p7ioR-35sCasD10A B396
Ligation of B353p7im2* into B358p9ioR-35sCasD10A*
- Promoter 1
- 35s
- Gene 1
- bar
- Promoter 2
- 35s
- Gene 2
- Cas9 nick
- Features
- U6At
- Other markers
- bar', 'bar, Sm/Sp
- Size
- 13,528 bp, circular
- Record date
- 13-MAY-2015
Construct
- 35S–bar · E9 · selection cassette, 309–1,782
- 35S-Pro–Cas9 · NOS-T · payload cassette, 1,837–6,834
- SfiI ×2 · flanks the 35S-Pro–Cas9 cassette · at 1,823, 7,413
- BsmBI Golden Gate · overhangs TGAT, GTTT · at 7,283, 7,315
- 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 |
|---|---|---|---|
| XbaI | T^CTAGA | 303 | between LB and 35S |
| XhoI (PaeR7I) | C^TCGAG | 727 | between 35S and bar' |
| AarI | CACCTGC(4/8) | 1,264 | in 'bar |
| MluI | A^CGCGT | 1,482 | between 'bar and E9 |
| PstI | CTGCA^G | 1,792 | between E9 and 35S-Pro |
| SbfI (SdaI) | CCTGCA^GG | 1,792 | between E9 and 35S-Pro |
| SpeI | A^CTAGT | 2,258 | between 35S-Pro and Cas9 |
| BamHI | G^GATCC | 2,265 | between 35S-Pro and Cas9 |
| SacI (Ecl136II) | GAGCT^C | 2,327 | in Cas9 |
| SmaI (XmaI, TspMI) | CCC^GGG | 6,578 | between Cas9 and NOS-T |
| EcoRI | G^AATTC | 6,838 | between NOS-T and U6At |
| BstBI (Bsp119I) | TT^CGAA | 7,296 | in targetRNA |
| HindIII | A^AGCTT | 7,299 | in targetRNA |
| SalI | G^TCGAC | 7,399 | between gRNA and RB |
| KpnI (Acc65I) | GGTAC^C | 7,436 | between gRNA and RB |
| PmeI (MssI) | GTTT^AAAC | 7,468 | between gRNA and RB |
| FseI | GGCCGG^CC | 10,666 | in pVS1 |
| BsiWI (Pfl23II) | C^GTACG | 10,894 | in pVS1 |
| NheI (BmtI) | G^CTAGC | 11,010 | in pVS1 |
| ClaI (BspDI) | AT^CGAT | 11,090 | in pVS1 |
| BstEII (Eco91I) | G^GTNACC | 12,375 | in Sm/Sp |
Cut twice: NdeI (8,974, 9,554), SfiI (1,823, 7,413), MfeI (14, 1,081), AgeI (11,504, 11,812), StuI (5,140, 7,407), NsiI (2,751, 6,701), AatII (2,962, 7,859), BsaI (11,413, 11,833), BsmBI (7,283, 7,315).
No site: AscI, PacI, SwaI, AvrII, HpaI, BsrGI.
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 | 309 | 724 | 416 | + |
| bar' | Selection marker CDS | 741 | 974 | 234 | + |
| STLS1 | Coding sequence CDS | 975 | 1,163 | 189 | + |
| ''bar | Selection marker CDS | 1,164 | 1,481 | 318 | + |
| 'E9 | Terminator misc_feature | 1,492 | 1,782 | 291 | + |
| 35S-Pro | Promoter misc_feature | 1,837 | 2,254 | 418 | + |
| TATA-box | Site CDS | 2,225 | 2,231 | 7 | + |
| 'Cas9 | Coding sequence CDS | 2,280 | 6,575 | 4,296 | + |
| NLS | Site misc_feature | 2,292 | 2,312 | 21 | + |
| D10A | Site misc_feature | 2,356 | 2,356 | 1 | + |
| int | Site misc_feature | 2,703 | 2,795 | 93 | + |
| NLS | Site misc_feature | 6,525 | 6,572 | 48 | + |
| NOS-T | Terminator misc_feature | 6,581 | 6,834 | 254 | + |
| U6At | Coding sequence CDS | 6,844 | 7,288 | 445 | + |
| targetRNA | Site CDS | 7,289 | 7,315 | 27 | + |
| hairpin | Site CDS | 7,316 | 7,345 | 30 | + |
| gRNA | Site misc_feature | 7,316 | 7,398 | 83 | + |
| RB | T-DNA border misc_feature | 7,484 | 7,507 | 24 | + |
| ColE1 | Origin / backbone misc_feature | 8,042 | 8,739 | 698 | + |
| 'pVS1 | Origin / backbone misc_feature | 9,027 | 11,814 | 2,788 | + |
| Sm/Sp | Selection marker CDS | 11,998 | 13,008 | 1,011 | + |
PromoterCoding sequenceTerminatorSelection markerOrigin / backboneT-DNA borderSite
Feature types are inferred from the labels in the GenBank file; the file itself is the reference.