LOCUS       sequence-318450-        6624 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Detect the short Plasma membrane-Endoplasmic reticulum contact .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6624)
  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 6624)
  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 Short P2A
FEATURES             Location/Qualifiers
     source          1..6624
                     /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             1246..1302
                     /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             1318..1341
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2176..2194)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2177..2194)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2214..2231)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2220..2444
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2490..2918
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2577..2596)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2787..2808
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2927..2947)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2932..3262
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3113..3248
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3175..3194
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3329..4123
                     /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(3383..3402)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3993..4012
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4297..4418
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4334..4353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4388..4407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4467..4483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4467..4483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4480..4502)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4491..4507
                     /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(4515..4521,4522..4539,4540..4545))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4560..4581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4698..4715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4869..5457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4949..4968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5628..6419,6420..6488))
                     /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     6251..6270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6489..6593)
                     /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 agctgagagt gttcctggcc
     1021 ctgcccatca tcatggtggt ggccttcagc atgtgcatca tctgcctgct gatggccggc
     1081 gacacctgga cagagacact ggcctacgtg ctgttctggg gcgtggccag catcggcacc
     1141 tttttcatca tcctgtacaa cggcaaggac ttcgtggacg cccccagact ggtgcagaaa
     1201 gagaagatcg accccgggct taaggagctc gcatgcggaa gcggagctac taacttcagc
     1261 ctgctgaagc aggctggaga cgtggaggag aaccctggac ctagatctga attcatggac
     1321 tacaaggacg acgacgacaa gtgcggcctg ttcatctgcc cctgcaacaa gctgaagtcc
     1381 agcgacgcct acaagaaggc ctggggcaac aaccaggatg gcgtggtggc ttcccagcct
     1441 gccagagtgg gaagcggcgg aagctctggc agcagaggct ccgatggctc tgacggcagc
     1501 agcaagggcg aggaactgtt cacaggcgtg gtgcctatcc tggtggaact ggacggcgac
     1561 gtgaacggcc acaagttttc tgtgcgggga gagggcgaag gcgacgccac aatcggaaag
     1621 ctgaccctga agttcatctg taccaccggc aagctgcccg tgccctggcc tacactcgtg
     1681 accacactgg gctacggcct gcagtgcttc gccagatacc ccgaccatat gaagcggcac
     1741 gacttcttca agagcgccat gcccgagggc tacgtgcagg atctgaccat cagcttcaag
     1801 gacgatggca agtacaagac cagagccgtc gtgaagttcg agggcgacac cctcgtgaac
     1861 cggatcgagc tgaagggcac cgatttcaaa gaggacggca acatcctggg ccacaagctg
     1921 gagtacaact tcaacagcca caacgtgtac atcaccgccg acaagcagaa gaacggcatc
     1981 aaggccaact tcaccgtgcg gcacaacgtg gaagatggca gcgtgcagct ggccgaccac
     2041 taccagcaga acacccccat cggagatggc cccgtgctgc tgcccgacaa ccactacctg
     2101 agctaccaga ccgtgctgag caaagatcca aatgaaaaag gaacataact cgagcatgca
     2161 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     2221 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2281 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2341 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2401 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2461 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2521 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2581 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2641 tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2701 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2761 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2821 ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt
     2881 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2941 ttagggtgtg gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa gcatgcatct
     3001 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3061 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3121 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3181 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     3241 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     3301 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3361 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3421 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     3481 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     3541 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     3601 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3661 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3721 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     3781 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     3841 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3901 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3961 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4021 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4081 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4141 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4201 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4261 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4321 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4381 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4441 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4501 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4561 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4621 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4681 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4741 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4801 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4861 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4921 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4981 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5041 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     5101 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5161 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5221 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5281 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     5341 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5401 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5461 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5521 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5581 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5641 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5701 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5761 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5821 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5881 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5941 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6001 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6061 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6121 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6181 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6241 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6301 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6361 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6421 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6481 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6541 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6601 ccccgaaaag tgccacctga cgtc
//
