LOCUS       sequence-443151-        6908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of GFP-5K4Q in mammalian cells to monitor membrane
            charge.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6908)
  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 6908)
  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-5K4Q
FEATURES             Location/Qualifiers
     source          1..6908
                     /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    2819..3385
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2835..3387
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2979..2996)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3206..3225
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3405..4004
                     /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(3405..3424)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3901..3921
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4234..4703
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4833..4850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5004..5592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5084..5103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5756..6547,6548..6616))
                     /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     6379..6398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6617..6721)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6789..6807
                     /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 cgggatccgg gaattcaagc aaagacggcc agcagcaaca
     2761 aaagaagtct aagaccaagt gtgtaatcat gtaactcgag agcggccgct acgtaaattc
     2821 cgcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
     2881 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
     2941 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg
     3001 tctgttgaat gtcgtgaagg aagcagttcc tctggaagtt tcttgaagac aaacaacgtc
     3061 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
     3121 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
     3181 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
     3241 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
     3301 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt
     3361 tttcctttga aaaacacgat gataatatgg ccacaaccgt cgacatgacc gagtacaagc
     3421 ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg
     3481 cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg
     3541 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     3601 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     3661 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     3721 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     3781 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     3841 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     3901 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     3961 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgagtcgac cgcccgcccc
     4021 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tggctcgacg ataaaataaa
     4081 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     4141 agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaga
     4201 agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg
     4261 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc
     4321 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc
     4381 cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc
     4441 ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct
     4501 tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggc
     4561 gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg
     4621 cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac
     4681 tacccgtcag cgggggtctt tcacatgcag catgtatcaa aattaatttg gttttttttc
     4741 ttaagtattt acattaaatg gccatagttg cattaatgaa tcggccaacg cgcggggaga
     4801 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4861 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4921 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4981 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5041 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5101 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5161 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5221 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5281 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5341 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5401 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5461 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5521 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5581 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5641 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5701 ttgcggccgg ccgcaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5761 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5821 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5881 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5941 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6001 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6061 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6121 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6181 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6241 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6301 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6361 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6421 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6481 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6541 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6601 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6661 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6721 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6781 cctataaaaa taggcgtatc acgaggccct ttcgtcttca ctataggagc ttggcccatt
     6841 gcatacgttg tatccatatc ataatatgta catttatatt ggctcatgtc caacattacc
     6901 gccatgtt
//
