LOCUS       sequence-432392-        6696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-tagged PPAY motif mutant (PAAA) of human SPG20 in 
            mammalian cells; this mutant does not interact with ITCH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6696)
  AUTHORS   Gahlot P, Kravic B, Rota G, van den Boom J, Levantovsky S, Schulze 
            N, Maspero E, Polo S, Behrends C, Meyer H
  TITLE     Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.
  JOURNAL   Mol Cell. 2024 Mar 14:S1097-2765(24)00172-2. doi: 
            10.1016/j.molcel.2024.02.029.
  PUBMED    38503285
REFERENCE   2  (bases 1 to 6696)
  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: "Mol Cell. 
            2024 Mar 14:S1097-2765(24)00172-2. doi: 
            10.1016/j.molcel.2024.02.029."
COMMENT     Sequence Label: peGFP C3-SPG20-PAAA
FEATURES             Location/Qualifiers
     source          1..6696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3484..3605
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3521..3540)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3575..3594
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3612..4067)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3749..3770)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3961..3980
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4094..4198
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4200..4557
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4200..4391
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4223..4243)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4408..4543
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4592..5386
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4592..5386
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4646..4665)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5256..5275
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5574..5593)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5618..5665
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5994..6582
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6483..6502
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt actcagatct cgagctcaag cttatggagc aagagccaca aaatggagaa
     1381 cctgctgaaa ttaagatcat cagagaagca tataagaagg cctttttatt tgttaacaaa
     1441 ggtctgaata cagatgaatt aggtcagaag gaagaagcaa agaactacta taagcaagga
     1501 ataggacacc tgctcagagg gatcagcatt tcatcaaaag agtctgaaca cacaggtcct
     1561 gggtgggaat ctgctagaca gatgcaacag aaaatgaaag aaactctaca gaatgtacgc
     1621 accaggctgg aaattctaga gaagggtctt gccacttctc tgcagaatga tcttcaggag
     1681 gtgcccaagt tatatccaga atttccacct aaagacatgt gtgaaaaatt accagagcct
     1741 cagtctttta gttcagctcc tcagcatgct gaagtaaatg gaaacacctc aactccaagt
     1801 gcaggggcag ttgctgcacc tgcttctctg tctttaccat cacaaagttg tccagcagaa
     1861 gctcctgctg ctgctactcc tcaagctgct gaaggtcact acactgtatc ctatggaaca
     1921 gattctgggg agttttcatc agttggagag gagttttata ggaatcattc tcagccaccg
     1981 cctcttgaga ccttagggct ggatgcagat gaattgattt tgataccaaa tggagtacag
     2041 attttttttg taaatcctgc aggggaggtt agtgcacctt cgtatcctgg gtaccttcga
     2101 attgtgaggt ttttggataa ttctctcgat acggttctaa accgtcctcc cgggtttctt
     2161 caggtttgtg actggttata tcctctagtt cctgatagat ctccggttct gaaatgtact
     2221 gcgggagcct acatgtttcc tgatacaatg ctacaagcag caggatgctt tgtgggggtc
     2281 gtcctgtcct ctgagttacc agaggatgat agagagctct ttgaggatct gttaaggcaa
     2341 atgtctgacc ttcggctcca ggccaactgg aacagagcag aagaagaaaa tgaattccaa
     2401 atccctggaa gaactagacc ctcctctgac caactaaaag aagcctctgg cactgatgtg
     2461 aaacagttgg accaaggcaa taaggatgta cgtcataaag gaaaacgtgg aaaaagggct
     2521 aaagatactt caagtgaaga agttaacctg agtcacattg taccatgtga gccagttcca
     2581 gaagaaaagc caaaagaatt acctgaatgg agtgaaaaag tggctcacaa cattttgtca
     2641 ggtgcttcct gggtgagttg gggtttagtc aaaggtgctg agattactgg taaggcaatc
     2701 cagaaaggtg cttctaaact ccgagagcgg attcaaccag aagaaaaacc cgtggaagtt
     2761 agtccagctg tcaccaaggg actttatata gcgaagcaag ctacaggagg agcagcaaaa
     2821 gtcagtcagt tcctggttga tggagtttgc actgtagcaa attgcgttgg aaaagaacta
     2881 gctccacatg tcaagaagca tggaagcaaa cttgttccag aatctcttaa aaaagacaaa
     2941 gatgggaaat ctcctctgga tggtgctatg gttgtagcag caagtagtgt tcaaggattt
     3001 tcaactgtct ggcaaggatt ggaatgtgca gctaaatgca tcgttaacaa tgtttcagca
     3061 gaaactgtac aaactgtcag atacaaatac ggatataatg caggagaagc tacccaccat
     3121 gcggtggatt ctgcggtcaa tgttggcgta actgcctaca atattaacaa cattggtatc
     3181 aaagcaatgg tgaagaaaac tgcaacacaa acaggacaca ctctccttga ggactatcag
     3241 atagttgata attctcagag ggaaaatcaa gaaggagcag caaatgtcaa cgtgagaggg
     3301 gagaaggatg agcagacgaa ggaagtaaag gaggcaaaga agaaagataa atgaggatcc
     3361 accggatcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     3421 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3481 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3541 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3601 tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3661 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3721 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3781 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3841 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3901 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3961 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4021 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     4081 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4141 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     4201 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     4261 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4321 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4381 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4441 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4501 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     4561 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4621 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4681 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4741 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     4801 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4861 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4921 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4981 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     5041 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     5101 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     5161 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     5221 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5281 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     5341 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     5401 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     5461 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5521 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     5581 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     5641 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     5701 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     5761 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     5821 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     5881 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5941 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6001 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6061 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6121 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6181 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6241 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6301 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6361 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6421 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6481 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6541 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6601 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6661 cctgattctg tggataaccg tattaccgcc atgcat
//
