LOCUS       sequence-266270-        6657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of SponGee-Kras, a plasma membrane-targeted but
            lipid raft-excluded variant of the cGMP buffer SponGee that prevents
            downstream signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6657)
  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 6657)
  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-Kras
FEATURES             Location/Qualifiers
     source          1..6657
                     /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      1775..1784
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1784..1807
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2858..2867
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2864..3538
                     /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(2994..3012)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3262..3281
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3456..3479
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3679..3698)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3744..3799
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3798..3817)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4160..4176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4160..4176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4173..4195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4184..4200
                     /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(4208..4214,4215..4232,4233..4238))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4253..4274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4333..4528
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4379..4514
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4534..4668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4571..4590)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4625..4644
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4736..4753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4907..5495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4987..5006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5666..6457,6458..6526))
                     /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     6289..6308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6527..6631)
                     /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 gggggsgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg 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 gtgctagctc cggagccacc atggactaca aagacgatga
     1801 cgacaagtcc ggaatcgata tgagcgagct ggaggaagac tttgccaaga ttctcatgct
     1861 caaggaggag aggatcaaag agctggagaa gcggctgtca gagaaggagg aagaaatcca
     1921 ggagctgaag aggaaactcc ataaatgcca gtcagtgctg cccgtgccct cgacccacat
     1981 cggcccccgg accacccggg cacagggcat ctcggccgag ccgcagacct acaggtcctt
     2041 ccacgacctc cgagtgaccc tgcccttcta ccccaagagt ccacagtcca agatcgatct
     2101 cataaaggag gccatccttg acaatgactt tatgaagaac ttggagctgt cacagatcca
     2161 agagattgtg gattgtatgt acccagtgga gtacggcaaa gacagctgca tcatcaaaga
     2221 aggagatgtg gggtcactgg tgtatgtcat ggaagatggt aaggttgaag ttacaaaaga
     2281 aggcgtgaag ctgtgcacaa tgggtcctgg taaagtgttt ggagagttgg ctatccttta
     2341 caactgtacc cggacggcga ccgtcaaaac tcttgtaaat gtgaaactct gggccattga
     2401 tcgacaatgt tttcagacga taatgatgag gacaggactt atcaagcata ccgagtatat
     2461 ggaattttta aaaagcgttc caacattcca gagccttcct gaagagatcc tcagtaaact
     2521 tgctgacgtc cttgaagaga cccactatga aaatggggaa tatatcatca ggcaaggtgc
     2581 aagaggggac accttcttta tcatcagtaa aggaaaggtt aatgtcactc gtgaagactc
     2641 gcccaatgaa gacccagtct ttcttagaac cttaggaaaa ggagattggt ttggagagaa
     2701 agccttgcag ggggaagatg tgagaacagc gaatgtaatt gcggcagaag ctgtaacctg
     2761 ccttgtgatc gacagagact ctttcaaaca tttgattgga ggattagatg atgtttctaa
     2821 taaagcatat gaagatgcag aagctaaggc ggatcccgcc accatggcct cctccgagga
     2881 cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag ggctccgtga acggccacga
     2941 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     3001 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcca
     3061 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     3121 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggatggcg gcgtggtgac
     3181 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     3241 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     3301 caccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaaga tgaggctgaa
     3361 gctgaaggac ggcggccact acgacgccga ggtcaagacc acctacatgg ccaagaagcc
     3421 cgtgcagctg cccggcgcct acaagaccga catcaagctg gacatcacct cccacaacga
     3481 ggactacacc atcgtggaac agtacgagcg cgccgagggc cgcccctcca ccggcgcctg
     3541 tacaaagaag aagaagaaga agagcaagac caagtgcgtg atcatgtagg ctagcgaatt
     3601 cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc
     3661 tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
     3721 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
     3781 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
     3841 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa
     3901 aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt
     3961 ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt
     4021 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc
     4081 ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc
     4141 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4201 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4261 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4321 gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4381 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4441 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4501 cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt
     4561 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4621 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat
     4681 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4741 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4801 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4861 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4921 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4981 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5041 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5101 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5161 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5221 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5281 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5341 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5401 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5461 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5521 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5581 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5641 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5701 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5761 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5821 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5881 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5941 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6001 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6061 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6121 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6181 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6241 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6301 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6361 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6421 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6481 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6541 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6601 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
