LOCUS       sequence-468021-        5658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RspA-CFAST on the outer mitochondrial membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5658)
  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 5658)
  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-CFAST
FEATURES             Location/Qualifiers
     source          1..5658
                     /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(1211..1229)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1212..1229)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1249..1266)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1255..1479
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1525..1953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1612..1631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1822..1843
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1962..1982)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1967..2296
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2147..2282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2209..2228
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2363..3157
                     /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(2417..2436)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3027..3046
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3331..3452
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3368..3387)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3422..3441
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3501..3517)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3501..3517)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3514..3536)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3525..3541
                     /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(3549..3555,3556..3573,3574..3579))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3594..3615
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3732..3749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3903..4491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3983..4002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4662..5453,5454..5522))
                     /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     5285..5304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5523..5627)
                     /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 tccggcgaca
     1141 gcttctggat cttcgtgaag agactgtaag atatcgcggc cgcctcgagc atgcatctag
     1201 agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc
     1261 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1321 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1381 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     1441 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     1501 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1561 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1621 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1681 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1741 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1801 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1861 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1921 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     1981 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2041 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2101 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     2161 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2221 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2281 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     2341 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     2401 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     2461 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     2521 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     2581 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     2641 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     2701 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     2761 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     2821 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     2881 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     2941 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3001 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3061 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3121 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     3181 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     3241 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3301 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     3361 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3421 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     3481 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3541 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3601 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3661 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3721 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3781 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3841 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3901 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3961 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4021 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4081 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4141 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4201 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4261 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4321 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4381 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4441 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4501 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4561 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4621 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4681 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4741 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4801 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4861 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4921 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4981 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5041 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5101 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5161 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5221 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5281 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5341 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5401 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5461 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5521 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5581 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5641 aaagtgccac ctgacgtc
//
