KAN(G418)-TET-OFF – ubiquitin – LEU – 4FLAG

193317 Saccharomyces cerevisiae Bacterial Expression

Plasmid Information

Purpose
Doxycycline-induced transcriptional regulation and Ubiquitin - LEU - 4xFLAG N-terminal tagging. Ubiquitin is cleaved off, resulting in an unstable protein with an N-terminal leucine.
Depositor
Rob Mitra
Article
Shively et al ACS Synth Biol. 2023 Jan 17. doi: 10.102
Insert
Ubiquitin - Leucine - 4FLAG N-terminal tagging
Tags
4FLAG
Available Since
Feb. 1, 2023
Availability
Available

Sequence Information

Length
5,295 bp
Topology
circular
Molecule Type
other DNA
View on AddGene.org

Features

CDS 7
Label Gene Product Position Strand
AmpR bla beta-lactamase 994..1855 Rev
DNA Sequence
ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA
Protein Sequence
MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW
KanR aph(3')-Ia aminoglycoside phosphotransferase 2852..3662 Rev
DNA Sequence
ATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA
Protein Sequence
MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF
ubiquitin Homo sapiens UBC human ubiquitin 4633..4861 Fwd
DNA Sequence
ATGCAAATTTTCGTCAAGACTTTAACCGGTAAGACTATTACCCTGGAAGTTGAATCTTCTGACACTATTGACAATGTCAAGTCCAAGATCCAAGACAAGGAAGGTATTCCACCTGACCAACAAAGATTGATCTTTGCTGGTAAGCAATTGGAAGATGGTAGAACTTTGTCCGACTACAACATCCAAAAGGAATCTACTCTACACTTGGTCTTGAGATTGAGAGGTGGT
Protein Sequence
MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
FLAG — FLAG(R) epitope tag, followed by an ent… 4879..4903 Fwd
DNA Sequence
GATTACAAAGACGATGATGACAAG
Protein Sequence
DYKDDDDK
FLAG — FLAG(R) epitope tag, followed by an ent… 4909..4933 Fwd
DNA Sequence
GACTATAAGGATGACGATGACAAG
Protein Sequence
DYKDDDDK
FLAG — FLAG(R) epitope tag, followed by an ent… 4939..4963 Fwd
DNA Sequence
GATTATAAAGATGATGACGATAAA
Protein Sequence
DYKDDDDK
FLAG — FLAG(R) epitope tag, followed by an ent… 4969..4993 Fwd
DNA Sequence
GACTACAAGGACGACGATGATAAA
Protein Sequence
DYKDDDDK
promoter 5
Label Gene Product Position Strand
AmpR promoter bla — 1855..1960 Rev
DNA Sequence
CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT
T7 promoter — — 2609..2628 Fwd
DNA Sequence
TAATACGACTCACTATAGG
TEF promoter — — 3662..4006 Rev
DNA Sequence
GACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACC
T3 promoter — — 5089..5108 Rev
DNA Sequence
AATTAACCCTCACTAAAGG
lac promoter — — 5176..5207 Rev
DNA Sequence
TTTACACTTTATGCTTCCGGCTCGTATGTTG
protein_bind 6
Label Gene Product Position Strand
tet operator tetO — 4281..4300 Fwd
DNA Sequence
TCCCTATCAGTGATAGAGA
tet operator tetO — 4323..4342 Fwd
DNA Sequence
TCCCTATCAGTGATAGAGA
tet operator tetO — 4365..4384 Fwd
DNA Sequence
TCCCTATCAGTGATAGAGA
tet operator tetO — 4407..4426 Fwd
DNA Sequence
TCCCTATCAGTGATAGAGA
lac operator — — 5152..5169 Fwd
DNA Sequence
TTGTTATCCGCTCACAA
CAP binding site — — 5221..5243 Fwd
DNA Sequence
ATGAGTGAGCTAACTCACATTA
rep_origin 2
Label Gene Product Position Strand
ori — — 235..824 Rev
DNA Sequence
TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
f1 ori — — 1986..2443 Fwd
DNA Sequence
ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTT
terminator 2
Label Gene Product Position Strand
TEF terminator — — 2649..2847 Rev
DNA Sequence
ACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTG
ADH1 terminator S. cerevisiae ADH1 — 4036..4224 Fwd
DNA Sequence
GCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGTATGAGGTCGCTCTTATTGACCACACCTCTACCGG