His_CL7_2NLS_iCas12a-4NLS

Plasmid Information

Purpose

Bacterial expression of NLS-rich iCas12a construct

Depositor

Jennifer Doudna

Article

Chen et al Nat Commun. 2024 Feb 26;15(1):1727. doi:

Insert

His_CL7_2NLS_iCas12a-4NLS

Sequence Information

Length

8,125 bp

Topology

circular

Molecule Type

other DNA

View on AddGene.org

Features

3'UTR 1

Label	Gene	Product	Position	Strand
cspA 3'UTR	cspA	—	1882..2027	Fwd
DNA Sequence TCTCTGCTTAAAAGCACAGAATCTAAGATCCCTGCCATTTGGCGGGGATTTTTTTATTTGTTTTCAGGAAATAAATAATCGATCGCGTAATAAAATCTATTATTATTTTTGTGAAGAATAAATTTGGGTGCAATGAGAATGCGCA

5'UTR 1

Label	Gene	Product	Position	Strand
cspA 5'UTR	cspA	—	5507..5641	Fwd
DNA Sequence CAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTGCACATTCCTTTAACGCTTCAAAATCTGTAAAGCACGCCATATCGCCGAAAGGCACACTTAATTATTAAGAGGTAATACACC

CDS 10

Label	Gene	Product	Position	Strand
SV40 NLS	—	nuclear localization signal of SV40 (si…	1761..1782	Fwd
DNA Sequence CCGAAGAAGAAACGCAAAGTC Protein Sequence PKKKRKV
SV40 NLS	—	nuclear localization signal of SV40 (si…	1791..1812	Fwd
DNA Sequence CCTAAGAAGAAGCGTAAGGTT Protein Sequence PKKKRKV
SV40 NLS	—	nuclear localization signal of SV40 (si…	1821..1842	Fwd
DNA Sequence CCAAAGAAGAAACGTAAAGTT Protein Sequence PKKKRKV
SV40 NLS	—	nuclear localization signal of SV40 (si…	1851..1872	Fwd
DNA Sequence CCGAAAAAGAAACGCAAGGTG Protein Sequence PKKKRKV
AmpR	bla	beta-lactamase	2384..3245	Fwd
DNA Sequence ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA Protein Sequence MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW
lacI	lacI	lac repressor	4175..5258	Rev
DNA Sequence GTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA Protein Sequence MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ
6xHis	—	6xHis affinity tag	5653..5671	Fwd
DNA Sequence CACCATCACCATCACCAT Protein Sequence HHHHHH
HRV 3C site	—	recognition and cleavage site for human…	6082..6106	Fwd
DNA Sequence TTGGAGGTTTTGTTCCAGGGTCCA Protein Sequence LEVLFQGP
SV40 NLS	—	nuclear localization signal of SV40 (si…	6115..6136	Fwd
DNA Sequence CCAAAGAAAAAGCGTAAAGTC Protein Sequence PKKKRKV
SV40 NLS	—	nuclear localization signal of SV40 (si…	6145..6166	Fwd
DNA Sequence CCGAAAAAGAAACGGAAAGTA Protein Sequence PKKKRKV

promoter 3

Label	Gene	Product	Position	Strand
AmpR promoter	bla	—	2279..2384	Fwd
DNA Sequence CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT
lacI promoter	lacI	—	5258..5336	Rev
DNA Sequence GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGTGCCCGGAAGAGAGTCAATTCAGGGTGGTGAAT
tac promoter	—	—	5435..5464	Fwd
DNA Sequence TTGACAATTAATCATCGGCTCGTATAATG

protein_bind 1

Label	Gene	Product	Position	Strand
lac operator	—	—	5469..5486	Fwd
DNA Sequence TTGTGAGCGGATAACAA

rep_origin 1

Label	Gene	Product	Position	Strand
ori	—	—	3415..4004	Fwd
DNA Sequence TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA

terminator 1

Label	Gene	Product	Position	Strand
rrnB T1 terminator	Escherichia coli rrnB	—	2102..2174	Fwd
DNA Sequence CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC