LOCUS       sequence-458401-        6733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a synthetic linker, tagged with EBFP2, Stabilizing the 
            ER-mitochondrial distance at 20 nm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6733)
  AUTHORS   Dematteis G, Tapella L, Casali C, Talmon M, Tonelli E, Reano S, 
            Ariotti A, Pessolano E, Malecka J, Chrostek G, Kulkoviene G, 
            Umbrasas D, Distasi C, Grilli M, Ladds G, Filigheddu N, Fresu LG, 
            Mikoshiba K, Matute C, Ramos-Gonzalez P, Jekabsone A, Cali T, Brini 
            M, Biggiogera M, Cavaliere F, Miggiano R, Genazzani AA, Lim D
  TITLE     ER-mitochondria distance is a critical parameter for efficient 
            mitochondrial Ca(2+) uptake and oxidative metabolism.
  JOURNAL   Commun Biol. 2024 Oct 10;7(1):1294. doi: 10.1038/s42003-024-06933-9.
  PUBMED    39390051
REFERENCE   2  (bases 1 to 6733)
  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: "Commun 
            Biol."; date: "2024-10-10"; volume: "7(1):1294. doi"; pages: " 
            10.1038/s42003-024-06933-9"
COMMENT     Sequence Label: pcDNA3.1-20nm-EBFP2
FEATURES             Location/Qualifiers
     source          1..6733
                     /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"
     primer_bind     complement(2324..2341)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2330..2554
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2600..3028
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2687..2706)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2897..2918
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3037..3057)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3042..3371
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3222..3357
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3284..3303
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3438..4232
                     /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(3492..3511)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4102..4121
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4406..4527
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4443..4462)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4497..4516
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4576..4592)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4576..4592)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4589..4611)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4600..4616
                     /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(4624..4630,4631..4648,4649..4654))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4669..4690
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4807..4824)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4978..5566)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5058..5077)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5737..6528,6529..6597))
                     /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     6360..6379
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6598..6702)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 gctggctagc
      901 atggccatcc agctgcgctc cctgtttcca ctggccctgc caggactgct ggccctgctg
      961 ggctggtggt ggttctttag caggaagaag gaccctaccc gctccgccaa taagcagcag
     1021 gaggaggagg cagagcggct gcggagaatc caggaggaga tggagaagga gagaaagagg
     1081 cgcgaggagg atgagcagcg gagaaggaaa gaggaggagg agcgccggat gaagctggag
     1141 atggaggcca agaggaagca ggaggaggag gagcggaaga agagagagga cgatgagaag
     1201 cggatccagg ccgagagaaa ctctgaccca cctgtggcca ccctggtgag caagggcgag
     1261 gagctgttca caggcgtggt gcccatcctg gtggagctgg acggcgatgt gaatggccac
     1321 aagttttctg tgagaggaga gggagaggga gatgcaacca acggaaagct gacactgaag
     1381 ttcatctgca ccacaggcaa gctgccagtg ccctggccta ccctggtgac cacactgtcc
     1441 cacggcgtgc agtgttttgc caggtacccc gaccacatga agcagcacga tttctttaag
     1501 tctgccatgc ctgagggcta cgtgcaggag cggaccatct tctttaagga cgatggcacc
     1561 tataagacaa gagccgaggt gaagttcgag ggcgacacac tggtgaacag gatcgagctg
     1621 aagggcgtgg acttcaagga ggatggcaat atcctgggcc acaagctgga gtacaacttt
     1681 aatagccaca acatctatat catggccgtg aagcagaaga acggcatcaa ggtgaacttc
     1741 aagatcagac acaatgtgga ggacggcagc gtgcagctgg ccgatcacta ccagcagaac
     1801 accccaatcg gagacggacc tgtgctgctg ccagattccc actatctgtc cacacagtct
     1861 gtgctgagca aggaccccaa tgagaagagg gatcacatgg tgctgctgga gttcaggacc
     1921 gcagcaggaa tcacactggg aatggacgag ctgtacaagt ccggactgag gtctcgcgcc
     1981 caggccagca actccaaaca gcaggaagag gaggccgaga ggctgagaag gattcaggaa
     2041 gagatggaga aggagcgcaa gcgccgggag gaggatgagc agagaaggcg caaagaagag
     2101 gaggagcgga gaatgaaact ggagatggaa gccaagcgca aacaggaaga ggaggagagg
     2161 aagaagcgcg aggacgatga gaagaggatc caggcagaga gcagagccca ggcctctaat
     2221 agccgggtga tggtgtatat cggcatcgcc atcttcctgt ttgtgggcct gttcatgaag
     2281 taatgactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     2341 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2401 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2461 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2521 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2581 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2641 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2701 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2761 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2821 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2881 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2941 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3001 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3061 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3121 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3181 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3241 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3301 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3361 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3421 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3481 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3541 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3601 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3661 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3721 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3781 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3841 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     3901 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     3961 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4021 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4081 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4141 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4201 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4261 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4321 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4381 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4441 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4501 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4561 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4621 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4681 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4741 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4801 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4861 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4921 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4981 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5041 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5101 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5161 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5221 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5281 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5341 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5401 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5461 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5521 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5581 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5641 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5701 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5761 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5821 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5881 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5941 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6001 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6061 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6121 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6181 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6241 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6301 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6361 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6421 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6481 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6541 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6601 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6661 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6721 gccacctgac gtc
//
