p6i2xoR-Ucas-U6Os B328
Ligation of A866p9iTE9oRes* into B294p6i2x-Ucas-U6Os*
- Promoter 1
- 2x35s
- Gene 1
- hptII
- Promoter 2
- UbiZm
- Gene 2
- Cas9 nick
- Features
- monocots
- Other markers
- hptII, Sm/Sp
- Size
- 15,473 bp, circular
- Record date
- 29-AUG-2016
Construct
- P2x35s–hptII · E9 · selection cassette, 67–2,387
- Ubi-int–Cas9 · NOS-T · payload cassette, 2,442–8,523
- U6Os–gRNA · gRNA cassette, 8,533–9,095
- MCS · annotated in the file · at 8,987, 9,012
- SfiI ×2 · flanks 2 cassettes · at 2,428, 9,110
- BsmBI Golden Gate · overhangs AGCT, GTTT · at 8,980, 9,012
- 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 |
|---|---|---|---|
| BamHI | G^GATCC | 857 | between 5´UTR and Kozak |
| MluI | A^CGCGT | 2,087 | between hptII and E9 |
| SbfI (SdaI) | CCTGCA^GG | 2,397 | between E9 and Ubi-int |
| SpeI | A^CTAGT | 2,436 | between E9 and Ubi-int |
| SacI (Ecl136II) | GAGCT^C | 4,016 | in Cas9 |
| SmaI (XmaI, TspMI) | CCC^GGG | 8,267 | between Cas9 and NOS-T |
| EcoRI | G^AATTC | 8,527 | between NOS-T and U6Os |
| BstBI (Bsp119I) | TT^CGAA | 8,993 | in MCS |
| HindIII | A^AGCTT | 8,996 | in MCS |
| SalI | G^TCGAC | 9,096 | between gRNA and RB |
| KpnI (Acc65I) | GGTAC^C | 9,133 | between gRNA and RB |
| PmeI (MssI) | GTTT^AAAC | 9,165 | between gRNA and RB |
| FseI | GGCCGG^CC | 12,363 | in pVS1' |
| BsiWI (Pfl23II) | C^GTACG | 12,591 | in pVS1' |
Cut twice: XhoI (851, 3,141), NheI (3,561, 12,707), ClaI (3,674, 12,787), SfiI (2,428, 9,110), AgeI (13,201, 13,509), AatII (4,651, 9,556), BstEII (1,162, 14,072), BsmBI (8,980, 9,012).
No site: AscI, PacI, SwaI, AvrII, HpaI, BsrGI, 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 |
|---|---|---|---|---|---|
| P2x35s | Promoter misc_feature | 67 | 814 | 748 | + |
| tata | Site misc_feature | 781 | 784 | 4 | + |
| 5´UTR | Site misc_feature | 815 | 850 | 36 | + |
| Kozak | Site misc_feature | 867 | 871 | 5 | + |
| hptII | Selection marker CDS | 872 | 1,441 | 570 | + |
| int | Site misc_feature | 1,442 | 1,630 | 189 | + |
| 'hptII | Selection marker CDS | 1,631 | 2,086 | 456 | + |
| E9 | Terminator misc_feature | 2,097 | 2,387 | 291 | + |
| transcription stop signal | Site misc_feature | 2,191 | 2,196 | 6 | + |
| transcription stop signal | Site misc_feature | 2,197 | 2,202 | 6 | + |
| transcription stop signal | Site misc_feature | 2,215 | 2,220 | 6 | + |
| Ubi-int | Promoter misc_feature | 2,442 | 3,950 | 1,509 | + |
| Intron | Site misc_feature | 3,429 | 3,948 | 520 | + |
| 'Cas9 | Coding sequence CDS | 3,976 | 8,264 | 4,289 | + |
| NLS | Site misc_feature | 3,981 | 4,001 | 21 | + |
| D10A | Site misc_feature | 4,045 | 4,045 | 1 | + |
| int | Site misc_feature | 4,392 | 4,484 | 93 | + |
| NLS | Site misc_feature | 8,214 | 8,261 | 48 | + |
| NOS-T | Terminator misc_feature | 8,270 | 8,523 | 254 | + |
| U6Os | Promoter misc_feature | 8,533 | 8,985 | 453 | + |
| MCS | Site CDS | 8,987 | 9,012 | 26 | + |
| hairpin | Site CDS | 9,013 | 9,042 | 30 | + |
| gRNA | Site misc_feature | 9,013 | 9,095 | 83 | + |
| RB | T-DNA border misc_feature | 9,181 | 9,204 | 24 | + |
| ColE1 | Origin / backbone misc_feature | 9,739 | 10,436 | 698 | + |
| 'pVS1' | Origin / backbone misc_feature | 10,724 | 13,511 | 2,788 | + |
| Sm/Sp | Selection marker CDS | 13,695 | 14,705 | 1,011 | + |
| LB | T-DNA border misc_feature | 15,226 | 15,249 | 24 | + |
PromoterCoding sequenceTerminatorSelection markerOrigin / backboneT-DNA borderSite
Feature types are inferred from the labels in the GenBank file; the file itself is the reference.