LOCUS       sequence-468022-        5870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RspA-NFAST on the ER membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5870)
  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 5870)
  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-RspA-NFAST
FEATURES             Location/Qualifiers
     source          1..5870
                     /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"
     promoter        complement(1423..1441)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1424..1441)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1461..1478)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1467..1691
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1737..2165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1824..1843)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2034..2055
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2174..2194)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2179..2508
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2359..2494
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2421..2440
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2575..3369
                     /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(2629..2648)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3239..3258
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3543..3664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3580..3599)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3634..3653
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3713..3729)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3713..3729)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3726..3748)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3737..3753
                     /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(3761..3767,3768..3785,3786..3791))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3806..3827
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3944..3961)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4115..4703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4195..4214)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4874..5665,5666..5734))
                     /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     5497..5516
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5735..5839)
                     /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 ccgatatcgt ctccggagga
     1021 ggcggcagcg gcggaggggg atccatggag accgtgagat tcggcggcga cgacatcgag
     1081 aacagcctgg ccaagatgga cgacaaggcc ctggacaagc tggccttcgg cgccatccag
     1141 ctggacggca acggcaagat catccactac aacgccgccg agggcaccat caccggcaga
     1201 gaccccaaga ccgtgatcgg caagaacttc ttcaccgacg tggcccccgg cacccagagc
     1261 aaggagttcc agggcagatt caaggagggc gtgcagaagg gcgacctgaa caccatgttc
     1321 gagtggatga tccccaccag cagaggcccc accaaggtga aggtgcacat gaagaaggcc
     1381 atgacctaag aattcctcga gcatgcatct agagggccct attctatagt gtcacctaaa
     1441 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     1501 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1561 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     1621 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     1681 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     1741 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1801 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1861 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1921 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1981 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2041 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2101 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2161 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2221 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2281 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2341 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2401 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     2461 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     2521 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     2581 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     2641 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     2701 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     2761 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     2821 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     2881 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     2941 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3001 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3061 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     3121 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3181 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3241 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     3301 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3361 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     3421 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3481 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3541 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3601 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3661 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3721 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3781 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3841 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3901 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3961 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4021 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4081 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4141 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4201 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4261 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4321 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4381 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4441 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4501 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4561 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4621 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4681 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4741 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4801 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4861 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4921 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4981 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5041 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5101 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5161 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5221 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5281 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5341 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5401 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5461 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5521 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5581 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5641 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5701 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5761 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5821 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
