LOCUS       sequence-326385-        7331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-Plaat3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7331)
  AUTHORS   Morishita H, Eguchi T, Tsukamoto S, Sakamaki Y, Takahashi S, Saito 
            C, Koyama-Honda I, Mizushima N
  TITLE     Organelle degradation in the lens by PLAAT phospholipases.
  JOURNAL   Nature. 2021 Apr;592(7855):634-638. doi: 10.1038/s41586-021-03439-w.
            Epub 2021 Apr 14.
  PUBMED    33854238
REFERENCE   2  (bases 1 to 7331)
  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"; doi: 
            "10.1038/s41586-021-03439-w"; journalName: "Nature"; date: 
            "2021-04"; volume: "592"; issue: "7855"; pages: "634-638"
COMMENT     Sequence Label: pMRX-IP-EGFP-mouse Plaat3 (WT)
FEATURES             Location/Qualifiers
     source          1..7331
                     /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"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1994..1996,1997..1999,2000..2710)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     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    3242..3808
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3258..3810
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3402..3419)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3629..3648
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3828..4427
                     /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(3828..3847)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4324..4344
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4657..5126
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5256..5273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5427..6015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5507..5526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6179..6970,6971..7039))
                     /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     6802..6821
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7040..7144)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7212..7230
                     /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 cgccctgagc aaagacccca acgagaagcg
     2641 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2701 gctgtacaag ggttcaggct cgggatccat gctagcaccc ataccagaac ccaagcctgg
     2761 agacctgatt gagattttcc gccctatgta cagacactgg gccatctatg ttggtgatgg
     2821 atacgtgatc cacctggctc ctccaagtga aatcgcagga gctggggcag ccagcatcat
     2881 gtctgctttg actgacaagg ccatagtgaa gaaagaactg ctgtgccatg tggccgggaa
     2941 ggacaagtac caggtcaata acaaacatga cgaggagtac accccactgc ctctgagcaa
     3001 gatcatccag cgggctgaga gactggtggg gcaggaggtg ctctacaggc tgaccagcga
     3061 gaactgtgag cactttgtga atgaactacg ctatggagtt cctcggagtg atcaggtcag
     3121 agatgcggtc aaggcggtag gcatcgctgg agtgggcttg gcggccttgg gcctcgttgg
     3181 agtcatgctc tccagaaaca agaaacagaa gcaatgactc gagagcggcc gctacgtaaa
     3241 ttccgccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     3301 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     3361 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     3421 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gtttcttgaa gacaaacaac
     3481 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     3541 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     3601 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     3661 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     3721 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     3781 ggttttcctt tgaaaaacac gatgataata tggccacaac cgtcgacatg accgagtaca
     3841 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg
     3901 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc
     3961 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     4021 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     4081 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     4141 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     4201 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     4261 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     4321 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     4381 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagtc gaccgcccgc
     4441 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatggctcg acgataaaat
     4501 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg
     4561 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag
     4621 agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat
     4681 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg
     4741 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     4801 gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     4861 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     4921 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     4981 ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc
     5041 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     5101 gactacccgt cagcgggggt ctttcacatg cagcatgtat caaaattaat ttggtttttt
     5161 ttcttaagta tttacattaa atggccatag ttgcattaat gaatcggcca acgcgcgggg
     5221 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5281 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5341 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5401 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5461 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5521 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5581 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5641 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5701 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5761 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5821 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5881 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5941 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6001 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6061 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6121 cttttgcggc cggccgcaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6181 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6241 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6301 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6361 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6421 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6481 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6541 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6601 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6661 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6721 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6781 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6841 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6901 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6961 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7021 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7081 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7141 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     7201 taacctataa aaataggcgt atcacgaggc cctttcgtct tcactatagg agcttggccc
     7261 attgcatacg ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt
     7321 accgccatgt t
//
