LOCUS       sequence-443435-        7221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of GFP-CERT(PHD)(W33A) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7221)
  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 7221)
  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-CERT(PHD)(W33A)
FEATURES             Location/Qualifiers
     source          1..7221
                     /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    3132..3698
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3148..3700
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3292..3309)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3519..3538
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3718..4317
                     /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(3718..3737)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4214..4234
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4547..5016
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5146..5163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5317..5905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5397..5416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6069..6860,6861..6929))
                     /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     6692..6711
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6930..7034)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7102..7120
                     /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 gtcggataat cagagctgga actcgtcggg
     2761 ctcggaggag gatccagaga cggagtctgg gccgcctgtg gagcgctgcg gggtcctcag
     2821 taaggccaca aactacattc atgggtggca ggatcgttgg gtagttttga aaaataatgc
     2881 tctgagttac tacaaatctg aagatgaaac agagtatggc tgcagaggat ccatctgtct
     2941 tagcaaggct gtcatcacac ctcacgattt tgatgaatgt cgatttgata ttagtgtaaa
     3001 tgatagtgtt tggtatcttc gtgctcagga tccagatcat agacagcaat ggatagatgc
     3061 cattgaacag cacaagactg aatctggata tggatgagaa ttcgggtctc gagagcggcc
     3121 gctacgtaaa ttccgccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     3181 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     3241 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     3301 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gtttcttgaa
     3361 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     3421 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     3481 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     3541 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     3601 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     3661 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac cgtcgacatg
     3721 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc
     3781 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc
     3841 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     3901 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     3961 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     4021 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     4081 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     4141 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     4201 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     4261 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagtc
     4321 gaccgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatggctcg
     4381 acgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc
     4441 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa
     4501 ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca
     4561 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca
     4621 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca
     4681 agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg
     4741 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag
     4801 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa
     4861 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca
     4921 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct
     4981 ctgagtgatt gactacccgt cagcgggggt ctttcacatg cagcatgtat caaaattaat
     5041 ttggtttttt ttcttaagta tttacattaa atggccatag ttgcattaat gaatcggcca
     5101 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5161 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5221 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5281 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5341 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5401 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5461 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5521 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5581 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5641 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5701 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5761 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5821 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5881 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5941 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6001 cacctagatc cttttgcggc cggccgcaaa tcaatctaaa gtatatatga gtaaacttgg
     6061 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6121 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6181 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6241 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6301 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6361 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6421 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6481 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6541 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6601 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6661 ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta
     6721 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6781 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6841 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6901 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6961 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7021 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     7081 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcactatagg
     7141 agcttggccc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat
     7201 gtccaacatt accgccatgt t
//
