pMCSG43 (linearized)
Linearized bacterial vector for ligation-independent cloning (LIC), encoding E. coli DsbA plus OsmY followed by an N-terminal TVMV site and C-terminal TEV-6xHis.
Sequence Author: Midwest Center for Structural Genomics
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Sticky ends from different BsaI sites may not be compatible.BsaI can be used between 37°C and 50°C. |
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The 1-base overhangs produced by AhdI may be hard to ligate. Sticky ends from different AhdI sites may not be compatible. |
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PciI is inhibited by nonionic detergents. |
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Sticky ends from different BspQI sites may not be compatible. |
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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. |
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Sticky ends from different PpuMI sites may not be compatible. |
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Sticky ends from different StyI sites may not be compatible. |
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Sticky ends from different AvaI sites may not be compatible. |
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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. |
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PaeR7I does not recognize the sequence CTCTCGAG. |
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After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility. |
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BsrGI is typically used at 37°C, but is even more active at 60°C. |
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Sticky ends from different DraIII sites may not be compatible. |
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Prolonged incubation with NdeI may lead to removal of additional nucleotides. |
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| * Blocked by Dam methylation. |
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| * Blocked by Dam methylation. |
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Efficient cleavage requires at least two copies of the SgrAI recognition sequence. |
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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. |
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Efficient cleavage requires at least two copies of the BspMI recognition sequence. Sticky ends from different BspMI sites may not be compatible. |
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The 1-base overhangs produced by EcoNI may be hard to ligate.Sticky ends from different EcoNI sites may not be compatible. |
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Sticky ends from different PflMI sites may not be compatible. |
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Sticky ends from different BstAPI sites may not be compatible. |
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| * Blocked by Dam methylation. BclI is typically used at 50-55°C, but is 50% active at 37°C. |
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ApaI can be used between 25°C and 37°C. |
| lacI 1999 .. 3081 = 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 1999 .. 3081 = 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 5270 .. 6130 = 861 bp 286 amino acids = 31.5 kDa 2 segments Segment 2: 5270 .. 6061 = 792 bp 263 amino acids = 28.9 kDa Product: β-lactamase confers resistance to ampicillin, carbenicillin, and related antibiotics |
| AmpR 5270 .. 6130 = 861 bp 286 amino acids = 31.5 kDa 2 segments Segment 1: signal sequence 6062 .. 6130 = 69 bp 23 amino acids = 2.6 kDa Product: β-lactamase confers resistance to ampicillin, carbenicillin, and related antibiotics |
| AmpR 5270 .. 6130 = 861 bp 286 amino acids = 31.5 kDa 2 segments Product: β-lactamase confers resistance to ampicillin, carbenicillin, and related antibiotics |
| 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 |
| OsmY 50 .. 652 = 603 bp 201 amino acids = 21.1 kDa Product: osmotically inducible protein OsmY from E. coli |
| OsmY 50 .. 652 = 603 bp 201 amino acids = 21.1 kDa Product: osmotically inducible protein OsmY from E. coli |
| DsbA 1190 .. 1816 = 627 bp 208 amino acids = 23.1 kDa Product: bacterial periplasmic oxidoreductase |
| DsbA 1190 .. 1816 = 627 bp 208 amino acids = 23.1 kDa Product: bacterial periplasmic oxidoreductase |
| ori 4511 .. 5099 = 589 bp high-copy-number ColE1/pMB1/pBR322/pUC origin of replication |
| ori 4511 .. 5099 = 589 bp high-copy-number ColE1/pMB1/pBR322/pUC origin of replication |
| dsbA promoter 958 .. 1189 = 232 bp promoter for the E. coli dsbA gene (Belin and Boquet, 1994) |
| dsbA promoter 958 .. 1189 = 232 bp promoter for the E. coli dsbA gene (Belin and Boquet, 1994) |
| rop 3890 .. 4081 = 192 bp 63 amino acids = 7.2 kDa Product: Rop protein, which maintains plasmids at low copy number |
| rop 3890 .. 4081 = 192 bp 63 amino acids = 7.2 kDa Product: Rop protein, which maintains plasmids at low copy number |
| AmpR promoter 6131 .. 6234 = 104 bp |
| AmpR promoter 6131 .. 6234 = 104 bp |
| lacI promoter 1921 .. 1998 = 78 bp |
| lacI promoter 1921 .. 1998 = 78 bp |
| T7 terminator 6544 .. 6591 = 48 bp transcription terminator for bacteriophage T7 RNA polymerase |
| T7 terminator 6544 .. 6591 = 48 bp transcription terminator for bacteriophage T7 RNA polymerase |
| 6xHis 6726 .. 6743 = 18 bp 6 amino acids = 840.9 Da Product: 6xHis affinity tag |
| 6xHis 6726 .. 6743 = 18 bp 6 amino acids = 840.9 Da Product: 6xHis affinity tag |
| TEV site 6750 .. 6770 = 21 bp 7 amino acids = 900.0 Da Product: tobacco etch virus (TEV) protease recognition and cleavage site |
| TEV site 6750 .. 6770 = 21 bp 7 amino acids = 900.0 Da Product: tobacco etch virus (TEV) protease recognition and cleavage site |
| lac operator 697 .. 721 = 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 697 .. 721 = 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 722 .. 740 = 19 bp promoter for bacteriophage T7 RNA polymerase |
| T7 promoter 722 .. 740 = 19 bp promoter for bacteriophage T7 RNA polymerase |
| 6xHis 6658 .. 6675 = 18 bp 6 amino acids = 840.9 Da Product: 6xHis affinity tag |
| 6xHis 6658 .. 6675 = 18 bp 6 amino acids = 840.9 Da Product: 6xHis affinity tag |
| RBS 661 .. 666 = 6 bp ribosome binding site |
| RBS 661 .. 666 = 6 bp ribosome binding site |
| ORF: 2122 .. 3081 = 960 bp ORF: 319 amino acids = 34.1 kDa |
| ORF: 2 .. 982 = 981 bp ORF: |