LOCUS       sequence-273569-        6187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of SpiCee, a buffer of calcium that prevents 
            downstream signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6187)
  AUTHORS   Ros O, Baudet S, Zagar Y, Loulier K, Roche F, Couvet S, Aghaie A, 
            Atkins M, Louail A, Petit C, Metin C, Mechulam Y, Nicol X
  TITLE     SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium 
            Manipulation.
  JOURNAL   Cell Rep. 2020 Jul 21;32(3):107934. doi: 
            10.1016/j.celrep.2020.107934.
  PUBMED    32697983
REFERENCE   2  (bases 1 to 6187)
  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: "Cell Rep.";
            date: "2020-07-21"; volume: "32(3):107934. doi"; pages: " 
            10.1016/j.celrep.2020.107934"
COMMENT     Sequence Label: pCX-SpiCee
FEATURES             Location/Qualifiers
     source          1..6187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1771..1780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1780..1803
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2413..2422
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2419..3093
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRPS
                     TGA"
     primer_bind     complement(2549..2567)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2817..2836
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3011..3034
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3211..3230)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3276..3331
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3330..3349)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3690..3706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3690..3706)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3703..3725)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3714..3730
                     /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(3738..3744,3745..3762,3763..3768))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3783..3804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3863..4058
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3909..4044
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4064..4198
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4101..4120)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4155..4174
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4266..4283)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4437..5025)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4517..4536)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5196..5987,5988..6056))
                     /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     5819..5838
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6057..6161)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattctg atatctttaa
     1741 ttaaaccccg ggaccggtgc tagctccgga gccaccatgg actacaaaga cgatgacgac
     1801 aagtccggaa tcgatatgtc gatgacagac ttgctcagcg ctgaggacat caagaaggcg
     1861 ataggagcct ttactgctgc agactccttc gaccacaaaa agttcttcca gatggtgggc
     1921 ctgaagaaaa agagtgcgga tgatgtgaag aaggtgttcc acattctgga caaagacaaa
     1981 gatggcttca ttgacgagga tgagctgggg tccattctga agggcttctc ctcagatgcc
     2041 agagacttgt ctgctaagga aacaaagacg ctgatggctg ctggagacaa ggacggggat
     2101 ggcaagattg gggttgaaga gttctccact ctggtggccg aaagcatcga tcttaagatg
     2161 gctgatcagc tgactgaaga gcagattgct gaatttaagg aggctttctc cctattcgat
     2221 aaagatggtg acggcaccat cacaaccaag gaactgggga ccgtcatgcg gtcactgggt
     2281 cagaacccaa cagaagccga gctgcaggat atgatcaacg aagtggatgc tgatggcaat
     2341 ggcaccattg acttcccaga gttcttgact atgatggcta gaaaaatgaa agacacactt
     2401 aaggcggatc ccgccaccat ggcctcctcc gaggacgtca tcaaggagtt catgcgcttc
     2461 aaggtgcgca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     2521 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg
     2581 cccttcgcct gggacatcct gtcccctcag ttccagtacg gctccaaggc ctacgtgaag
     2641 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     2701 cgcgtgatga acttcgagga tggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     2761 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     2821 gtaatgcaga agaagaccat gggctgggag gcctccaccg agcggatgta ccccgaggac
     2881 ggcgccctga agggcgagat caagatgagg ctgaagctga aggacggcgg ccactacgac
     2941 gccgaggtca agaccaccta catggccaag aagcccgtgc agctgcccgg cgcctacaag
     3001 accgacatca agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     3061 gagcgcgccg agggccgccc ctccaccggc gcctgtacaa gtaagaattc tgcagataaa
     3121 cgctagcgaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     3181 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     3241 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     3301 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     3361 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgcca tatgctggct
     3421 gccatgaaca aaggtggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc
     3481 attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg
     3541 tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt
     3601 tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatga agatccctcg
     3661 acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3721 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3781 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3841 aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa
     3901 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3961 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     4021 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc
     4081 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4141 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg
     4201 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4261 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4321 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4381 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4441 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4501 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4561 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4621 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4681 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4741 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4801 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4861 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4921 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4981 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5041 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5101 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5161 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5221 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5281 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5341 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5401 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5461 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5521 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5581 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5641 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5701 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5761 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5821 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5881 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5941 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6001 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6061 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6121 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6181 ccacctg
//
