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DNA Cloning Service

CRISPR/Cas

p7ioR-CBE-v2 F836

Ligation of p2* into F673p7ioR-CBEhA-basta-mut*

246810121416'nCas9pVS1Ubq10nCas9''Sm/SpColE1ORF-1'35S''NptIIo'U6'35SbaríntronNptIIo''E9NOS-TNOS-TUGIUGIbarSTLS1'intronXhoI (727)MluI (1486)BsrGI (1795)SbfI (3622)SpeI (5137)BamHI (10033)SmaI (10650)KpnI, PmeIBsiWI, NheI, ClaIF836p7ioR-CBE-v217,594 bp
Gene 1
bar
Gene 2
CBE
Other markers
NptIIo', 'NptIIo', Sm/Sp
Size
17,594 bp, circular
Record date
26-OCT-2023

Construct

  • 35S–bar · E9 · selection cassette, 309–1,786
  • 35S–NptIIo · NOS-T · selection cassette, 1,813–3,607
  • Ubq10–ORF-1–nCas9–UGI · NOS-T · payload cassette, 3,661–10,906
  • U6–gRNA · gRNA cassette, 10,916–11,464
  • SfiI ×2 · flanks 2 cassettes · at 3,653, 11,479
  • 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^TCGAG727between 35S and target Basta
MluIA^CGCGT1,486between bar and E9
BsrGI (Bsp1407I)T^GTACA1,795between E9 and 35S
SbfI (SdaI)CCTGCA^GG3,622between NOS-T and Ubq10
SpeIA^CTAGT5,137between Ubq10 and Gam
BamHIG^GATCC10,033in nCas9
SmaI (XmaI, TspMI)CCC^GGG10,650between NLS and NOS-T
KpnI (Acc65I)GGTAC^C11,502between gRNA and RB
PmeI (MssI)GTTT^AAAC11,534between gRNA and RB
BsiWI (Pfl23II)C^GTACG14,960in pVS1
NheI (BmtI)G^CTAGC15,076in pVS1
ClaI (BspDI)AT^CGAT15,156in pVS1

Cut twice: EcoRI (4,321, 10,910), PstI (3,622, 5,480), NdeI (13,040, 13,620), SfiI (3,653, 11,479), FseI (2,992, 14,732), AgeI (15,570, 15,878), StuI (8,642, 11,473), NsiI (3,474, 10,773), AatII (6,464, 11,925), BstEII (3,095, 16,441).

No site: HindIII, AscI, PacI, SwaI, AvrII, 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
LBT-DNA border misc_feature12424+
35SPromoter misc_feature309724416+
target BastaSite CDS74076223+
barSelection marker CDS745978234+
mutSite misc_feature7467461+
STLS1Other misc_feature9791,167189+
barSelection marker CDS1,1681,483316+
'E9Terminator misc_feature1,4961,786291+
'35SPromoter misc_feature1,8132,320508+
NptIIo'Selection marker CDS2,3432,641299+
'intronCoding sequence CDS2,6422,828187+
''NptIIo'Selection marker CDS2,8293,332504+
NOS-TTerminator misc_feature3,3543,607254+
Ubq10Other misc_feature3,6615,1361,476+
Ubi cds 5`Coding sequence CDS4,7654,82864+
íntronOther misc_feature4,8315,134304+
'GamSite CDS5,1835,1842+
LinkerCoding sequence CDS5,1855,23248+
ORF-1Coding sequence CDS5,2335,829597+
LinkerSite misc_feature5,8305,92596+
nCas9''Coding sequence CDS5,9267,2321,307+
'nCas9Coding sequence CDS7,23310,0562,824+
UGICoding sequence CDS10,05710,305249+
LinkerSite CDS10,30610,33530+
UGICoding sequence CDS10,33610,584249+
LinkerSite CDS10,58510,59612+
NLSCoding sequence CDS10,59710,64448+
NOS-TTerminator misc_feature10,65310,906254+
U6'Promoter misc_feature10,91611,360445+
gRNA BastaSite CDS11,36211,38120+
hairpinSite CDS11,38211,41130+
gRNASite misc_feature11,38211,46483+
RBT-DNA border misc_feature11,55011,57324+
ColE1Origin / backbone misc_feature12,10812,805698+
pVS1Origin / backbone misc_feature13,09315,8802,788+
Sm/SpSelection marker CDS16,06417,0741,011+

PromoterCoding sequenceTerminatorSelection markerOrigin / backboneT-DNA borderSite

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