LOCUS       sequence-490789-        7593 bp    DNA     circular SYN 27-OCT-2025
DEFINITION  Rubicon mutated at four residues in the RH domain to disrupt Rab7 
            binding, tagged at the N-terminus with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7593)
  AUTHORS   Basak B, Holzbaur ELF
  TITLE     Mitochondrial damage triggers the concerted degradation of negative 
            regulators of neuronal autophagy.
  JOURNAL   Nat Commun. 2025 Aug 9;16(1):7367. doi: 10.1038/s41467-025-62379-5.
  PUBMED    40783388
REFERENCE   2  (bases 1 to 7593)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 27, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-08-9"; volume: "16(1):7367. doi"; pages: " 
            10.1038/s41467-025-62379-5"
COMMENT     Sequence Label: mCherry-Rubicon-CGHL
FEATURES             Location/Qualifiers
     source          1..7593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     545..564
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(653..1010)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      667..802
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        819..1010
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     967..987
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(1012..1116)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1143..1598
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1230..1249)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1440..1461
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1605..1726
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1616..1635)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1670..1689
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4787..5494)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5062..5081)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5331..5349
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      5491..5500
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5539..5742)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5568..5588)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5743..6046
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(6221..6809)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6301..6320)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    7138..7185
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     7210..7229
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(join(7417..7593,1..618))
                     /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             complement(join(7417..7593,1..618))
                     /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(7528..7547)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt
       61 cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg
      121 cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg
      181 agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc
      241 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag
      301 gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc
      361 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag
      421 ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag
      481 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg
      541 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa
      601 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa
      661 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
      721 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
      781 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
      841 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
      901 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
      961 ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag gactcttcct
     1021 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     1081 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     1141 tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1201 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1261 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1321 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1381 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1441 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1501 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1561 taacgcgaat tttaacaaaa tattaacgct tacaatttac gcgttaagat acattgatga
     1621 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1681 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1741 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     1801 cctctacaaa tgtggtatgg ctgattatga tcagttatct agatccggtg gatccctcga
     1861 gtcaggtagc ctccagcacg gcagcttcga gggccagagc ttcagcgggt tcttcctcat
     1921 agtcgctcag gtagctctcc aggctttgtc tggccagggc ttccctcctg gcctggagtc
     1981 tctcgcacct aggacagctg ccgctcttga atccggcttt caggtaacaa gcctttccct
     2041 cctctccggt cctgcactta tgcagctcga aggggaagat gatgtcgtcc tcgttctggc
     2101 agaactcgca aatgaatccc ttggcctgac acagcatgca tctctccacg tgggtagctc
     2161 cggctctggt cagttcggcc agcctgggtc ccagctctcc ctttctggtg gcggtcaggt
     2221 cgttcaggct gtacaggtgc agatcttcgg tcaggtggcc ggggacggtg tcaaagctgt
     2281 ccagcagctc cttagccagc ctacaggttt tgaacatatt cttcatgtgg cacagctgca
     2341 ctctcagcag cctgacctgg ttcagcagct tgaccttcct gtacagggcg ctattaatgt
     2401 cctgcacgtt aaacagaggg tcgttccaga tcttgatcag caggtctttg ctaaagttgc
     2461 tgacgtaata tttgctgaaa tcccactttc tcagcaccct gctagggata gccatctgag
     2521 cgttctcgtg acagcactgg caaaagtact tgcccaggta ctcgcagtat ctcagtctct
     2581 tgatgtagtc ggggtcggtc ctgattccgc agccagcaca tctatagttc tgcttggcca
     2641 cggcaatctt tcttgtaggg gcggggtgca cgttgaagat gatttggggc ctgggaggag
     2701 cccactccag gttgcctctc accctgatcc tcagcttata aatgtcggcg tgctggccgt
