p9ioR-35sCasWToi B764
Ligation of Cas9WToi Bam, Nru* into B357p9ioR-35sCasWT*
- Promoter 1
- 35s
- Gene 1
- nptII
- Promoter 2
- 35s
- Gene 2
- Cas9
- Features
- U6At
- Other markers
- Sm/Sp
- Size
- 13,681 bp, circular
- Record date
- 11-SEP-2017
Construct
- 35S–NptII · E9 · selection cassette, 309–2,034
- 35S-Pro–Cas9WT · NOS-T · payload cassette, 2,089–6,987
- MCS · 4 unique sites · at 7,552, 7,621
- SfiI ×2 · flanks the 35S-Pro–Cas9WT cassette · at 2,075, 7,566
- BsmBI Golden Gate · overhangs TGAT, GTTT · at 7,436, 7,468
- 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 NptII |
| MluI | A^CGCGT | 1,734 | between NptII and E9 |
| SbfI (SdaI) | CCTGCA^GG | 2,044 | between E9 and 35S-Pro |
| SpeI | A^CTAGT | 2,510 | between 35S-Pro and NLS |
| BamHI | G^GATCC | 2,517 | between 35S-Pro and NLS |
| SacI (Ecl136II) | GAGCT^C | 2,573 | in Cas9WT |
| SmaI (XmaI, TspMI) | CCC^GGG | 6,731 | between Cas9WT and NOS-T |
| NsiI (Mph1103I) | ATGCA^T | 6,854 | in NOS-T |
| EcoRI | G^AATTC | 6,991 | between NOS-T and RNA' |
| BstBI (Bsp119I) | TT^CGAA | 7,449 | in targetRNA |
| HindIII | A^AGCTT | 7,452 | in targetRNA |
| SalI | G^TCGAC | 7,552 | between RNA' and RB |
| ApaI (PspOMI) | GGGCC^C | 7,583 | between RNA' and RB |
| KpnI (Acc65I) | GGTAC^C | 7,589 | between RNA' and RB |
| PmeI (MssI) | GTTT^AAAC | 7,621 | between RNA' and RB |
| FseI | GGCCGG^CC | 10,819 | in pVS1 |
| BsiWI (Pfl23II) | C^GTACG | 11,047 | in pVS1 |
| NheI (BmtI) | G^CTAGC | 11,163 | in pVS1 |
| ClaI (BspDI) | AT^CGAT | 11,243 | in pVS1 |
| BstEII (Eco91I) | G^GTNACC | 12,528 | in Sm/Sp |
Cut twice: PstI (1,120, 2,044), NdeI (9,127, 9,707), BglII (2,824, 7,572), SfiI (2,075, 7,566), MfeI (14, 884), AgeI (11,657, 11,965), StuI (5,293, 7,560), SacII (3,068, 5,165), AatII (3,115, 8,012), BsaI (11,566, 11,986), BsmBI (7,436, 7,468).
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 |
|---|---|---|---|---|---|
| LB | T-DNA border misc_feature | 1 | 24 | 24 | + |
| 35S | Promoter misc_feature | 309 | 724 | 416 | + |
| NptII | Site CDS | 741 | 777 | 37 | + |
| int | Site misc_feature | 778 | 966 | 189 | + |
| NptII | Selection marker CDS | 967 | 1,733 | 767 | + |
| 'E9 | Terminator misc_feature | 1,744 | 2,034 | 291 | + |
| 35S-Pro | Promoter misc_feature | 2,089 | 2,506 | 418 | + |
| TATA-box | Site CDS | 2,477 | 2,483 | 7 | + |
| NLS | Site misc_feature | 2,538 | 2,558 | 21 | + |
| 'Cas9WT | Coding sequence CDS | 2,538 | 6,728 | 4,191 | + |
| int' | Site misc_feature | 2,949 | 2,956 | 8 | + |
| NLS | Site misc_feature | 6,678 | 6,725 | 48 | + |
| NOS-T | Terminator misc_feature | 6,734 | 6,987 | 254 | + |
| 'RNA' | Other misc_feature | 6,992 | 7,552 | 561 | + |
| U6At | Coding sequence CDS | 6,997 | 7,441 | 445 | + |
| targetRNA | Site CDS | 7,442 | 7,468 | 27 | + |
| hairpin | Site CDS | 7,469 | 7,498 | 30 | + |
| gRNA | Site misc_feature | 7,469 | 7,551 | 83 | + |
| RB | T-DNA border misc_feature | 7,637 | 7,660 | 24 | + |
| ColE1 | Origin / backbone misc_feature | 8,195 | 8,892 | 698 | + |
| 'pVS1 | Origin / backbone misc_feature | 9,180 | 11,967 | 2,788 | + |
| Sm/Sp | Selection marker CDS | 12,151 | 13,161 | 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.