p9ioR-35sCasDead D182
Ligation of dCas-Nru-Xma* into B358p9ioR-35sCasD10A*
- Promoter 1
- 35s
- Gene 1
- nptII
- Promoter 2
- 35s
- Gene 2
- dead
- Features
- Dicots
- Other markers
- Sm/Sp
- Size
- 13,780 bp, circular
- Record date
- 19-AUG-2020
Construct
- P35S–NptII · TE9 · selection cassette, 309–2,034
- P35S–Cas9 · NOS-T · payload cassette, 2,089–7,086
- U6At–gRNA · gRNA cassette, 7,096–7,650
- MCS · 4 unique sites · at 7,651, 7,720
- SfiI ×2 · flanks 2 cassettes · at 2,075, 7,665
- BsmBI Golden Gate · overhangs TGAT, GTTT · at 7,535, 7,567
- 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 P35S |
| XhoI (PaeR7I) | C^TCGAG | 727 | between P35S and NptII |
| MluI | A^CGCGT | 1,734 | between NptII and TE9 |
| SbfI (SdaI) | CCTGCA^GG | 2,044 | between TE9 and P35S |
| SpeI | A^CTAGT | 2,510 | between P35S and Cas9 |
| BamHI | G^GATCC | 2,517 | between P35S and Cas9 |
| SacI (Ecl136II) | GAGCT^C | 2,579 | in Cas9 |
| SmaI (XmaI, TspMI) | CCC^GGG | 6,830 | between Cas9 and NOS-T |
| EcoRI | G^AATTC | 7,090 | between NOS-T and U6At |
| BstBI (Bsp119I) | TT^CGAA | 7,548 | in targetRNA |
| HindIII | A^AGCTT | 7,551 | in targetRNA |
| SalI | G^TCGAC | 7,651 | between gRNA and RB |
| ApaI (PspOMI) | GGGCC^C | 7,682 | between gRNA and RB |
| KpnI (Acc65I) | GGTAC^C | 7,688 | between gRNA and RB |
| PmeI (MssI) | GTTT^AAAC | 7,720 | between gRNA and RB |
| FseI | GGCCGG^CC | 10,918 | in pVS1 |
| BsiWI (Pfl23II) | C^GTACG | 11,146 | in pVS1 |
| NheI (BmtI) | G^CTAGC | 11,262 | in pVS1 |
| ClaI (BspDI) | AT^CGAT | 11,342 | in pVS1 |
| BstEII (Eco91I) | G^GTNACC | 12,627 | in Sm/Sp |
Cut twice: PstI (1,120, 2,044), NdeI (9,226, 9,806), BglII (2,830, 7,671), SfiI (2,075, 7,665), MfeI (14, 884), AgeI (11,756, 12,064), StuI (5,392, 7,659), NsiI (3,003, 6,953), SacII (3,167, 5,264), AatII (3,214, 8,111), BsaI (11,665, 12,085), BsmBI (7,535, 7,567).
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 | + |
| P35S | 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 | + |
| TE9 | Terminator misc_feature | 1,744 | 2,034 | 291 | + |
| P35S | Promoter misc_feature | 2,089 | 2,506 | 418 | + |
| TATA-box | Site misc_feature | 2,477 | 2,483 | 7 | + |
| 'Cas9 | Coding sequence CDS | 2,532 | 6,827 | 4,296 | + |
| NLS | Site misc_feature | 2,544 | 2,564 | 21 | + |
| D10A | Site misc_feature | 2,608 | 2,608 | 1 | + |
| intron from rbcs Nicotiana tobaccum | Site misc_feature | 2,955 | 3,047 | 93 | + |
| H840A | Site misc_feature | 5,190 | 5,191 | 2 | + |
| NLS | Site misc_feature | 6,777 | 6,824 | 48 | + |
| NOS-T | Terminator misc_feature | 6,833 | 7,086 | 254 | + |
| U6At | Promoter misc_feature | 7,096 | 7,540 | 445 | + |
| targetRNA | Site CDS | 7,541 | 7,567 | 27 | + |
| hairpin | Site CDS | 7,568 | 7,597 | 30 | + |
| gRNA | Site misc_feature | 7,568 | 7,650 | 83 | + |
| RB | T-DNA border misc_feature | 7,736 | 7,759 | 24 | + |
| ColE1 | Origin / backbone misc_feature | 8,294 | 8,991 | 698 | + |
| pVS1 | Origin / backbone misc_feature | 9,279 | 12,066 | 2,788 | + |
| Sm/Sp | Selection marker CDS | 12,250 | 13,260 | 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.