LOCUS       sequence-468027-        5987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RspA-NFAST on the ER membrane after a 55 aminoacid 
            linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5987)
  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 5987)
  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: ER-long-RspA-NFAST
FEATURES             Location/Qualifiers
     source          1..5987
                     /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"
     regulatory      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1003..1026
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1540..1558)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1541..1558)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1578..1595)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1584..1808
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1854..2282
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1941..1960)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2151..2172
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2291..2311)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2296..2625
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2476..2611
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2538..2557
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2692..3486
                     /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(2746..2765)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3356..3375
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3660..3781
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3697..3716)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3751..3770
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3830..3846)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3830..3846)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3843..3865)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3854..3870
                     /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(3878..3884,3885..3902,3903..3908))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3923..3944
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4061..4078)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4232..4820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4312..4331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4991..5782,5783..5851))
                     /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     5614..5633
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5852..5956)
                     /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 gcttgccacc
      901 atggaccctg tggtggtgct ggggctctgt ctctcctgtt tgcttctcct ttcactctgg
      961 aaacagagct atgggggagg gtctggtgga tctggaggta ccgactacaa agacgatgac
     1021 gacaagggca gcggaggagg ctccggagga ggcggcagcg gcggagggaa gaaaagcgga
     1081 ggcggaggct ccggaggagg cggcagcggc ggagggaaga aatccggagg aggcggcagc
     1141 ggcggagggt ctggtggatc catggagacc gtgagattcg gcggcgacga catcgagaac
     1201 agcctggcca agatggacga caaggccctg gacaagctgg ccttcggcgc catccagctg
     1261 gacggcaacg gcaagatcat ccactacaac gccgccgagg gcaccatcac cggcagagac
     1321 cccaagaccg tgatcggcaa gaacttcttc accgacgtgg cccccggcac ccagagcaag
     1381 gagttccagg gcagattcaa ggagggcgtg cagaagggcg acctgaacac catgttcgag
     1441 tggatgatcc ccaccagcag aggccccacc aaggtgaagg tgcacatgaa gaaggccatg
     1501 acctaagaat tcctcgagca tgcatctaga gggccctatt ctatagtgtc acctaaatgc
     1561 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1621 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1681 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     1741 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     1801 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     1861 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1921 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1981 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2041 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2101 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2161 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2221 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2281 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2341 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2401 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2461 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2521 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     2581 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     2641 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     2701 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2761 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2821 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     2881 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     2941 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3001 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3061 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3121 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3181 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3241 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3301 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3361 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3421 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3481 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3541 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     3601 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     3661 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     3721 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3781 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     3841 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3901 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3961 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4021 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4081 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4141 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4201 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4261 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4321 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4381 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4441 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4501 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4561 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4621 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4681 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4741 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4801 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4861 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4921 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4981 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5041 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5101 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5161 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5221 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5281 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5341 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5401 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5461 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5521 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5581 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5641 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5701 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5761 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5821 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5881 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5941 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
