LOCUS       sequence-322825-        6977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Detect the long Plasma membrane-Endoplasmic reticulum contact .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6977)
  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 6977)
  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 PM-ER Long P2A
FEATURES             Location/Qualifiers
     source          1..6977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             924..971
                     /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             1008..1274
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="MWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF
                     NQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             1599..1655
                     /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             1671..1694
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2532..2550)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2533..2550)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2570..2587)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2576..2800
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2846..3274
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2933..2952)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3143..3164
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3283..3303)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3288..3617
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3468..3603
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3530..3549
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3684..4478
                     /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(3738..3757)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4348..4367
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4652..4773
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4689..4708)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4743..4762
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4822..4838)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4822..4838)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4835..4857)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4846..4862
                     /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(4870..4876,4877..4894,4895..4900))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4915..4936
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5053..5070)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5224..5812)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5304..5323)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5983..6774,6775..6843))
                     /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     6606..6625
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6844..6948)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttggta
      901 ccgagctcgg atccgccacc atgcgggacc acatggtgct gcacgagtac gtgaacgccg
      961 ctggcatcac aggcggagat ggcggatctg gcggcggaag caaactgatg tggcacgagg
     1021 gactggaaga ggccagcaga ctgtacttcg gcgagcggaa cgtgaagggc atgttcgagg
     1081 tgctggaacc cctgcacgcc atgatggaaa gaggccccca gaccctgaaa gagacaagct
     1141 tcaaccaggc ctacggccgg gacctgatgg aagcccagga atggtgccgg aagtacatga
     1201 agtccggcaa tgtgaaggac ctgacacagg cctgggacct gtactaccac gtgttccggc
     1261 ggatcagcaa gcagggctct gaagccgccg ctagagaagc tgctgctaga ggcggagctt
     1321 ctggcgctgg cgcaggcgct ggggccatcc tgaatagcag agtgttcctg gccctgccca
     1381 tcatcatggt ggtggccttc agcatgtgca tcatctgcct gctgatggcc ggcgacacct
     1441 ggacagagac actggcctac gtgctgttct ggggcgtggc cagcatcggc acctttttca
     1501 tcatcctgta caacggcaag gacttcgtgg acgcccccag gctggtgcag aaagagaaga
     1561 tcgaccccgg gcttaaggag ctcgcatgcg gaagcggagc tactaacttc agcctgctga
     1621 agcaggctgg agacgtggag gagaaccctg gacctagatc tgaattcatg gactacaagg
     1681 acgacgacga caagtgcggc ctgttcatct gcccctgcaa caagctgaag tccagcgacg
     1741 cctacaagaa ggcctggggc aacaaccagg atggcgtggt ggcttcccag cctgccagag
     1801 tgggaagcgg cggaagctct ggcagcagag gctccgatgg ctctgacggc agcagcaagg
     1861 gcgaggaact gttcacaggc gtggtgccta tcctggtgga actggacggc gacgtgaacg
     1921 gccacaagtt ttctgtgcgg ggagagggcg aaggcgacgc cacaatcgga aagctgaccc
     1981 tgaagttcat ctgtaccacc ggcaagctgc ccgtgccctg gcctacactc gtgaccacac
     2041 tgggctacgg cctgcagtgc ttcgccagat accccgacca tatgaagcgg cacgacttct
     2101 tcaagagcgc catgcccgag ggctacgtgc aggaacggac catcagcttc aaggacgatg
     2161 gcaagtacaa gaccagagcc gtcgtgaagt tcgagggcga caccctcgtg aaccggatcg
     2221 agctgaaggg caccgatttc aaagaggacg gcaacatcct gggccacaag ctggagtaca
     2281 acttcaacag ccacaacgtg tacatcaccg ccgacaagca gaagaacggc atcaaggcca
     2341 acttcaccgt gcggcacaac gtggaagatg gcagcgtgca gctggccgac cactaccagc
     2401 agaacacccc catcggagat ggccccgtgc tgctgcccga caaccactac ctgagctacc
     2461 agaccgtgct gagcaaggac cccaacgaga agggcacatg agcggccgct cgagcctcta
     2521 gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg
     2581 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2641 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2701 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2761 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2821 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2881 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2941 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3001 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3061 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3121 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     3181 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3241 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     3301 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3361 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3421 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3481 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3541 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3601 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     3661 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3721 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3781 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3841 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3901 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3961 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4021 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4081 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4141 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4201 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4261 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4321 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4381 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4441 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4501 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4561 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4621 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4681 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4741 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4801 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4861 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4921 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4981 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5041 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5101 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5161 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5221 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5281 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5341 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5401 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5461 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5521 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5581 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5641 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5701 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5761 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5821 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5881 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5941 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6001 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6061 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6121 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6181 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6241 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6301 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6361 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6421 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6481 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6541 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6601 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6661 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6721 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6781 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6841 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6901 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6961 aaaagtgcca cctgacg
//
