LOCUS       sequence-458397-        6577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a synthetic linker, tagged with EBFP2, Stabilizing the 
            ER-mitochondrial distance at 10 nm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6577)
  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 6577)
  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: pcDNA3.1-10nm-EBFP2
FEATURES             Location/Qualifiers
     source          1..6577
                     /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(2168..2185)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2174..2398
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2444..2872
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2531..2550)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2741..2762
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2881..2901)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2886..3215
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3066..3201
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3128..3147
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3282..4076
                     /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(3336..3355)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3946..3965
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4250..4371
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4287..4306)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4341..4360
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4420..4436)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4420..4436)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4433..4455)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4444..4460
                     /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(4468..4474,4475..4492,4493..4498))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4513..4534
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4651..4668)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4822..5410)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4902..4921)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5581..6372,6373..6441))
                     /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     6204..6223
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6442..6546)
                     /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 agctgaggtc tctgtttcca ctggccctgc caggactgct ggccctgctg
      961 ggctggtggt ggttctttag cagaaagaag gaccctacca ggtctgccaa tagcgaggcc
     1021 gccgcccggg aggccgccgc cagagaggcc gccgccaggg aggcagcagc ccgcgaggca
     1081 gcagcccggg aggctgctgc cagagaggct gctgccaggg aggccgccgc ccgcgaggcc
     1141 gccgcccgga acagcgaccc acctgtggcc accctggtgt ccaagggcga ggagctgttc
     1201 acaggcgtgg tgcccatcct ggtggagctg gacggcgatg tgaatggcca caagttttct
     1261 gtgcggggag agggagaggg agatgcaacc aacggcaagc tgacactgaa gttcatctgc
     1321 accacaggca agctgccagt gccctggcct accctggtga ccacactgtc ccacggcgtg
     1381 cagtgttttg ccagataccc cgaccacatg aagcagcacg atttctttaa gtctgccatg
     1441 cctgagggct acgtgcagga gagaaccatc ttctttaagg acgatggcac ctataagaca
     1501 agggccgagg tgaagttcga gggcgacaca ctggtgaacc gcatcgagct gaagggcgtg
     1561 gacttcaagg aggatggcaa tatcctgggc cacaagctgg agtacaactt taatagccac
     1621 aacatctata tcatggccgt gaagcagaag aacggcatca aggtgaactt caagatcaga
     1681 cacaatgtgg aggacggcag cgtgcagctg gccgatcact accagcagaa caccccaatc
     1741 ggagacggac ctgtgctgct gccagattcc cactatctgt ccacacagtc tgtgctgagc
     1801 aaggacccca atgagaagcg cgatcacatg gtgctgctgg agttcaggac cgcagcagga
     1861 atcacactgg gcatggatga gctgtacaag tccggactgc ggtctagagc ccaggccagc
     1921 aactccgaag ctgctgccag agaggcagct gccagggagg ccgctgcccg cgaggccgca
     1981 gcccgggagg ctgcagccag agaggctgca gccagggagg ctgctgcccg cgaggctgct
     2041 gcccgggagg ctgcagctag gaattcccgc gtgatggtgt atatcggcat cgccatcttc
     2101 ctgtttgtgg gcctgttcat gaagtaatga ctcgagtcta gagggcccgt ttaaacccgc
     2161 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2221 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2281 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2341 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2401 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2461 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2521 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2581 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2641 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2701 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2761 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2821 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     2881 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2941 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3001 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3061 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3121 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3181 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3241 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3301 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3361 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3421 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3481 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3541 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3601 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3661 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3721 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3781 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3841 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     3901 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     3961 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4021 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4081 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4141 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4201 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4261 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4321 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4381 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4441 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4501 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4561 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4621 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4681 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4741 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4801 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4861 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4921 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4981 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5041 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5101 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5161 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5221 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5281 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5341 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5401 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5461 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5521 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5581 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5641 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5701 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5761 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5821 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5881 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5941 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6001 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6061 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6121 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6181 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6241 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6301 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6361 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6421 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6481 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6541 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
