LOCUS       sequence-318448-        6975 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 6975)
  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 6975)
  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..6975
                     /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"
     CDS             922..969
                     /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             1006..1272
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="MWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF
                     NQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             1597..1653
                     /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             1669..1692
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2527..2545)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2528..2545)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2565..2582)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2571..2795
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2841..3269
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2928..2947)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3138..3159
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3278..3298)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3283..3613
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3464..3599
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3526..3545
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3680..4474
                     /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(3734..3753)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4344..4363
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4648..4769
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4685..4704)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4739..4758
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4818..4834)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4818..4834)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4831..4853)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4842..4858
                     /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(4866..4872,4873..4890,4891..4896))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4911..4932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5049..5066)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5220..5808)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5300..5319)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5979..6770,6771..6839))
                     /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     6602..6621
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6840..6944)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 ccgccaccat gcgggaccac atggtgctgc acgagtacgt gaacgccgct
      961 ggcatcacag gcggagatgg cggatctggc ggcggaagca aactgatgtg gcacgaggga
     1021 ctggaagagg ccagcagact gtacttcggc gagcggaacg tgaagggcat gttcgaggtg
     1081 ctggaacccc tgcacgccat gatggaaaga ggcccccaga ccctgaaaga gacaagcttc
     1141 aaccaggcct acggccggga cctgatggaa gcccaggaat ggtgccggaa gtacatgaag
     1201 tccggcaatg tgaaggacct gacacaggcc tgggacctgt actaccacgt gttccggcgg
     1261 atcagcaagc agggctctga agccgccgct agagaagctg ctgctagagg cggagcttct
     1321 ggcgctggcg caggcgctgg ggccatcctg aatagcagag tgttcctggc cctgcccatc
     1381 atcatggtgg tggccttcag catgtgcatc atctgcctgc tgatggccgg cgacacctgg
     1441 acagagacac tggcctacgt gctgttctgg ggcgtggcca gcatcggcac ctttttcatc
     1501 atcctgtaca acggcaagga cttcgtggac gcccccaggc tggtgcagaa agagaagatc
     1561 gaccccgggc ttaaggagct cgcatgcgga agcggagcta ctaacttcag cctgctgaag
     1621 caggctggag acgtggagga gaaccctgga cctagatctg aattcatgga ctacaaggac
     1681 gacgacgaca agtgcggcct gttcatctgc ccctgcaaca agctgaagtc cagcgacgcc
     1741 tacaagaagg cctggggcaa caaccaggat ggcgtggtgg cttcccagcc tgccagagtg
     1801 ggaagcggcg gaagctctgg cagcagaggc tccgatggct ctgacggcag cagcaagggc
     1861 gaggaactgt tcacaggcgt ggtgcctatc ctggtggaac tggacggcga cgtgaacggc
     1921 cacaagtttt ctgtgcgggg agagggcgaa ggcgacgcca caatcggaaa gctgaccctg
     1981 aagttcatct gtaccaccgg caagctgccc gtgccctggc ctacactcgt gaccacactg
     2041 ggctacggcc tgcagtgctt cgccagatac cccgaccata tgaagcggca cgacttcttc
     2101 aagagcgcca tgcccgaggg ctacgtgcag gaacggacca tcagcttcaa ggacgatggc
     2161 aagtacaaga ccagagccgt cgtgaagttc gagggcgaca ccctcgtgaa ccggatcgag
     2221 ctgaagggca ccgatttcaa agaggacggc aacatcctgg gccacaagct ggagtacaac
     2281 ttcaacagcc acaacgtgta catcaccgcc gacaagcaga agaacggcat caaggccaac
     2341 ttcaccgtgc ggcacaacgt ggaagatggc agcgtgcagc tggccgacca ctaccagcag
     2401 aacaccccca tcggagatgg ccccgtgctg ctgcccgaca accactacct gagctaccag
     2461 accgtgctga gcaaagatcc aaatgaaaaa ggaacataac tcgagcatgc atctagaggg
     2521 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc
     2581 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2641 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2701 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2761 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2821 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2881 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2941 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcggggcat
     3001 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3061 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3121 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3181 ggtctattct tttgatttat aagggatttt ggggatttcg gcctattggt taaaaaatga
     3241 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3301 ggaaagtccc caggctcccc aggcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3361 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3421 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3481 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3541 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3601 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     3661 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3721 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3781 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3841 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3901 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3961 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4021 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4081 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4141 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4201 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4261 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4321 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4381 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4441 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4501 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4561 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4621 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4681 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4741 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4801 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4861 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4921 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4981 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5041 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5101 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5161 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5221 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5281 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5341 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5401 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5461 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5521 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5581 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5641 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5701 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5761 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5821 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5881 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5941 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6001 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6061 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6121 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6181 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6241 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6301 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6361 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6421 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6481 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6541 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6601 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6661 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6721 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6781 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6841 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6901 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6961 gtgccacctg acgtc
//
