LOCUS       sequence-259086-        6651 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 6651)
  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 6651)
  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..6651
                     /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(3675..3694)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3740..3795
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3794..3813)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4154..4170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4154..4170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4167..4189)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4178..4194
                     /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(4202..4208,4209..4226,4227..4232))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4247..4268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4327..4522
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4373..4508
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4528..4662
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4565..4584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4619..4638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4730..4747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4901..5489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4981..5000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5660..6451,6452..6520))
                     /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     6283..6302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6521..6625)
                     /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 aagaagaaga agaagaagag caagaccaag tgcgtgatca tgtaggctag cgaattcact
     3601 cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac
     3661 aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     3721 gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat
     3781 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat
     3841 gagtatttgg tttagagttt ggcaacatat gccatatgct ggctgccatg aacaaaggtg
     3901 gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag
     3961 aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt
     4021 aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact
     4081 actcccagtc atagctgtcc ctcttctctt atgaagatcc ctcgacctgc agcccaagct
     4141 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4201 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4261 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4321 ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4381 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4441 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4501 tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa
     4561 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4621 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg
     4681 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4741 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4801 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4861 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4921 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4981 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5041 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     5101 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5161 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5221 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5281 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5341 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5401 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5461 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5521 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5581 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5641 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5701 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5761 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5821 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5881 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5941 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6001 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6061 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6121 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6181 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6241 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6301 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6361 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6421 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6481 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6541 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6601 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
