LOCUS       sequence-379062-        7766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of 3xFLAH-NCOA4 (delta IDR2) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7766)
  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 7766)
  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 (dIDR2)
FEATURES             Location/Qualifiers
     source          1..7766
                     /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    3677..4243
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3693..4245
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3837..3854)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4064..4083
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4263..4862
                     /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(4263..4282)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4759..4779
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5092..5561
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5691..5708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5862..6450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5942..5961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6614..7405,7406..7474))
                     /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     7237..7256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7475..7579)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7647..7665
                     /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 cataagccgt 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 aggatcctgg tgttccttta acacagctga
     3421 ctgggtcctg ccaggaaaga agatgggcaa cctcagccag ttatcttctg gagaagacaa
     3481 gtggctgctt cgaaagaagg cccaggaagt attacttaat tcacctctac aggaggaaca
     3541 taacttcccc ccagaccatt atggcctccc tgcagtttgt gatctctttg cctgtatgca
     3601 gcttaaagtt gataaagaga agtggttata tcgaactcct ctacagatgt agctcgagag
     3661 cggccgctac gtaaattccg cccccccccc taacgttact ggccgaagcc gcttggaata
     3721 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
     3781 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
     3841 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagtttc
     3901 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
     3961 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
     4021 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
     4081 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
     4141 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc
     4201 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccgtcg
     4261 acatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg
     4321 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg
     4381 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     4441 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     4501 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     4561 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     4621 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     4681 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     4741 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     4801 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     4861 gagtcgaccg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     4921 gctcgacgat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
     4981 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata
     5041 cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg
     5101 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     5161 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag
     5221 ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc
     5281 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca
     5341 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc
     5401 cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt
     5461 atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt
     5521 ctcctctgag tgattgacta cccgtcagcg ggggtctttc acatgcagca tgtatcaaaa
     5581 ttaatttggt tttttttctt aagtatttac attaaatggc catagttgca ttaatgaatc
     5641 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5701 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5761 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5821 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5881 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5941 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6001 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6061 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6121 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6181 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6241 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6301 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6361 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6421 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6481 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6541 atcttcacct agatcctttt gcggccggcc gcaaatcaat ctaaagtata tatgagtaaa
     6601 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6661 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6721 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6781 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6841 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6901 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6961 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7021 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7081 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7141 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7201 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     7261 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7321 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7381 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7441 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7501 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7561 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     7621 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcact
     7681 ataggagctt ggcccattgc atacgttgta tccatatcat aatatgtaca tttatattgg
     7741 ctcatgtcca acattaccgc catgtt
//
