LOCUS       sequence-308263-        6154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of a mutated form of SpiCee, that does not 
            affect calcium signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6154)
  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 6154)
  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-mutSpiCee
FEATURES             Location/Qualifiers
     source          1..6154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          161..1177
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1101..1123
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1185..1204
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1284..1293
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1293..1316
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1926..1935
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1932..2606
                     /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(2062..2080)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2330..2349
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2524..2547
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2675..2694)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2740..2795
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2794..2813)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3156..3172)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3156..3172)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3169..3191)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3180..3196
                     /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(3204..3210,3211..3228,3229..3234))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3249..3270
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3329..3524
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3375..3510
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3530..3664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3567..3586)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3621..3640
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3732..3749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3903..4491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3983..4002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4662..5453,5454..5522))
                     /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     5285..5304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5523..5627)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5658..6037
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(6039..6154,1..160)
                     /label=chicken beta-actin promoter
ORIGIN
        1 ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg
       61 tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg
      121 gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct
      181 tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc
      241 gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt
      301 ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga
      361 gggccctttg tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag
      421 cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct
      481 ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg
      541 ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg
      601 gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc gagttgctga
      661 gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc
      721 gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga
      781 gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag
      841 ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc ctttgtccca
      901 aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc
      961 gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg
     1021 ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg gctgccttcg
     1081 ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc
     1141 tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat
     1201 tgtgctgtct catcattttg gcaaagaatt ctgatatctt taattaagaa ttcgctagcg
     1261 gtaccaccgg tgctagctcc ggagccacca tggactacaa agacgatgac gacaagtccg
     1321 gaatcgatat gtcgatgaca gacttgctca gcgctgagga catcaagaag gcgataggag
     1381 cctttactgc tgcagactcc ttcgaccaca aaaagttctt ccagatggtg ggcctgaaga
     1441 aaaagagtgc ggatgatgtg aagaaggtgt tccacattct ggccaaagac aaagatggct
     1501 tcattgacga ggatcagctg gggtccattc tgaagggctt ctcctcagat gccagagact
     1561 tgtctgctaa ggaaacaaag acgctgatgg ctgctggagc gaaggacggg gatggcaaga
     1621 ttggggttga acagttctcc actctggtgg ccgaaagcat cgatcttaag atggctgatc
     1681 agctgactga agagcagatt gctgaattta aggaggcttt ctccctattc gctaaagatg
     1741 gtgacggcac catcacaacc aagcaactgg ggaccgtcat gcggtcactg ggtcagaacc
     1801 caacagaagc cgagctgcag gatatgatca acgaagtggc tgctgatggc aatggcacca
     1861 ttgacttccc acagttcttg actatgatgg ctagaaaaat gaaagacaca cttaaggcgg
     1921 atcccgccac catggcctcc tccgaggacg tcatcaagga gttcatgcgc ttcaaggtgc
     1981 gcatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     2041 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
     2101 cctgggacat cctgtcccct cagttccagt acggctccaa ggcctacgtg aagcaccccg
     2161 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     2221 tgaacttcga ggatggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     2281 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     2341 agaagaagac catgggctgg gaggcctcca ccgagcggat gtaccccgag gacggcgccc
     2401 tgaagggcga gatcaagatg aggctgaagc tgaaggacgg cggccactac gacgccgagg
     2461 tcaagaccac ctacatggcc aagaagcccg tgcagctgcc cggcgcctac aagaccgaca
     2521 tcaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgcg
     2581 ccgagggccg cccctccacc ggcgcctgta caagtaagaa ttctgcagat atccatcaca
     2641 ctggcggccg ccctgcagga ctagttctag aagatctttt tccctctgcc aaaaattatg
     2701 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     2761 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     2821 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc
     2881 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg
     2941 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt
     3001 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca
     3061 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat
     3121 ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3181 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3241 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3301 tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc
     3361 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3421 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     3481 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat
     3541 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3601 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3661 gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3721 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3781 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3841 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3901 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3961 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4021 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4081 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4141 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4201 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4261 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4321 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4381 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4441 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4501 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4561 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4621 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4681 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4741 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4801 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4861 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4921 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4981 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5041 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5101 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5161 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5221 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5281 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5341 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5401 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5461 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5521 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5581 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5641 aaagtgccac ctgggtcgac attgattatt gactagttat taatagtaat caattacggg
     5701 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     5761 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     5821 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     5881 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     5941 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     6001 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
     6061 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
     6121 attattttgt gcagcgatgg gggcgggggg gggg
//
