pET-28b-T7-bLbCas12a-NLS(nucleoplasmin)-6xHis (RTW645)

114070 Synthetic Bacterial Expression

Plasmid Information

Purpose
T7 promoter bacterial expression plasmid for bacterial codon optimized LbCas12a with C-terminal NLS and His-tag
Depositor
Keith Joung, Benjamin Kleinstiver
Article
Kleinstiver et al Nat Biotechnol. 2019 Feb 11. pii: 10.103
Insert
bacteria codon optimized LbCas12a with C-terminal NLS and His-tag
Tags
NLS(nucleoplasmin) and 6xHis
Available Since
Dec. 11, 2018
Availability
Available

Sequence Information

Length
8,982 bp
Topology
circular
Molecule Type
other DNA
View on AddGene.org

Features

CDS 6
Label Gene Product Position Strand
rop rop Rop protein, which maintains plasmids a… 245..437 Rev
DNA Sequence
GTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA
Protein Sequence
MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHADELYRSCLARFGDDGENL
lacI lacI lac repressor 1245..2328 Rev
DNA Sequence
GTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA
Protein Sequence
MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ
LbCpf1 — CRISPR-associated protein from Lachnosp… 2801..6488 Fwd
DNA Sequence
ATGGGCAGCAAACTGGAAAAATTTACGAATTGTTATAGCCTGTCCAAGACCCTGCGTTTCAAAGCCATCCCCGTTGGCAAAACCCAGGAGAATATTGATAATAAACGTCTGCTGGTTGAGGATGAAAAAAGAGCAGAAGACTATAAGGGAGTCAAAAAACTGCTGGATCGGTACTACCTGAGCTTTATAAATGACGTGCTGCATAGCATTAAACTGAAAAATCTGAATAACTATATTAGTCTGTTCCGCAAGAAAACCCGAACAGAGAAAGAAAATAAAGAGCTGGAAAACCTGGAGATCAATCTGCGTAAAGAGATCGCAAAAGCTTTTAAAGGAAATGAAGGTTATAAAAGCCTGTTCAAAAAAGACATTATTGAAACCATCCTGCCGGAATTTCTGGATGATAAAGACGAGATAGCGCTCGTGAACAGCTTCAACGGGTTCACGACCGCCTTCACGGGCTTTTTCGATAACAGGGAAAATATGTTTTCAGAGGAAGCCAAAAGCACCTCGATAGCGTTCCGTTGCATTAATGAAAATTTGACAAGATATATCAGCAACATGGATATTTTCGAGAAAGTTGATGCGATCTTTGACAAACATGAAGTGCAGGAGATTAAGGAAAAAATTCTGAACAGCGATTATGATGTTGAGGATTTTTTCGAGGGGGAATTTTTTAACTTTGTACTGACACAGGAAGGTATAGATGTGTATAATGCTATTATCGGCGGGTTCGTTACCGAATCCGGCGAGAAAATTAAGGGTCTGAATGAGTACATCAATCTGTATAACCAAAAGACCAAACAGAAACTGCCAAAATTCAAACCGCTGTACAAGCAAGTCCTGAGCGATCGGGAAAGCTTGAGCTTTTACGGTGAAGGTTATACCAGCGACGAGGAGGTACTGGAGGTCTTTCGCAATACCCTGAACAAGAACAGCGAAATTTTCAGCTCCATTAAAAAGCTGGAGAAACTGTTTAAGAATTTTGACGAGTACAGCAGCGCAGGTATTTTTGTGAAGAACGGACCTGCCATAAGCACCATTAGCAAGGATATTTTTGGAGAGTGGAATGTTATCCGTGATAAATGGAACGCGGAATATGATGACATACACCTGAAAAAGAAGGCTGTGGTAACTGAGAAATATGAAGACGATCGCCGCAAAAGCTTTAAAAAAATCGGCAGCTTTAGCCTGGAGCAGCTGCAGGAATATGCGGACGCCGACCTGAGCGTGGTCGAGAAACTGAAGGAAATTATTATCCAAAAAGTGGATGAGATTTACAAGGTATATGGTAGCAGCGAAAAACTGTTTGATGCGGACTTCGTTCTGGAAAAAAGCCTGAAAAAAAATGATGCTGTTGTTGCGATCATGAAAGACCTGCTCGATAGCGTTAAGAGCTTTGAAAATTACATTAAAGCATTCTTTGGCGAGGGCAAAGAAACAAACAGAGACGAAAGCTTTTATGGCGACTTCGTCCTGGCTTATGACATCCTGTTGAAGGTAGATCATATATATGATGCAATTCGTAATTACGTAACCCAAAAGCCGTACAGCAAAGATAAGTTCAAACTGTATTTCCAGAACCCGCAGTTTATGGGTGGCTGGGACAAAGACAAGGAGACAGACTATCGCGCCACTATTCTGCGTTACGGCAGCAAGTACTATCTCGCCATCATGGACAAAAAATATGCAAAGTGTCTGCAGAAAATCGATAAAGACGACGTGAACGGAAATTACGAAAAGATTAATTATAAGCTGCTGCCAGGGCCCAACAAGATGTTACCGAAAGTATTTTTTTCCAAAAAATGGATGGCATACTATAACCCGAGCGAGGATATACAGAAGATTTACAAAAATGGGACCTTCAAAAAGGGGGATATGTTCAATCTGAATGACTGCCACAAACTGATCGATTTTTTTAAAGATAGCATCAGCCGTTATCCTAAATGGTCAAACGCGTATGATTTTAATTTCTCCGAAACGGAGAAATATAAAGACATTGCTGGTTTCTATCGCGAAGTCGAAGAACAGGGTTATAAAGTTAGCTTTGAATCGGCCAGCAAGAAAGAGGTTGATAAACTGGTGGAGGAGGGTAAGCTGTATATGTTTCAGATTTATAACAAAGACTTTAGCGACAAAAGCCACGGTACTCCTAATCTGCATACGATGTACTTTAAACTGCTGTTTGATGAGAATAACCACGGCCAAATCCGTCTCTCCGGTGGAGCAGAACTTTTTATGCGGCGTGCGAGCCTAAAAAAGGAAGAACTGGTGGTGCATCCCGCCAACAGCCCGATTGCTAACAAAAATCCAGATAATCCTAAGAAGACCACCACACTGTCGTACGATGTCTATAAGGATAAACGTTTCTCGGAAGACCAGTATGAATTGCATATACCGATAGCAATTAATAAATGCCCAAAAAACATTTTCAAAATCAACACTGAAGTTCGTGTGCTGCTGAAACATGATGATAATCCGTATGTGATCGGAATTGACCGTGGGGAGAGAAATCTGCTGTATATTGTAGTCGTTGATGGCAAGGGCAACATCGTTGAGCAGTATAGCCTGAATGAAATAATTAATAATTTTAACGGTATACGTATTAAAACCGACTATCATAGCCTGCTGGATAAAAAGGAGAAAGAGCGTTTTGAGGCACGCCAAAATTGGACGAGCATCGAAAACATCAAGGAACTGAAGGCAGGATATATCAGCCAAGTAGTCCATAAAATCTGTGAACTGGTGGAGAAGTACGACGCTGTCATTGCCCTGGAAGACCTCAATAGCGGCTTTAAAAACAGCCGGGTGAAGGTGGAGAAACAGGTATACCAAAAGTTTGAAAAGATGCTCATTGATAAGCTGAACTATATGGTTGATAAAAAGAGCAACCCGTGCGCCACTGGCGGTGCACTGAAAGGGTACCAAATTACCAATAAATTTGAAAGCTTTAAAAGCATGAGCACGCAGAATGGGTTTATTTTTTATATACCAGCATGGCTGACGAGCAAGATTGACCCCAGCACTGGTTTTGTCAATCTGCTGAAAACCAAATACACAAGCATTGCGGATAGCAAAAAATTTATTTCGAGCTTCGACCGTATTATGTATGTTCCGGAGGAAGATCTGTTTGAATTTGCCCTGGATTATAAAAACTTCAGCCGCACCGATGCAGATTATATCAAAAAATGGAAGCTGTACAGTTATGGTAATCGTATACGTATCTTCCGTAATCCGAAGAAAAACAATGTGTTCGATTGGGAAGAGGTCTGTCTGACCAGCGCGTATAAAGAACTGTTCAACAAGTACGGAATAAATTATCAGCAAGGTGACATTCGCGCACTGCTGTGTGAACAGTCAGATAAAGCATTTTATAGCAGCTTTATGGCGCTGATGAGCCTGATGCTCCAGATGCGCAACAGCATAACCGGTCGCACAGATGTTGACTTTCTGATCAGCCCTGTGAAGAATAGCGACGGCATCTTCTACGATTCCAGGAACTATGAAGCACAGGAAAACGCTATTCTGCCTAAAAATGCCGATGCCAACGGCGCCTATAATATTGCACGGAAGGTTCTGTGGGCGATTGGACAGTTCAAGAAAGCGGAAGATGAGAAGCTGGATAAGGTAAAAATTGCTATTAGCAATAAGGAATGGCTGGAGTACGCACAGACATCGGTTAAACAC
Protein Sequence
MGSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
nucleoplasmin NLS — bipartite nuclear localization signal f… 6494..6542 Fwd
DNA Sequence
AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG
Protein Sequence
KRPAATKKAGQAKKKK
6xHis — 6xHis affinity tag 6560..6578 Fwd
DNA Sequence
CACCACCACCACCACCAC
Protein Sequence
HHHHHH
KanR aph(3')-Ia aminoglycoside phosphotransferase 7275..8091 Rev
DNA Sequence
ATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA
Protein Sequence
MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF
promoter 2
Label Gene Product Position Strand
lacI promoter lacI — 2328..2406 Rev
DNA Sequence
GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAAT
T7 promoter — — 2714..2733 Fwd
DNA Sequence
TAATACGACTCACTATAGG
protein_bind 1
Label Gene Product Position Strand
lac operator — — 2733..2758 Fwd
DNA Sequence
GGAATTGTGAGCGGATAACAATTCC
rep_origin 2
Label Gene Product Position Strand
f1 ori — — 6728..7184 Fwd
DNA Sequence
ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTT
ori — — 8212..8801 Fwd
DNA Sequence
TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
terminator 1
Label Gene Product Position Strand
T7 terminator — — 6644..6692 Fwd
DNA Sequence
CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG