design_knockout_package

The complete build package for a knockout or knock-in, as one object: guides inside the target, an off-target census for the chosen one within this molecule, a repair template with real homology arms, a screening PCR whose primers sit OUTSIDE the arms, the protocol in order, the fragments and oligos to buy, and every coordinate ready for the map. Two donor modes: by default the arms sit on the cut (a point insertion — the target's own sequence is kept on both sides), and with replace_target the arms flank the whole target and it is deleted, which is the mode for replacing one CDS with another (an empty insert then deletes the target cleanly). It refuses rather than guessing: no PAM in the target, arms that run off a linear molecule, a screening amplicon that cannot clear the arms, or a non-DNA insert; and in the default mode it WARNS when an insert lands inside the target, because that is a fusion rather than a replacement. Arm length, screening flank and guide count are yours to state, because each follows the delivery method and the organism.

Name	Required	Description	Default
`_files`	No	The project: record references shaped like _subject. Their records become library documents a tool names in sequences/parts/document; a trace or alignment is found by file name.
`guides`	No	how many guides to carry into the package, best-scoring first. The rest are the alternatives, not filler.
`insert`	No	sequence to place at the cut (a tag, a selection cassette). Leave empty for a scarless rejoin — the package then WARNS that the two screening products are the same length.
`subject`	No	the molecule to edit — wire a record or name a library document; leave unset to edit the open record.
`_subject`	No	The record to act on: exactly one of sequence, accession (+source), sample (an id from list_samples) or file ({name, text} or {name, base64}); record_index picks one record of several.
`nuclease`	No	the nuclease (PAM + spacer length) to scan the target for. Values: SpCas9 (SpCas9: NGG (3')), SpCas9-NG (SpCas9-NG: NG (3')), SpG (SpG: NGN (3')), SpRY (SpRY: NRN (3')), xCas9 (xCas9: NG (3')), SaCas9 (SaCas9: NNGRRT (3')), SaCas9-KKH (SaCas9-KKH: NNNRRT (3')), AsCas12a (AsCas12a: TTTV (5')), LbCas12a (LbCas12a: TTTV (5')), enAsCas12a (enAsCas12a: TTTV (5')).	SpCas9
`primer_tm`	No	target melting temperature for the screening primers [°C] — follows your polymerase and buffer. In °C. Advanced.
`arm_length`	Yes	homology-arm length on EACH side of the cut, in bp. A protocol decision that follows the delivery method, so there is no default: state what yours needs. The published working range is 100-800 (100-400 nt synthetic, up to ~800 bp cloned). In bp.
`target_end`	Yes	end of the gene to knock out (0-based, exclusive). In bp.
`insert_name`	No	what the insert is called in the protocol and on the order form.
`target_name`	No	what the target is called, so every mark, primer and order line names its subject.	target
`screen_flank`	Yes	how far OUTSIDE the arms the screening primers must bind, in bp. Not decoration: a primer inside the arms amplifies the repair template as readily as the locus, so the assay cannot tell an edited colony from plasmid carry-over. In bp.
`target_start`	Yes	start of the gene to knock out (0-based, inclusive). In bp.
`max_mismatches`	No	mismatch ceiling for the off-target search within this molecule. Advanced.
`replace_target`	No	replace the WHOLE target region with the insert, arms flanking the target — the gene-replacement donor. Off (default): the arms sit on the cut and the target's own sequence is kept on both sides of the insert (a point insertion), so this must be ON to knock one CDS in for another. Empty insert + this = a clean deletion of the target. Advanced.
`restriction_enzymes`	No	cloning enzymes whose recognition site must NOT appear in a spacer. Advanced.

Name

Required

Description

Default

_files

The project: record references shaped like _subject. Their records become library documents a tool names in sequences/parts/document; a trace or alignment is found by file name.

guides

how many guides to carry into the package, best-scoring first. The rest are the alternatives, not filler.

insert

sequence to place at the cut (a tag, a selection cassette). Leave empty for a scarless rejoin — the package then WARNS that the two screening products are the same length.

subject

the molecule to edit — wire a record or name a library document; leave unset to edit the open record.

