LOCUS       sequence-473618-        7415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient overexpression of KEAP1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7415)
  AUTHORS   Marzio A, Kurz E, Sahni JM, Di Feo G, Puccini J, Jiang S, Hirsch CA,
            Arbini AA, Wu WL, Pass HI, Bar-Sagi D, Papagiannakopoulos T, Pagano 
            M
  TITLE     EMSY inhibits homologous recombination repair and the interferon 
            response, promoting lung cancer immune evasion.
  JOURNAL   Cell. 2022 Jan 6;185(1):169-183.e19. doi: 
            10.1016/j.cell.2021.12.005. Epub 2021 Dec 27.
  PUBMED    34963055
REFERENCE   2  (bases 1 to 7415)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2021.12.005"; journalName: "Cell"; date: "2022-01-6-
            6"; volume: "185"; issue: "1"; pages: "169-183.e19"
COMMENT     Sequence Label: pcDNA3.1 2xFLAG-2xSTREP-Keap1 (mouse)
FEATURES             Location/Qualifiers
     source          1..7415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      643..1231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1132..1151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1385..1402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1519..1540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1555..1560,1561..1578,1579..1585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1593..1609
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     1598..1620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1617..1633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1617..1633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1682..1803
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1693..1712)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1747..1766
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1977..2771)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2088..2107)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2698..2717
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2838..3167)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2852..2987
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2906..2925)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3152..3172
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3181..3609)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3291..3312)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3503..3522
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3655..3879
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3868..3885
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5807..5830)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(5867..5890)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(5906..5929)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(5945..5968)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(6009..6028)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6010..6028)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6073..6276)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6102..6122)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6277..6656
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6828..6847
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6922..7026
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7027..7095,7096..7415,1..472)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7245..7264)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
       61 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
      121 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
      181 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
      241 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
      301 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
      361 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
      421 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
      481 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
      541 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
      601 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
      661 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
      721 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
      781 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
      841 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
      901 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
      961 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     1021 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     1081 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     1141 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     1201 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     1261 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     1321 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     1381 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     1441 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     1501 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     1561 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     1621 aaacagctat gaccatgatt acgccaagct ctagctagag gtcgacggta tacagacatg
     1681 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     1741 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     1801 gttggggtgg gcgaagaact ccagcatgag atccccgcgc tggaggatca tccagccggc
     1861 gtcccggaaa acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc
     1921 tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg aaccccagag tcccgctcag
     1981 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
     2041 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
     2101 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
     2161 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
     2221 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
     2281 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
     2341 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
