LOCUS       sequence-273564-        6595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of NTOM-SpiCee, a mitochondria-targeted variant
            of the calcium buffer SpiCee that prevents downstream signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6595)
  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 6595)
  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-NTOM-SpiCee
FEATURES             Location/Qualifiers
     source          1..6595
                     /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      2821..2830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2827..3501
                     /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(2957..2975)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3225..3244
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3419..3442
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3619..3638)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3684..3739
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3738..3757)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4098..4114)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4098..4114)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4111..4133)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4122..4138
                     /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(4146..4152,4153..4170,4171..4176))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4191..4212
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4271..4466
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4317..4452
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4472..4606
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4509..4528)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4563..4582
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4674..4691)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4845..5433)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4925..4944)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5604..6395,6396..6464))
                     /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     6227..6246
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6465..6569)
                     /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 ggccaccata catggtgggc cgcaacagcg cgattgcagc tggagtgtgc
     1801 ggcgcgctgt tcattggcta ttgcatttac tttgatcgca aacgccgcag cgatccgaac
     1861 tttaaatcta gattggctac tacacttgaa cgtattgaga agaactttgt cattactgac
     1921 ccaagattgc cagataatcc cattatattc gcgtccgata gtttcttgca gttgacagaa
     1981 tatagccgtg aagaaatttt gggaagaaac tgcaggtttc tacaaggtcc tgaaactgat
     2041 cgcgcgacag tgagaaaaat tagagatgcc atagataacc aaacagaggt cactgttcag
     2101 ctgattaatt atacaaagag tggtaaaaag ttctggaacc tctttcactt gcagcctatg
     2161 cgagatcaga agggagatgt ccagtacttt attggggttc agttggatgg aactgagatc
     2221 gatatgtcga tgacagactt gctcagcgct gaggacatca agaaggcgat aggagccttt
     2281 actgctgcag actccttcga ccacaaaaag ttcttccaga tggtgggcct gaagaaaaag
     2341 agtgcggatg atgtgaagaa ggtgttccac attctggaca aagacaaaga tggcttcatt
     2401 gacgaggatg agctggggtc cattctgaag ggcttctcct cagatgccag agacttgtct
     2461 gctaaggaaa caaagacgct gatggctgct ggagacaagg acggggatgg caagattggg
     2521 gttgaagagt tctccactct ggtggccgaa agcatcgatc ttaagatggc tgatcagctg
     2581 actgaagagc agattgctga atttaaggag gctttctccc tattcgataa agatggtgac
     2641 ggcaccatca caaccaagga actggggacc gtcatgcggt cactgggtca gaacccaaca
     2701 gaagccgagc tgcaggatat gatcaacgaa gtggatgctg atggcaatgg caccattgac
     2761 ttcccagagt tcttgactat gatggctaga aaaatgaaag acacacttaa ggcggatccc
     2821 gccaccatgg cctcctccga ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg
     2881 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     2941 gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg
     3001 gacatcctgt cccctcagtt ccagtacggc tccaaggcct acgtgaagca ccccgccgac
     3061 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     3121 ttcgaggatg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     3181 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     3241 aagaccatgg gctgggaggc ctccaccgag cggatgtacc ccgaggacgg cgccctgaag
     3301 ggcgagatca agatgaggct gaagctgaag gacggcggcc actacgacgc cgaggtcaag
     3361 accacctaca tggccaagaa gcccgtgcag ctgcccggcg cctacaagac cgacatcaag
     3421 ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga gcgcgccgag
     3481 ggccgcccct ccaccggcgc ctgtacaagt aagaattctg cagataaacg ctagcgaatt
     3541 cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc
     3601 tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
     3661 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
     3721 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
     3781 gaatgagtat ttggtttaga gtttggcaac atatgccata tgctggctgc catgaacaaa
     3841 ggtggctata aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc
     3901 atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt
     3961 ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct
     4021 gactactccc agtcatagct gtccctcttc tcttatgaag atccctcgac ctgcagccca
     4081 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4141 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4201 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4261 cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4321 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4381 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4441 tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta
     4501 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4561 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga
     4621 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4681 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4741 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4801 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4861 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4921 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4981 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     5041 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5101 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5161 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5221 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5281 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5341 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5401 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5461 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5521 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5581 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5641 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5701 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5761 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5821 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5881 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5941 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6001 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6061 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6121 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6181 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6241 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6301 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6361 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6421 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6481 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6541 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctg
//
