LOCUS       sequence-468033-        5959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RspA-NFAST on the outer mitochondrial membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5959)
  AUTHORS   Garcia Casas P, Rossini M, Pavenius L, Saeed M, Arnst N, Sonda S, 
            Fernandes T, D'Arsie I, Bruzzone M, Berno V, Raimondi A, Sassano ML,
            Naia L, Barbieri E, Sigismund S, Agostinis P, Sturlese M, Niemeyer 
            BA, Brismar H, Ankarcrona M, Gautier A, Pizzo P, Filadi R
  TITLE     Simultaneous detection of membrane contact dynamics and associated 
            Ca(2+) signals by reversible chemogenetic reporters.
  JOURNAL   Nat Commun. 2024 Nov 12;15(1):9775. doi: 10.1038/s41467-024-52985-0.
  PUBMED    39532847
REFERENCE   2  (bases 1 to 5959)
  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: "2024-11-12"; volume: "15(1):9775. doi"; pages: " 
            10.1038/s41467-024-52985-0"
COMMENT     Sequence Label: OMM-short-RspA-NFAST
FEATURES             Location/Qualifiers
     source          1..5959
                     /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"
     promoter        complement(1512..1530)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1513..1530)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1550..1567)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1556..1780
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1826..2254
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1913..1932)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2123..2144
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2263..2283)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2268..2597
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2448..2583
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2510..2529
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2664..3458
                     /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(2718..2737)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3328..3347
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3632..3753
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3669..3688)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3723..3742
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3802..3818)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3802..3818)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3815..3837)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3826..3842
                     /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(3850..3856,3857..3874,3875..3880))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3895..3916
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4033..4050)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4204..4792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4284..4303)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4963..5754,5755..5823))
                     /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     5586..5605
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5824..5928)
                     /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 gcttcgcaga
      901 ggacacttgt catggccgcc tctaaacctg tggaggcagc ggtggtcgca gccgctgtac
      961 cgagctccgg gagtggggtg ggcggcggcg ggactgcggg cccgggcacg ggggggctgc
     1021 cgcgatggca gctggctctg gcggtcgggg cacccctgct gctgggcgcg ggtgccatat
     1081 acctgtggag tcggcagcaa cggcgccggg aggccagagg ccggggcgga tccatggaga
     1141 ccgtgagatt cggcggcgac gacatcgaga acagcctggc caagatggac gacaaggccc
     1201 tggacaagct ggccttcggc gccatccagc tggacggcaa cggcaagatc atccactaca
     1261 acgccgccga gggcaccatc accggcagag accccaagac cgtgatcggc aagaacttct
     1321 tcaccgacgt ggcccccggc acccagagca aggagttcca gggcagattc aaggagggcg
     1381 tgcagaaggg cgacctgaac accatgttcg agtggatgat ccccaccagc agaggcccca
     1441 ccaaggtgaa ggtgcacatg aagaaggcca tgacctaaga attcctcgag catgcatcta
     1501 gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg
     1561 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1621 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1681 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1741 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     1801 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1861 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1921 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     1981 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2041 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2101 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2161 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2221 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2281 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2341 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2401 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2461 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2521 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2581 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     2641 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     2701 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     2761 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     2821 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     2881 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     2941 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3001 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3061 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3121 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3181 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3241 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3301 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3361 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3421 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3481 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3541 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3601 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3661 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3721 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3781 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3841 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3901 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3961 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4021 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4081 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4141 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4201 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4261 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4321 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4381 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4441 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4501 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4561 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4621 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4681 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4741 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4801 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4861 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4921 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4981 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5041 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5101 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5161 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5221 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5281 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5341 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5401 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5461 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5521 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5581 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5641 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5701 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5761 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5821 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5881 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5941 aaaagtgcca cctgacgtc
//
