LOCUS       sequence-490795-        8468 bp    DNA     circular SYN 27-OCT-2025
DEFINITION  Rubicon tagged N-terminally with EGFP; clone a gift from the Hurley 
            Lab at UC Berkeley.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8468)
  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 8468)
  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: EGFP-Rubicon
FEATURES             Location/Qualifiers
     source          1..8468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        997..1376
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1378..1654
                     /label=chicken beta-actin promoter
     intron          1655..2671
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2595..2617
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2679..2698
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2745..2754
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2751..2753,2754..2756,2757..3467)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2796..2817)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3057..3076)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3404..3425
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6482..6501)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6547..6602
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6601..6620)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6963..6979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6963..6979)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6976..6998)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6987..7003
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7011..7017,7018..7035,7036..7041))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7056..7077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7136..7331
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7182..7317
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7337..7471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7374..7393)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7428..7447
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7539..7556)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7710..8298)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7790..7809)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagttatt
     1021 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1081 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1141 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1201 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1261 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1321 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     1381 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     1441 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg ggggggggcg
     1501 ggcgccaggc gggggggggc ggggcggggg ggggggcggg gcgaggcgga gaggtgcggc
     1561 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
     1621 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
     1681 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
     1741 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta
     1801 atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc
     1861 tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc
     1921 gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc
     1981 gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg
     2041 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     2101 gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg
     2161 cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg
     2221 ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc
     2281 gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     2341 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     2401 tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg
     2461 gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg
     2521 tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg
     2581 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     2641 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
     2701 gtctcatcat tttggcaaag aattcctcga tcgacggtat cgatgccacc atggtgagca
     2761 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2821 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2881 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2941 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     3001 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     3061 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     3121 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3181 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     3241 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     3301 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     3361 cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     3421 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggt accatgaggc
     3481 ctgaaggcgc cggaatggag ctgggcggag gcgaagaaag gctgcctgag gagagcagaa
     3541 gagagcattg gcagctgctg ggcaacctga aaaccaccgt ggagggactg gtgagcacca
     3601 atagccccaa cgtctggagc aagtacggcg gcctggagag gctgtgcagg gacatgcaat
     3661 ccatcctgta tcatggactg atcagggacc aggcctgcag gagacaaacc gactactggc
     3721 agttcgtgaa ggacatcagg tggctctccc cccactccgc tctgcacgtg gagaagttca
     3781 tttccgtcca cgaaaacgat cagagctccg ccgacggagc ctccgaaaga gccgtggccg
