LOCUS       sequence-322810-        7808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Detect the long Plasma membrane--mitochondria-Endoplasmic reticulum 
            contact .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7808)
  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 7808)
  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 Long P2A
FEATURES             Location/Qualifiers
     source          1..7808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(62..81)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        253..632
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        633..836
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     787..807
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     881..900
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        881..899
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             940..987
                     /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             1024..1290
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="MWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF
                     NQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             1615..1671
                     /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             2461..2517
                     /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             2533..2556
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(3379..3397)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3380..3397)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3417..3434)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3423..3647
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3693..4121
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3780..3799)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3990..4011
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4130..4150)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4135..4464
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4315..4450
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4377..4396
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4531..5325
                     /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(4585..4604)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5195..5214
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5499..5620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5536..5555)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5590..5609
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5669..5685)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5669..5685)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5682..5704)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5693..5709
                     /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(5717..5723,5724..5741,5742..5747))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5762..5783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5900..5917)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6071..6659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6151..6170)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6830..7621,7622..7690))
                     /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     7453..7472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7691..7795)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
       61 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
      121 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
      181 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      241 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      301 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      361 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      421 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      481 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      541 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      601 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      661 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      721 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      781 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
      841 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
      901 agacccaagc ttggtaccga gctcggatcc gccaccatgc gggaccacat ggtgctgcac
      961 gagtacgtga acgccgctgg catcacaggc ggagatggcg gatctggcgg cggaagcaaa
     1021 ctgatgtggc acgagggact ggaagaggcc agcagactgt acttcggcga gcggaacgtg
     1081 aagggcatgt tcgaggtgct ggaacccctg cacgccatga tggaaagagg cccccagacc
     1141 ctgaaagaga caagcttcaa ccaggcctac ggccgggacc tgatggaagc ccaggaatgg
     1201 tgccggaagt acatgaagtc cggcaatgtg aaggacctga cacaggcctg ggacctgtac
     1261 taccacgtgt tccggcggat cagcaagcag ggctctgaag ccgccgctag agaagctgct
     1321 gctagaggcg gagcttctgg cgctggcgca ggcgctgggg ccatcctgaa tagcagagtg
     1381 ttcctggccc tgcccatcat catggtggtg gccttcagca tgtgcatcat ctgcctgctg
     1441 atggccggcg acacctggac agagacactg gcctacgtgc tgttctgggg cgtggccagc
     1501 atcggcacct ttttcatcat cctgtacaac ggcaaggact tcgtggacgc ccccaggctg
     1561 gtgcagaaag agaagatcga ccccgggctt aaggagctcg catgcggaag cggagctact
     1621 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tagatctgaa
     1681 ttcatggtgg gccggaacag cgccatcgcc gcgggcgtgt gcggtgccct cttcataggg
     1741 tactgcatct actttgaccg caaaaggcgg agtgacccca actccaaagg agaagaactg
     1801 tttaccggcg tggtgccaat tctcgtggaa ctggatggcg atgtgaatgg ccacaaattt
     1861 tctgtcagag gagagggtga aggtgatgcc acaatcggaa agctcaccct gaaattcatc
     1921 tgcaccactg gaaagctccc tgtgccatgg ccaacactgg tcactaccct gacctacggc
     1981 gtgcagtgct tttccagata cccagaccat atgaagaggc atgacttttt caagagcgcc
     2041 atgcccgagg gctatgtgca ggagagaacc atctctttca aagatgacgg gaaatacaag
     2101 acccgcgctg tggtcaagtt cgaaggagac acactggtga atagaatcga gttgaagggc
     2161 acagacttta aggaagatgg aaacattctc ggccacaagc tggaatacaa ctttaactcc
     2221 cacaatgtgt acatcacagc cgacaagcaa aagaatggca tcaaggctaa cttcacagtc
     2281 agacacaacg tcgaggatgg aagcgtgcag ctggccgacc attatcaaca gaacactcca
     2341 atcggcgacg gccctgtgct cctcccagac aaccattacc tgtccaccca gacagtcctg
     2401 agcaaagatc caaatgaaaa aggaacactc gagcttaagg agctcgcatg cggaagcgga
     2461 gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctaga
     2521 tctgaattca tggactacaa ggacgacgac gacaagtgcg gcctgttcat ctgcccctgc
     2581 aacaagctga agtccagcga cgcctacaag aaggcctggg gcaacaacca ggatggcgtg
     2641 gtggcttccc agcctgccag agtgggaagc ggcggaagct ctggcagcag aggctccgat
     2701 ggctctgacg gcagcagcaa gggcgaggaa ctgttcacag gcgtggtgcc tatcctggtg
     2761 gaactggacg gcgacgtgaa cggccacaag ttttctgtgc ggggagaggg cgaaggcgac
     2821 gccacaatcg gaaagctgac cctgaagttc atctgtacca ccggcaagct gcccgtgccc
     2881 tggcctacac tcgtgaccac actgggctac ggcctgcagt gcttcgccag ataccccgac
     2941 catatgaagc ggcacgactt cttcaagagc gccatgcccg agggctacgt gcaggaacgg
     3001 accatcagct tcaaggacga tggcaagtac aagaccagag ccgtcgtgaa gttcgagggc
     3061 gacaccctcg tgaaccggat cgagctgaag ggcaccgatt tcaaagagga cggcaacatc
     3121 ctgggccaca agctggagta caacttcaac agccacaacg tgtacatcac cgccgacaag
     3181 cagaagaacg gcatcaaggc caacttcacc gtgcggcaca acgtggaaga tggcagcgtg
     3241 cagctggccg accactacca gcagaacacc cccatcggag atggccccgt gctgctgccc
     3301 gacaaccact acctgagcta ccagaccgtg ctgagcaaag atccaaatga aaaaggaaca
     3361 taatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     3421 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3481 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3541 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3601 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3661 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3721 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3781 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3841 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3901 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3961 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4021 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4081 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     4141 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4201 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4261 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4321 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4381 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4441 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     4501 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4561 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4621 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4681 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4741 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4801 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4861 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4921 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4981 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     5041 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     5101 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5161 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5221 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     5281 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5341 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5401 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5461 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5521 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5581 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5641 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5701 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5761 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5821 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5881 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5941 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6001 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6061 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6121 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6181 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6241 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6301 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6361 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6421 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6481 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6541 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6601 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6661 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6721 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6781 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6841 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6901 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6961 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7021 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7081 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7141 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7201 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7261 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7321 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7381 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7441 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7501 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7561 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7621 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7681 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7741 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7801 ttccccga
//
