LOCUS       sequence-304814-        6271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Lyn-SpiCee, a lipid raft-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 6271)
  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 6271)
  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-Lyn-SpiCee
FEATURES             Location/Qualifiers
     source          1..6271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     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      1769..1778
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1853..1862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1862..1885
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2495..2504
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2501..3175
                     /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(2631..2649)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2899..2918
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3093..3116
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3293..3312)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3358..3413
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3412..3431)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3774..3790)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3774..3790)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3787..3809)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3798..3814
                     /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(3822..3828,3829..3846,3847..3852))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3867..3888
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3947..4142
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3993..4128
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4148..4282
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4185..4204)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4239..4258
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4350..4367)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4521..5109)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4601..4620)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5280..6071,6072..6140))
                     /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     5903..5922
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6141..6245)
                     /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 gggggggcgc gccrgggggg gggggggggg gsgggggggg
      541 gggsggggsg aggcggagag 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 gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg 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 gtgctagcgc caccatgggc tgcatcaaga gcaagcgcaa
     1801 ggacaagatg ggctgcatca agagcaagcg caaggacaag gctagctccg gagccaccat
     1861 ggactacaaa gacgatgacg acaagtccgg aatcgatatg tcgatgacag acttgctcag
     1921 cgctgaggac atcaagaagg cgataggagc ctttactgct gcagactcct tcgaccacaa
     1981 aaagttcttc cagatggtgg gcctgaagaa aaagagtgcg gatgatgtga agaaggtgtt
     2041 ccacattctg gacaaagaca aagatggctt cattgacgag gatgagctgg ggtccattct
     2101 gaagggcttc tcctcagatg ccagagactt gtctgctaag gaaacaaaga cgctgatggc
     2161 tgctggagac aaggacgggg atggcaagat tggggttgaa gagttctcca ctctggtggc
     2221 cgaaagcatc gatcttaaga tggctgatca gctgactgaa gagcagattg ctgaatttaa
     2281 ggaggctttc tccctattcg ataaagatgg tgacggcacc atcacaacca aggaactggg
     2341 gaccgtcatg cggtcactgg gtcagaaccc aacagaagcc gagctgcagg atatgatcaa
     2401 cgaagtggat gctgatggca atggcaccat tgacttccca gagttcttga ctatgatggc
     2461 tagaaaaatg aaagacacac ttaaggcgga tcccgccacc atggcctcct ccgaggacgt
     2521 catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg gccacgagtt
     2581 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     2641 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc agttccagta
     2701 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     2761 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gatggcggcg tggtgaccgt
     2821 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     2881 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac
     2941 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga ggctgaagct
     3001 gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca agaagcccgt
     3061 gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc acaacgagga
     3121 ctacaccatc gtggaacagt acgagcgcgc cgagggccgc ccctccaccg gcgcctgtac
     3181 aagtaagaat tctgcagata aacgctagcg aattcactcc tcaggtgcag gctgcctatc
     3241 agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc
     3301 cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact tctggctaat
     3361 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     3421 ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg
     3481 caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt
     3541 atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt
     3601 agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct
     3661 tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc
     3721 ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc atggtcatag
     3781 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3841 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3901 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat ctcaattagt
     3961 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4021 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4081 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4141 aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4201 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4261 tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc
     4321 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4381 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4441 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4501 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4561 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4621 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4681 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4741 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4801 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4861 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4921 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4981 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5041 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5101 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5161 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5221 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5281 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5341 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5401 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5461 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5521 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5581 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5641 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5701 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5761 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5821 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5881 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5941 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6001 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6061 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6121 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6181 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6241 ccgcgcacat ttccccgaaa agtgccacct g
//
