LOCUS       sequence-379063-        7700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of 3xFLAH-NCOA4 (delta M) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7700)
  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 7700)
  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 (dM)
FEATURES             Location/Qualifiers
     source          1..7700
                     /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    3611..4177
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3627..4179
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3771..3788)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3998..4017
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4197..4796
                     /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(4197..4216)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4693..4713
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5026..5495
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5625..5642)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5796..6384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5876..5895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6548..7339,7340..7408))
                     /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     7171..7190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7409..7513)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7581..7599
                     /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 aatggcagta aggataaaaa
     3061 tgggatgcct gtggaaccca aacctgagcc tgagaagcat aaagattccc tgaatatgtg
     3121 gctctgtcct agaaaagaag taatagaaca aactaaagca ccaaaggcaa tgactccttc
     3181 tagaattgct gattccttcc aagtcataaa gaacagcccc ttgtcggagt ggcttatcag
     3241 gcccccatac aaagaaggaa gtcccaagga agtgcctggt actgaagaca gagctggcaa
     3301 acagaagttt aaaagcccca tgaatacttc ctggtgttcc tttaacacag ctgactgggt
     3361 cctgccagga aagaagatgg gcaacctcag ccagttatct tctggagaag acaagtggct
     3421 gcttcgaaag aaggcccagg aagtattact taattcacct ctacaggagg aacataactt
     3481 ccccccagac cattatggcc tccctgcagt ttgtgatctc tttgcctgta tgcagcttaa
     3541 agttgataaa gagaagtggt tatatcgaac tcctctacag atgtagctcg agagcggccg
     3601 ctacgtaaat tccgcccccc cccctaacgt tactggccga agccgcttgg aataaggccg
     3661 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
     3721 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa
     3781 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag tttcttgaag
     3841 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg
     3901 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg
     3961 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa
     4021 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg
     4081 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca
     4141 cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc gtcgacatga
     4201 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca
     4261 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc
     4321 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     4381 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     4441 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     4501 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     4561 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     4621 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     4681 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     4741 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgagtcg
     4801 accgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catggctcga
     4861 cgataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     4921 gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac
     4981 tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa
     5041 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag
     5101 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     5161 gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt
     5221 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
     5281 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
     5341 ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa
     5401 taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc
     5461 tgagtgattg actacccgtc agcgggggtc tttcacatgc agcatgtatc aaaattaatt
     5521 tggttttttt tcttaagtat ttacattaaa tggccatagt tgcattaatg aatcggccaa
     5581 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5641 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5701 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5761 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5821 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5881 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5941 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6001 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6061 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6121 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6181 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6241 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6301 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6361 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6421 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6481 acctagatcc ttttgcggcc ggccgcaaat caatctaaag tatatatgag taaacttggt
     6541 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6601 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6661 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6721 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6781 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6841 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6901 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6961 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7021 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7081 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7141 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     7201 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7261 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7321 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7381 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7441 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7501 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     7561 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt cactatagga
     7621 gcttggccca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg
     7681 tccaacatta ccgccatgtt
//
