LOCUS       sequence-379056-        8045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of 3xFLAH-NCOA4 (I56E mutant) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8045)
  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 8045)
  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 (I56E)
FEATURES             Location/Qualifiers
     source          1..8045
                     /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    3956..4522
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3972..4524
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4116..4133)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4343..4362
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4542..5141
                     /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(4542..4561)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5038..5058
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5371..5840
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5970..5987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6141..6729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6221..6240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6893..7684,7685..7753))
                     /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     7516..7535
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7754..7858)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7926..7944
                     /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 gggaattcga ataccttcca
     2101 agaccagagt ggcagctcca gtaatagaga accccttttg aggtgtagtg atgcacggag
     2161 ggacttggag cttgctattg gtggagttct ccgggctgaa cagcaaatta aagataactt
     2221 gcgagaggtc aaagctcaga ttcacagttg cgaaagccgt cacctggaat gtcttagaag
     2281 ccgtgaggta tggctgtatg aacaggtgga ccttatttat cagcttaaag aggagacact
     2341 tcaacagcag gctcagcagc tctactcgtt attgggccag ttcaattgtc ttactcatca
     2401 actggagtgt acccaaaaca aagatctagc caatcaagtc tctgtgtgcc tggagagact
     2461 gggcagtttg acccttaagc ctgaagattc aactgtcctg ctctttgaag ctgacacaat
     2521 tactctgcgc cagaccatca ccacatttgg gtctctcaaa accattcaaa ttcctgagca
     2581 cttgatggct catgctagtt cagcaaatat tgggcccttc ctggagaaga gaggctgtat
     2641 ctccatgcca gagcagaagt cagcatccgg tattgtagct gtccctttca gcgaatggct
     2701 ccttggaagc aaacctgcca gtggttatca agctccttac atacccagca ccgaccccca
     2761 ggactggctt acccaaaagc agaccttgga gaacagtcag acttcttcca gagcctgcaa
     2821 tttcttcaat aatgtcgggg gaaacctaaa gggcttagaa aactggctcc tcaagagtga
     2881 aaaatcaagt tatcaaaagt gtaacagcca ttccactact agttctttct ccattgaaat
     2941 ggaaaaggtt ggagatcaag agcttcctga tcaagatgag atggacctat cagattggct
     3001 agtgactccc caggaatccc ataagctgcg gaagcctgag aatggcagtc gtgaaacttc
     3061 tgaaaaattc aaattattgt tccagtccta taatgtgaat gattggcttg tcaagactga
     3121 ctcctgtacc aactgtcagg gaaaccagcc caaaggtgtg gagattgaaa acctgggcaa
     3181 tctgaagtgc ctgaatgacc acttggaggc caagaaacca ttgtccaccc ccagcatggt
     3241 tacagaggat tggcttgtcc agaaccatca ggacccatgt aaggtagagg aggtgtgcag
     3301 agccaatgag ccctgcacaa gctttgcaga gtgtgtgtgt gatgagaatt gtgagaagga
     3361 ggctctgtat aagtggcttc tgaagaaaga aggaaaggat aaaaatggga tgcctgtgga
     3421 acccaaacct gagcctgaga agcataaaga ttccctgaat atgtggctct gtcctagaaa
     3481 agaagtaata gaacaaacta aagcaccaaa ggcaatgact ccttctagaa ttgctgattc
     3541 cttccaagtc ataaagaaca gccccttgtc ggagtggctt atcaggcccc catacaaaga
     3601 aggaagtccc aaggaagtgc ctggtactga agacagagct ggcaaacaga agtttaaaag
     3661 ccccatgaat acttcctggt gttcctttaa cacagctgac tgggtcctgc caggaaagaa
     3721 gatgggcaac ctcagccagt tatcttctgg agaagacaag tggctgcttc gaaagaaggc
     3781 ccaggaagta ttacttaatt cacctctaca ggaggaacat aacttccccc cagaccatta
     3841 tggcctccct gcagtttgtg atctctttgc ctgtatgcag cttaaagttg ataaagagaa
     3901 gtggttatat cgaactcctc tacagatgta gctcgagagc ggccgctacg taaattccgc
     3961 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     4021 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     4081 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     4141 gttgaatgtc gtgaaggaag cagttcctct ggaagtttct tgaagacaaa caacgtctgt
     4201 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     4261 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     4321 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     4381 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     4441 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     4501 cctttgaaaa acacgatgat aatatggcca caaccgtcga catgaccgag tacaagccca
     4561 cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt
     4621 tcgccgacta ccccgccacg cgccacaccg tcgacccgga ccgccacatc gagcgggtca
     4681 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     4741 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     4801 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     4861 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     4921 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     4981 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     5041 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     5101 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg agtcgaccgc ccgccccacg
     5161 acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctcgacgata aaataaaaga
     5221 ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc
     5281 tagcttaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagagaagt
     5341 tcagatcaag gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg
     5401 taagcagttc ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa
     5461 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc
     5521 agatgcggtc cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca
     5581 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
     5641 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg
     5701 ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag
     5761 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
     5821 ccgtcagcgg gggtctttca catgcagcat gtatcaaaat taatttggtt ttttttctta
     5881 agtatttaca ttaaatggcc atagttgcat taatgaatcg gccaacgcgc ggggagaggc
     5941 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6001 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6061 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6121 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6181 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6241 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6301 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6361 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6421 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6481 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6541 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6601 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6661 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6721 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6781 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttttg
     6841 cggccggccg caaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6901 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6961 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7021 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7081 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7141 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7201 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7261 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7321 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7381 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7441 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7501 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7561 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7621 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7681 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7741 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7801 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7861 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     7921 ataaaaatag gcgtatcacg aggccctttc gtcttcacta taggagcttg gcccattgca
     7981 tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc
     8041 atgtt
//
