LOCUS       sequence-468031-        7815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of hSTIM1-RspA-NFAST.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7815)
  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 7815)
  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: STIM1-RspA-NFAST
FEATURES             Location/Qualifiers
     source          1..7815
                     /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(3368..3386)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3369..3386)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3406..3423)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3412..3636
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3682..4110
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3769..3788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3979..4000
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4119..4139)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4124..4453
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4304..4439
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4366..4385
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4520..5314
                     /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(4574..4593)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5184..5203
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5488..5609
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5525..5544)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5579..5598
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5658..5674)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5658..5674)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5671..5693)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5682..5698
                     /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(5706..5712,5713..5730,5731..5736))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5751..5772
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5889..5906)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6060..6648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6140..6159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6819..7610,7611..7679))
                     /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     7442..7461
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7680..7784)
                     /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 atggatgtat gcgtccgtct tgccctgtgg ctcctctggg gactcctcct gcaccagggc
      961 cagagcctca gccatagtca cagtgagaag gcgacaggaa ccagctcggg ggccaactct
     1021 gaggagtcca ctgcagcaga gttttgccga attgacaagc ccctgtgtca cagtgaggat
     1081 gagaaactca gcttcgaggc agtccgtaac atccacaaac tgatggacga tgatgccaat
     1141 ggtgatgtgg atgtggaaga aagtgatgag ttcctgaggg aagacctcaa ttaccatgac
     1201 ccaacagtga aacacagcac cttccatggt gaggataagc tcatcagcgt ggaggacctg
     1261 tggaaggcat ggaagtcatc agaagtatac aattggaccg tggatgaggt ggtacagtgg
     1321 ctgatcacat atgtggagct gcctcagtat gaggagacct tccggaagct gcagctcagt
     1381 ggccatgcca tgccaaggct ggctgtcacc aacaccacca tgacagggac tgtgctgaag
     1441 atgacagacc ggagtcatcg gcagaagctg cagctgaagg ctctggatac agtgctcttt
     1501 gggcctcctc tcttgactcg ccataatcac ctcaaggact tcatgctggt ggtgtctatc
     1561 gttattggtg tgggcggctg ctggtttgcc tatatccaga accgttactc caaggagcac
     1621 atgaagaaga tgatgaagga cttggagggg ttacaccgag ctgagcagag tctgcatgac
     1681 cttcaggaaa ggctgcacaa ggcccaggag gagcaccgca cagtggaggt ggagaaggtc
     1741 catctggaaa agaagctgcg cgatgagatc aaccttgcta agcaggaagc ccagcggctg
     1801 aaggagctgc gggagggtac tgagaatgag cggagccgcc aaaaatatgc tgaggaggag
     1861 ttggagcagg ttcgggaggc cttgaggaaa gcagagaagg agctagaatc tcacagctca
     1921 tggtatgctc cagaggccct tcagaagtgg ctgcagctga cacatgaggt ggaggtgcaa
     1981 tattacaaca tcaagaagca aaatgctgag aagcagctgc tggtggccaa ggagggggct
     2041 gagaagataa aaaagaagag aaacacactc tttggcacct tccacgtggc ccacagctct
     2101 tccctggatg atgtagatca taaaattcta acagctaagc aagcactgag cgaggtgaca
     2161 gcagcattgc gggagcgcct gcaccgctgg caacagatcg agatcctctg tggcttccag
     2221 attgtcaaca accctggcat ccactcactg gtggctgccc tcaacataga ccccagctgg
     2281 atgggcagta cacgccccaa ccctgctcac ttcatcatga ctgacgacgt ggatgacatg
     2341 gatgaggaga ttgtgtctcc cttgtccatg cagtccccta gcctgcagag cagtgttcgg
     2401 cagcgcctga cggagccaca gcatggcctg ggatctcaga gggatttgac ccattccgat
     2461 tcggagtcct ccctccacat gagtgaccgc cagcgtgtgg cccccaaacc tcctcagatg
     2521 agccgtgctg cagacgaggc tctcaatgcc atgacttcca atggcagcca ccggctgatc
     2581 gagggggtcc acccagggtc tctggtggag aaactgcctg acagccctgc cctggccaag
     2641 aaggcattac tggcgctgaa ccatgggctg gacaaggccc acagcctgat ggagctgagc
     2701 ccctcagccc cacctggtgg ctctccacat ttggattctt cccgttctca cagccccagc
     2761 tccccagacc cagacacacc atctccagtt ggggacagcc gagccctgca agccagccga
     2821 aacacacgca ttccccacct ggctggcaag aaggctgtgg ctgaggagga taatggctct
     2881 attggcgagg aaacagactc cagcccaggc cggaagaagt ttcccctcaa aatctttaag
     2941 aagcctctta agaagggcag cggaggaggc ggatccatgg agaccgtgag attcggcggc
     3001 gacgacatcg agaacagcct ggccaagatg gacgacaagg ccctggacaa gctggccttc
     3061 ggcgccatcc agctggacgg caacggcaag atcatccact acaacgccgc cgagggcacc
     3121 atcaccggca gagaccccaa gaccgtgatc ggcaagaact tcttcaccga cgtggccccc
     3181 ggcacccaga gcaaggagtt ccagggcaga ttcaaggagg gcgtgcagaa gggcgacctg
     3241 aacaccatgt tcgagtggat gatccccacc agcagaggcc ccaccaaggt gaaggtgcac
     3301 atgaagaagg ccatgaccta agaattcctc gaggcggccg ctcgagcatg catctagagg
     3361 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     3421 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3481 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3541 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3601 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3661 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3721 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3781 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3841 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3901 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3961 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4021 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4081 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     4141 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4201 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4261 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4321 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4381 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4441 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     4501 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     4561 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4621 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4681 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4741 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4801 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4861 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4921 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4981 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     5041 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     5101 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     5161 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     5221 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     5281 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     5341 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     5401 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     5461 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     5521 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5581 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     5641 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5701 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5761 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5821 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5881 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5941 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6001 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6061 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6121 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6181 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6241 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6301 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6361 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6421 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6481 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6541 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6601 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6661 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6721 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6781 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6841 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6901 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6961 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7021 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7081 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7141 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7201 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7261 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7321 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7381 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7441 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7501 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7561 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7621 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7681 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7741 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7801 gtgccacctg acgtc
//
