LIC Generator, a resource to generate primers for Ligation Independent Cloning (LIC)

This program generates the sequence of a set of 4 primers to amplify both insert and vector for LIC cloning.
Easily introduce tags or protease cleavage sites in your vector/insert.

LIC requires only three steps Compact display

1- Choose your LIC1 sequence (5' to your insert)

check one or several boxes

Option	Vector part		LIC1		Insert part	dNTP (insert)	Remarks	Linker*
Shine-Dalgarno		C	TAAGGAGGTTAAT	C	ATG	dGTP	No extra sequence
Kozak		A	GGGCCGCCGCC	A	CCATG	dTTP	No extra sequence
Kozak		T	AAGGAGGCCACCA	T	G	dATP	No extra sequence
Kozak		G	AAACCACCACCAT	G		dCTP	No extra sequence
Simple LIC	AC	C	ATGGGTGGTGGTGG	C		dGTP	A at -3
Simple LIC	AC	C	ATGGGTGGTGGTGG	C	ATG	dGTP	ATG conserved
Simple LIC	ACCA	T	GAGCGGCGCAA	T	G	dATP	ATG conserved
Simple LIC	ACCA	T	GAGCGGCGCAG	T	G	dATP	A at -3
Simple LIC	ACC	A	TGTCGGGCGGC	A	TG	dTTP	ATG conserved
Simple LIC	ACC	A	TGTCGGGCGGC	A		dTTP	A at -3
Simple LIC	ACCAT	G	ACCCTCTCCCTTTC	G		dCTP	A at -3
Simple LIC	ACCAT	G	ACCCTCTCCCTTTC	G	ATG	dCTP	ATG conserved
His Tag	AT	G	CATCACCACCACCACCAC	G	GT	dCTP	NsiI
His Tag	ACCATGAGCGC	G	CATCACCACCACCACCAC	G	GT	dCTP	A at -3	Spacer
His Tag	ACCAT	G	CATCACCACCACCACCAC	G	GT	dCTP	A at -3
His Tag	GC	G	CATCACCACCACCACCAC	G	GT	dCTP	No ATG
His Tag	AGCGC	G	CATCACCACCACCACCAC	G	GT	dCTP	No ATG	Spacer
His Tag	AT	G	CATCACCACCACCACCAC	G	GTATG	dCTP	ATG conserved
His Tag	ACCATGAGCGC	G	CATCACCACCACCACCAC	G	GTATG	dCTP	A at -3	Spacer
His Tag	ATGCA	T	CACCACCACCACCAC	T	CT	dATP	NsiI
His Tag	ACCATGAGCGCGCA	T	CACCACCACCACCAC	T	CT	dATP	A at -3	Spacer
His Tag	ATGAGCGCGCA	T	CACCACCACCACCAC	T	CT	dATP		Spacer
His Tag	AGCGCGCA	T	CACCACCACCACCAC	T	CT	dATP	No ATG	Spacer
