LOCUS       sequence-468024-        5846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RspA-CFAST on the PM facing the cytosol.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5846)
  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 5846)
  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: PM-RspA-CFAST
FEATURES             Location/Qualifiers
     source          1..5846
                     /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"
     CDS             937..966
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             973..1293
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     promoter        complement(1399..1417)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1400..1417)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1437..1454)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1443..1667
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1713..2141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1800..1819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2010..2031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2150..2170)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2155..2484
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2335..2470
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2397..2416
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2551..3345
                     /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(2605..2624)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3215..3234
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3519..3640
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3556..3575)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3610..3629
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3689..3705)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3689..3705)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3702..3724)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3713..3729
                     /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(3737..3743,3744..3761,3762..3767))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3782..3803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3920..3937)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4091..4679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4171..4190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4850..5641,5642..5710))
                     /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     5473..5492
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5711..5815)
                     /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 gcttatgggc
      901 tgcatcaagt ccaagggcaa ggactccgcc ggcggcgaac aaaaacttat ttctgaagag
      961 gacttggaat tcggagtgca ggtggaaacc atctccccag gagacgggcg caccttcccc
     1021 aagcgcggcc agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaattt
     1081 gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc
     1141 cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata
     1201 tctccagatt atgcctatgg tgccactggg cacccaggca tcatcccacc acatgccact
     1261 ctcgtcttcg atgtggagct tctaaaactg gaagaatccg gaggaggcgg cagcggcgga
     1321 gggggatccg gcgacagctt ctggatcttc gtgaagagac tgtaagatat cctcgagcat
     1381 gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     1441 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1501 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1561 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1621 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1681 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     1741 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1801 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1861 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1921 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1981 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2041 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2101 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2161 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2221 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2281 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2341 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2401 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     2461 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     2521 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     2581 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2641 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2701 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     2761 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2821 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2881 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     2941 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3001 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3061 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3121 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3181 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3241 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3301 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     3361 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     3421 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3481 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     3541 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3601 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     3661 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3721 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3781 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3841 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3901 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3961 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4021 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4081 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4141 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4201 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4261 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4321 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4381 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4441 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4501 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4561 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4621 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4681 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4741 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4801 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4861 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4921 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4981 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5041 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5101 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5161 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5221 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5281 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5341 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5401 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5461 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5521 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5581 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5641 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5701 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5761 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5821 ttccccgaaa agtgccacct gacgtc
//