     2761 cgtcggggga gataggcagg ctatcgggaa tagggagcag cttttgaggg gcgtcgtgtt
     2821 caggcacgag ccattccagc tcgctggcag cgggcagctg catgccttcg aactgcttca
     2881 gcaggcccat agccacagct tcggcgctgg tggagtggag aaagcagtgg gaaaagcttt
     2941 gggaggacac aaaggacttg gagctggaag cggtgtttct cctgatgtcg gcatcttgga
     3001 tctcaaactc atcgacctca tcggcggagc cggaatcgct gagttgggcg gaatctctgc
     3061 tggaaaattg ggagctggag ctggtgaccc tgaaggagcc atgctcggcc tcttggtaca
     3121 tgggcaggag attcttggtc ctgatctgtt gtctcctgag cctgatcttc tgcttcagtt
     3181 cttgaatctc cctgtcggag tcttcctctt ccacctcttc ctcctccaga cactgggaca
     3241 tcatgttaca tttcatcagt tcgatagcag cgatcaggga ctcgctgatg ctaaagtggg
     3301 cgttctcctt ttcgaggtcg gcgcagcttc cgaagtcctg ctcgctgaga taggaaatca
     3361 ggctctggcc ttcggaaggt cttctgaaca ttccctcgcc gctacagaga tacctgcctc
     3421 cctcatactc catgtacagg ctggagggtg tggagacctc gctgctctgt ccggagtagg
     3481 gcagggggcc tctcagctta gccttatcgt tacagctttc gggggctccc ctggaggcga
     3541 tgctggtatc ggaatggctt ctgatgtggc tttttttggc gccggatgtc acggtgaggg
     3601 tctggccctc ggagaaggag ctccttctca gcacggagga gagctggctc tcgcctccgc
     3661 cttcttggtc tcccaggctg ctagcggcgg agtcaggttt ctggctgctg gaggaggaga
     3721 acaggttgct gctgctggat tcagcattgc tgctgtggga ctggcaggag gctgtcctgc
     3781 ccctaggatc gaggttgccg atagcgaggt actcgggtcc ctcggaagcg tctccagggc
     3841 tgtcattttg gctgctcacc caggaagcct cgatggggga ggtcagtgtg ctgctggaca
     3901 tctcgttagg tgtgagaggg gagtccctgg ccagagcgga cacgctcaca ggaggagcct
     3961 ggattgtctg atcttcagcg ggggacacgt ggcctctgga ctcttgaggt tttctgggag
     4021 gtccgctcag agggaaggag gtggacctcc tttcggagcc attattaggc acggactggt
     4081 gcagggagct aaagctgccg aagtagctgt gctgagcgta gctgttgggg ggggtgtagg
     4141 tgctgctggg cagagcggtc agggactggc tctttgtcac caggaggggg gactcgtgtt
     4201 tcctagcgaa catgctagcg tcgatctggg ccagcagcct ggggttgttc tgttccacag
     4261 cttcgaggca ctggagcatg gctgtcacgt gggcgtcgct cagcaggaag gcggcgtctg
     4321 tgtagaattt cctgatgtac tgtctgtctc cgagcagggg tctcagctga gcgctcaggc
     4381 agtggtactg gagggaatgc tggagccaga gctcggccac ggctctttcg gaggctccgt
     4441 cggcggagct ctgatcgttt tcgtggacgg aaatgaactt ctccacgtgc agagcggagt
     4501 ggggggagag ccacctgatg tccttcacga actgccagta gtcggtttgt ctcctgcagg
     4561 cctggtccct gatcagtcca tgatacagga tggattgcat gtccctgcac agcctctcca
     4621 ggccgccgta cttgctccag acgttggggc tattggtgct caccagtccc tccacggtgg
     4681 ttttcaggtt gcccagcagc tgccaatgct ctcttctgct ctcctcaggc agcctttctt
     4741 cgcctccgcc cagctccatt ccggcgcctt caggcctcat ggtacccttg tacagctcgt
     4801 ccatgccgcc ggtggagtgg cggccctcgg cgcgttcgta ctgttccacg atggtgtagt
     4861 cctcgttgtg ggaggtgatg tccaacttga tgttgacgtt gtaggcgccg ggcagctgca
     4921 cgggcttctt ggccttgtag gtggtcttga cctcagcgtc gtagtggccg ccgtccttca
     4981 gcttcagcct ctgcttgatc tcgcccttca gggcgccgtc ctcggggtac atccgctcgg
     5041 aggaggcctc ccagcccatg gtcttcttct gcattacggg gccgtcggag gggaagttgg
     5101 tgccgcgcag cttcaccttg tagatgaact cgccgtcctg cagggaggag tcctgggtca
     5161 cggtcaccac gccgccgtcc tcgaagttca tcacgcgctc ccacttgaag ccctcgggga
     5221 aggacagctt caagtagtcg gggatgtcgg cggggtgctt cacgtaggcc ttggagccgt
     5281 acatgaactg aggggacagg atgtcccagg cgaagggcag ggggccaccc ttggtcacct
     5341 tcagcttggc ggtctgggtg ccctcgtagg ggcggccctc gccctcgccc tcgatctcga
     5401 actcgtggcc gttcacggag ccctccatgt gcaccttgaa gcgcatgaac tccttgatga
     5461 tggccatgtt atcctcctcg cccttgctca ccatggtggc gaccggtagc gctagcggat
     5521 ctgacggttc actaaaccag ctctgcttat atagacctcc caccgtacac gcctaccgcc
     5581 catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg
     5641 ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa
     5701 ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac
     5761 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
     5821 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     5881 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     5941 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
     6001 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
     6061 tgggctatga actaatgacc ccgtaattga ttactattaa taactaatgc atggcggtaa
     6121 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6181 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6241 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6301 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6361 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6421 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6481 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6541 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6601 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6661 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6721 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6781 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6841 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6901 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6961 agtaacctga ggctatggca gggcctgccg ccccgacgtt ggctgcgagc cctgggcctt
     7021 cacccgaact tggggggtgg ggtggggaaa aggaagaaac gcgggcgtat tggccccaat
     7081 ggggtctcgg tggggtatcg acagagtgcc agccctggga ccgaaccccg cgtttatgaa
     7141 caaacgaccc aacaccgtgc gttttattct gtctttttat tgccgtcata gcgcgggttc
     7201 cttccggtat tgtctccttc cgtgtttcag ttagcctccc cctagggtgg gcgaagaact
     7261 ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga
     7321 agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg
     7381 tcgcttggtc ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg
     7441 atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc
     7501 agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata
     7561 gcggtccgcc acacccagcc ggccacagtc gat
//
