LOCUS       sequence-423006-        7129 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Detect the short-range Lysosome-Mitochondria contact.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7129)
  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 7129)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 Short P2A
FEATURES             Location/Qualifiers
     source          1..7129
                     /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             1759..1806
                     /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             1843..1899
                     /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(2682..2700)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2683..2700)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2720..2737)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2726..2950
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2996..3424
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3083..3102)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3293..3314
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3433..3453)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3438..3767
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3618..3753
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3680..3699
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3834..4628
                     /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(3888..3907)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4498..4517
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4802..4923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4839..4858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4893..4912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4972..4988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4972..4988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4985..5007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4996..5012
                     /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(5020..5026,5027..5044,5045..5050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5065..5086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5203..5220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5374..5962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5454..5473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6133..6924,6925..6993))
                     /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     6756..6775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6994..7098)
                     /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 gtagaacggg cgacggcggc
     1741 agcggcggcg gcagcggccg ggaccacatg gtgctgcacg agtacgtgaa cgccgctggc
     1801 atcacaggcc ccgggcttaa ggagctcgca tgcggaagcg gagctactaa cttcagcctg
     1861 ctgaagcagg ctggagacgt ggaggagaac cctggaccta gatctgaatt catggtgggc
     1921 cggaacagcg ccatcgccgc gggcgtgtgc ggtgccctct tcatagggta ctgcatctac
     1981 tttgaccgca aaaggcggag tgaccccaac tccaaaggag aagaactgtt taccggcgtg
     2041 gtgccaattc tcgtggaact ggatggcgat gtgaatggcc acaaattttc tgtcagagga
     2101 gagggtgaag gtgatgccac aatcggaaag ctcaccctga aattcatctg caccactgga
     2161 aagctccctg tgccatggcc aacactggtc actaccctga cctacggcgt gcagtgcttt
     2221 tccagatacc cagaccatat gaagaggcat gactttttca agagcgccat gcccgagggc
     2281 tatgtgcagg agagaaccat ctctttcaaa gatgacggga aatacaagac ccgcgctgtg
     2341 gtcaagttcg aaggagacac actggtgaat agaatcgagt tgaagggcac agactttaag
     2401 gaagatggaa acattctcgg ccacaagctg gaatacaact ttaactccca caatgtgtac
     2461 atcacagccg acaagcaaaa gaatggcatc aaggctaact tcacagtcag acacaacgtc
     2521 gaggatggaa gcgtgcagct ggccgaccat tatcaacaga acactccaat cggcgacggc
     2581 cctgtgctcc tcccagacaa ccattacctg tccacccaga cagtcctgag caaagatcca
     2641 aatgaaaaag gaacataact cgagcctcta gagggcccta ttctatagtg tcacctaaat
     2701 gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2761 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2821 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2881 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2941 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3001 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3061 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3121 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3181 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3241 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3301 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3361 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3421 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3481 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3541 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3601 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3661 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3721 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctaccggtag
     3781 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3841 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3901 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3961 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4021 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4081 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4141 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4201 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4261 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4321 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4381 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4441 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4501 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4561 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4621 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4681 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4741 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4801 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4861 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4921 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4981 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5041 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5101 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5161 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5221 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5281 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5341 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5401 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5461 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5521 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5581 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5641 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5701 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5761 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5821 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5881 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5941 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6001 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6061 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6121 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6181 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6241 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6301 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6361 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6421 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6481 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6541 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6601 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6661 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6721 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6781 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6841 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6901 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6961 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7021 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7081 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
