p6D35s-nSpRY-hA3Ai G059

Ligation of hpt-Xho-Mlu* into G007p7D35s-nSpRY-hA3Ai*

Promoter 1: 35s
Gene 1: hptII
Gene 2: ABE
Features: dicots
Other markers: hptII'', SpRY'', strep/spec
Size: 15,194 bp, circular
Record date: 23-FEB-2024

Construct

35S–hptII · E9 · selection cassette, 169–2,120
Ubq10–Gam–ORF-1–SpRY–UGI · NOS-T · payload cassette, 2,169–10,047
U6Gm–gRNA · gRNA cassette, 10,057–10,471
SfiI ×2 · flanks 2 cassettes · at 2,161, 10,486
BsaI Golden Gate · overhangs GGAT, GTTT · at 10,356, 10,388
T-DNA borders · LB, RB

Read out of the GenBank file; compare every vector in the multiple expression cassettes and cloning sites table.

Files

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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
XhoI (PaeR7I)	C^TCGAG	587	between 35S and Kozak
MluI	A^CGCGT	1,820	between hptII'' and E9
SbfI (SdaI)	CCTGCA^GG	2,130	between E9 and Ubq10
NotI	GC^GGCCGC	2,162	between E9 and Ubq10
SpeI	A^CTAGT	3,645	between Ubq10 and NLS
AvrII (XmaJI)	C^CTAGG	6,833	in SpRY''
BamHI	G^GATCC	9,174	in Linker
SmaI (XmaI, TspMI)	CCC^GGG	9,791	between NLS and NOS-T
BsrGI (Bsp1407I)	T^GTACA	10,201	in U6Gm
BsmBI (Esp3I)	CGTCTC(1/5)	10,214	in U6Gm
BstBI (Bsp119I)	TT^CGAA	10,369	in insert protospacer
HindIII	A^AGCTT	10,372	in insert protospacer
NheI (BmtI)	G^CTAGC	10,472	between gRNA and Right Border Region
KpnI (Acc65I)	GGTAC^C	10,509	between gRNA and Right Border Region
PmeI (MssI)	GTTT^AAAC	10,541	in Right Border Region

Cut twice: EcoRI (2,829, 10,051), SalI (3,007, 9,620), ApaI (9,680, 10,503), SfiI (2,161, 10,486), MfeI (14, 1,281), NsiI (4,550, 9,914), AatII (5,605, 10,932), BsaI (10,356, 10,388), SapI (3,267, 6,217).

No site: NdeI, ClaI, AscI, PacI, FseI, SwaI, BsiWI, AgeI, HpaI, 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	169	584	416	+
Kozak	Site misc_feature	600	604	5	+
hptII	Selection marker CDS	605	1,174	570	+
int	Site misc_feature	1,175	1,363	189	+
'hptII''	Selection marker CDS	1,364	1,819	456	+
'E9	Terminator misc_feature	1,830	2,120	291	+
Ubq10	Other misc_feature	2,169	3,644	1,476	+
Ubi cds 5`	Coding sequence CDS	3,273	3,336	64	+
íntron	Other misc_feature	3,339	3,642	304	+
NLS	Site misc_feature	3,672	3,692	21	+
Linker	Site CDS	3,693	3,710	18	+
Gam	Coding sequence CDS	3,711	4,232	522	+
Linker	Coding sequence CDS	4,233	4,280	48	+
ORF-1	Coding sequence CDS	4,281	4,462	182	+
int	Site misc_feature	4,502	4,594	93	+
ORF-1	Coding sequence CDS	4,621	4,970	350	+
Linker	Site misc_feature	4,971	5,066	96	+
'SpRY''	Selection marker CDS	5,067	9,167	4,101	+
D10A	Site misc_feature	5,091	5,093	3	+
Linker	Site CDS	9,168	9,197	30	+
UGI	Coding sequence CDS	9,198	9,446	249	+
Linker	Site CDS	9,447	9,476	30	+
UGI	Coding sequence CDS	9,477	9,725	249	+
Linker	Site CDS	9,726	9,737	12	+
NLS	Coding sequence CDS	9,738	9,785	48	+
NOS-T	Terminator misc_feature	9,794	10,047	254	+
U6Gm	Promoter misc_feature	10,057	10,361	305	+
insert protospacer	Site CDS	10,362	10,388	27	+
hairpin	Site CDS	10,389	10,418	30	+
gRNA	Site misc_feature	10,389	10,471	83	+
Right Border Region	Other misc_feature	10,512	11,114	603	+
RB	T-DNA border misc_feature	10,557	10,580	24	+
'ColE1''	Origin / backbone misc_feature	11,128	11,786	659	+
''pVS1 ORI'''	Origin / backbone misc_feature	11,815	13,778	1,964	+
ParB	Coding sequence CDS	12,141	13,316	1,176	+
strep/spec	Selection marker CDS	13,967	14,977	1,011	+
Left Border Region	T-DNA border misc_feature	15,045	163	0	+

PromoterCoding sequenceTerminatorSelection markerOrigin / backboneT-DNA borderSite

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