p9ioR-35sCasD10A B358
Ligation of 35s-Sbf-Bam* into A872p9ioR-Cas9-U6At*
- Promoter 1
- 35s
- Gene 1
- nptII
- Promoter 2
- 35s
- Gene 2
- Cas9 nick
- Features
- U6At
- Other markers
- Sm/Sp
- Size
- 13,780 bp, circular
- Record date
- 06-MAY-2015
Construct
- 35S–NptII · E9 · selection cassette, 309–2,034
- 35S–CasD10A–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 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 |
| SpeI | A^CTAGT | 2,510 | between 35S and CasD10A |
| BamHI | G^GATCC | 2,517 | between 35S and CasD10A |
| SacI (Ecl136II) | GAGCT^C | 2,579 | in CasD10A |
| 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 | + |
| 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 | Promoter misc_feature | 2,089 | 2,506 | 418 | + |
| TATA-box | Site CDS | 2,477 | 2,483 | 7 | + |
| CasD10A | Coding sequence CDS | 2,532 | 2,954 | 423 | + |
| NLS | Site misc_feature | 2,544 | 2,564 | 21 | + |
| D10A | Site misc_feature | 2,608 | 2,608 | 1 | + |
| int | Site CDS | 2,955 | 3,047 | 93 | + |
| 'Cas9 | Coding sequence CDS | 3,048 | 6,827 | 3,780 | + |
| 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.