Skip to content
DNA Cloning Service

CRISPR/Cas

p7-5ca-CBEhAi-Ung G017

Ligation of Ung + Schnitt* into F980p7bm-CBEhAi-2x5ca*

246810121416nCas9'''pVS1 ORI'''Ubq10ParBstrep/specNptIIo'ORF-1ORF Ung Opti'ColE1''Right Border RegionGam'35SU6At'35Sbaríntron'E9NOS-TNOS-Tshort''barSTLS1XhoI (587)MluI (1347)FseI (2853)SbfI, NotISpeI (4998)BamHI (10527)NheI (10959)SmaI (11303)KpnI, PmeIG017p7-5ca-CBEhAi-U…16,840 bp
Gene 1
bar
Gene 2
CBE
Other markers
bar, NptIIo', strep/spec
Size
16,840 bp, circular
Record date
21-FEB-2024

Construct

  • 35S–bar · E9 · selection cassette, 169–1,647
  • 35S–NptIIo · NOS-T · selection cassette, 1,674–3,468
  • Ubq10–Gam–ORF-1–nCas9–ORF Ung Opti · NOS-T · payload cassette, 3,522–11,559
  • SfiI ×2 · flanks the Ubq10–Gam–ORF-1–nCas9–ORF Ung Opti cassette · at 3,514, 12,132
  • 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 cutterSiteCuts atLocation
XhoI (PaeR7I)C^TCGAG587between 35S and target 5ca
MluIA^CGCGT1,347between bar and E9
FseIGGCCGG^CC2,853in NptIIo'
SbfI (SdaI)CCTGCA^GG3,483between NOS-T and Ubq10
NotIGC^GGCCGC3,515between NOS-T and Ubq10
SpeIA^CTAGT4,998between Ubq10 and NLS
BamHIG^GATCC10,527in 3x Linker
NheI (BmtI)G^CTAGC10,959in ORF Ung Opti
SmaI (XmaI, TspMI)CCC^GGG11,303between NLS and NOS-T
KpnI (Acc65I)GGTAC^C12,155between gRNA and Right Border Region
PmeI (MssI)GTTT^AAAC12,187in Right Border Region

Cut twice: EcoRI (4,182, 11,563), SalI (4,360, 12,118), PstI (3,483, 5,974), SfiI (3,514, 12,132), StuI (9,136, 12,126), BsrGI (1,656, 10,825), BstEII (2,956, 15,990), BsaI (687, 2,743), SapI (4,620, 7,570).

No site: HindIII, NdeI, ClaI, AscI, PacI, SwaI, AvrII, BsiWI, AgeI, BstBI, HpaI, BsmBI.

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

FeatureTypeStartEndLengthStrand
LB'T-DNA border misc_feature12424+
35SPromoter misc_feature169584416+
target 5caSite CDS6156239+
'barSelection marker CDS615839225+
PAMSite CDS6216233+
STLS1Other misc_feature8401,028189+
barSelection marker CDS1,0291,344316+
'E9Terminator misc_feature1,3571,647291+
'35SPromoter misc_feature1,6742,181508+
NptIIo'Selection marker CDS2,2043,193990+
'intronCoding sequence CDS2,5032,689187+
NOS-TTerminator misc_feature3,2153,468254+
Ubq10Other misc_feature3,5224,9971,476+
Ubi cds 5`Coding sequence CDS4,6264,68964+
íntronOther misc_feature4,6924,995304+
NLSSite misc_feature5,0255,04521+
LinkerSite CDS5,0465,06318+
GamCoding sequence CDS5,0645,585522+
LinkerCoding sequence CDS5,5865,63348+
ORF-1Coding sequence CDS5,6346,323690+
intSite misc_feature5,8555,94793+
LinkerSite misc_feature6,3246,41996+
nCas9'Coding sequence CDS6,42010,5204,101+
3x LinkerSite CDS10,52110,55030+
ORF Ung OptiCoding sequence CDS10,55111,237687+
linkerSite CDS11,23811,24912+
NLSCoding sequence CDS11,25011,29748+
NOS-TTerminator misc_feature11,30611,559254+
U6At'Coding sequence CDS11,56912,008440+
short'Coding sequence CDS11,76212,008247+
cutSite misc_feature12,00912,0124+
U6AtSite misc_feature12,00912,0135+
hairpinSite CDS12,03512,06430+
gRNASite misc_feature12,03512,11783+
Right Border RegionOther misc_feature12,15812,760603+
RBT-DNA border misc_feature12,20312,22624+
'ColE1''Origin / backbone misc_feature12,77413,432659+
''pVS1 ORI'''Origin / backbone misc_feature13,46115,4241,964+
ParBCoding sequence CDS13,78714,9621,176+
strep/specSelection marker CDS15,61316,6231,011+
Left Border RegionT-DNA border misc_feature16,6911630+

PromoterCoding sequenceTerminatorSelection markerOrigin / backboneT-DNA borderSite

Feature types are inferred from the labels in the GenBank file; the file itself is the reference.