LOCUS       sequence-454110-        8291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of GFP-PI4K2A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8291)
  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 8291)
  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-PI4K2A
FEATURES             Location/Qualifiers
     source          1..8291
                     /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    4202..4768
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4218..4770
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4362..4379)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4589..4608
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4788..5387
                     /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(4788..4807)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5284..5304
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5617..6086
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6216..6233)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6387..6975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6467..6486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7139..7930,7931..7999))
                     /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     7762..7781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8000..8104)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8172..8190
                     /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 gaattcgatg gacgagacga gcccactagt
     2761 gtcccccgag cgggcccaac ccccggacta caccttcccg tcgggctcgg gcgctcactt
     2821 tccgcaggtg cccgggggcg cggtccgagt ggcggcggcg gccggctcgg gcccctctcc
     2881 gccgggctcg ccgggccacg accgcgagcg gcagccactg ttggatcggg cccggggcgc
     2941 ggcggcccag ggccagaccc aaaccgtggc ggcgcaggcc caggctctgg ccgctcaggc
     3001 cgcggcggca gcccacgccg ctcaggccca ccgcgagcgg aacgagttcc cggaggatcc
     3061 tgagttcgag gcggtggtgc ggcaggccga gctggccatc gagcgctgca tctttcccga
     3121 gcgcatctac cagggctcca gcggaagcta cttcgtcaag gaccctcagg ggaggatcat
     3181 tgctgtcttc aaacccaaga atgaagagcc ctatgggcat cttaatccta agtggaccaa
     3241 gtggctgcag aagctgtgct gtccttgctg ctttggccgt gactgccttg tccttaacca
     3301 gggctatctc tcagaagcag gggccagcct ggtggaccaa aaactggaac tcaacattgt
     3361 tccccgtaca aaggtagtat acctggccag tgagaccttc aactatagtg ccattgaccg
     3421 agtgaagtcc aggggcaagc ggcttgcact agagaaagtg ccaaaagttg gacagcggtt
     3481 taaccgcatc gggctaccac caaaggttgg ttcattccag ctctttgttg aaggctacaa
     3541 agatgcagac tattggctgc ggcgttttga agcagaacct cttcctgaga acactaaccg
     3601 gcaactactg ctccagtttg agcggttggt ggtgctggat tacatcatcc gcaacactga
     3661 tcgaggcaat gacaactggc tgattaaata tgactgtcca atggatagtt ctagctctcg
     3721 ggacacagac tgggtggtgg tgaaggagcc tgttatcaag gtggctgcca tagacaatgg
     3781 gctggccttc ccactgaagc atcctgactc ctggagggca tatccttttt actgggcctg
     3841 gttgccccag gcgaaagtcc cattttctca ggagatcaaa gatctgatcc ttccaaagat
     3901 atcggaccct aacttcgtca aggacttgga agaggaccta tatgaactct tcaagaaaga
     3961 tcctggtttc gacaggggcc agttccataa gcagattgct gtcatgcggg gccagatctt
     4021 aaatctgacc caggccttga aagacaacaa gagtcccctg cacctcgtcc agatgccacc
     4081 tgtgattgtc gagacggccc gttcccacca gcggtcttct agcgagtcct acacacagag
     4141 ctttcagagc cggaagccct tcttttcatg gtggtagctc gagagcggcc gctacgtaaa
     4201 ttccgccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     4261 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     4321 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     4381 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gtttcttgaa gacaaacaac
     4441 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     4501 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     4561 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     4621 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     4681 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     4741 ggttttcctt tgaaaaacac gatgataata tggccacaac cgtcgacatg accgagtaca
     4801 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg
     4861 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc
     4921 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     4981 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     5041 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     5101 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     5161 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     5221 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     5281 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     5341 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagtc gaccgcccgc
     5401 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatggctcg acgataaaat
     5461 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg
     5521 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag
     5581 agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat
     5641 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg
     5701 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     5761 gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     5821 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     5881 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     5941 ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc
     6001 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     6061 gactacccgt cagcgggggt ctttcacatg cagcatgtat caaaattaat ttggtttttt
     6121 ttcttaagta tttacattaa atggccatag ttgcattaat gaatcggcca acgcgcgggg
     6181 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     6241 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     6301 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     6361 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6421 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6481 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6541 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6601 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6661 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6721 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6781 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6841 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6901 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6961 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     7021 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     7081 cttttgcggc cggccgcaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7141 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7201 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7261 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7321 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7381 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7441 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7501 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7561 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7621 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7681 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7741 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7801 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7861 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7921 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7981 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     8041 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8101 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     8161 taacctataa aaataggcgt atcacgaggc cctttcgtct tcactatagg agcttggccc
     8221 attgcatacg ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt
     8281 accgccatgt t
//