     3841 agctctggct ccagcattcc ctccagtacc actgcctgag cgctcagctg agacccctgc
     3901 tcggagacag acagtacatc aggaaattct acacagacgc cgccttcctg ctgagcgacg
     3961 cccacgtgac agccatgctc cagtgcctcg aagctgtgga acagaacaac cccaggctgc
     4021 tggcccagat cgacgctagc atgttcgcta ggaaacacga gtcccccctc ctggtgacaa
     4081 agagccagtc cctgaccgct ctgcccagca gcacctacac cccccccaac agctacgctc
     4141 agcacagcta cttcggcagc tttagctccc tgcaccagtc cgtgcctaat aatggctccg
     4201 aaaggaggtc cacctccttc cctctgagcg gacctcccag aaaacctcaa gagtccagag
     4261 gccacgtgtc ccccgctgaa gatcagacaa tccaggctcc tcctgtgagc gtgtccgctc
     4321 tggccaggga ctcccctctc acacctaacg agatgtccag cagcacactg acctccccca
     4381 tcgaggcttc ctgggtgagc agccaaaatg acagccctgg agacgcttcc gagggacccg
     4441 agtacctcgc tatcggcaac ctcgatccta ggggcaggac agcctcctgc cagtcccaca
     4501 gcagcaatgc tgaatccagc agcagcaacc tgttctcctc ctccagcagc cagaaacctg
     4561 actccgccgc tagcagcctg ggagaccaag aaggcggagg cgagagccag ctctcctccg
     4621 tgctgagaag gagctccttc tccgagggcc agaccctcac cgtgacatcc ggcgccaaaa
     4681 aaagccacat cagaagccat tccgatacca gcatcgcctc caggggagcc cccgaaagct
     4741 gtaacgataa ggctaagctg agaggccccc tgccctactc cggacagagc agcgaggtct
     4801 ccacaccctc cagcctgtac atggagtatg agggaggcag gtatctctgt agcggcgagg
     4861 gaatgttcag aagaccttcc gaaggccaga gcctgatttc ctatctcagc gagcaggact
     4921 tcggaagctg cgccgacctc gaaaaggaga acgcccactt tagcatcagc gagtccctga
     4981 tcgctgctat cgaactgatg aaatgtaaca tgatgtccca gtgtctggag gaggaagagg
     5041 tggaagagga agactccgac agggagattc aagaactgaa gcagaagatc aggctcagga
     5101 gacaacagat caggaccaag aatctcctgc ccatgtacca agaggccgag catggctcct
     5161 tcagggtcac cagctccagc tcccaatttt ccagcagaga ttccgcccaa ctcagcgatt
     5221 ccggctccgc cgatgaggtc gatgagtttg agatccaaga tgccgacatc aggagaaaca
     5281 ccgcttccag ctccaagtcc tttgtgtcct cccaaagctt ttcccactgc tttctccact
     5341 ccaccagcgc cgaagctgtg gctatgggcc tgctgaagca gttcgaaggc atgcagctgc
     5401 ccgctgccag cgagctggaa tggctcgtgc ctgaacacga cgcccctcaa aagctgctcc
     5461 ctattcccga tagcctgcct atctcccccg acgacggcca gcacgccgac atttataagc
     5521 tgaggatcag ggtgagaggc aacctggagt gggctcctcc caggccccaa atcatcttca
     5581 acgtgcaccc cgcccctaca agaaagattg ccgtggccaa gcagaactat agatgtgctg
     5641 gctgcggaat caggaccgac cccgactaca tcaagagact gagatactgc gagtacctgg
     5701 gcaagtactt ttgccagtgc tgtcacgaga acgctcagat ggctatccct agcagggtgc
     5761 tgagaaagtg ggatttcagc aaatattacg tcagcaactt tagcaaagac ctgctgatca
     5821 agatctggaa cgaccctctg tttaacgtgc aggacattaa tagcgccctg tacaggaagg
     5881 tcaagctgct gaaccaggtc aggctgctga gagtgcagct gtgccacatg aagaatatgt
     5941 tcaaaacctg taggctggct aaggagctgc tggacagctt tgacaccgtc cccggccacc
     6001 tgaccgaaga tctgcacctg tacagcctga acgacctgac cgccaccaga aagggagagc
     6061 tgggacccag gctggccgaa ctgaccagag ccggagctac ccacgtggag agatgcatgc
     6121 tgtgtcaggc caagggattc atttgcgagt tctgccagaa cgaggacgac atcatcttcc
     6181 ccttcgagct gcataagtgc aggacctgtg aggagtgtaa ggcttgttac cacaaagcct
     6241 gcttcaagag cggcagctgt cctaggtgcg agagactcca ggccaggagg gaagccctgg
     6301 ccagacaaag cctggagagc tacctgagcg actatgagga agaacccgct gaagctctgg
     6361 ccctcgaagc tgccgtgctg gaggctacct gactcgagga attcactcct caggtgcagg
     6421 ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag
     6481 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt
     6541 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct
     6601 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt
     6661 agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag
     6721 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     6781 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     6841 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     6901 tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca
     6961 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     7021 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     7081 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     7141 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     7201 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     7261 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     7321 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     7381 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     7441 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     7501 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     7561 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     7621 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     7681 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     7741 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     7801 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     7861 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     7921 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     7981 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     8041 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     8101 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     8161 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     8221 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     8281 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     8341 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     8401 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     8461 tctgacag
//
