LOCUS       sequence-379066-        8696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of mGFP-NCOA4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8696)
  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 8696)
  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-mGFP-NCOA4
FEATURES             Location/Qualifiers
     source          1..8696
                     /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             join(1994..1996,1997..1999,2000..2710)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2039..2060)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2300..2319)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2647..2668
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4607..5173
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4623..5175
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4767..4784)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4994..5013
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5193..5792
                     /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(5193..5212)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5689..5709
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             6022..6491
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6621..6638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6792..7380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6872..6891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7544..8335,8336..8404))
                     /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     8167..8186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8405..8509)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8577..8595
                     /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 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2041 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     2101 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     2161 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     2221 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2281 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     2341 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2401 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2461 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2521 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2581 gcccgacaac cactacctga gcacccagtc caaactgagc aaagacccca acgagaagcg
     2641 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2701 gctgtacaag ggttcaggct cgggatccgg gaattcgaat accttccaag accagagtgg
     2761 cagctccagt aatagagaac cccttttgag gtgtagtgat gcacggaggg acttggagct
     2821 tgctattggt ggagttctcc gggctgaaca gcaaattaaa gataacttgc gagaggtcaa
     2881 agctcagatt cacagttgca taagccgtca cctggaatgt cttagaagcc gtgaggtatg
     2941 gctgtatgaa caggtggacc ttatttatca gcttaaagag gagacacttc aacagcaggc
     3001 tcagcagctc tactcgttat tgggccagtt caattgtctt actcatcaac tggagtgtac
     3061 ccaaaacaaa gatctagcca atcaagtctc tgtgtgcctg gagagactgg gcagtttgac
     3121 ccttaagcct gaagattcaa ctgtcctgct ctttgaagct gacacaatta ctctgcgcca
     3181 gaccatcacc acatttgggt ctctcaaaac cattcaaatt cctgagcact tgatggctca
     3241 tgctagttca gcaaatattg ggcccttcct ggagaagaga ggctgtatct ccatgccaga
     3301 gcagaagtca gcatccggta ttgtagctgt ccctttcagc gaatggctcc ttggaagcaa
     3361 acctgccagt ggttatcaag ctccttacat acccagcacc gacccccagg actggcttac
     3421 ccaaaagcag accttggaga acagtcagac ttcttccaga gcctgcaatt tcttcaataa
     3481 tgtcggggga aacctaaagg gcttagaaaa ctggctcctc aagagtgaaa aatcaagtta
     3541 tcaaaagtgt aacagccatt ccactactag ttctttctcc attgaaatgg aaaaggttgg
     3601 agatcaagag cttcctgatc aagatgagat ggacctatca gattggctag tgactcccca
     3661 ggaatcccat aagctgcgga agcctgagaa tggcagtcgt gaaacttctg aaaaattcaa
     3721 attattgttc cagtcctata atgtgaatga ttggcttgtc aagactgact cctgtaccaa
     3781 ctgtcaggga aaccagccca aaggtgtgga gattgaaaac ctgggcaatc tgaagtgcct
     3841 gaatgaccac ttggaggcca agaaaccatt gtccaccccc agcatggtta cagaggattg
     3901 gcttgtccag aaccatcagg acccatgtaa ggtagaggag gtgtgcagag ccaatgagcc
     3961 ctgcacaagc tttgcagagt gtgtgtgtga tgagaattgt gagaaggagg ctctgtataa
     4021 gtggcttctg aagaaagaag gaaaggataa aaatgggatg cctgtggaac ccaaacctga
     4081 gcctgagaag cataaagatt ccctgaatat gtggctctgt cctagaaaag aagtaataga
     4141 acaaactaaa gcaccaaagg caatgactcc ttctagaatt gctgattcct tccaagtcat
     4201 aaagaacagc cccttgtcgg agtggcttat caggccccca tacaaagaag gaagtcccaa
     4261 ggaagtgcct ggtactgaag acagagctgg caaacagaag tttaaaagcc ccatgaatac
     4321 ttcctggtgt tcctttaaca cagctgactg ggtcctgcca ggaaagaaga tgggcaacct
     4381 cagccagtta tcttctggag aagacaagtg gctgcttcga aagaaggccc aggaagtatt
     4441 acttaattca cctctacagg aggaacataa cttcccccca gaccattatg gcctccctgc
     4501 agtttgtgat ctctttgcct gtatgcagct taaagttgat aaagagaagt ggttatatcg
     4561 aactcctcta cagatgtagc tcgagtaaag cggccgctac gtaaattccg cccccccccc
     4621 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     4681 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     4741 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     4801 cgtgaaggaa gcagttcctc tggaagtttc ttgaagacaa acaacgtctg tagcgaccct
     4861 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     4921 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     4981 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     5041 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     5101 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     5161 aacacgatga taatatggcc acaaccgtcg acatgaccga gtacaagccc acggtgcgcc
     5221 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact
     5281 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
     5341 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     5401 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     5461 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     5521 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     5581 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     5641 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     5701 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     5761 cctggtgcat gacccgcaag cccggtgcct gagtcgaccg cccgccccac gacccgcagc
     5821 gcccgaccga aaggagcgca cgaccccatg gctcgacgat aaaataaaag attttattta
     5881 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag
     5941 taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa
     6001 ggtcaggaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt
     6061 cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca aacaggatat
     6121 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt
     6181 ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga
     6241 aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg
     6301 cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc gccagtcctc
     6361 cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg
     6421 acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg
     6481 ggggtctttc acatgcagca tgtatcaaaa ttaatttggt tttttttctt aagtatttac
     6541 attaaatggc catagttgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6601 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     6661 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     6721 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     6781 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6841 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6901 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6961 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     7021 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     7081 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     7141 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     7201 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     7261 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     7321 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     7381 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     7441 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt gcggccggcc
     7501 gcaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7561 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7621 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7681 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7741 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7801 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7861 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7921 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7981 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     8041 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     8101 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     8161 cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     8221 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     8281 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     8341 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     8401 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8461 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8521 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     8581 ggcgtatcac gaggcccttt cgtcttcact ataggagctt ggcccattgc atacgttgta
     8641 tccatatcat aatatgtaca tttatattgg ctcatgtcca acattaccgc catgtt
//
