LOCUS       sequence-493025-        7265 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  Expresses the SPLICa reporter for ER-Mitochondria contacts and 
            Calcium transients at the contact.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7265)
  AUTHORS   Yamaguchi I, Barazzuol L, Dematteis G, Zhu W, Wen Y, Drobizhev M, 
            Lim D, Campbell RE, Cali T, Nasu Y
  TITLE     Bipartite Genetically Encoded Biosensors to Sense Calcium Ion 
            Dynamics at Membrane-Membrane Contact Sites.
  JOURNAL   Anal Chem. 2025 Sep 16;97(36):19848-19861. doi: 
            10.1021/acs.analchem.5c03831. Epub 2025 Sep 1.
  PUBMED    40888292
REFERENCE   2  (bases 1 to 7265)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acs.analchem.5c03831"; journalName: "Anal Chem"; date: 
            "2025-09-16- 16"; volume: "97"; issue: "36"; pages: "19848-19861"
COMMENT     Sequence Label: SPLICa:ER-MT (High Affinity)
FEATURES             Location/Qualifiers
     source          1..7265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    27..48
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(63..68,69..86,87..93)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    101..117
                     /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)."
     primer_bind     106..128
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     125..141
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     125..141
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    190..311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(201..220)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     255..274
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(485..1279)
                     /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(596..615)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1206..1225
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1346..1675)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1360..1495
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1414..1433)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1660..1680
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1689..2117)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1799..1820)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2011..2030
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2163..2387
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2376..2393
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3194..3250)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      3802..3811
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4367..4386)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4368..4386)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4431..4634)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4460..4480)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4635..5014
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5186..5205
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5280..5384
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5385..5453,5454..6245)
                     /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     complement(5603..5622)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6416..7004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6905..6924
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7158..7175
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
       61 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      121 cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt cgacggtata
      181 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
      241 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
      301 ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc
      361 cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa
      421 tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc
      481 ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg
      541 cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat
      601 cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga
      661 tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg
      721 tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg
      781 gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc
      841 gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat
      901 caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa
      961 ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg
     1021 cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata
     1081 gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa
     1141 gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct
     1201 gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca
     1261 atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatccgaaaa
     1321 tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa aagcctcctc
     1381 actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa
     1441 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg
     1501 gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc
     1561 ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt
     1621 gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc
     1681 acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1741 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     1801 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1861 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     1921 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     1981 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2041 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2101 gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt ccgcctcaga
     2161 agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat cctccccctt
     2221 gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac aatgcgatgc
     2281 aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg tcaaggaagg
     2341 cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg aggctgatca
     2401 gcgggtttaa acgggccctc tagactcgag cggccgctca tttggctgtc atcatccgca
     2461 cgaattcctc gtagttcact tggccgtcgc catcgatatc ggcttctctc agcatctcgt
     2521 ccacttcttc gtcggtcagc ttctctccca ggttggtcac gatatgccgc agctctgcgg
     2581 cggagatgta gctattgccg tctttgtcga acacgccata agcctctcta agctcctcct
     2641 ctgtgtcccg gtatttcatc tttctggcca tcatggccag gaactcgggg aaatcgattg
     2701 ttccgtcgcc ctcggcgtcc acctcgttga tcatgtcctg cagttcggcc tcggtggggt
     2761 tttggcccag acttctcatc acggtgccca gctctctggt ggtgattgtt ccatccccgt
     2821 ccttgtcgaa cagagagaac agttccttga tctcggcgat ctgctcctcg gtcagctggt
     2881 cgctctcgcc tgtggtgtag gaccacttgc tggtagaaat gattgtcttg tcgttggggt
     2941 aggtcatagt gcttctgttc cagtccaggc cgctgctcag ccgccgcagc acccgcacgg
     3001 cgtggccggt cttgtaccac ttccttctag agctgtcgac catgaattcg gccatgttgt
     3061 tatcctcgct gctggctgtg ccgttggggt cgctccgtct ctttctatcg aagtagatac
     3121 aatagccgat aaacagggcg ccgcacacgc cggcggcgat ggcggagttt ctgcccacca
     3181 taatgtcgga tctagggcca gggttttctt ccacgtctcc ggcctgtttc agcaggctga
     3241 agtttgtagc gccgctgcca caagccagtt ccttcaggct tccgtcgatc ttttctttct
     3301 gcaccagtct aggggcatcc acgaaatcct ttccgttgta caggatgatg aagaaggtgc
     3361 cgatgctagc cacgccccag aacagcacgt aggccagtgt ctcggtccag gtgtcgccgg
     3421 ccatcagcag gcagatgatg cacatgctga aggccacgac catgatgata gggagagcca
     3481 ggaaaacgcg cagctttgat ccgccaccgc taccgccatc gcctccggtt ccttccagca
     3541 cccggagctt agatccaccg ccagagccgc cgtcgccacc ggtgcccact ctcagcttag
     3601 agccacctcc ggagccgcca tctcctccgg tgcctgtaat gctgttggtc atcacaggtc
     3661 catcagcagg aaatccggtg ccttccacct gggcttcgcc gatgatgtgg ctcccctcgt
     3721 atgtgtagcg gtagttgacg gtgaggctgg cgccgtcctc gaactgcatg gttctgtgca
     3781 cctggtagcc gctgccgtcc accatggcgg cctggaaggg gctcatgccg tcagggtaag
     3841 gcaggtactg gtggaagccg tagccgatgt gggggaccag gatccatgga gaaaattgca
     3901 ggtcgcccat ggtgcttttc agattcagtt cctcgtagcc gccattaggg ttgccagagc
     3961 cctgtcccac catgtcaaaa tcaaccccgt tgatgctgcc gaaaatgtgc agctcgtggg
     4021 tggctggcag agaagccatg ttgtcctcct cgcccttgga caccatgcct gtgccgccag
     4081 agccgcccac caccttataa agctcatcca tccccatcac atcggtcacg gccttctgcc
     4141 attccttgaa attcagctct gtcttgctgt gcttcagctc ggtctttctg aacacgtaca
     4201 taggctggtt cttcaggtaa ttagcggcca taggcttggc aaaggtgtag gttgttctca
     4261 ctgtgcaccg gtatcttttg ccgtccatgg tgaattccac cacactggac tagtggatcc
     4321 gagctcggta ccaagcttaa gtttaaacgc tagccagctt gggtctccct atagtgagtc
     4381 gtattaattt cgataagcca gtaagcagtg ggttctctag ttagccagag agctctgctt
     4441 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta
     4501 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
     4561 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
     4621 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
     4681 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     4741 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     4801 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     4861 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     4921 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     4981 gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc ccgtacatcg
     5041 cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaattgtc ggtcaagcct
     5101 tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa cacacaagca
     5161 gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5221 aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc
     5281 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5341 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5401 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5461 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5521 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5581 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5641 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5701 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5761 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5821 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5881 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5941 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6001 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6061 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6121 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6181 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6241 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6301 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6361 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6421 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6481 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6541 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6601 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6661 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6721 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6781 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6841 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6901 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6961 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7021 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     7081 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     7141 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     7201 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     7261 actgg
//
