pcDNA6.2 V5 GW D-TOPO
Mammalian directional TOPO® cloning vector with a blasticidin resistance gene and a C-terminal V5 tag, for creating a Gateway® expression clone.
Sequence Author: Thermo Fisher (Invitrogen)
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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 BsmI sites may not be compatible. |
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Sticky ends from different PfoI sites may not be compatible. |
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Sticky ends from different BlpI sites may not be compatible. |
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| * Blocked by Dam methylation. |
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Efficient cleavage requires at least two copies of the BsgI recognition sequence. Sticky ends from different BsgI sites may not be compatible.For full activity, add fresh S-adenosylmethionine (SAM). |
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Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present. This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.Sticky ends from different Bpu10I sites may not be compatible. |
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Prolonged incubation with NdeI may lead to removal of additional nucleotides. |
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BssHII is typically used at 50°C, but is 75% active at 37°C. |
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| * Blocked by Dam methylation. |
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ApaI can be used between 25°C and 37°C. |
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Sticky ends from different CsiI sites may not be compatible. |
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| * Blocked by Dcm methylation. Sticky ends from different SexAI 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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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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| * 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 BbsI sites may not be compatible.BbsI gradually loses activity when stored at -20°C. |
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Sticky ends from different PpuMI sites may not be compatible. |
| AmpR 3211 .. 4071 = 861 bp 286 amino acids = 31.6 kDa 2 segments Segment 2: 3211 .. 4002 = 792 bp 263 amino acids = 28.9 kDa Product: β-lactamase confers resistance to ampicillin, carbenicillin, and related antibiotics |
| AmpR 3211 .. 4071 = 861 bp 286 amino acids = 31.6 kDa 2 segments Segment 1: signal sequence 4003 .. 4071 = 69 bp 23 amino acids = 2.6 kDa Product: β-lactamase confers resistance to ampicillin, carbenicillin, and related antibiotics |
| AmpR 3211 .. 4071 = 861 bp 286 amino acids = 31.6 kDa 2 segments Product: β-lactamase confers resistance to ampicillin, carbenicillin, and related antibiotics |
| ori 2455 .. 3040 = 586 bp high-copy-number ColE1/pMB1/pBR322/pUC origin of replication |
| ori 2455 .. 3040 = 586 bp high-copy-number ColE1/pMB1/pBR322/pUC origin of replication |
| f1 ori 442 .. 870 = 429 bp f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis |
| f1 ori 442 .. 870 = 429 bp f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis |
| BSD 1327 .. 1725 = 399 bp 132 amino acids = 13.7 kDa Product: blasticidin S deaminase confers resistance to blasticidin |
| BSD 1327 .. 1725 = 399 bp 132 amino acids = 13.7 kDa Product: blasticidin S deaminase confers resistance to blasticidin |
| CMV enhancer 4442 .. 4821 = 380 bp human cytomegalovirus immediate early enhancer |
| CMV enhancer 4442 .. 4821 = 380 bp human cytomegalovirus immediate early enhancer |
| SV40 promoter 884 .. 1213 = 330 bp SV40 enhancer and early promoter |
| SV40 promoter 884 .. 1213 = 330 bp SV40 enhancer and early promoter |
| CMV promoter 4822 .. 5025 = 204 bp human cytomegalovirus (CMV) immediate early promoter |
| CMV promoter 4822 .. 5025 = 204 bp human cytomegalovirus (CMV) immediate early promoter |
| SV40 poly(A) signal 1883 .. 2004 = 122 bp SV40 polyadenylation signal |
| SV40 poly(A) signal 1883 .. 2004 = 122 bp SV40 polyadenylation signal |
| AmpR promoter 4072 .. 4176 = 105 bp |
| AmpR promoter 4072 .. 4176 = 105 bp |
| HSV TK poly(A) signal 192 .. 240 = 49 bp herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985) |
| HSV TK poly(A) signal 192 .. 240 = 49 bp herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985) |
| EM7 promoter 1261 .. 1308 = 48 bp synthetic bacterial promoter |
| EM7 promoter 1261 .. 1308 = 48 bp synthetic bacterial promoter |
| V5 tag 67 .. 108 = 42 bp 14 amino acids = 1.4 kDa Product: epitope tag from simian virus 5 |
| V5 tag 67 .. 108 = 42 bp 14 amino acids = 1.4 kDa Product: epitope tag from simian virus 5 |
| attB2 17 .. 41 = 25 bp recombination site for the Gateway® BP reaction |
| attB2 17 .. 41 = 25 bp recombination site for the Gateway® BP reaction |
| attB1 5118 .. 5142 = 25 bp recombination site for the Gateway® BP reaction |
| attB1 5118 .. 5142 = 25 bp recombination site for the Gateway® BP reaction |
| T7 promoter 5070 .. 5088 = 19 bp promoter for bacteriophage T7 RNA polymerase |
| T7 promoter 5070 .. 5088 = 19 bp promoter for bacteriophage T7 RNA polymerase |
| SV40 ori 1064 .. 1199 = 136 bp SV40 origin of replication |
| SV40 ori 1064 .. 1199 = 136 bp SV40 origin of replication |
| ORF: 1327 .. 1725 = 399 bp ORF: 132 amino acids = 13.7 kDa |
| ORF: 3341 .. 3607 = 267 bp |