LOCUS       sequence-266288-        6633 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 6633)
  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 6633)
  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..6633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..659
                     /label=chicken beta-actin promoter
     intron          660..1676
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1600..1622
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1684..1703
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1773..1782
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1782..1805
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2856..2865
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2862..3536
                     /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(2992..3010)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3260..3279
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3454..3477
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3654..3673)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3719..3774
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3773..3792)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4135..4151)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4135..4151)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4148..4170)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4159..4175
                     /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(4183..4189,4190..4207,4208..4213))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4228..4249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4308..4503
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4354..4489
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4509..4643
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4546..4565)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4600..4619
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4711..4728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4882..5470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4962..4981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5641..6432,6433..6501))
                     /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     6264..6283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6502..6606)
                     /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 tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg
      541 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
      601 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
      661 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
      721 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
      781 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
      841 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
      961 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
     1021 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1081 gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc
     1141 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg
     1201 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
     1261 ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg
     1321 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     1381 agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc
     1441 cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg
     1501 gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc
     1561 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     1621 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     1681 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc tgatatcttt
     1741 aattaaaccc cgggaccggt gctagctccg gagccaccat ggactacaaa gacgatgacg
     1801 acaagtccgg aatcgatatg agcgagctgg aggaagactt tgccaagatt ctcatgctca
     1861 aggaggagag gatcaaagag ctggagaagc ggctgtcaga gaaggaggaa gaaatccagg
     1921 agctgaagag gaaactccat aaatgccagt cagtgctgcc cgtgccctcg acccacatcg
     1981 gcccccggac cacccgggca cagggcatct cggccgagcc gcagacctac aggtccttcc
     2041 acgacctccg agtgaccctg cccttctacc ccaagagtcc acagtccaag atcgatctca
     2101 taaaggaggc catccttgac aatgacttta tgaagaactt ggagctgtca cagatccaag
     2161 agattgtgga ttgtatgtac ccagtggagt acggcaaaga cagctgcatc atcaaagaag
     2221 gagatgtggg gtcactggtg tatgtcatgg aagatggtaa ggttgaagtt acaaaagaag
     2281 gcgtgaagct gtgcacaatg ggtcctggta aagtgtttgg agagttggct atcctttaca
     2341 actgtacccg gacggcgacc gtcaaaactc ttgtaaatgt gaaactctgg gccattgatc
     2401 gacaatgttt tcagacgata atgatgagga caggacttat caagcatacc gagtatatgg
     2461 aatttttaaa aagcgttcca acattccaga gccttcctga agagatcctc agtaaacttg
     2521 ctgacgtcct tgaagagacc cactatgaaa atggggaata tatcatcagg caaggtgcaa
     2581 gaggggacac cttctttatc atcagtaaag gaaaggttaa tgtcactcgt gaagactcgc
     2641 ccaatgaaga cccagtcttt cttagaacct taggaaaagg agattggttt ggagagaaag
     2701 ccttgcaggg ggaagatgtg agaacagcga atgtaattgc ggcagaagct gtaacctgcc
     2761 ttgtgatcga cagagactct ttcaaacatt tgattggagg attagatgat gtttctaata
     2821 aagcatatga agatgcagaa gctaaggcgg atcccgccac catggcctcc tccgaggacg
     2881 tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac ggccacgagt
     2941 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     3001 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttccagt
     3061 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     3121 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggatggcggc gtggtgaccg
     3181 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     3241 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     3301 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagatg aggctgaagc
     3361 tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacatggcc aagaagcccg
     3421 tgcagctgcc cggcgcctac aagaccgaca tcaagctgga catcacctcc cacaacgagg
     3481 actacaccat cgtggaacag tacgagcgcg ccgagggccg cccctccacc ggcgcctgta
     3541 caagtaagaa ttctgcagat aaacgctagc gaattcactc ctcaggtgca ggctgcctat
     3601 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt
     3661 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa
     3721 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga
     3781 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg
     3841 gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag
     3901 tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt
     3961 tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc
     4021 ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc
     4081 cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat catggtcata
     4141 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4201 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4261 ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag
     4321 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4381 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4441 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4501 aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4561 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4621 atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg
     4681 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4741 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4801 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4861 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4921 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4981 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5041 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5101 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5161 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5221 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5281 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5341 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5401 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5461 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5521 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5581 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5641 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5701 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5761 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5821 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5881 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5941 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6001 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6061 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6121 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6181 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6241 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6301 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6361 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6421 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6481 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6541 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6601 tccgcgcaca tttccccgaa aagtgccacc tgg
//
