LOCUS       sequence-379065-        7559 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of 3xFLAH-NCOA4 (delta N) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7559)
  AUTHORS   Ohshima T, Yamamoto H, Sakamaki Y, Saito C, Mizushima N
  TITLE     NCOA4 drives ferritin phase separation to facilitate 
            macroferritinophagy and microferritinophagy.
  JOURNAL   J Cell Biol. 2022 Oct 3;221(10). pii: 213442. doi: 
            10.1083/jcb.202203102. Epub 2022 Sep 6.
  PUBMED    36066504
REFERENCE   2  (bases 1 to 7559)
  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: "J Cell 
            Biol."; date: "2022-10-3"; volume: "221(10). pii: 213442. doi"; 
            pages: " 10.1083/jcb.202203102. Epub 2022 Sep 6"
COMMENT     Sequence Label: pMRX-IP-3xFLAG-NCOA4 (dN)
FEATURES             Location/Qualifiers
     source          1..7559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    739..1096
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1161..1577
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1507..1529
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1545..1561
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1587..1961
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1899..1915
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      1988..1997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1997..2062
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    3470..4036
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3486..4038
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3630..3647)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3857..3876
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4056..4655
                     /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(4056..4075)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4552..4572
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4885..5354
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5484..5501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5655..6243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5735..5754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6407..7198,7199..7267))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7030..7049
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7268..7372)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7440..7458
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg ctagagaacc atcagatgtt tccagggtgc cccaaggacc
      421 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
      481 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
      541 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
      601 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
      661 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc
      721 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt
      781 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg
      841 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc
      901 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg
      961 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc
     1021 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg
     1081 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt
     1141 tgtctgaaaa tatgggcccg ggccagactg ttaccactcc cttaagtttg accttaggtc
     1201 actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt
     1261 gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca
     1321 cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg
     1381 gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc
     1441 cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt
     1501 ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca
     1561 ctccttctct aggcgccccc atatggccat atgagatctt atatggggca cccccgcccc
     1621 ttgtaaactt ccctgaccct gacatgacaa gagttactaa cagcccctct ctccaagctc
     1681 acttacaggc tctctactta gtccagcacg aagtctggag acctctggcg gcagcctacc
     1741 aagaacaact ggaccgaccg gtggtacctc acccttaccg agtcggcgac acagtgtggg
     1801 tccgccgaca ccagactaag aacctagaac ctcgctggaa aggaccttac acagtcctgc
     1861 tgaccacccc caccgccctc aaagtagacg gcatcgcagc ttggatacac gccgcccacg
     1921 tgaaggctgc cgaccccggg ggtggaccat cctctagact gccggatctg ccatcgatat
     1981 aaagcttgcc accatggact acaaagacca tgacggtgat tataaagatc atgacatcga
     2041 ctacaaggat gacgatgaca agggttcagg ctcgggatcc gggaattcgg agcacttgat
     2101 ggctcatgct agttcagcaa atattgggcc cttcctggag aagagaggct gtatctccat
     2161 gccagagcag aagtcagcat ccggtattgt agctgtccct ttcagcgaat ggctccttgg
     2221 aagcaaacct gccagtggtt atcaagctcc ttacataccc agcaccgacc cccaggactg
     2281 gcttacccaa aagcagacct tggagaacag tcagacttct tccagagcct gcaatttctt
     2341 caataatgtc gggggaaacc taaagggctt agaaaactgg ctcctcaaga gtgaaaaatc
     2401 aagttatcaa aagtgtaaca gccattccac tactagttct ttctccattg aaatggaaaa
     2461 ggttggagat caagagcttc ctgatcaaga tgagatggac ctatcagatt ggctagtgac
     2521 tccccaggaa tcccataagc tgcggaagcc tgagaatggc agtcgtgaaa cttctgaaaa
     2581 attcaaatta ttgttccagt cctataatgt gaatgattgg cttgtcaaga ctgactcctg
     2641 taccaactgt cagggaaacc agcccaaagg tgtggagatt gaaaacctgg gcaatctgaa
     2701 gtgcctgaat gaccacttgg aggccaagaa accattgtcc acccccagca tggttacaga
     2761 ggattggctt gtccagaacc atcaggaccc atgtaaggta gaggaggtgt gcagagccaa
     2821 tgagccctgc acaagctttg cagagtgtgt gtgtgatgag aattgtgaga aggaggctct
     2881 gtataagtgg cttctgaaga aagaaggaaa ggataaaaat gggatgcctg tggaacccaa
     2941 acctgagcct gagaagcata aagattccct gaatatgtgg ctctgtccta gaaaagaagt
     3001 aatagaacaa actaaagcac caaaggcaat gactccttct agaattgctg attccttcca
     3061 agtcataaag aacagcccct tgtcggagtg gcttatcagg cccccataca aagaaggaag
     3121 tcccaaggaa gtgcctggta ctgaagacag agctggcaaa cagaagttta aaagccccat
     3181 gaatacttcc tggtgttcct ttaacacagc tgactgggtc ctgccaggaa agaagatggg
     3241 caacctcagc cagttatctt ctggagaaga caagtggctg cttcgaaaga aggcccagga
     3301 agtattactt aattcacctc tacaggagga acataacttc cccccagacc attatggcct
     3361 ccctgcagtt tgtgatctct ttgcctgtat gcagcttaaa gttgataaag agaagtggtt
     3421 atatcgaact cctctacaga tgtagctcga gagcggccgc tacgtaaatt ccgccccccc
     3481 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     3541 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     3601 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     3661 tgtcgtgaag gaagcagttc ctctggaagt ttcttgaaga caaacaacgt ctgtagcgac
     3721 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     3781 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     3841 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     3901 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
     3961 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     4021 aaaaacacga tgataatatg gccacaaccg tcgacatgac cgagtacaag cccacggtgc
     4081 gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg
     4141 actaccccgc cacgcgccac accgtcgacc cggaccgcca catcgagcgg gtcaccgagc
     4201 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     4261 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     4321 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     4381 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     4441 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     4501 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     4561 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     4621 gcacctggtg catgacccgc aagcccggtg cctgagtcga ccgcccgccc cacgacccgc
     4681 agcgcccgac cgaaaggagc gcacgacccc atggctcgac gataaaataa aagattttat
     4741 ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt
     4801 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag aagttcagat
     4861 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca
     4921 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga
     4981 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc
     5041 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc
     5101 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
     5161 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc
     5221 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
     5281 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
     5341 gcgggggtct ttcacatgca gcatgtatca aaattaattt ggtttttttt cttaagtatt
     5401 tacattaaat ggccatagtt gcattaatga atcggccaac gcgcggggag aggcggtttg
     5461 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5521 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5581 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5641 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5701 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5761 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5821 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5881 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5941 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6001 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6061 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6121 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6181 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6241 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6301 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttgcggccg
     6361 gccgcaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6421 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6481 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6541 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6601 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6661 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6721 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6781 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6841 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6901 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6961 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7021 caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7081 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7141 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7201 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7261 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7321 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7381 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     7441 ataggcgtat cacgaggccc tttcgtcttc actataggag cttggcccat tgcatacgtt
     7501 gtatccatat cataatatgt acatttatat tggctcatgt ccaacattac cgccatgtt
//
