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DNA Cloning Service

CRISPR/Cas

p7ioR-nCasNG-CBEhA-basta-mut F841

Ligation of Fra-Cas9-NG* into F673p7ioR-CBEhA-basta-mut*

24681012141618ORF-1pVS1Ubq10Sm/SpColE1ORF-1Gam'35S''NptIIo'U6'35SbaríntronNptIIo''E9NOS-TNOS-TUGIUGIbarSTLS1'intronXhoI (727)MluI (1486)BsrGI (1795)SbfI (3622)SpeI (5137)BamHI (10573)SmaI (11190)KpnI, PmeIBsiWI, NheI, ClaIF841p7ioR-nCasNG-CB…18,134 bp
Gene 1
bar
Gene 2
CBE
Other markers
NptIIo', 'NptIIo', Sm/Sp
Size
18,134 bp, circular
Record date
31-OCT-2023

Construct

  • 35S–bar · E9 · selection cassette, 309–1,786
  • 35S–NptIIo · NOS-T · selection cassette, 1,813–3,607
  • Ubq10–Gam–ORF-1–UGI · NOS-T · payload cassette, 3,661–11,446
  • U6–gRNA · gRNA cassette, 11,456–12,004
  • SfiI ×2 · flanks 2 cassettes · at 3,653, 12,019
  • 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 NLS
BamHIG^GATCC10,573in ORF-1
SmaI (XmaI, TspMI)CCC^GGG11,190between NLS and NOS-T
KpnI (Acc65I)GGTAC^C12,042between gRNA and RB
PmeI (MssI)GTTT^AAAC12,074between gRNA and RB
BsiWI (Pfl23II)C^GTACG15,500in pVS1
NheI (BmtI)G^CTAGC15,616in pVS1
ClaI (BspDI)AT^CGAT15,696in pVS1

Cut twice: EcoRI (4,321, 11,450), PstI (3,622, 6,020), NdeI (13,580, 14,160), SfiI (3,653, 12,019), FseI (2,992, 15,272), AgeI (16,110, 16,418), StuI (9,182, 12,013), NsiI (3,474, 11,313), AatII (7,004, 12,465), BstEII (3,095, 16,981).

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+
PAMSite CDS7607623+
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+
NLSSite misc_feature5,1645,18421+
LinkerSite CDS5,1855,20218+
GamCoding sequence CDS5,2035,724522+
LinkerCoding sequence CDS5,7255,77248+
ORF-1Coding sequence CDS5,7736,369597+
LinkerSite misc_feature6,3706,46596+
ORF-1Coding sequence CDS6,46610,5964,131+
UGICoding sequence CDS10,59710,845249+
LinkerSite CDS10,84610,87530+
UGICoding sequence CDS10,87611,124249+
LinkerSite CDS11,12511,13612+
NLSCoding sequence CDS11,13711,18448+
NOS-TTerminator misc_feature11,19311,446254+
U6'Promoter misc_feature11,45611,900445+
gRNA BastaSite CDS11,90211,92120+
hairpinSite CDS11,92211,95130+
gRNASite misc_feature11,92212,00483+
RBT-DNA border misc_feature12,09012,11324+
ColE1Origin / backbone misc_feature12,64813,345698+
pVS1Origin / backbone misc_feature13,63316,4202,788+
Sm/SpSelection marker CDS16,60417,6141,011+

PromoterCoding sequenceTerminatorSelection markerOrigin / backboneT-DNA borderSite

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