pNIC28-Bsa4
Bacterial vector encoding an N-terminal 6xHis-TEV cassette and kanamycin resistance plus a SacB negative selection marker, for purification of recombinant proteins.
Sequence Author: Structural Genomics Consortium
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Efficient cleavage requires at least two copies of the SgrAI recognition sequence. |
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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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Sticky ends from different BstEII sites may not be compatible.BstEII is typically used at 60°C, but is 50% active at 37°C. |
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Efficient cleavage requires at least two copies of the NmeAIII recognition sequence. Sticky ends from different NmeAIII sites may not be compatible.For full activity, add fresh S-adenosylmethionine (SAM). |
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ApaI can be used between 25°C and 37°C. |
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BssHII is typically used at 50°C, but is 75% active at 37°C. |
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Sticky ends from different BglI sites may not be compatible. |
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Sticky ends from different PpuMI sites may not be compatible. |
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The 1-base overhangs produced by PflFI may be hard to ligate.Sticky ends from different PflFI sites may not be compatible. |
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The 1-base overhangs produced by Tth111I may be hard to ligate.Sticky ends from different Tth111I sites may not be compatible. |
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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 AlwNI sites may not be compatible. |
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Prolonged incubation with NdeI may lead to removal of additional nucleotides. |
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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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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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| * Blocked by Dcm methylation. |
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BsrGI is typically used at 37°C, but is even more active at 60°C. |
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Efficient cleavage requires at least two copies of the SacII recognition sequence. |
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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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After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility. |
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PaeR7I does not recognize the sequence CTCTCGAG. |
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Sticky ends from different BlpI sites may not be compatible. |
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Sticky ends from different DraIII sites may not be compatible. |
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Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present. |
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SmaI can be used at 37°C for brief incubations. |
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| SacB 616 .. 2037 = 1422 bp 473 amino acids = 53.0 kDa 2 segments Segment 1: signal peptide 616 .. 702 = 87 bp 29 amino acids = 3.0 kDa Product: secreted levansucrase that renders bacterial growth sensitive to sucrose negative selection marker |
| SacB 616 .. 2037 = 1422 bp 473 amino acids = 53.0 kDa 2 segments Segment 2: 703 .. 2037 = 1335 bp 444 amino acids = 50.0 kDa Product: secreted levansucrase that renders bacterial growth sensitive to sucrose negative selection marker |
| SacB 616 .. 2037 = 1422 bp 473 amino acids = 53.0 kDa 2 segments Product: secreted levansucrase that renders bacterial growth sensitive to sucrose negative selection marker |
| lacI 5816 .. 6898 = 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 5816 .. 6898 = 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). |
| KanR 2861 .. 3676 = 816 bp 271 amino acids = 31.0 kDa Product: aminoglycoside phosphotransferase confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes |
| KanR 2861 .. 3676 = 816 bp 271 amino acids = 31.0 kDa Product: aminoglycoside phosphotransferase confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes |
| ori 3798 .. 4386 = 589 bp high-copy-number ColE1/pMB1/pBR322/pUC origin of replication |
| ori 3798 .. 4386 = 589 bp high-copy-number ColE1/pMB1/pBR322/pUC origin of replication |
| f1 ori 2313 .. 2768 = 456 bp f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis |
| f1 ori 2313 .. 2768 = 456 bp f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis |
| sacB promoter 170 .. 615 = 446 bp sacB promoter and control region |
| sacB promoter 170 .. 615 = 446 bp sacB promoter and control region |
| rop 4816 .. 5007 = 192 bp 63 amino acids = 7.2 kDa Product: Rop protein, which maintains plasmids at low copy number |
| rop 4816 .. 5007 = 192 bp 63 amino acids = 7.2 kDa Product: Rop protein, which maintains plasmids at low copy number |
| lacI promoter 6899 .. 6976 = 78 bp |
| lacI promoter 6899 .. 6976 = 78 bp |
| ATG 89 .. 91 = 3 bp 1 amino acid = 149.2 Da |
| ATG 89 .. 91 = 3 bp 1 amino acid = 149.2 Da |
| 6xHis 92 .. 109 = 18 bp 6 amino acids = 840.9 Da Product: 6xHis affinity tag |
| 6xHis 92 .. 109 = 18 bp 6 amino acids = 840.9 Da Product: 6xHis affinity tag |
| TEV site 134 .. 154 = 21 bp 7 amino acids = 900.0 Da Product: tobacco etch virus (TEV) protease recognition and cleavage site |
| TEV site 134 .. 154 = 21 bp 7 amino acids = 900.0 Da Product: tobacco etch virus (TEV) protease recognition and cleavage site |
| T7 terminator 2229 .. 2276 = 48 bp transcription terminator for bacteriophage T7 RNA polymerase |
| T7 terminator 2229 .. 2276 = 48 bp transcription terminator for bacteriophage T7 RNA polymerase |
| lac operator 20 .. 44 = 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 20 .. 44 = 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 1 .. 19 = 19 bp promoter for bacteriophage T7 RNA polymerase |
| T7 promoter 1 .. 19 = 19 bp promoter for bacteriophage T7 RNA polymerase |
| 6xHis 2145 .. 2162 = 18 bp 6 amino acids = 840.9 Da Product: 6xHis affinity tag |
| 6xHis 2145 .. 2162 = 18 bp 6 amino acids = 840.9 Da Product: 6xHis affinity tag |
| RBS 75 .. 80 = 6 bp ribosome binding site |
| RBS 75 .. 80 = 6 bp ribosome binding site |