LOCUS       sequence-304815-        6137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of SpiCee-NES, a nucleus-excluded 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 6137)
  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 6137)
  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-NES
FEATURES             Location/Qualifiers
     source          1..6137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          116..1132
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1056..1078
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1140..1159
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1229..1238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1238..1261
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1871..1880
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1877..2551
                     /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(2007..2025)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2275..2294
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2469..2492
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2663..2682)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2728..2783
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2782..2801)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3144..3160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3144..3160)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3157..3179)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3168..3184
                     /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(3192..3198,3199..3216,3217..3222))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3237..3258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3317..3512
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3363..3498
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3518..3652
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3555..3574)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3609..3628
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3720..3737)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3891..4479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3971..3990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4650..5441,5442..5510))
                     /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     5273..5292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5511..5615)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5646..6025
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
       61 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
      121 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
      181 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
      241 ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct
      301 tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg
      361 tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg
      421 gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg
      481 tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt
      541 gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc
      601 tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc
      661 ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc
      721 gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg
      781 tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg
      841 acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc
      901 tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct
      961 tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg
     1021 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     1081 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     1141 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattctgat atctttaatt
     1201 aaaccccggg accggtgcta gctccggagc caccatggac tacaaagacg atgacgacaa
     1261 gtccggaatc gatatgtcga tgacagactt gctcagcgct gaggacatca agaaggcgat
     1321 aggagccttt actgctgcag actccttcga ccacaaaaag ttcttccaga tggtgggcct
     1381 gaagaaaaag agtgcggatg atgtgaagaa ggtgttccac attctggaca aagacaaaga
     1441 tggcttcatt gacgaggatg agctggggtc cattctgaag ggcttctcct cagatgccag
     1501 agacttgtct gctaaggaaa caaagacgct gatggctgct ggagacaagg acggggatgg
     1561 caagattggg gttgaagagt tctccactct ggtggccgaa agcatcgatc ttaagatggc
     1621 tgatcagctg actgaagagc agattgctga atttaaggag gctttctccc tattcgataa
     1681 agatggtgac ggcaccatca caaccaagga actggggacc gtcatgcggt cactgggtca
     1741 gaacccaaca gaagccgagc tgcaggatat gatcaacgaa gtggatgctg atggcaatgg
     1801 caccattgac ttcccagagt tcttgactat gatggctaga aaaatgaaag acacacttaa
     1861 ggcggatccc gccaccatgg cctcctccga ggacgtcatc aaggagttca tgcgcttcaa
     1921 ggtgcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     1981 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc
     2041 cttcgcctgg gacatcctgt cccctcagtt ccagtacggc tccaaggcct acgtgaagca
     2101 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     2161 cgtgatgaac ttcgaggatg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     2221 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     2281 aatgcagaag aagaccatgg gctgggaggc ctccaccgag cggatgtacc ccgaggacgg
     2341 cgccctgaag ggcgagatca agatgaggct gaagctgaag gacggcggcc actacgacgc
     2401 cgaggtcaag accacctaca tggccaagaa gcccgtgcag ctgcccggcg cctacaagac
     2461 cgacatcaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     2521 gcgcgccgag ggccgcccct ccaccggcgc ctgtacactt cagctattcg ttcttcggct
     2581 cgcttaggcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     2641 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     2701 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     2761 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     2821 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     2881 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     2941 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     3001 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     3061 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
     3121 agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3181 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3241 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3301 tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3361 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3421 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3481 gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa
     3541 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3601 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat
     3661 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3721 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3781 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3841 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3901 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3961 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4021 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4081 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4141 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4201 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4261 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4321 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4381 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4441 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4501 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4561 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4621 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4681 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4741 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4801 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4861 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4921 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4981 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5041 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5101 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5161 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5221 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5281 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5341 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5401 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5461 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5521 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5581 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5641 gggtcgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     5701 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     5761 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     5821 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     5881 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     5941 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     6001 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
     6061 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
     6121 agcgatgggg gcggggg
//