His Tag	CA	T	CACCACCACCACCAC	T	CTATG	dATP
His Tag	ACCATGAGCGCGCA	T	CACCACCACCACCAC	T	CTATG	dATP	A at -3	Spacer
His Tag	CA	T	CACCACCACCACCAC	T	CT	dATP	No ATG
Strep Tag	ACCATGAGCGCTTGGA	G	CCACCCACAATTC	G	AGAAG	dCTP	A at -3	Spacer
Strep Tag	ACCATGTGGA	G	CCACCCACAATTC	G	AGAAG	dCTP	A at -3
Strep Tag	ACCATGAGCGCTTGGA	G	CCACCCACAATTC	G	AGAAGATG	dCTP	ATG conserved	Spacer
Strep Tag	ACCATGTGGA	G	CCACCCACAATTC	G	AGAAGATG	dCTP	ATG conserved
Strep Tag	ACCATGAGCGCT	T	GGAGCCACCCGCAG	T	TCGAGAAG	dATP	A at -3	Spacer
Strep Tag	ACCATG	T	GGAGCCACCCGCAG	T	TCGAGAAG	dATP	A at -3
Strep Tag	ACCATGAGCGCT	T	GGAGCCACCCGCAG	T	TCGAGAAG	dATP	A at -3	Spacer
Strep Tag	ACCATG	T	GGAGCCACCCGCAG	T	TCGAGAAG	dATP	A at -3
Strep Tag	AGCGCT	T	GGAGCCACCCGCAG	T	TCGAGAAG	dATP	No ATG	Spacer
Strep Tag		T	GGAGCCACCCGCAG	T	TCGAGAAG	dATP	No ATG
Tev	AGCGCTGA	G	AACTTATACTTCCAAA	G	CTTC	dCTP	No peptidase	Spacer
Tev	GA	G	AACTTATACTTCCAAA	G	CTTC	dCTP	No peptidase
Tev	AGCGCTGA	G	AACTTATACTTCCAAA	G	C	dCTP		Spacer
Tev	GA	G	AACTTATACTTCCAAA	G	C	dCTP
Prescission	GC	C	GGTTTGGAGGTG	C	TCTTCCAGGGTCCC	dGTP
Prescission	AGCGCTGC	C	GGTTTGGAGGTG	C	TCTTCCAGGGTCCC	dGTP		Spacer
Prescission	GC	C	GGTTTGGAGGTG	C	TCTTCCAGGGTCCCATG	dGTP	ATG conserved
Prescission	AGCGCTGC	C	GGTTTGGAGGTG	C	TCTTCCAGGGTCCCATG	dGTP	ATG conserved	Spacer
Thrombin	A	A	CGCTCTGGTGCC	A	CGT	dTTP
Factor Xa	AGCGCTGG	C	GGTATTGAGGGT	C	GC	dGTP		Spacer
Factor Xa	GG	C	GGTATTGAGGGT	C	GC	dGTP
Factor Xa	GG	C	GGTATTGAGGGT	C	GCATG	dGTP	ATG conserved
Enterokinase	AGCGCTGG	T	GACGACGACAAGGG	T		dATP		Spacer
Enterokinase	AGCGCTGG	T	GACGACGACAAGA	T	G	dATP	ATG conserved	Spacer
Enterokinase	GG	T	GACGACGACAAGGG	T		dATP
Enterokinase	GG	T	GACGACGACAAGA	T	G	dATP	ATG conserved

