LOCUS       sequence-443426-        7308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of GFP-TRPML1(PHD) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7308)
  AUTHORS   Shinoda S, Sakai Y, Matsui T, Uematsu M, Koyama-Honda I, Sakamaki 
            JI, Yamamoto H, Mizushima N
  TITLE     Syntaxin 17 recruitment to mature autophagosomes is temporally 
            regulated by PI4P accumulation.
  JOURNAL   Elife. 2024 Jun 4;12:RP92189. doi: 10.7554/eLife.92189.
  PUBMED    38831696
REFERENCE   2  (bases 1 to 7308)
  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: "Elife."; 
            date: "2024-06-4"; volume: "12:RP92189. doi"; pages: " 
            10.7554/eLife.92189"
COMMENT     Sequence Label: pMRXIP-GFP-TRPML1(PHD)
FEATURES             Location/Qualifiers
     source          1..7308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    667..1024
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1089..1505
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1435..1457
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1473..1489
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1515..1889
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1827..1843
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             join(1901..1903,1904..1906,1907..2617)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1901..1903,1904..1906,1907..2617)
                     /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(1946..1967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2207..2226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2554..2575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3159..3725
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3175..3727
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3319..3336)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3546..3565
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3739..4338
                     /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(3739..3758)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4235..4255
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4562..5031
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5161..5178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5332..5920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5412..5431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6084..6875,6876..6944))
                     /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     6707..6726
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6945..7049)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7117..7135
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7237..7308,1..308)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      241 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      301 ttaccatgct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
      361 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
      421 cccgagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgattgact
      481 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
      541 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
      601 catttggggg ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg
      661 ggaggtaagc tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga
      721 ttttatgcgc ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg
      781 tggaactgac gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg
      841 gggccgtttt tgtggcccga cctgagtcca aaaatcccga tcgttttgga ctctttggtg
      901 cacccccctt agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc
      961 cgcctccgtc tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct
     1021 gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata
     1081 tgggcccggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     1141 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     1201 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     1261 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     1321 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     1381 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     1441 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     1501 gcgcccccat atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc
     1561 ctgaccctga catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc
     1621 tctacttagt ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg
     1681 accgaccggt ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc
     1741 agactaagaa cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca
     1801 ccgccctcaa agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg
     1861 accccggggg tggaccatcc tctagactgc cggatctgcc atggtgagca agggcgagga
     1921 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1981 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2041 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2101 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2161 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2221 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2281 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2341 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     2401 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     2461 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     2521 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2581 cgccgggatc actctcggca tggacgagct gtacaagggt ggcggtggct cgggcggtgg
     2641 tgggtcgggt ggcggcggat ctatcgatga attggaattc atgacagccc cggcgggtcc
     2701 gcgcggctca gagaccgagc ggcttctgac ccccaacccc gggtatggga cccaggcggg
     2761 gccttcaccg gcccctccga cacccccaga agaggaagac cttcgccgtc gtctcaaata
     2821 ctttttcatg agtccctgcg acaagtttcg agccaagggc cgcaagccct gcaagctgat
     2881 gctgggtggc ggtggctcga tgacagcccc ggcgggtccg cgcggctcag agaccgagcg
     2941 gcttctgacc cccaaccccg ggtatgggac ccaggcgggg ccttcaccgg cccctccgac
     3001 acccccagaa gaggaagacc ttcgccgtcg tctcaaatac tttttcatga gtccctgcga
     3061 caagtttcga gccaagggcc gcaagccctg caagctgatg ctgtgagaat tcacgcgttc
     3121 gcgagttaac ggatccctcg agcggccgct acgtaaattc cgcccccccc cctaacgtta
     3181 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     3241 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     3301 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     3361 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     3421 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     3481 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     3541 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     3601 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
     3661 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     3721 gataatatgg ccacaaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     3781 gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     3841 cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     3901 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     3961 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     4021 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     4081 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     4141 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     4201 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     4261 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     4321 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     4381 cgaccccatg gctcgacgat aaaataaaag attttattta gtctccagaa aaagggggga
     4441 atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca
     4501 tggaaaaata cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca
     4561 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca
     4621 agaacagatg gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     4681 cccggctcag ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta
     4741 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt
     4801 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     4861 taaaagagcc cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg
     4921 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc
     4981 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc acatgcagca
     5041 tgtatcaaaa ttaatttggt tttttttctt aagtatttac attaaatggc catagttgca
     5101 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5161 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5221 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5281 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5341 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5401 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5461 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5521 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5581 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5641 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5701 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5761 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5821 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5881 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5941 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6001 atcaaaaagg atcttcacct agatcctttt gcggccggcc gcaaatcaat ctaaagtata
     6061 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6121 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6181 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6241 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6301 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6361 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6421 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6481 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6541 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6601 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6661 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     6721 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6781 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6841 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6901 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6961 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7021 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     7081 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     7141 cgtcttcact cgaggagctt ggcccattgc atacgttgta tccatatcat aatatgtaca
     7201 tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt
     7261 aatagtaatc aattacgggg tcattagttc atagcccata tatggagt
//
