeq-Amp (linearized)
pMAL-c6T
pSpot4
LITMUS38
pEZSeq-Kan
pMAL-p5E
pSTBlue-1
LITMUS38i
pEZSeq-Kan (linearized)
pMAL-p5G
pSTV28
LITMUS39
pEZ BAC
pMAL-p5X
pSTV29
M13mp18
pEZ BAC (BamHI linearized)
pMCS5
pT7Blue
M13mp19
pEZ BAC (HpaI linearized)
pMiniT
pT7Blue-2
MBP
pF1A T7
pMiniT (linearized)
pT7Blue-3
pACYC177
pF1K T7
pMiniT 2.0
pT7CFE1-CFtag
pACYC184
pF25A ICE T7
pMiniT 2.0 (linearized)
pT7CFE1-CGFP-HA-His
pAN7-1
pF25K ICE T7
pMMB190
pT7CFE1-CGST-HA-His
pANT7_cGST
pF3A WG (BYDV)
pMMB206
pT7CFE1-CHA
pANT7_nHA
pF3K WG (BYDV)
pMMB207
pT7CFE1-CHis
pAUR316
pFC20A HaloTag T7 SP6
pMMB208
pT7CFE1-CMyc
pBC KS(+)
pFC20K HaloTag T7 SP6
pMMB66EH
pT7CFE1-NFtag
pBC KS(-)
pFC30A His6HaloTag T7
pMMB66HE
pT7CFE1-NHA
pBC SK(+)
pFC30K His6HaloTag T7
pMMB67EH
pT7CFE1-NHA-CHis
pBC SK(-)
pFC7A (HQ)
pMMB67HE
pT7CFE1-NHis
pBeloBAC11
pFC7K (HQ)
pNEB193
pT7CFE1-NHis-GST
pBluescript II KS(+)
pFN18A HaloTag T7
pNEB206A (linearized)
pT7CFE1-NHis-GST-CHA
pBluescript II KS(-)
pFN18K HaloTag T7
pOSIP-CH
pT7CFE1-NMyc
pBluescript II SK(+)
pFN19A HaloTag T7 SP6
pOSIP-CO
pTOP Blunt V2 (linearized)
pBluescript II SK(-)
pFN19K HaloTag T7 SP6
pOSIP-CT
pTrcHis A
pBluescript KS(+)
pFN29A His6HaloTag T7
pOSIP-KC
pTrcHis B
pBluescript KS(-)
pFN29K His6HaloTag T7
pOSIP-KH
pTrcHis C
pBluescript SK(+)
pFN2A (GST)
pOSIP-KL
pTrcHis2 A
pBluescript SK(-)
pFN2K (GST)
pOSIP-KO
pTrcHis2 B
pBR322
pFN6A (HQ)
pOSIP-KP
pTrcHis2 C
pBS KS(+)
pFN6K (HQ)
pOSIP-KT
pTV 118N
pBS KS(-)
pGEM-11Zf(+)
pOSIP-TH
pTWV228
pBS SK(+)
pGEM-11Zf(-)
pOSIP-TO
pTXB1
pBS SK(-)
pGEM-3Z
pOSIP-TT
pTYB21
pBS(+)
pGEM-3Zf(+)
pPCR-Script Amp SK(+)
pType IIs
pBS(-)
pGEM-3Zf(-)
pPCR-Script Cam SK(+)
pTZ19R
pBSK(+) Simple-Amp
pGEM-4Z
pPTR I
pTZ19U
pBSK(+) Simple-Kan
pGEM-5Zf(+)
pPTR II
pTZ57R
pcDNA3
pGEM-7Zf(+)
pRACE (linearized)
pUAST
pcDNA3.1(+)
pGEM-7Zf(-)
pRANGER-BTB-1
pUC118
pcDNA3.1(-)
pGEM-9Zf(-)
pRANGER-BTB-2
pUC119
pCold I
pGEM-T
pRANGER-BTB-3
pUC18
pCold II
pGEM-T (linearized)
pRANGER-BTB-5
pUC19
pCold III
pGEM-T Easy
pRHA-109
pUC19L
pCold IV
pGEM-T Easy (linearized)
pRHA-113
pUC57
pCold TF
pGGA
pRHA-67
pUC57-Kan
pCR-XL-2-TOPO
pGGAselect
pRSET A
pUC57-Simple
pCR-XL-2-TOPO (linearized)
PGK promoter
pRSET B
pUCmu
pDD
pGS-21a
pRSET C
pVLT31
pDNR-Dual
pH6HTC His6HaloTag T7
pSB1C3
pVLT33
pDNR-LIB
pH6HTN His6HaloTag T7
pSC101
pVLT35
pE-SUMOpro Amp
pHP45(omega)
pSEAP2-Basic
pYES1L
pE-SUMOpro Kan
pHP45(omega)-Km
pSF-Core
pZErO-2
pE-SUMOpro3 Amp
pHP45(omega)-Tc
pSMART BAC v2.0
SNAP-tag
pE-SUMOpro3 Kan
pHSG298
pSMART BAC v2.0 (BamHI linearized)
SP6 promoter
pE-SUMOstar Amp
pHSG299
pSMART BAC v2.0 (EcoRI linearized)
SV40 promoter
pE-SUMOstar Kan
pHSG396
pSMART BAC v2.0 (HindIII linearized)
T3 promoter
pEASY-Blunt (linearized)
pHSG398
pSMART FOS v2.0 (linearized)
T5 promoter
pEASY-Blunt E1 (linearized)
pHY300PLK
pSMART HCAmp
T7 promoter
pEASY-Blunt E2 (linearized)
pICOz
pSMART HCAmp (linearized)
tac promoter
pEASY-Blunt Simple (linearized)
pInvRecA
pSMART HCKan
trc promoter
pEASY-Blunt Zero (linearized)
pJAZZ-OC
pSMART HCKan (linearized)
phiX174
pEASY-Blunt3 (linearized)
pJAZZ-OK

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