LOCUS       sequence-304820-        6601 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 6601)
  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 6601)
  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..6601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          112..1128
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1052..1074
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1136..1155
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2275..2284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2281..2955
                     /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(2411..2429)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2679..2698
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2873..2896
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3073..3092)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3138..3193
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3192..3211)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3554..3570)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3554..3570)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3567..3589)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3578..3594
                     /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(3602..3608,3609..3626,3627..3632))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3647..3668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3727..3922
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3773..3908
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3928..4062
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3965..3984)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4019..4038
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4130..4147)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4301..4889)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4381..4400)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5060..5851,5852..5920))
                     /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     5683..5702
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5921..6025)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6056..6435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(6437..6601,1..111)
                     /label=chicken beta-actin promoter
ORIGIN
        1 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
       61 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
      121 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
      181 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
      241 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
      301 gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
      361 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
      421 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
      481 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
      541 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc
      601 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
      661 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
      721 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
      781 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
      841 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
      901 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
      961 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     1021 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     1081 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     1141 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tctgatatct ttaattaaac
     1201 cccgggccac catacatggt gggccgcaac agcgcgattg cagctggagt gtgcggcgcg
     1261 ctgttcattg gctattgcat ttactttgat cgcaaacgcc gcagcgatcc gaactttaaa
     1321 tctagattgg ctactacact tgaacgtatt gagaagaact ttgtcattac tgacccaaga
     1381 ttgccagata atcccattat attcgcgtcc gatagtttct tgcagttgac agaatatagc
     1441 cgtgaagaaa ttttgggaag aaactgcagg tttctacaag gtcctgaaac tgatcgcgcg
     1501 acagtgagaa aaattagaga tgccatagat aaccaaacag aggtcactgt tcagctgatt
     1561 aattatacaa agagtggtaa aaagttctgg aacctctttc acttgcagcc tatgcgagat
     1621 cagaagggag atgtccagta ctttattggg gttcagttgg atggaactga gatcgatatg
     1681 tcgatgacag acttgctcag cgctgaggac atcaagaagg cgataggagc ctttactgct
     1741 gcagactcct tcgaccacaa aaagttcttc cagatggtgg gcctgaagaa aaagagtgcg
     1801 gatgatgtga agaaggtgtt ccacattctg gacaaagaca aagatggctt cattgacgag
     1861 gatgagctgg ggtccattct gaagggcttc tcctcagatg ccagagactt gtctgctaag
     1921 gaaacaaaga cgctgatggc tgctggagac aaggacgggg atggcaagat tggggttgaa
     1981 gagttctcca ctctggtggc cgaaagcatc gatcttaaga tggctgatca gctgactgaa
     2041 gagcagattg ctgaatttaa ggaggctttc tccctattcg ataaagatgg tgacggcacc
     2101 atcacaacca aggaactggg gaccgtcatg cggtcactgg gtcagaaccc aacagaagcc
     2161 gagctgcagg atatgatcaa cgaagtggat gctgatggca atggcaccat tgacttccca
     2221 gagttcttga ctatgatggc tagaaaaatg aaagacacac ttaaggcgga tcccgccacc
     2281 atggcctcct ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc
     2341 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     2401 acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc
     2461 ctgtcccctc agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     2521 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     2581 gatggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     2641 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     2701 atgggctggg aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag
     2761 atcaagatga ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc
     2821 tacatggcca agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac
     2881 atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc
     2941 ccctccaccg gcgcctgtac aagtaagaat tctgcagata aacgctagcg aattcactcc
     3001 tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa
     3061 ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     3121 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     3181 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     3241 gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg
     3301 gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag
     3361 aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt
     3421 aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact
     3481 actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct
     3541 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3601 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3661 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3721 ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3781 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3841 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3901 tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa
     3961 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4021 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg
     4081 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4141 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4201 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4261 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4321 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4381 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4441 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4501 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4561 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4621 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4681 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4741 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4801 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4861 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4921 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4981 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5041 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5101 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5161 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5221 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5281 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5341 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5401 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5461 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5521 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5581 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5641 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5701 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5761 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5821 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5881 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5941 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6001 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcgacat
     6061 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     6121 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     6181 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     6241 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     6301 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     6361 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     6421 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     6481 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     6541 gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
     6601 g
//
