LOCUS       sequence-304813-        6193 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 6193)
  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 6193)
  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..6193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1775..1784
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1784..1807
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2417..2426
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2423..3097
                     /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(2553..2571)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2821..2840
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3015..3038
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3215..3234)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3280..3335
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3334..3353)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3696..3712)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3696..3712)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3709..3731)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3720..3736
                     /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(3744..3750,3751..3768,3769..3774))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3789..3810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3869..4064
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3915..4050
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4070..4204
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4107..4126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4161..4180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4272..4289)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4443..5031)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4523..4542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5202..5993,5994..6062))
                     /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     5825..5844
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6063..6167)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggsgcgc gccrggsggg gggggggggg gsgrggggsg
      541 gggsggggsg rggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gkgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg rgggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tctgatatct
     1741 ttaattaaac cccgggaccg gtgctagctc cggagccacc atggactaca aagacgatga
     1801 cgacaagtcc ggaatcgata tgtcgatgac agacttgctc agcgctgagg acatcaagaa
     1861 ggcgatagga gcctttactg ctgcagactc cttcgaccac aaaaagttct tccagatggt
     1921 gggcctgaag aaaaagagtg cggatgatgt gaagaaggtg ttccacattc tggacaaaga
     1981 caaagatggc ttcattgacg aggatgagct ggggtccatt ctgaagggct tctcctcaga
     2041 tgccagagac ttgtctgcta aggaaacaaa gacgctgatg gctgctggag acaaggacgg
     2101 ggatggcaag attggggttg aagagttctc cactctggtg gccgaaagca tcgatcttaa
     2161 gatggctgat cagctgactg aagagcagat tgctgaattt aaggaggctt tctccctatt
     2221 cgataaagat ggtgacggca ccatcacaac caaggaactg gggaccgtca tgcggtcact
     2281 gggtcagaac ccaacagaag ccgagctgca ggatatgatc aacgaagtgg atgctgatgg
     2341 caatggcacc attgacttcc cagagttctt gactatgatg gctagaaaaa tgaaagacac
     2401 acttaaggcg gatcccgcca ccatggcctc ctccgaggac gtcatcaagg agttcatgcg
     2461 cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     2521 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
     2581 cctgcccttc gcctgggaca tcctgtcccc tcagttccag tacggctcca aggcctacgt
     2641 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     2701 ggagcgcgtg atgaacttcg aggatggcgg cgtggtgacc gtgacccagg actcctccct
     2761 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     2821 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgga tgtaccccga
     2881 ggacggcgcc ctgaagggcg agatcaagat gaggctgaag ctgaaggacg gcggccacta
     2941 cgacgccgag gtcaagacca cctacatggc caagaagccc gtgcagctgc ccggcgccta
     3001 caagaccgac atcaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     3061 gtacgagcgc gccgagggcc gcccctccac cggcgcctgt acaagtaaga attctgcaga
     3121 taaacgctag cgaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     3181 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg
     3241 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     3301 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     3361 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc
     3421 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg
     3481 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt
     3541 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca
     3601 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat
     3661 ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3721 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3781 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3841 tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc
     3901 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3961 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4021 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat
     4081 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4141 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4201 gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4261 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4321 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4381 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4441 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4501 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4561 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4621 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4681 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4741 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4801 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4861 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4921 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4981 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5041 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5101 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5161 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5221 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5281 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5341 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5401 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5461 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5521 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5581 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5641 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5701 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5761 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5821 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5881 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5941 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6001 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6061 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6121 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6181 aaagtgccac ctg
//
