LOCUS       sequence-304816-        6216 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of SpiCee-Kras, a plasma membrane-targeted but 
            lipid raft-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 6216)
  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 6216)
  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-Kras
FEATURES             Location/Qualifiers
     source          1..6216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          162..1178
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1102..1124
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1186..1205
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1275..1284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1284..1307
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1917..1926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1923..2597
                     /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(2053..2071)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2321..2340
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2515..2538
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2738..2757)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2803..2858
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2857..2876)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3219..3235)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3219..3235)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3232..3254)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3243..3259
                     /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(3267..3273,3274..3291,3292..3297))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3312..3333
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3392..3587
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3438..3573
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3593..3727
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3630..3649)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3684..3703
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3795..3812)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3966..4554)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4046..4065)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4725..5516,5517..5585))
                     /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     5348..5367
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5586..5690)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5721..6100
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggggggcgc gccggggggg gggggggggg gggggggggg ggggggggsg rggsggrgag
       61 gkgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      121 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      181 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      241 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      301 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      361 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      421 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      481 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
      541 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
      601 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
      661 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
      721 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
      781 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
      841 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
      901 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
      961 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1021 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1081 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1141 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1201 ttgtgctgtc tcatcatttt ggcaaagaat tctgatatct ttaattaaac cccgggaccg
     1261 gtgctagctc cggagccacc atggactaca aagacgatga cgacaagtcc ggaatcgata
     1321 tgtcgatgac agacttgctc agcgctgagg acatcaagaa ggcgatagga gcctttactg
     1381 ctgcagactc cttcgaccac aaaaagttct tccagatggt gggcctgaag aaaaagagtg
     1441 cggatgatgt gaagaaggtg ttccacattc tggacaaaga caaagatggc ttcattgacg
     1501 aggatgagct ggggtccatt ctgaagggct tctcctcaga tgccagagac ttgtctgcta
     1561 aggaaacaaa gacgctgatg gctgctggag acaaggacgg ggatggcaag attggggttg
     1621 aagagttctc cactctggtg gccgaaagca tcgatcttaa gatggctgat cagctgactg
     1681 aagagcagat tgctgaattt aaggaggctt tctccctatt cgataaagat ggtgacggca
     1741 ccatcacaac caaggaactg gggaccgtca tgcggtcact gggtcagaac ccaacagaag
     1801 ccgagctgca ggatatgatc aacgaagtgg atgctgatgg caatggcacc attgacttcc
     1861 cagagttctt gactatgatg gctagaaaaa tgaaagacac acttaaggcg gatcccgcca
     1921 ccatggcctc ctccgaggac gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg
     1981 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     2041 gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca
     2101 tcctgtcccc tcagttccag tacggctcca aggcctacgt gaagcacccc gccgacatcc
     2161 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     2221 aggatggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     2281 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     2341 ccatgggctg ggaggcctcc accgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     2401 agatcaagat gaggctgaag ctgaaggacg gcggccacta cgacgccgag gtcaagacca
     2461 cctacatggc caagaagccc gtgcagctgc ccggcgccta caagaccgac atcaagctgg
     2521 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc gccgagggcc
     2581 gcccctccac cggcgcctgt acaaagaaga agaagaagaa gagcaagacc aagtgcgtga
     2641 tcatgtaggc tagcgaattc actcctcagg tgcaggctgc ctatcagaag gtggtggctg
     2701 gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt
     2761 atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt
     2821 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     2881 caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata
     2941 tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc
     3001 ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat
     3061 tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag
     3121 ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga
     3181 gatccctcga cctgcagccc aagcttggcg taatcatggt catagctgtt tcctgtgtga
     3241 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3301 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3361 cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca accatagtcc
     3421 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3481 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3541 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt
     3601 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     3661 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     3721 ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3781 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3841 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3901 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3961 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4021 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4081 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4141 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4201 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4261 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4321 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4381 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4441 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4501 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4561 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4621 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4681 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4741 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4801 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4861 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4921 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4981 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5041 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5101 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5161 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5221 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5281 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5341 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5401 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5461 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5521 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5581 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5641 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5701 cgaaaagtgc cacctgggtc gacattgatt attgactagt tattaatagt aatcaattac
     5761 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     5821 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     5881 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     5941 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     6001 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     6061 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt
     6121 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt
     6181 ttaattattt tgtgcagcga tgggggcggg gggggg
//
