LOCUS       sequence-379045-        7790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of 3xFLAH-NCOA4 (delta C) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7790)
  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 7790)
  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 (dC)
FEATURES             Location/Qualifiers
     source          1..7790
                     /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    3701..4267
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3717..4269
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3861..3878)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4088..4107
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4287..4886
                     /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(4287..4306)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4783..4803
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5116..5585
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5715..5732)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5886..6474)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5966..5985)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6638..7429,7430..7498))
                     /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     7261..7280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7499..7603)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7671..7689
                     /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 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 acttagctcg agagcggccg ctacgtaaat tccgcccccc cccctaacgt
     3721 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
     3781 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag
     3841 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa
     3901 ggaagcagtt cctctggaag tttcttgaag acaaacaacg tctgtagcga ccctttgcag
     3961 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga
     4021 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
     4081 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc
     4141 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag
     4201 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg
     4261 atgataatat ggccacaacc gtcgacatga ccgagtacaa gcccacggtg cgcctcgcca
     4321 cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     4381 ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     4441 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     4501 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     4561 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     4621 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     4681 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     4741 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     4801 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     4861 gcatgacccg caagcccggt gcctgagtcg accgcccgcc ccacgacccg cagcgcccga
     4921 ccgaaaggag cgcacgaccc catggctcga cgataaaata aaagatttta tttagtctcc
     4981 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
     5041 cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga tcaaggtcag
     5101 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     5161 ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg
     5221 taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtccagcc
     5281 ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac
     5341 cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg
     5401 ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt cctccgattg
     5461 actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt
     5521 ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
     5581 tttcacatgc agcatgtatc aaaattaatt tggttttttt tcttaagtat ttacattaaa
     5641 tggccatagt tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5701 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5761 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5821 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5881 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5941 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6001 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6061 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6121 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6181 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6241 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6301 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6361 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6421 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6481 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6541 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttgcggcc ggccgcaaat
     6601 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6661 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6721 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6781 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6841 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6901 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6961 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7021 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7081 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7141 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7201 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg
     7261 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7321 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7381 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7441 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7501 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7561 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7621 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     7681 tcacgaggcc ctttcgtctt cactatagga gcttggccca ttgcatacgt tgtatccata
     7741 tcataatatg tacatttata ttggctcatg tccaacatta ccgccatgtt
//
