LOCUS       sequence-403943-        6589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-p62 PB1mut in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6589)
  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 6589)
  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 PB1mut
FEATURES             Location/Qualifiers
     source          1..6589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        94..473
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        474..677
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     628..648
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     722..741
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        722..740
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             838..903
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      913..922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          2320..2549
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2532..2551
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2622..3196,3197..3199,3200..3208)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2789..2806)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3016..3035
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3228..3827
                     /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(3228..3247)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3724..3744
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4065..4186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4102..4121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4291..4307)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4291..4307)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4304..4326)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4315..4331
                     /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(4339..4345,4346..4363,4364..4369))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4384..4405
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4522..4539)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4693..5281)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4773..4792)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5452..6243,6244..6312))
                     /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     6075..6094
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6313..6417)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6492..6511)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
       61 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
      121 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      181 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      241 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      301 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      361 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      421 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      481 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      541 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      601 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      661 tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa
      721 ttaatacgac tcactatagg gagacccaag cttggtaccg agctcggatc gatatctgcg
      781 gcctagctag cgcttaaggc ctgttaaccg gtcgtacgtc tccggattcg aattatggac
      841 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
      901 aaggaattcg ccgccaccat ggcgtcgttc acggtggccg cctatcttct gggcaaggag
      961 gaggcgaccc gcgagatccg ccgcttcagc ttctgcttca gcccggagcc ggaggcggaa
     1021 gcccaagccg cggccggccc ggggccctgc gagaggctgc tgagccgagt ggctgtgctg
     1081 ttccccacgc tgaggcctgg cggcttccag gcgcactacc gcgccgagga tggggacttg
     1141 gttgcctttt ccagtgatga ggagctgaca atggctatgt cctatgtgaa agatgacatc
     1201 ttccgcatct acattaaaga gaagaaggag tgccggcggg aacatcgccc accatgtgct
     1261 caggaggcac cccgaaacat ggtgcacccc aatgtgatct gtgatggttg caacgggcct
     1321 gtggtgggaa ctcgctataa gtgcagtgtg tgcccagact acgacctgtg cagcgtgtgc
     1381 gaggggaagg gcctgcacag ggaacacagc aagctcatct ttcccaaccc ctttggccac
     1441 ctctctgata gcttctctca tagccgctgg cttcggaagc tgaaacatgg acactttggc
     1501 tggcctggct gggagatggg cccaccgggg aactggagcc cacgtcctcc tcgtgcaggg
     1561 gatggccgcc cttgccctac agctgagtca gcttctgctc caccagaaga tcccaatgtc
     1621 aatttcctga agaatgtggg ggagagtgtg gcagctgccc tcagccctct aggcattgag
     1681 gttgacattg atgtggaaca tggagggaag agaagccgcc tgacacccac taccccagaa
     1741 agttccagca caggcacaga agacaagagt aacactcagc caagcagctg ctcttcggaa
     1801 gtcagcaaac ctgacggggc tggggagggc cctgctcagt ctctgacaga gcaaatgaaa
     1861 aagatagcct tggagtcggt gggacagcca gaggaacaga tggagtcggg aaactgctca
     1921 ggaggagacg atgactggac acatttgtct tcaaaagaag tggacccatc tacaggtgaa
     1981 ctccagtctc tacagatgcc agaatcggaa gggccaagct ctctagaccc ctcacaggaa
     2041 ggacccacag ggctgaagga agctgcccta tacccacatc tcccaccaga ggctgatccc
     2101 cggctgattg agtccctctc ccagatgctg tccatgggtt tctcggatga aggcggctgg
     2161 ctcaccaggc tcctacagac caagaattac gacatcgggg ctgctctgga cacgatccag
     2221 tattcgaagc accctccacc attgtgactc gagggccgca tagataactg atccagtgtg
     2281 ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctcaaaag
     2341 cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca
     2401 cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa aagacaatct
     2461 ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact tgagtgacaa
     2521 tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac tgcaggtcga
     2581 gcatgcatct agggcggcca attccgcccc tctccccccc ccccctctcc ctcccccccc
     2641 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     2701 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     2761 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     2821 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     2881 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     2941 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     3001 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     3061 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     3121 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     3181 aaaacacgat gataagcttg ccacaaccca caaggagacg accttccatg accgagtaca
     3241 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg
     3301 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc
     3361 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     3421 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     3481 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     3541 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     3601 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     3661 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     3721 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     3781 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg
     3841 acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctccgaccga agccacccgg
     3901 ggcggccccg ccgaccccgc acccgccccc gaggcccacc gactctagct agataactga
     3961 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     4021 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     4081 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4141 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc tcgatcgagt
     4201 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4261 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4321 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4381 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4441 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4501 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4561 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4621 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4681 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4741 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4801 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4861 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4921 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4981 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5041 agcagccact ggtaacagga ttagcggagc gaggtatgta ggcggtgcta cagagttctt
     5101 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5161 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5221 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5281 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5341 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5401 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5461 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5521 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5581 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5641 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5701 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5761 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5821 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5881 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5941 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6001 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6061 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6121 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6181 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6241 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6301 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6361 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6421 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     6481 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     6541 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaa
//
