LOCUS       sequence-322811-        7457 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Detect the short Plasma membrane-mitochondria-Endoplasmic reticulum 
            contact .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7457)
  AUTHORS   Vallese F, Catoni C, Cieri D, Barazzuol L, Ramirez O, Calore V, 
            Bonora M, Giamogante F, Pinton P, Brini M, Cali T
  TITLE     An expanded palette of improved SPLICS reporters detects multiple 
            organelle contacts in vitro and in vivo.
  JOURNAL   Nat Commun. 2020 Nov 27;11(1):6069. doi: 10.1038/s41467-020-19892-6.
  PUBMED    33247103
REFERENCE   2  (bases 1 to 7457)
  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: "2020-11-27"; volume: "11(1):6069. doi"; pages: " 
            10.1038/s41467-020-19892-6"
COMMENT     Sequence Label: SPLICS Mt-ER- PM Short P2A
FEATURES             Location/Qualifiers
     source          1..7457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..39)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        211..590
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        591..794
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     745..765
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     839..858
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        839..857
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             898..945
                     /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             1222..1278
                     /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"
     CDS             2068..2124
                     /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"
     CDS             2140..2163
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2986..3004)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2987..3004)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3024..3041)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3030..3254
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3300..3728
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3387..3406)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3597..3618
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3737..3757)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3742..4071
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3922..4057
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3984..4003
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4138..4932
                     /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(4192..4211)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4802..4821
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5106..5227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5143..5162)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5197..5216
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5276..5292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5276..5292)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5289..5311)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5300..5316
                     /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(5324..5330,5331..5348,5349..5354))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5369..5390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5507..5524)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5678..6266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5758..5777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6437..7228,7229..7297))
                     /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     7060..7079
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7298..7402)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
       61 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
      121 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
      181 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      241 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      301 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      361 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      421 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      481 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      541 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      601 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      661 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      721 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      781 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
      841 atacgactca ctatagggag acccaagctt ggtaccgagc tcggatccgc caccatgcgg
      901 gaccacatgg tgctgcacga gtacgtgaac gccgctggca tcacaggcgg agatggcgga
      961 tctggcggcg gaagcaagct gagagtgttc ctggccctgc ccatcatcat ggtggtggcc
     1021 ttcagcatgt gcatcatctg cctgctgatg gccggcgaca cctggacaga gacactggcc
     1081 tacgtgctgt tctggggcgt ggccagcatc ggcacctttt tcatcatcct gtacaacggc
     1141 aaggacttcg tggacgcccc cagactggtg cagaaagaga agatcgaccc cgggcttaag
     1201 gagctcgcat gcggaagcgg agctactaac ttcagcctgc tgaagcaggc tggagacgtg
     1261 gaggagaacc ctggacctag atctgaattc atggtgggcc ggaacagcgc catcgccgcg
     1321 ggcgtgtgcg gtgccctctt catagggtac tgcatctact ttgaccgcaa aaggcggagt
     1381 gaccccaact ccaaaggaga agaactgttt accggcgtgg tgccaattct cgtggaactg
     1441 gatggcgatg tgaatggcca caaattttct gtcagaggag agggtgaagg tgatgccaca
     1501 atcggaaagc tcaccctgaa attcatctgc accactggaa agctccctgt gccatggcca
     1561 acactggtca ctaccctgac ctacggcgtg cagtgctttt ccagataccc agaccatatg
     1621 aagaggcatg actttttcaa gagcgccatg cccgagggct atgtgcagga gagaaccatc
     1681 tctttcaaag atgacgggaa atacaagacc cgcgctgtgg tcaagttcga aggagacaca
     1741 ctggtgaata gaatcgagtt gaagggcaca gactttaagg aagatggaaa cattctcggc
     1801 cacaagctgg aatacaactt taactcccac aatgtgtaca tcacagccga caagcaaaag
     1861 aatggcatca aggctaactt cacagtcaga cacaacgtcg aggatggaag cgtgcagctg
     1921 gccgaccatt atcaacagaa cactccaatc ggcgacggcc ctgtgctcct cccagacaac
     1981 cattacctgt ccacccagac agtcctgagc aaagatccaa atgaaaaagg aacactcgag
     2041 cttaaggagc tcgcatgcgg aagcggagct actaacttca gcctgctgaa gcaggctgga
     2101 gacgtggagg agaaccctgg acctagatct gaattcatgg actacaagga cgacgacgac
     2161 aagtgcggcc tgttcatctg cccctgcaac aagctgaagt ccagcgacgc ctacaagaag
     2221 gcctggggca acaaccagga tggcgtggtg gcttcccagc ctgccagagt gggaagcggc
     2281 ggaagctctg gcagcagagg ctccgatggc tctgacggca gcagcaaggg cgaggaactg
     2341 ttcacaggcg tggtgcctat cctggtggaa ctggacggcg acgtgaacgg ccacaagttt
     2401 tctgtgcggg gagagggcga aggcgacgcc acaatcggaa agctgaccct gaagttcatc
     2461 tgtaccaccg gcaagctgcc cgtgccctgg cctacactcg tgaccacact gggctacggc
     2521 ctgcagtgct tcgccagata ccccgaccat atgaagcggc acgacttctt caagagcgcc
     2581 atgcccgagg gctacgtgca ggaacggacc atcagcttca aggacgatgg caagtacaag
     2641 accagagccg tcgtgaagtt cgagggcgac accctcgtga accggatcga gctgaagggc
     2701 accgatttca aagaggacgg caacatcctg ggccacaagc tggagtacaa cttcaacagc
     2761 cacaacgtgt acatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaccgtg
     2821 cggcacaacg tggaagatgg cagcgtgcag ctggccgacc actaccagca gaacaccccc
     2881 atcggagatg gccccgtgct gctgcccgac aaccactacc tgagctacca gaccgtgctg
     2941 agcaaagatc caaatgaaaa aggaacataa tctagagggc cctattctat agtgtcacct
     3001 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3061 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3121 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3181 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3241 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3301 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3361 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3421 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3481 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3541 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3601 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3661 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3721 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3781 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3841 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3901 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3961 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     4021 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     4081 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     4141 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4201 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4261 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     4321 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4381 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4441 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4501 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4561 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4621 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     4681 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4741 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4801 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4861 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4921 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4981 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     5041 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     5101 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5161 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5221 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5281 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5341 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5401 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5461 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5521 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5581 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5641 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5701 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5761 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5821 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5881 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5941 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6001 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6061 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6121 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6181 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6241 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6301 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6361 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6421 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6481 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6541 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6601 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6661 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6721 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6781 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6841 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6901 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6961 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7021 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7081 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7141 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7201 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7261 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7321 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7381 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     7441 cgggagatct cccgatc
//
