LOCUS       sequence-432391-        5481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-tagged SC domain of human SPG20 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5481)
  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 5481)
  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 C1-SPG20 SC domain
FEATURES             Location/Qualifiers
     source          1..5481
                     /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    2269..2390
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2306..2325)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2360..2379
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2397..2852)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2534..2555)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2746..2765
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2879..2983
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2985..3342
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2985..3176
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3008..3028)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3193..3328
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3377..4171
                     /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             3377..4171
                     /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(3431..3450)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4041..4060
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4359..4378)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4403..4450
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4779..5367
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5268..5287
                     /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 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc
     1381 gaatggagtg aaaaagtggc tcacaacatt ttgtcaggtg cttcctgggt gagttggggt
     1441 ttagtcaaag gtgctgagat tactggtaag gcaatccaga aaggtgcttc taaactccga
     1501 gagcggattc aaccagaaga aaaacccgtg gaagttagtc cagctgtcac caagggactt
     1561 tatatagcga agcaagctac aggaggagca gcaaaagtca gtcagttcct ggttgatgga
     1621 gtttgcactg tagcaaattg cgttggaaaa gaactagctc cacatgtcaa gaagcatgga
     1681 agcaaacttg ttccagaatc tcttaaaaaa gacaaagatg ggaaatctcc tctggatggt
     1741 gctatggttg tagcagcaag tagtgttcaa ggattttcaa ctgtctggca aggattggaa
     1801 tgtgcagcta aatgcatcgt taacaatgtt tcagcagaaa ctgtacaaac tgtcagatac
     1861 aaatacggat ataatgcagg agaagctacc caccatgcgg tggattctgc ggtcaatgtt
     1921 ggcgtaactg cctacaatat taacaacatt ggtatcaaag caatggtgaa gaaaactgca
     1981 acacaaacag gacacactct ccttgaggac tatcagatag ttgataattc tcagagggaa
     2041 aatcaagaag gagcagcaaa tgtcaacgtg agaggggaga aggatgagca gacgaaggaa
     2101 gtaaaggagg caaagaagaa agataaatga gcgggcccgg gatccaccgg atctagataa
     2161 ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     2221 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     2281 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2341 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat
     2401 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     2461 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2521 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2581 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2641 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2701 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2761 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2821 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     2881 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2941 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     3001 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3061 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3121 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3181 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3241 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     3301 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     3361 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3421 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3481 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3541 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3601 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3661 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3721 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3781 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3841 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3901 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3961 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4021 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4081 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4141 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4201 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4261 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4321 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     4381 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     4441 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     4501 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     4561 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     4621 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4681 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4741 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4801 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4861 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4921 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4981 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5041 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5101 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5161 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5221 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5281 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5341 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5401 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5461 aaccgtatta ccgccatgca t
//
