LOCUS       sequence-443183-        7560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of GFP-P4M-SidMx2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7560)
  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 7560)
  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-P4M-SidMx2
FEATURES             Location/Qualifiers
     source          1..7560
                     /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(1997..1999,2000..2002,2003..2713)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1997..1999,2000..2002,2003..2713)
                     /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(2042..2063)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2303..2322)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2650..2671
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2777..3082
                     /codon_start=1
                     /product="high-affinity phosphatidylinositol 4-phosphate 
                     binding module of SidM (DrrA) from Legionella pneumophila 
                     (Hammond et al., 2014)"
                     /label=P4M
                     /translation="TASTENFKNVKEKYQQMRGDALKTEILADFKDKLAEATDEQSLKQ
                     IVAELKSKDEYRILAKGQGLTTQLLGLKTSSVSSFEKMVEETRESIKSQERQTIKIK"
     CDS             3128..3433
                     /codon_start=1
                     /product="high-affinity phosphatidylinositol 4-phosphate 
                     binding module of SidM (DrrA) from Legionella pneumophila 
                     (Hammond et al., 2014)"
                     /label=P4M
                     /translation="TASTENFKNVKEKYQQMRGDALKTEILADFKDKLAEATDEQSLKQ
                     IVAELKSKDEYRILAKGQGLTTQLLGLKTSSVSSFEKMVEETRESIKSQERQTIKIK"
     misc_feature    3471..4037
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3487..4039
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3631..3648)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3858..3877
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4057..4656
                     /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(4057..4076)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4553..4573
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4886..5355
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5485..5502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5656..6244)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5736..5755)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6408..7199,7200..7268))
                     /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     7031..7050
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7269..7373)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7441..7459
                     /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 aaagctttcg ggatccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2041 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2101 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2161 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2221 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2281 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2341 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2401 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2461 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2521 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2581 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2641 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2701 cgagctgtac aagtccggac tcagatctcg agctcaagct tcgaattctg gtggtagtgc
     2761 ttccggactc agatccacgg caagcacgga aaactttaaa aatgttaaag aaaaatatca
     2821 gcaaatgcga ggtgatgctt taaaaacaga aatcctggct gatttcaagg ataaactggc
     2881 tgaagctacc gatgaacaga gccttaagca aattgttgct gaattgaaaa gtaaagatga
     2941 atatagaatt ttggctaagg ggcaagggtt aacaacccag cttttagggt taaagacaag
     3001 ttcagtgtct tcatttgaga aaatggttga ggaaacaaga gaaagtatta agtctcaaga
     3061 aagacaaacc attaagataa aaggtggtag tgctgtcgac ggtggtagtg cttccggact
     3121 cagatccacg gcaagcacgg aaaactttaa aaatgttaaa gaaaaatatc agcaaatgcg
     3181 aggtgatgct ttaaaaacag aaatcctggc tgatttcaag gataaactgg ctgaagctac
     3241 cgatgaacag agccttaagc aaattgttgc tgaattgaaa agtaaagatg aatatagaat
     3301 tttggctaag gggcaagggt taacaaccca gcttttaggg ttaaagacaa gttcagtgtc
     3361 ttcatttgag aaaatggttg aggaaacaag agaaagtatt aagtctcaag aaagacaaac
     3421 cattaagata aaataagaat tcgggtctcg agagcggccg ctacgtaaat tccgcccccc
     3481 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     3541 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     3601 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     3661 atgtcgtgaa ggaagcagtt cctctggaag tttcttgaag acaaacaacg tctgtagcga
     3721 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     3781 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     3841 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     3901 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     3961 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     4021 gaaaaacacg atgataatat ggccacaacc gtcgacatga ccgagtacaa gcccacggtg
     4081 cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc
     4141 gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag
     4201 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     4261 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     4321 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     4381 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     4441 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     4501 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     4561 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     4621 cgcacctggt gcatgacccg caagcccggt gcctgagtcg accgcccgcc ccacgacccg
     4681 cagcgcccga ccgaaaggag cgcacgaccc catggctcga cgataaaata aaagatttta
     4741 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     4801 taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga
     4861 tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc
     4921 agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg
     4981 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
     5041 cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
     5101 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     5161 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt
     5221 cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
     5281 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc
     5341 agcgggggtc tttcacatgc agcatgtatc aaaattaatt tggttttttt tcttaagtat
     5401 ttacattaaa tggccatagt tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5461 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5521 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5581 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5641 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5701 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5761 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5821 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5881 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5941 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6001 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6061 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6121 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6181 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6241 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6301 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttgcggcc
     6361 ggccgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6421 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6481 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6541 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6601 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6661 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6721 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6781 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6841 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6901 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6961 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7021 tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7081 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7141 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7201 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7261 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7321 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7381 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     7441 aataggcgta tcacgaggcc ctttcgtctt cactatagga gcttggccca ttgcatacgt
     7501 tgtatccata tcataatatg tacatttata ttggctcatg tccaacatta ccgccatgtt
//
