LOCUS       sequence-403942-        6475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-p62 var in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6475)
  AUTHORS   Kageyama S, Saito T, Obata M, Koide RH, Ichimura Y, Komatsu M
  TITLE     Negative regulation of the Keap1-Nrf2 pathway by a p62/Sqstm1 
            splicing variant.
  JOURNAL   Mol Cell Biol. 2018 Jan 16. pii: MCB.00642-17. doi: 
            10.1128/MCB.00642-17.
  PUBMED    29339380
REFERENCE   2  (bases 1 to 6475)
  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"; journalName: "Mol Cell 
            Biol. 2018 Jan 16. pii: MCB.00642-17. doi: 10.1128/MCB.00642-17."
COMMENT     Sequence Label: p3xFLAG-p62 var2
FEATURES             Location/Qualifiers
     source          1..6475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        90..469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        470..673
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     624..644
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..737
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        718..736
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             834..899
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      909..918
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          2202..2431
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2414..2433
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2504..3078,3079..3081,3082..3090)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2671..2688)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2898..2917
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3110..3709
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3110..3129)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3606..3626
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3947..4068
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3984..4003)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4173..4189)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4173..4189)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4186..4208)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4197..4213
                     /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)."
     promoter        complement(join(4221..4227,4228..4245,4246..4251))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4266..4287
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4404..4421)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4575..5163)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4655..4674)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5334..6125,6126..6194))
                     /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     5957..5976
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6195..6299)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6374..6393)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
       61 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      121 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      181 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      241 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      301 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      361 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      421 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      481 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      541 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      601 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      661 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
      721 tacgactcac tatagggaga cccaagcttg gtaccgagct cggatcgata tctgcggcct
      781 agctagcgct taaggcctgt taaccggtcg tacgtctccg gattcgaatt atggactaca
      841 aagaccatga cggtgattat aaagatcatg acatcgatta caaggatgac gatgacaagg
      901 aattcgccgc caccatggcg tcgttcacgg tgaaggccta tcttctgggc aaggaggagg
      961 cgacccgcga gatccgccgc ttcagcttct gcttcagccc ggagccggag gcggaagccc
     1021 aagccgcggc cggcccgggg ccctgcgaga ggctgctgag ccgagtggct gtgctgttcc
     1081 ccacgctgag gcctggcggc ttccaggcgc actaccgcga tgaggatggg gacttggttg
     1141 ccttttccag tgatgaggag ctgacaatgg ctatgtccta tgtgaaagat gacatcttcc
     1201 gcatctacat taaagagaag aaggagtgcc ggcgggaaca tcgcccacca tgtgctcagg
     1261 aggcaccccg aaacatggtg caccccaatg tgatctgtga tggttgcaac gggcctgtgg
     1321 tgggaactcg ctataagtgc agtgtgtgcc cagactacga cctgtgcagc gtgtgcgagg
     1381 ggaagggcct gcacagggaa cacagcaagc tcatctttcc caaccccttt ggccacctct
     1441 ctgatagctt ctctcatagc cgctggcttc ggaagctgaa acatggacac tttggctggc
     1501 ctggctggga gatgggccca ccggggaact ggagcccacg tcctcctcgt gcaggggatg
     1561 gccgcccttg ccctacagct gagtcagctt ctgctccacc agaagatccc aatgtcaatt
     1621 tcctgaagaa tgtgggggag agtgtggcag ctgccctcag ccctctaggc attgaggttg
     1681 acattgatgt ggaacatgga gggaagagaa gccgcctgac acccactacc ccagaaagtt
     1741 ccagcacagg cacagaagac aagagtaaca ctcagccaag cagctgctct tcggaagtca
     1801 gcaaacctga cggggctggg gagggccctg ctcagtctct gacagagcaa atgaaaaaga
     1861 tagccttgga gtcggtggga cagccagagg aacagatgga gtcgggaaac tgctcaggag
     1921 gagacgatga ctggacacat ttgtcttcaa aagaagtgga cccatctaca gaggctgatc
     1981 cccggctgat tgagtccctc tcccagatgc tgtccatggg tttctcggat gaaggcggct
     2041 ggctcaccag gctcctacag accaagaatt acgacatcgg ggctgctctg gacacgatcc
     2101 agtattcgaa gcaccctcca ccattgtgac tcgagggccg catagataac tgatccagtg
     2161 tgctggaatt aattcgctgt ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa
     2221 agcgggcatg acttctgcgc taagattgtc agtttccaaa aacgaggagg atttgatatt
     2281 cacctggccc gcggtgatgc ctttgagggt ggccgcgtcc atctggtcag aaaagacaat
     2341 ctttttgttg tcaagcttga ggtgtggcag gcttgagatc tggccataca cttgagtgac
     2401 aatgacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca actgcaggtc
     2461 gagcatgcat ctagggcggc caattccgcc cctctccccc ccccccctct ccctcccccc
     2521 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     2581 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     2641 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     2701 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     2761 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     2821 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     2881 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     2941 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     3001 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     3061 gaaaaacacg atgataagct tgccacaacc cacaaggaga cgaccttcca tgaccgagta
     3121 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc
     3181 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga
     3241 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     3301 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     3361 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     3421 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     3481 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     3541 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     3601 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     3661 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca
     3721 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat ggctccgacc gaagccaccc
     3781 ggggcggccc cgccgacccc gcacccgccc ccgaggccca ccgactctag ctagataact
     3841 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     3901 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     3961 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4021 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac gctcgatcga
     4081 gtgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4141 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4201 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4261 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4321 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4381 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4441 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4501 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4561 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4621 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4681 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4741 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4801 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4861 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4921 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4981 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5041 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5101 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5161 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5221 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5281 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5341 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5401 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5461 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5521 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5581 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5641 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5701 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5761 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5821 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5881 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5941 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6001 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6061 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6121 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6181 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6241 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6301 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     6361 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     6421 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaag
//