_subject

The record to act on: exactly one of sequence, accession (+source), sample (an id from list_samples) or file ({name, text} or {name, base64}); record_index picks one record of several.

nuclease

the nuclease (PAM + spacer length) to scan the target for. Values: SpCas9 (SpCas9: NGG (3')), SpCas9-NG (SpCas9-NG: NG (3')), SpG (SpG: NGN (3')), SpRY (SpRY: NRN (3')), xCas9 (xCas9: NG (3')), SaCas9 (SaCas9: NNGRRT (3')), SaCas9-KKH (SaCas9-KKH: NNNRRT (3')), AsCas12a (AsCas12a: TTTV (5')), LbCas12a (LbCas12a: TTTV (5')), enAsCas12a (enAsCas12a: TTTV (5')).

SpCas9

primer_tm

target melting temperature for the screening primers [°C] — follows your polymerase and buffer. In °C. Advanced.

arm_length

Yes

homology-arm length on EACH side of the cut, in bp. A protocol decision that follows the delivery method, so there is no default: state what yours needs. The published working range is 100-800 (100-400 nt synthetic, up to ~800 bp cloned). In bp.

target_end

Yes

end of the gene to knock out (0-based, exclusive). In bp.

insert_name

what the insert is called in the protocol and on the order form.

target_name

what the target is called, so every mark, primer and order line names its subject.

target

screen_flank

Yes

how far OUTSIDE the arms the screening primers must bind, in bp. Not decoration: a primer inside the arms amplifies the repair template as readily as the locus, so the assay cannot tell an edited colony from plasmid carry-over. In bp.

target_start

Yes

start of the gene to knock out (0-based, inclusive). In bp.

max_mismatches

mismatch ceiling for the off-target search within this molecule. Advanced.

replace_target

replace the WHOLE target region with the insert, arms flanking the target — the gene-replacement donor. Off (default): the arms sit on the cut and the target's own sequence is kept on both sides of the insert (a point insertion), so this must be ON to knock one CDS in for another. Empty insert + this = a clean deletion of the target. Advanced.

restriction_enzymes

cloning enzymes whose recognition site must NOT appear in a spacer. Advanced.

Name	Required	Description
`donor`	Yes	the repair template: both arms with their genomic spans, the insert, and the junctions to sequence across
`marks`	Yes	every span this package names, ready for the map
`order`	Yes	the fragments and oligos to buy, each with the design decision it comes from
`steps`	Yes	the protocol in the order it is done
`chosen`	Yes	the guide the package was built around
`guides`	Yes	every guide inside the target, scored, best first
`record`	Yes	the repair template as a sequence record, so the run leaves the user holding the thing to order
`report`	Yes	the whole package as Markdown — protocol, order form, guides, template, screening and marks — so it survives outside the transcript
`screening`	Yes	the screening PCR: the pair, the two product lengths, and the span they cover
`offTargets`	Yes	the off-target census for the chosen spacer WITHIN this molecule, with the scope stated

Name

Required

Description

Default

donor

Yes

the repair template: both arms with their genomic spans, the insert, and the junctions to sequence across

marks

Yes

every span this package names, ready for the map

order

Yes

the fragments and oligos to buy, each with the design decision it comes from

steps

Yes

the protocol in the order it is done

chosen

Yes

the guide the package was built around

guides

Yes

every guide inside the target, scored, best first

record

Yes

the repair template as a sequence record, so the run leaves the user holding the thing to order

report

Yes

the whole package as Markdown — protocol, order form, guides, template, screening and marks — so it survives outside the transcript

screening

Yes

the screening PCR: the pair, the two product lengths, and the span they cover

offTargets

Yes

the off-target census for the chosen spacer WITHIN this molecule, with the scope stated

jean — Molecular Biology & Genomics

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