LOCUS       sequence-443432-        7146 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of GFP-Spo20(PABD) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7146)
  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 7146)
  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-Spo20(PABD)
FEATURES             Location/Qualifiers
     source          1..7146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    667..1024
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1089..1505
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1435..1457
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1473..1489
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1515..1889
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1827..1843
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             join(1901..1903,1904..1906,1907..2617)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1901..1903,1904..1906,1907..2617)
                     /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(1946..1967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2207..2226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2554..2575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2997..3563
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3013..3565
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3157..3174)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3384..3403
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3577..4176
                     /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(3577..3596)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4073..4093
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4400..4869
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4999..5016)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5170..5758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5250..5269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5922..6713,6714..6782))
                     /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     6545..6564
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6783..6887)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6955..6973
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7075..7146,1..308)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      241 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      301 ttaccatgct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
      361 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
      421 cccgagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgattgact
      481 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
      541 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
      601 catttggggg ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg
      661 ggaggtaagc tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga
      721 ttttatgcgc ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg
      781 tggaactgac gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg
      841 gggccgtttt tgtggcccga cctgagtcca aaaatcccga tcgttttgga ctctttggtg
      901 cacccccctt agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc
      961 cgcctccgtc tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct
     1021 gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata
     1081 tgggcccggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     1141 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     1201 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     1261 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     1321 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     1381 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     1441 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     1501 gcgcccccat atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc
     1561 ctgaccctga catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc
     1621 tctacttagt ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg
     1681 accgaccggt ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc
     1741 agactaagaa cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca
     1801 ccgccctcaa agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg
     1861 accccggggg tggaccatcc tctagactgc cggatctgcc atggtgagca agggcgagga
     1921 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1981 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2041 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2101 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2161 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2221 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2281 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2341 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     2401 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     2461 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     2521 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2581 cgccgggatc actctcggca tggacgagct gtacaagggt ggcggtggct cgggcggtgg
     2641 tgggtcgggt ggcggcggat ctatcgatga attggaattc atggacaatt gttcaggaag
     2701 cagaagacgt gataggctac atgtgaagct taaatccttg aggaataaaa tccacaaaca
     2761 acttcaccca aactgtcggt tcgatgacgc cactaagact agtggtggcg gtggctcgat
     2821 ggacaattgt tcaggaagca gaagacgtga taggctacat gtgaagctta aatccttgag
     2881 gaataaaatc cacaaacaac ttcacccaaa ctgtcggttc gatgacgcca ctaagactag
     2941 ttgagaattc acgcgttcgc gagttaacgg atccctcgag cggccgctac gtaaattccg
     3001 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     3061 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     3121 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     3181 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     3241 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     3301 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     3361 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     3421 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     3481 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     3541 tcctttgaaa aacacgatga taatatggcc acaaccatga ccgagtacaa gcccacggtg
     3601 cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc
     3661 gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag
     3721 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     3781 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     3841 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     3901 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     3961 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     4021 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     4081 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     4141 cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc
     4201 ccgaccgaaa ggagcgcacg accccatggc tcgacgataa aataaaagat tttatttagt
     4261 ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta
     4321 acgccatttt gcaaggcatg gaaaaataca taactgagaa tagagaagtt cagatcaagg
     4381 tcaggaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc
     4441 tgccccggct cagggccaag aacagatgga acagctgaat atgggccaaa caggatatct
     4501 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc
     4561 agccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa
     4621 tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct
     4681 tctgctcccc gagctcaata aaagagccca caacccctca ctcggggcgc cagtcctccg
     4741 attgactgag tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt tgcatccgac
     4801 ttgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc cgtcagcggg
     4861 ggtctttcac atgcagcatg tatcaaaatt aatttggttt tttttcttaa gtatttacat
     4921 taaatggcca tagttgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4981 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5041 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5101 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5161 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5221 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5281 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5341 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5401 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5461 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5521 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5581 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5641 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5701 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5761 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5821 gattttggtc atgagattat caaaaaggat cttcacctag atccttttgc ggccggccgc
     5881 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5941 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6001 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6061 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6121 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6181 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6241 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6301 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6361 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6421 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6481 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     6541 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6601 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6661 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6721 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6781 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6841 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6901 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     6961 cgtatcacga ggccctttcg tcttcactcg aggagcttgg cccattgcat acgttgtatc
     7021 catatcataa tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt
     7081 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     7141 tggagt
//
