LOCUS       sequence-171122-        7076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of PLAA with N-terminal GFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7076)
  AUTHORS   Papadopoulos C, Kirchner P, Bug M, Grum D, Koerver L, Schulze N, 
            Poehler R, Dressler A, Fengler S, Arhzaouy K, Lux V, Ehrmann M, 
            Weihl CC, Meyer H
  TITLE     VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of 
            ruptured lysosomes by autophagy.
  JOURNAL   EMBO J. 2016 Oct 17. pii: e201695148.
  PUBMED    27753622
REFERENCE   2  (bases 1 to 7076)
  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: "EMBO J. 
            2016 Oct 17. pii: e201695148."
COMMENT     Sequence Label: pEGFP-PLAA
FEATURES             Location/Qualifiers
     source          1..7076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1..3,4..6,7..717)
                     /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(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3252..3373
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3289..3308)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3343..3362
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3380..3835)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3517..3538)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3729..3748
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3862..3966
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3968..4325
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3968..4159
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3991..4011)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4176..4311
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4360..5154
                     /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             4360..5154
                     /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(4414..4433)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5024..5043
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5342..5361)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5386..5433
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5762..6350
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6251..6270
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6525..6828
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6829..7032
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6983..7003
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7071..7076,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      481 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc
      721 ggactcagat ctcgagctac aagcggcgca accaggtacc ggctgagctg ctcgctccgg
      781 ggccacgagc tggacgtacg gggcctggtg tgctgcgcct atccgccggg agcctttgtg
      841 tccgtgtccc gagaccgcac cacccgcctc tgggccccag acagtccaaa caggagcttt
      901 acagaaatgc actgtatgag tggccattcc aattttgtat cttgtgtatg catcataccc
      961 tcaagtgaca tctaccctca tggcctaatt gccaccggtg gaaatgacca caatatatgc
     1021 attttctcac tggacagtcc aatgccactt tatattctaa aaggccacaa aaatactgtt
     1081 tgtagtctat catctggaaa atttgggaca ttacttagtg gttcatggga caccactgct
     1141 aaagtctggc tgaatgacaa gtgcatgatg accttgcagg gtcatacagc tgcagtgtgg
     1201 gcggtaaaga tcttacctga acagggctta atgttgactg gatcagcaga caagactgtt
     1261 aaactgtgga aggctggaag atgtgagagg actttttcag ggcatgaaga ctgtgtaaga
     1321 ggtttggcaa ttttgagtga aacagaattt ctttcctgtg caaatgatgc tagtattaga
     1381 aggtggcaaa tcactggcga gtgtcttgaa gtatattatg gacatacaaa ttatatttat
     1441 agcatatccg tttttccaaa ttgtagagac tttgtgacaa cagcagagga cagatctctg
     1501 agaatctgga aacatgggga atgtgctcaa actatccgac ttccagctca gtctatatgg
     1561 tgctgctgtg tgctcgacaa tggtgacatt gtggttggtg cgagtgatgg cattattaga
     1621 gtgtttacag aatcagaaga tcgaacagca agtgctgaag aaatcaaggc ttttgaaaaa
     1681 gaactgtctc acgcaaccat tgattctaaa actggcgatt taggggacat caatgctgag
     1741 cagcttcctg ggagggaaca tcttaatgaa cctggtacta gagaaggaca gactcgtcta
     1801 atcagagatg gggagaaagt cgaagcctat cagtggagtg ttagtgaagg gaggtggata
     1861 aaaattggtg atgttgttgg ctcatctggt gctaatcagc aaacatctgg aaaagtttta
     1921 tatgaaggga aagaatttga ttatgttttc tcaattgatg tcaatgaagg tggaccatca
     1981 tataaattgc catataatac cagtgatgac ccttggttaa ctgcatacaa cttcttacag
     2041 aagaatgatt tgaatcctat gtttctggat caagtagcta aatttattat tgataacaca
     2101 aaaggtcaaa tgttgggact tgggaatccc agcttttcag atccatttac aggtggtggt
     2161 cggtatgttc cgggctcttc gggatcttct aacacactac ccacagcaga tccttttaca
     2221 ggtgctggtc gttatgtacc aggttctgca agtatgggaa ctaccatggc cggagttgat
     2281 ccatttacag ggaatagtgc ctaccgatca gctgcatcta aaacaatgaa tatttatttc
     2341 cctaaaaaag aggctgtcac atttgaccaa gcaaacccta cacaaatatt aggtaaactg
     2401 aaggaactta atggaactgc acctgaagag aagaagttaa ctgaggatga cttgatactt
     2461 cttgagaaga tactgtctct aatatgtaat agttcttcag aaaaacccac agtccagcaa
     2521 cttcagattt tgtggaaagc tattaactgt cctgaagata ttgtctttcc tgcacttgac
     2581 attcttcggt tgtcaattaa acaccccagt gtgaatgaga acttctgcaa tgaaaaggaa
     2641 ggggctcagt tcagcagtca tcttatcaat cttctgaacc ctaaaggaaa gccagcaaac
     2701 cagctgcttg ctctcaggac tttttgcaat tgttttgttg gccaggcagg acaaaaactc
     2761 atgatgtccc agagggaatc actgatgtcc catgcaatag aactgaaatc agggagcaat
     2821 aagaacattc acattgctct ggctacattg gccctgaact attctgtttg ttttcataaa
     2881 gaccataaca ttgaagggaa agcccaatgt ttgtcactaa ttagcacaat cttggaagta
     2941 gtacaagacc tagaagccac ttttagactt cttgtggctc ttggaacact tatcagtgat
     3001 gattcaaatg ctgtacaatt agccaagtct ttaggtgttg attctcaaat aaaaaagtat
     3061 tcctcagtat cagaaccagc taaagtaagt gaatgctgta gatttatcct aaatttgctg
     3121 taggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     3181 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     3241 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3301 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3361 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3421 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3481 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3541 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3601 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3661 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3721 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3781 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3841 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3901 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3961 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     4021 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4081 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4141 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     4201 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4261 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4321 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4381 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4441 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4501 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     4561 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4621 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4681 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4741 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4801 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4861 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4921 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4981 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     5041 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     5101 gctcccgatt cgcaacgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     5161 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     5221 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5281 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     5341 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     5401 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     5461 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     5521 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     5581 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     5641 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5701 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5761 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5821 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5881 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5941 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6001 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6061 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6121 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6181 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6241 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6301 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6361 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6421 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa
     6481 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     6541 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     6601 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     6661 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     6721 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     6781 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     6841 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     6901 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     6961 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     7021 ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc gccacc
//