2- Choose your LIC2 sequence (3' from your insert)

check one or several boxes

Remark: LIC1 and LIC2 must match with respect to the nucleotide (same color code)

Choice	Insert part		LIC2		Vector part	dNTP (insert)	Remarks	Linker*
Simple LIC	TAG	C	GTTGTGAGGGTG	C		dCTP
Simple LIC	AG	C	GGTGGTGGTGG	C	TAG	dCTP	extra sequence
Simple LIC	TG	A	GTTGTGCGGGTG	A		dATP
Simple LIC	TA	G	CCTAACCTAACCC	G		dGTP
Simple LIC		T	AGGCAGCAAGCC	T		dTTP
His Tag	AT	G	CATCACCACCACCACCAC	G	GT	dGTP	Stop missing
His Tag	GT	G	CACCACCACCACCACCAC	G	GT	dGTP	Stop missing
His Tag	AGCGCGAT	G	CATCACCACCACCACCAC	G	GT	dGTP	Stop missing	Spacer
His Tag	AGCGCGGT	G	CACCACCACCACCACCAC	G	GT	dGTP	Stop missing	Spacer
His Tag	AT	G	CATCACCACCACCACCAC	G	GTTAG	dGTP	NsiI in tag
His Tag	GT	G	CACCACCACCACCACCAC	G	GTTAG	dGTP	SnoI in tag
His Tag	ATGCA	T	CACCACCACCACCAC	T	CT	dTTP	Stop missing
His Tag	CA	T	CACCACCACCACCAC	T	CT	dTTP	Stop missing
His Tag	AGCGCGCA	T	CACCACCACCACCAC	T	CT	dTTP	Stop missing	Spacer
His Tag	AGCGCGCA	T	CACCACCACCACCAC	T	CT	dTTP	Stop missing	Spacer
His Tag	ATGCA	T	CACCACCACCACCAC	T	AG	dTTP	NsiI in tag
His Tag	CA	T	CACCACCACCACCAC	T	AG	dTTP	NoATG but Stop
Strep Tag	AGCGCTTGGA	G	CCACCCACAATTC	G	AGAAGTAG	dGTP		Spacer
Strep Tag	TGGA	G	CCACCCACAATTC	G	AGAAGTAG	dGTP
Strep Tag	AGCGCTTGGA	G	CCACCCACAATTC	G	AGAAG	dGTP	Stop missing	Spacer
Strep Tag	TGGA	G	CCACCCACAATTC	G	AGAAG	dGTP	Stop missing
Strep Tag	AGCGCT	T	GGAGCCACCCGCAG	T	TCGAGAAGTAG	dTTP		Spacer
Strep Tag		T	GGAGCCACCCGCAG	T	TCGAGAAGTAG	dTTP
Strep Tag	AGCGCT	T	GGAGCCACCCGCAG	T	TCGAGAAG	dTTP	Stop missing	Spacer
Strep Tag		T	GGAGCCACCCGCAG	T	TCGAGAAG	dTTP	Stop missing
Tev	AGCGCTGA	G	AACTTATACTTCCAAA	G	CGCG	dGTP		Spacer
Tev	AGCGCTGA	G	AACTTATACTTCCAAA	G	CGCG	dGTP		Spacer
Tev	GA	G	AACTTATACTTCCAAA	G	C	dGTP
Tev	AGCGCTGA	G	AACTTATACTTCCAAA	G	CTTC	dGTP	No peptidase	Spacer
Tev	GA	G	AACTTATACTTCCAAA	G	CTTC	dGTP	No peptidase
Prescission	AGCGCTGC	C	GGTTTGGAGGTG	C	TCTTCCAGGGTCCC	dCTP		Spacer
Prescission	GC	C	GGTTTGGAGGTG	C	TCTTCCAGGGTCCC	dCTP
Thrombin	A	A	CGCTCTGGTGCC	A	CGT	dATP
Factor Xa	AGCGCTGG	C	GGTATTGAGGGT	C	GC	dCTP		Spacer
Factor Xa	GG	C	GGTATTGAGGGT	C	GC	dCTP
Enterokinase	AGCGCTGG	T	GACGACGACAAGGG	T		dTTP		Spacer
Enterokinase	AGCGCTGG	T	GACGACGACAAGA	T	G	dTTP	ATG after EK	Spacer
Enterokinase	GG	T	GACGACGACAAGGG	T		dTTP
Enterokinase	GG	T	GACGACGACAAGA	T	G	dTTP	ATG after EK
Enterokinase	AGCGCTGG	C	GATGATGATAAGGG	C		dCTP		Spacer
Enterokinase	AGCGCTGG	C	GATGATGATAAGATGGG	C		dCTP	ATG after EK	Spacer
Enterokinase	GG	C	GATGATGATAAGGG	C		dCTP
Enterokinase	GG	C	GATGATGATAAGATGGG	C		dCTP	ATG after EK

*Kozak means that a Kozak consensus is present before the Start
*Linker means that a Ser-Ala linker was added for example between the initiation Met and the tag

3- Add the informations about your project below

(not recorded)


Enter the sequence of your insert (or at least the extremities comprising ATG and Stop codon) plain text, numbers and spaces will be removed
Demo sequence
Enter a name for your project (recommended)

Enter the sequence of your vector preceding the insert (30 signs min, 100 signs max, plain text)

Enter the sequence of your vector following the insert (30 signs min, 100 signs max, plain text)

More about LIC ? | Protocols | Contact: mcdagher(at)chu(dash)grenoble(dot)fr
Marie-Claire Dagher - Centre Diagnostic et Recherche sur la Granulomatose Septique Chronique, CHU Grenoble, UMR CNRS 5525, TIM-C IMAG 38043 Grenoble Cedex 9 France logo cnrs

Acknowledgements: logo cea