p6D35s-C9wt-U6GmNhe F651
Ligation of D784p7D35s-C9wt-U6GmNhe* into D010p6D35s*
- Promoter 1
- 35s
- Gene 1
- hptII
- Gene 2
- Cas9
- Features
- dicots, U6Gm
- Other markers
- hptII'', strep/spec
- Size
- 13,245 bp, circular
- Record date
- 15-JUN-2023
Construct
- 35S–hptII · E9 · selection cassette, 169–2,120
- Pro-Ubq10At–Cas9WT · NOS-T · payload cassette, 2,146–8,098
- U6Gm–gRNA · gRNA cassette, 8,108–8,522
- MCS · 4 unique sites · at 8,523, 8,592
- SfiI ×2 · flanks 2 cassettes · at 2,138, 8,537
- BsaI Golden Gate · overhangs GGAT, GTTT · at 8,407, 8,439
- 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 Kozak |
| MluI | A^CGCGT | 1,820 | between hptII'' and E9 |
| SbfI (SdaI) | CCTGCA^GG | 2,130 | between E9 and Pro-Ubq10At |
| NotI | GC^GGCCGC | 2,139 | between E9 and Pro-Ubq10At |
| SalI | G^TCGAC | 2,984 | in Pro-Ubq10At |
| SpeI | A^CTAGT | 3,622 | between Pro-Ubq10At and Cas9WT |
| BamHI | G^GATCC | 3,628 | between Pro-Ubq10At and Cas9WT |
| NcoI | C^CATGG | 5,107 | in Cas9WT |
| SmaI (XmaI, TspMI) | CCC^GGG | 7,842 | between Cas9WT and NOS-T |
| NsiI (Mph1103I) | ATGCA^T | 7,965 | in NOS-T |
| BsrGI (Bsp1407I) | T^GTACA | 8,252 | in U6Gm |
| BsmBI (Esp3I) | CGTCTC(1/5) | 8,265 | in U6Gm |
| BstBI (Bsp119I) | TT^CGAA | 8,420 | in targetRNA |
| HindIII | A^AGCTT | 8,423 | in targetRNA |
| NheI (BmtI) | G^CTAGC | 8,523 | between gRNA and Right Border Region |
| ApaI (PspOMI) | GGGCC^C | 8,554 | between gRNA and Right Border Region |
| KpnI (Acc65I) | GGTAC^C | 8,560 | between gRNA and Right Border Region |
| PmeI (MssI) | GTTT^AAAC | 8,592 | in Right Border Region |
Cut twice: EcoRI (2,806, 8,102), PstI (2,130, 8,146), SphI (6,951, 8,717), SacI (3,239, 3,684), SfiI (2,138, 8,537), MfeI (14, 1,281), EagI (2,139, 8,974), AatII (4,226, 8,983), BsaI (8,407, 8,439), SapI (3,244, 4,838).
No site: NdeI, ClaI, AscI, PacI, FseI, SwaI, AvrII, BsiWI, AgeI, HpaI, AarI.
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 | + |
| Kozak | Site misc_feature | 600 | 604 | 5 | + |
| hptII | Selection marker CDS | 605 | 1,174 | 570 | + |
| int | Site misc_feature | 1,175 | 1,363 | 189 | + |
| 'hptII'' | Selection marker CDS | 1,364 | 1,819 | 456 | + |
| 'E9 | Terminator misc_feature | 1,830 | 2,120 | 291 | + |
| Pro-Ubq10At | Promoter misc_feature | 2,146 | 3,621 | 1,476 | + |
| Ubi cds 5` | Coding sequence CDS | 3,250 | 3,313 | 64 | + |
| íntron | Other misc_feature | 3,316 | 3,619 | 304 | + |
| 'Cas9WT | Coding sequence CDS | 3,643 | 7,839 | 4,197 | + |
| NLS | Site misc_feature | 3,649 | 3,669 | 21 | + |
| NLS | Site misc_feature | 7,789 | 7,836 | 48 | + |
| NOS-T | Terminator misc_feature | 7,845 | 8,098 | 254 | + |
| U6Gm | Promoter misc_feature | 8,108 | 8,412 | 305 | + |
| targetRNA | Site CDS | 8,413 | 8,439 | 27 | + |
| hairpin | Site CDS | 8,440 | 8,469 | 30 | + |
| gRNA | Site misc_feature | 8,440 | 8,522 | 83 | + |
| Right Border Region | Other misc_feature | 8,563 | 9,165 | 603 | + |
| RB | T-DNA border misc_feature | 8,608 | 8,631 | 24 | + |
| 'ColE1'' | Origin / backbone misc_feature | 9,179 | 9,837 | 659 | + |
| ''pVS1 ORI''' | Origin / backbone misc_feature | 9,866 | 11,829 | 1,964 | + |
| ParB | Coding sequence CDS | 10,192 | 11,367 | 1,176 | + |
| strep/spec | Selection marker CDS | 12,018 | 13,028 | 1,011 | + |
| Left Border Region | T-DNA border misc_feature | 13,096 | 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.