LOCUS       sequence-415624-        6474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-p62 var2 PB1mut in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6474)
  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 6474)
  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 PB1mut
FEATURES             Location/Qualifiers
     source          1..6474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        96..475
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        476..679
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     630..650
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     724..743
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        724..742
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             840..905
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      915..924
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          2208..2437
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2420..2439
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2509..3083,3084..3086,3087..3095)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2676..2693)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2903..2922
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3115..3714
                     /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(3115..3134)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3611..3631
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3952..4073
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3989..4008)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4178..4194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4178..4194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4191..4213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4202..4218
                     /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(4226..4232,4233..4250,4251..4256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4271..4292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4409..4426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4580..5168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4660..4679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5339..6130,6131..6199))
                     /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     5962..5981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6200..6304)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6379..6398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
       61 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
      121 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      181 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      241 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      301 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      361 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      421 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      481 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      541 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      601 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      661 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
      721 aattaatacg actcactata gggagaccca agcttggtac cgagctcgga tcgatatctg
      781 cggcctagct agcgcttaag gcctgttaac cggtcgtacg tctccggatt cgaattatgg
      841 actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag gatgacgatg
      901 acaaggaatt cgccgccacc atggcgtcgt tcacggtggc cgcctatctt ctgggcaagg
      961 aggaggcgac ccgcgagatc cgccgcttca gcttctgctt cagcccggag ccggaggcgg
     1021 aagcccaagc cgcggccggc ccggggccct gcgagaggct gctgagccga gtggctgtgc
     1081 tgttccccac gctgaggcct ggcggcttcc aggcgcacta ccgcgccgag gatggggact
     1141 tggttgcctt ttccagtgat gaggagctga caatggctat gtcctatgtg aaagatgaca
     1201 tcttccgcat ctacattaaa gagaagaagg agtgccggcg ggaacatcgc ccaccatgtg
     1261 ctcaggaggc accccgaaac atggtgcacc ccaatgtgat ctgtgatggt tgcaacgggc
     1321 ctgtggtggg aactcgctat aagtgcagtg tgtgcccaga ctacgacctg tgcagcgtgt
     1381 gcgaggggaa gggcctgcac agggaacaca gcaagctcat ctttcccaac ccctttggcc
     1441 acctctctga tagcttctct catagccgct ggcttcggaa gctgaaacat ggacactttg
     1501 gctggcctgg ctgggagatg ggcccaccgg ggaactggag cccacgtcct cctcgtgcag
     1561 gggatggccg cccttgccct acagctgagt cagcttctgc tccaccagaa gatcccaatg
     1621 tcaatttcct gaagaatgtg ggggagagtg tggcagctgc cctcagccct ctaggcattg
     1681 aggttgacat tgatgtggaa catggaggga agagaagccg cctgacaccc actaccccag
     1741 aaagttccag cacaggcaca gaagacaaga gtaacactca gccaagcagc tgctcttcgg
     1801 aagtcagcaa acctgacggg gctggggagg gccctgctca gtctctgaca gagcaaatga
     1861 aaaagatagc cttggagtcg gtgggacagc cagaggaaca gatggagtcg ggaaactgct
     1921 caggaggaga cgatgactgg acacatttgt cttcaaaaga agtggaccca tctacagagg
     1981 ctgatccccg gctgattgag tccctctccc agatgctgtc catgggtttc tcggatgaag
     2041 gcggctggct caccaggctc ctacagacca agaattacga catcggggct gctctggaca
     2101 cgatccagta ttcgaagcac cctccaccat tgtgactcga gggccgcata gataactgat
     2161 ccagtgtgct ggaattaatt cgctgtctgc gagggccagc tgttggggtg agtactccct
     2221 ctcaaaagcg ggcatgactt ctgcgctaag attgtcagtt tccaaaaacg aggaggattt
     2281 gatattcacc tggcccgcgg tgatgccttt gagggtggcc gcgtccatct ggtcagaaaa
     2341 gacaatcttt ttgttgtcaa gcttgaggtg tggcaggctt gagatctggc catacacttg
     2401 agtgacaatg acatccactt tgcctttctc tccacaggtg tccactccca ggtccaactg
     2461 caggtcgagc atgcatctag ggcggccaat tccgcccctc tccccccccc cctctccctc
     2521 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     2581 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     2641 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     2701 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     2761 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     2821 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     2881 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     2941 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     3001 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     3061 cctttgaaaa acacgatgat aagcttgcca caacccacaa ggagacgacc ttccatgacc
     3121 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc
     3181 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac
     3241 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     3301 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     3361 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     3421 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     3481 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     3541 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     3601 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     3661 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg
     3721 ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatggctc cgaccgaagc
     3781 cacccggggc ggccccgccg accccgcacc cgcccccgag gcccaccgac tctagctaga
     3841 taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     3901 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     3961 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4021 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgctcg
     4081 atcgagtgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4141 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4201 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4261 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4321 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4381 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4441 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4501 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4561 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4621 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4681 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4741 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4801 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4861 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4921 cactggcagc agccactggt aacaggatta gcggagcgag gtatgtaggc ggtgctacag
     4981 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5041 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5101 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5161 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5221 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5281 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5341 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5401 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5461 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5521 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5581 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5641 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5701 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5761 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5821 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5881 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5941 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6001 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6061 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6121 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6181 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6241 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6301 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6361 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     6421 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctac
//
