LOCUS       sequence-423008-        7195 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Detect the long-range Lysosome-Mitochondria contact.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7195)
  AUTHORS   Giamogante F, Barazzuol L, Maiorca F, Poggio E, Esposito A, Masato 
            A, Napolitano G, Vagnoni A, Cali T, Brini M
  TITLE     A SPLICS reporter reveals [Formula: see text]-synuclein regulation 
            of lysosome-mitochondria contacts which affects TFEB nuclear 
            translocation.
  JOURNAL   Nat Commun. 2024 Feb 19;15(1):1516. doi: 10.1038/s41467-024-46007-2.
  PUBMED    38374070
REFERENCE   2  (bases 1 to 7195)
  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: "Nat 
            Commun."; date: "2024-02-19"; volume: "15(1):1516. doi"; pages: " 
            10.1038/s41467-024-46007-2"
COMMENT     Sequence Label: SPLICS LY-MT Long P2A
FEATURES             Location/Qualifiers
     source          1..7195
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      914..923
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1825..1872
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             1909..1965
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     promoter        complement(2748..2766)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2749..2766)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2786..2803)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2792..3016
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3062..3490
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3149..3168)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3359..3380
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3499..3519)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3504..3833
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3684..3819
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3746..3765
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3900..4694
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3954..3973)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4564..4583
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4868..4989
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4905..4924)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4959..4978
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5038..5054)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5038..5054)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5051..5073)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5062..5078
                     /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(5086..5092,5093..5110,5111..5116))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5131..5152
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5269..5286)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5440..6028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5520..5539)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6199..6990,6991..7059))
                     /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     6822..6841
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7060..7164)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccaccatggc ggcggggggc aggatggagg acggttcctt ggatatcacc
      961 cagagtattg aagacgaccc acttctggat gcccagcttc tcccacacca ctcattacaa
     1021 gctcacttta gaccccgatt ccatcctctt cctacagtca tcatagtgaa tcttctgtgg
     1081 tttattcatc tcgtgtttgt tgttttagca tttttaacag gtgtgctttg ttcttatcct
     1141 aatccaaatg aggacaagtg cccaggaaat tacacaaacc cattgaaagt tcagacggtt
     1201 ataatccttg ggaaagttat tttgtggatt ctccatttac tccttgaatg ctacatccag
     1261 tatcaccaca gcaaaatcag aaaccgaggc tataacttga tctaccgatc aacaaggcat
     1321 ctcaagagac ttgcgttgat gatacagtcc tctggcaaca cagtgcttct cctcatactg
     1381 tgcatgcagc actccttccc agagcctggc agattgtatc ttgacctcat tctggccatc
     1441 ttggcactgg aactcatctg ttccctgata tgtctcctca tttacacagt gaaaatccgg
     1501 agatttaata aagctaaacc agagcctgat atacttgaag aagaaaaaat ctatgcttac
     1561 cccagcaata ttacctcgga gactggattc agaactattt caagcctaga agaaattgtt
     1621 gaaaagcaag gagacaccat tgaatacctg aagcgacaca atgcgctgct gagtaagcga
     1681 ttgttggctc tcacttcctc agacctgggc tgtcagccaa gtagaacgga cgttggtggt
     1741 ggcggatcag aaggaggcgg tagcgggggc cctggttcgg gaggggaagg ttctgctggg
     1801 ggagggagcg ctggcggggg gtctcgggac cacatggtgc tgcacgagta cgtgaacgcc
     1861 gctggcatca caggccccgg gcttaaggag ctcgcatgcg gaagcggagc tactaacttc
     1921 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctagatc tgaattcatg
     1981 gtgggccgga acagcgccat cgccgcgggc gtgtgcggtg ccctcttcat agggtactgc
     2041 atctactttg accgcaaaag gcggagtgac cccaactcca aaggagaaga actgtttacc
     2101 ggcgtggtgc caattctcgt ggaactggat ggcgatgtga atggccacaa attttctgtc
     2161 agaggagagg gtgaaggtga tgccacaatc ggaaagctca ccctgaaatt catctgcacc
     2221 actggaaagc tccctgtgcc atggccaaca ctggtcacta ccctgaccta cggcgtgcag
     2281 tgcttttcca gatacccaga ccatatgaag aggcatgact ttttcaagag cgccatgccc
     2341 gagggctatg tgcaggagag aaccatctct ttcaaagatg acgggaaata caagacccgc
     2401 gctgtggtca agttcgaagg agacacactg gtgaatagaa tcgagttgaa gggcacagac
     2461 tttaaggaag atggaaacat tctcggccac aagctggaat acaactttaa ctcccacaat
     2521 gtgtacatca cagccgacaa gcaaaagaat ggcatcaagg ctaacttcac agtcagacac
     2581 aacgtcgagg atggaagcgt gcagctggcc gaccattatc aacagaacac tccaatcggc
     2641 gacggccctg tgctcctccc agacaaccat tacctgtcca cccagacagt cctgagcaaa
     2701 gatccaaatg aaaaaggaac ataactcgag cctctagagg gccctattct atagtgtcac
     2761 ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2821 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2881 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2941 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     3001 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     3061 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3121 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3181 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3241 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3301 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3361 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3421 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3481 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3541 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3601 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3661 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3721 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3781 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctac
     3841 cggtagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     3901 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3961 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4021 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4081 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4141 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4201 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4261 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4321 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4381 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4441 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4501 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4561 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4621 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4681 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4741 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4801 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4861 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4921 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4981 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     5041 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5101 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5161 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5221 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5281 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5341 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5401 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5461 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5521 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5581 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5641 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5701 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5761 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5821 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5881 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5941 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6001 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6061 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6121 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6181 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6241 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6301 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6361 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6421 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6481 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6541 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6601 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6661 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6721 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6781 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6841 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6901 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6961 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7021 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7081 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7141 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
