LOCUS       sequence-259083-        6628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of SponGee, a buffer of cGMP that prevents 
            downstream signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6628)
  AUTHORS   Ros O, Zagar Y, Ribes S, Baudet S, Loulier K, Couvet S, Ladarre D, 
            Aghaie A, Louail A, Petit C, Mechulam Y, Lenkei Z, Nicol X
  TITLE     SponGee: A Genetic Tool for Subcellular and Cell-Specific cGMP 
            Manipulation.
  JOURNAL   Cell Rep. 2019 Jun 25;27(13):4003-4012.e6. doi: 
            10.1016/j.celrep.2019.05.102.
  PUBMED    31242429
REFERENCE   2  (bases 1 to 6628)
  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"; doi: 
            "10.1016/j.celrep.2019.05"; journalName: "Cell Rep"; date: 
            "2019-06-25- 25"; volume: "27"; issue: "13"; pages: "4003-4012.e6"
COMMENT     Sequence Label: pCX-SponGee
FEATURES             Location/Qualifiers
     source          1..6628
                     /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      1771..1780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1780..1803
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2854..2863
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2860..3534
                     /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(2990..3008)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3258..3277
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3452..3475
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3652..3671)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3717..3772
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3771..3790)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4131..4147)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4131..4147)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4144..4166)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4155..4171
                     /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(4179..4185,4186..4203,4204..4209))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4224..4245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4304..4499
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4350..4485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4505..4639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4542..4561)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4596..4615
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4707..4724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4878..5466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4958..4977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5637..6428,6429..6497))
                     /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     6260..6279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6498..6602)
                     /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 ggaccggtgc tagctccgga gccaccatgg actacaaaga cgatgacgac
     1801 aagtccggaa tcgatatgag cgagctggag gaagactttg ccaagattct catgctcaag
     1861 gaggagagga tcaaagagct ggagaagcgg ctgtcagaga aggaggaaga aatccaggag
     1921 ctgaagagga aactccataa atgccagtca gtgctgcccg tgccctcgac ccacatcggc
     1981 ccccggacca cccgggcaca gggcatctcg gccgagccgc agacctacag gtccttccac
     2041 gacctccgag tgaccctgcc cttctacccc aagagtccac agtccaagat cgatctcata
     2101 aaggaggcca tccttgacaa tgactttatg aagaacttgg agctgtcaca gatccaagag
     2161 attgtggatt gtatgtaccc agtggagtac ggcaaagaca gctgcatcat caaagaagga
     2221 gatgtggggt cactggtgta tgtcatggaa gatggtaagg ttgaagttac aaaagaaggc
     2281 gtgaagctgt gcacaatggg tcctggtaaa gtgtttggag agttggctat cctttacaac
     2341 tgtacccgga cggcgaccgt caaaactctt gtaaatgtga aactctgggc cattgatcga
     2401 caatgttttc agacgataat gatgaggaca ggacttatca agcataccga gtatatggaa
     2461 tttttaaaaa gcgttccaac attccagagc cttcctgaag agatcctcag taaacttgct
     2521 gacgtccttg aagagaccca ctatgaaaat ggggaatata tcatcaggca aggtgcaaga
     2581 ggggacacct tctttatcat cagtaaagga aaggttaatg tcactcgtga agactcgccc
     2641 aatgaagacc cagtctttct tagaacctta ggaaaaggag attggtttgg agagaaagcc
     2701 ttgcaggggg aagatgtgag aacagcgaat gtaattgcgg cagaagctgt aacctgcctt
     2761 gtgatcgaca gagactcttt caaacatttg attggaggat tagatgatgt ttctaataaa
     2821 gcatatgaag atgcagaagc taaggcggat cccgccacca tggcctcctc cgaggacgtc
     2881 atcaaggagt tcatgcgctt caaggtgcgc atggagggct ccgtgaacgg ccacgagttc
     2941 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     3001 gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccctca gttccagtac
     3061 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     3121 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg atggcggcgt ggtgaccgtg
     3181 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     3241 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctccacc
     3301 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagatgag gctgaagctg
     3361 aaggacggcg gccactacga cgccgaggtc aagaccacct acatggccaa gaagcccgtg
     3421 cagctgcccg gcgcctacaa gaccgacatc aagctggaca tcacctccca caacgaggac
     3481 tacaccatcg tggaacagta cgagcgcgcc gagggccgcc cctccaccgg cgcctgtaca
     3541 agtaagaatt ctgcagataa acgctagcga attcactcct caggtgcagg ctgcctatca
     3601 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     3661 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     3721 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     3781 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     3841 aacatatgcc atatgctggc tgccatgaac aaaggtggct ataaagaggt catcagtata
     3901 tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga
     3961 ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac
     4021 atgttttact agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc
     4081 ttctcttatg aagatccctc gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg
     4141 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4201 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4261 ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc aattagtcag
     4321 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4381 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4441 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     4501 agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4561 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4621 atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4681 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4741 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4801 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4861 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4921 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4981 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5041 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     5101 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5161 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5221 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5281 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5341 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5401 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5461 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5521 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5581 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5641 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5701 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5761 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5821 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5881 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5941 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6001 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6061 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6121 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6181 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6241 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6301 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6361 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6421 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6481 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6541 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6601 cgcacatttc cccgaaaagt gccacctg
//