     2401 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
     2461 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
     2521 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
     2581 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg
     2641 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
     2701 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
     2761 tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatccgaa aatggatata
     2821 caagctcccg ggagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc
     2881 tggaatagct cagaggcaga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag
     2941 ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg
     3001 ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     3061 gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt
     3121 ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccacagaatt
     3181 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     3241 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     3301 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     3361 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     3421 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     3481 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     3541 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     3601 agggcgcgtg gggatacccc ctagagcccc agctggttct ttccgcctca gaagccatag
     3661 agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct
     3721 gccccacccc accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct
     3781 cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg
     3841 aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgggttt
     3901 aaacgggccc tctagactcg agtcagcagg tacagttttg ttgatcaatt tgcttccgac
     3961 agggttccat ggtgacggcc acacccaccc cgctgcggcc agatgtcatg cgggtcacct
     4021 cactccaggt atcactgtcc gggtcatagc attccacact gtccagaaaa gtgtggccat
     4081 catagcctcc gaggacgtag atcttgccct ggtgcacagt aatccccagc gcactacggt
     4141 gatgcctcat gggggctacg aaagtccagg tctctgtctc cacgtcgtag cgctccacac
     4201 tgttcaactg gtcctgccca tcgtagcccc ctgctgcata gatacagttg tgcagcacgc
     4261 agaccccggc cccgctccgg atggtattca tcggtgtgat catccgccac tcattcctct
     4321 ctggatagta acattctgcg gagttaagcc ggttagtccc gtcaaagccc cccactgcat
     4381 acagcaagcg gttgagcact gccacgccca cgccaatcct ccgtgtcaac attggcgcga
     4441 ctagatgcca ctcgtcccgc tctggctcat atctctccac gctgctgtgg tggatgcagc
     4501 cgtgggaacc cccgactgcg tagatgtggc catctatgac ccccaccccg atgcggttgc
     4561 gtggcacgct catagaggca cagggcgacc actggttggt catggggttg tagcagtcca
     4621 gggcgctgga gtcagtgttg ccatccggag agttgttgcg gccgcccaca gcgtatagca
     4681 gcccacccac cacgcagcct gccagcccac tgcgcggcac ctgtagatcg gccaggcgca
     4741 gccaggagcc attgctcggg ttgtaggcct ccaggtagct gagcgactgt cggaagtaac
     4801 cgcccgctgt gtagatgagg cggcccactt tgggcgcgcg gcagggcact gcctgcgtgg
     4861 gcttgtgcag cgtgagctcc tggaatatct gcaccaggta gtccttgcag cgcgcgtcgg
     4921 cctgcaggat ctcacacttc tgcagctgtg tctgcaggaa gcgcggcgtg agcgcatggc
     4981 agcgcacggc ccgcagcagt gcctgcacgt agaagcgccg ctgcgggcag tcgtatttga
     5041 cccagtcgat gcacgcgtgg aacacctcgg actcgcagcg tacgttcaga tcatcccggc
     5101 tgatgagcgt ggccagctgg cagtgtgaca ggttgaagaa ctcctcctgc ttggccacct
     5161 ccccgaagtg catgtagata tactcccggg cacgctggtg cagttcagtg cagccgatct
     5221 gctccgcgaa gttggcgatg ccaatggcgt tgctggggtc cagctgctgc acgaggaagt
     5281 cgctgcaggc tcgaaccacg ctgtcaatct ggtacatgac cgccccgttc atcacgtgca
     5341 ggacacactt ctcgcccacg gagatggagg ccgtgtaggc gaactcaata agcctttcca
     5401 tgaccttagg gtggatgcct tcgatggaca ccacctccat gccctgctcc cgaagcccgt
     5461 tggtgaacat ggctttaaag actgggctgg aggaggccag caccactttg tgagccatga
     5521 attgggcagc tgggatgtcc tcatatttga cctgcagggt cacgtcacag agttgctggc
     5581 tcaggcgaag ctcgttcatg acgccaaaag cctgcttggt gtgatcctct agtgtgtagc
     5641 tgaaggttcg gttaccgtcc tgcgagggcg tcacctctgc cttgcactcc gtggaggcat
     5701 acatcactgc gtccccggcc ccctcggggc actttgacca caggggcagg aactggctgc
     5761 tgcggggagc cccgctaagc ttgggttcgg gctgggatcc agctcctttc tcgaactgcg
     5821 ggtggctcca gcttcctcct ccgctgcctc cgccgcttcc tcctcccttc tcgaactgag
     5881 ggtggctcca gctggcggcc gaccctttat catcatcatc tttataatcc atagatcctg
     5941 gtaccttgtc atcgtcgtcc ttgtagtcca tggtaagctt aagtttaaac gctagccagc
     6001 ttgggtctcc ctatagtgag tcgtattaat ttcgataagc cagtaagcag tgggttctct
     6061 agttagccag agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
     6121 cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
     6181 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
     6241 tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac
     6301 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     6361 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     6421 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     6481 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     6541 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     6601 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg
     6661 cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt
     6721 catgcaattg tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc
     6781 agcgacctcc aacacacaag cagggagcag atactggctt aactatgcgg catcagagca
     6841 gattgtactg agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt
     6901 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6961 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     7021 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     7081 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     7141 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     7201 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     7261 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     7321 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     7381 gaattatgca gtgctgccat aaccatgagt gataa
//
