LOCUS       sequence-443192-        7529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of GFP-2xFYVE in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7529)
  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 7529)
  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-2xFYVE
FEATURES             Location/Qualifiers
     source          1..7529
                     /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"
     CDS             join(1982..1984,1985..1987,1988..2698)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1982..1984,1985..1987,1988..2698)
                     /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(2027..2048)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2288..2307)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2635..2656
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     gap             3083..3182
                     /estimated_length=100
     misc_feature    3313..3879
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3329..3881
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3473..3490)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3700..3719
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3899..4498
                     /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(3899..3918)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4395..4415
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4728..5197
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5454..5471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5625..6213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5705..5724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6377..7168,7169..7237))
                     /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     7000..7019
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7238..7342)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7410..7428
                     /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 ccatcgatac
     1981 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2041 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2101 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2161 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2221 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2281 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2341 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2401 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2461 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     2521 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2581 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2641 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagat
     2701 cgatgaattc gaaagtgatg ccatgttcgc tgctgaaaga gcccctgact gggtggatgc
     2761 tgaggaatgc catcggtgca gagtacagtt tggggtggtg acccgcaagc atcactgccg
     2821 agcatgtggg cagatcttct gtggcaagtg ctcctccaag tactccacca tccccaagtt
     2881 cggcattgag aaggaggtgc gcgtgtgtga gccctgctat gagcagctga acaagaaggc
     2941 acaaggacaa ggatccgaaa gtgatgccat gttcgctgct gaaagagccc ctgactgggt
     3001 ggatgctgag gaatgccatc ggtgcagagt acagtttggg gtggtgaccc gcaagcatca
     3061 ctgccgagca tgtgggcaga tcnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     3121 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     3181 nnttctgtgg caagtgctcc tccaagtact ccaccatccc caagttcggc attgagaagg
     3241 aggtgcgcgt gtgtgagccc tgctatgagc agctgaacaa gaaggcataa gtcgagcggc
     3301 cgctacgtaa attccgcccc cccccctaac gttactggcc gaagccgctt ggaataaggc
     3361 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
     3421 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
     3481 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agtttcttga
     3541 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     3601 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     3661 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     3721 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     3781 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac
     3841 cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa ccgtcgacat
     3901 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg
     3961 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg
     4021 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     4081 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     4141 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     4201 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     4261 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     4321 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     4381 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     4441 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgagt
     4501 cgaccgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatggctc
     4561 gacgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac
     4621 ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga aaaatacata
     4681 actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagctg aatatgggcc
     4741 aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggaac
     4801 agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
     4861 aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga accatcagat
     4921 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca
     4981 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca
     5041 acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc
     5101 aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc
     5161 tctgagtgat tgactacccg tcagcggggg tctttcatga ctgagtcgcc cgggtacccg
     5221 tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag
     5281 ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcacatgca gcatgtatca
     5341 aaattaattt ggtttttttt cttaagtatt tacattaaat ggccatagtt gcattaatga
     5401 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5461 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5521 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5581 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5641 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5701 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5761 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5821 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5881 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5941 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6001 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6061 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6121 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6181 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6241 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6301 aggatcttca cctagatcct tttgcggccg gccgcaaatc aatctaaagt atatatgagt
     6361 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6421 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6481 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6541 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6601 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6661 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6721 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6781 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6841 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6901 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6961 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     7021 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7081 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7141 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7201 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7261 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7321 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     7381 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     7441 actataggag cttggcccat tgcatacgtt gtatccatat cataatatgt acatttatat
     7501 tggctcatgt ccaacattac cgccatgtt
//
