p9D35s-C9D10A-U6GmNhe E734
Ligation of D029p9ioR-U10CasD10A* into E733p9D35s-C9wt-U6GmNhe*
- Promoter 1
- 35s
- Gene 1
- nptII
- Gene 2
- Cas9 nick
- Features
- dicots, U6Gm
- Other markers
- NptIIo, strep/spec
- Size
- 13,029 bp, circular
- Record date
- 26-SEP-2024
Construct
- 35S–NptIIo · E9 · selection cassette, 169–1,898
- Pro-Ubq10At–D10A · NOS-T · payload cassette, 1,924–7,882
- U6Gm–gRNA · gRNA cassette, 7,892–8,306
- SfiI ×2 · flanks 2 cassettes · at 1,916, 8,321
- BsaI Golden Gate · overhangs GGAT, GTTT · at 8,191, 8,223
- T-DNA borders · LB, RB
Read out of the GenBank file; compare every vector in the multiple expression cassettes and cloning sites table.
Files
Order
Vectors from the list cost 250 €, Cas9 vectors 400 €, net. Send the vector name to info@dna-cloning.com and we ship on invoice.
Restriction sites
| Unique cutter | Site | Cuts at | Location |
|---|---|---|---|
| XhoI (PaeR7I) | C^TCGAG | 587 | between 35S and NptIIo |
| FseI | GGCCGG^CC | 1,254 | in NptIIo |
| MluI | A^CGCGT | 1,598 | between NptIIo and E9 |
| SbfI (SdaI) | CCTGCA^GG | 1,908 | between E9 and Pro-Ubq10At |
| NotI | GC^GGCCGC | 1,917 | between E9 and Pro-Ubq10At |
| SalI | G^TCGAC | 2,762 | in Pro-Ubq10At |
| SpeI | A^CTAGT | 3,400 | between Pro-Ubq10At and D10A' |
| BamHI | G^GATCC | 3,406 | between Pro-Ubq10At and D10A' |
| SmaI (XmaI, TspMI) | CCC^GGG | 7,626 | between D10A' and NOS-T |
| NsiI (Mph1103I) | ATGCA^T | 7,749 | in NOS-T |
| BsrGI (Bsp1407I) | T^GTACA | 8,036 | in U6Gm |
| BsmBI (Esp3I) | CGTCTC(1/5) | 8,049 | in U6Gm |
| BstBI (Bsp119I) | TT^CGAA | 8,204 | in targetRNA |
| HindIII | A^AGCTT | 8,207 | in targetRNA |
| NheI (BmtI) | G^CTAGC | 8,307 | between gRNA and Right Border Region |
| KpnI (Acc65I) | GGTAC^C | 8,344 | between gRNA and Right Border Region |
| PmeI (MssI) | GTTT^AAAC | 8,376 | in Right Border Region |
Cut twice: EcoRI (2,584, 7,886), PstI (1,908, 7,930), SphI (6,735, 8,501), SacI (1,115, 3,017), NcoI (3,453, 4,891), ApaI (688, 8,338), SfiI (1,916, 8,321), MfeI (14, 1,008), EagI (1,917, 8,758), SacII (3,963, 6,060), AatII (4,010, 8,767), BstEII (1,357, 12,179), BsaI (8,191, 8,223), SapI (3,022, 4,622), AarI (1,078, 1,199).
No site: NdeI, ClaI, AscI, PacI, SwaI, AvrII, BsiWI, AgeI, HpaI.
Computed from the GenBank sequence for 50 common enzymes; positions are the cut in the top strand, without Dam/Dcm methylation. Compare vectors in the restriction site finder.
Annotated features
| Feature | Type | Start | End | Length | Strand |
|---|---|---|---|---|---|
| LB' | T-DNA border misc_feature | 1 | 24 | 24 | + |
| 35S | Promoter misc_feature | 169 | 584 | 416 | + |
| NptIIo | Selection marker CDS | 605 | 903 | 299 | + |
| 'intron | Coding sequence CDS | 904 | 1,090 | 187 | + |
| NptIIo | Selection marker CDS | 1,091 | 1,594 | 504 | + |
| 'E9 | Terminator misc_feature | 1,608 | 1,898 | 291 | + |
| Pro-Ubq10At | Promoter misc_feature | 1,924 | 3,399 | 1,476 | + |
| Ubi cds 5` | Coding sequence CDS | 3,028 | 3,091 | 64 | + |
| íntron | Other misc_feature | 3,094 | 3,397 | 304 | + |
| D10A' | Coding sequence CDS | 3,421 | 7,623 | 4,203 | + |
| NLS | Site misc_feature | 3,427 | 3,447 | 21 | + |
| D10A | Site misc_feature | 3,497 | 3,497 | 1 | + |
| NLS | Site misc_feature | 7,573 | 7,620 | 48 | + |
| NOS-T | Terminator misc_feature | 7,629 | 7,882 | 254 | + |
| U6Gm | Promoter misc_feature | 7,892 | 8,196 | 305 | + |
| targetRNA | Site CDS | 8,197 | 8,223 | 27 | + |
| hairpin | Site CDS | 8,224 | 8,253 | 30 | + |
| gRNA | Site misc_feature | 8,224 | 8,306 | 83 | + |
| Right Border Region | Other misc_feature | 8,347 | 8,949 | 603 | + |
| RB | T-DNA border misc_feature | 8,392 | 8,415 | 24 | + |
| 'ColE1'' | Origin / backbone misc_feature | 8,963 | 9,621 | 659 | + |
| ''pVS1 ORI''' | Origin / backbone misc_feature | 9,650 | 11,613 | 1,964 | + |
| ParB | Coding sequence CDS | 9,976 | 11,151 | 1,176 | + |
| strep/spec | Selection marker CDS | 11,802 | 12,812 | 1,011 | + |
| Left Border Region | T-DNA border misc_feature | 12,880 | 163 | 0 | + |
PromoterCoding sequenceTerminatorSelection markerOrigin / backboneT-DNA borderSite
Feature types are inferred from the labels in the GenBank file; the file itself is the reference.