LOCUS       sequence-322838-        6626 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 6626)
  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 6626)
  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..6626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..62)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     768..788
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     862..881
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        862..880
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             921..968
                     /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             1245..1301
                     /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             1317..1340
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2178..2196)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2179..2196)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2216..2233)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2222..2446
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2492..2920
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2579..2598)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2789..2810
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2929..2949)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2934..3263
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3114..3249
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3176..3195
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3330..4124
                     /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(3384..3403)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3994..4013
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4298..4419
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4335..4354)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4389..4408
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4468..4484)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4468..4484)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4481..4503)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4492..4508
                     /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(4516..4522,4523..4540,4541..4546))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4561..4582
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4699..4716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4870..5458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4950..4969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5629..6420,6421..6489))
                     /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     6252..6271
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6490..6594)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
       61 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      121 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
      181 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      781 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
      841 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag cttggtaccg
      901 agctcggatc cgccaccatg cgggaccaca tggtgctgca cgagtacgtg aacgccgctg
      961 gcatcacagg cggagatggc ggatctggcg gcggaagcaa gctgagagtg ttcctggccc
     1021 tgcccatcat catggtggtg gccttcagca tgtgcatcat ctgcctgctg atggccggcg
     1081 acacctggac agagacactg gcctacgtgc tgttctgggg cgtggccagc atcggcacct
     1141 ttttcatcat cctgtacaac ggcaaggact tcgtggacgc ccccagactg gtgcagaaag
     1201 agaagatcga ccccgggctt aaggagctcg catgcggaag cggagctact aacttcagcc
     1261 tgctgaagca ggctggagac gtggaggaga accctggacc tagatctgaa ttcatggact
     1321 acaaggacga cgacgacaag tgcggcctgt tcatctgccc ctgcaacaag ctgaagtcca
     1381 gcgacgccta caagaaggcc tggggcaaca accaggatgg cgtggtggct tcccagcctg
     1441 ccagagtggg aagcggcgga agctctggca gcagaggctc cgatggctct gacggcagca
     1501 gcaagggcga ggaactgttc acaggcgtgg tgcctatcct ggtggaactg gacggcgacg
     1561 tgaacggcca caagttttct gtgcggggag agggcgaagg cgacgccaca atcggaaagc
     1621 tgaccctgaa gttcatctgt accaccggca agctgcccgt gccctggcct acactcgtga
     1681 ccacactggg ctacggcctg cagtgcttcg ccagataccc cgaccatatg aagcggcacg
     1741 acttcttcaa gagcgccatg cccgagggct acgtgcagga acggaccatc agcttcaagg
     1801 acgatggcaa gtacaagacc agagccgtcg tgaagttcga gggcgacacc ctcgtgaacc
     1861 ggatcgagct gaagggcacc gatttcaaag aggacggcaa catcctgggc cacaagctgg
     1921 agtacaactt caacagccac aacgtgtaca tcaccgccga caagcagaag aacggcatca
     1981 aggccaactt caccgtgcgg cacaacgtgg aagatggcag cgtgcagctg gccgaccact
     2041 accagcagaa cacccccatc ggagatggcc ccgtgctgct gcccgacaac cactacctga
     2101 gctaccagac cgtgctgagc aaggacccca acgagaaggg cacatgagcg gccgctcgag
     2161 cctctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg
     2221 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2281 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2341 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2401 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2461 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2521 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2581 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2641 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2701 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2761 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2821 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2881 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     2941 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3001 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3061 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3121 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3181 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3241 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3301 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3361 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3421 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3481 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3541 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3601 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3661 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3721 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3781 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3841 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3901 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3961 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4021 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4081 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4141 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4201 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4261 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4321 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4381 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     4441 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4501 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4561 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4621 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4681 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4741 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4801 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4861 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4921 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4981 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5041 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5101 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5161 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5221 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5281 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5341 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5401 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5461 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5521 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5581 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5641 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5701 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5761 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5821 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5881 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5941 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6001 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6061 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6121 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6181 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6241 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6301 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6361 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6421 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6481 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6541 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6601 tccccgaaaa gtgccacctg acgtcg
//
