LOCUS       sequence-347285-        7399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of G-alpha-q(L254A) fused to 
            mNeonGreen-eDHFR(69K6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7399)
  AUTHORS   Suzuki S, Nakamura A, Hatano Y, Yoshikawa M, Yoshii T, Sawada S, 
            Atsuta-Tsunoda K, Aoki K, Tsukiji S
  TITLE     A chemogenetic platform for controlling plasma membrane signaling 
            and synthetic signal oscillation.
  JOURNAL   Cell Chem Biol. 2022 Sep 15;29(9):1446-1464.e10. doi: 
            10.1016/j.chembiol.2022.06.005. Epub 2022 Jul 13.
  PUBMED    35835118
REFERENCE   2  (bases 1 to 7399)
  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.chembiol.2022.06.005"; journalName: "Cell Chem Biol"; 
            date: "2022-09-15- 15"; volume: "29"; issue: "9"; pages: 
            "1446-1464.e10"
COMMENT     Sequence Label: pCAGGS-mNG-eDHFR(69K6)-G-alpha-q(L254A)
FEATURES             Location/Qualifiers
     source          1..7399
                     /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..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1725..1734
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1731..2438
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1986..1995
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4264..4284)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4264..4282)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4303..4319)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4421..4440)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4486..4541
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4540..4559)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4902..4918)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4926..4942
                     /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(4950..4956,4957..4974,4975..4980))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4995..5016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5075..5270
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5121..5256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5276..5410
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5313..5332)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5367..5386
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5478..5495)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5649..6237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5729..5748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6408..7199,7200..7268))
                     /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     7031..7050
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7269..7373)
                     /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 ggcgagggcg
      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 ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attggccacc atggtgagca
     1741 agggcgagga ggataacatg gcctctctcc cagcgacaca tgagttacac atctttggct
     1801 ccatcaacgg tgtggacttt gacatggtgg gtcagggcac cggcaatcca aatgatggtt
     1861 atgaggagtt aaacctgaag tccaccaagg gtgacctcca gttctccccc tggattctgg
     1921 tccctcatat cgggtatggc ttccatcagt acctgcccta ccctgacggg atgtcgcctt
     1981 tccaggccgc catggtagat ggctccggct accaagtcca tcgcacaatg cagtttgaag
     2041 atggtgcctc ccttactgtt aactaccgct acacctacga gggaagccac atcaaaggag
     2101 aggcccaggt gaaggggact ggtttccctg ctgacggtcc tgtgatgacc aactcgctga
     2161 ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa cgacaaaacc atcatcagta
     2221 cctttaagtg gagttacacc actggaaatg gcaagcgcta ccggagcact gcgcggacca
     2281 cctacacctt tgccaagcca atggcggcta actatctgaa gaaccagccg atgtacgtgt
     2341 tccgtaagac ggagctcaag cactccaaga ccgagctcaa cttcaaggag tggcaaaagg
     2401 cctttaccga tgtgatgggc atggacgagc tctacaagtc tgggtccgca gccagtagcg
     2461 ccggaagtat gatcagtctg attgcggcgt tagcggtaga tcgcgttatc ggcatggaaa
     2521 acgccatgcc gtggaacctg cctgccgatc tcgcctggtt taaacgcaac accttaaata
     2581 aacccgtgat tatgggccgc catacctggg aatcaatcgg tcgtccgttg ccaggacgca
     2641 aaaatattat cctcagcagt caaccgggta cggacaaaaa aaagaaaaag aaagatcgcg
     2701 taacgtgggt gaagtcggtg gatgaagcca tcgcggcgtg tggtgacgta ccagaaatca
     2761 tggtgattgg cggcggtcgc gtttatgaac agttcttgcc aaaagcgcaa aaactgtatc
     2821 tgacgcatat cgacgcagaa gtggaaggcg acacccattt cccggattac gagccggatg
     2881 actgggaatc ggtattcagc gaattccacg atgctgatgc gcagaactct cacagctatt
     2941 gctttgagat tctggagcgg cggctgtact ctggtggcgg aggctcgggc ggaggtgggt
     3001 cgggtggcgg cggatcttca gctggagctt cgttgtacaa ggcaagagga gccgctgcgg
     3061 gtgccggagg cgctggtagg agcggcaaaa aggggaagaa agggaaaaag ggcaccacaa
     3121 tgactctgga gtccatcatg gcatcctccc tgagcgagga ggccaaggaa gcccggagga
     3181 tcaacgacga gatcgagcgg cagctgcgca gggacaagcg cgacgcccgc cgggagctca
     3241 agctgctgct gctggggaca ggggagagtg gcaagtcgac cttcatcaag cagatgagga
     3301 tcatccacgg gtcgggctac tctgacgaag acaagcgcgg cttcaccaag ctggtgtatc
     3361 agaacatctt cacggccatg caggccatga tcagagcgat ggacacactc aagatcccat
     3421 acaagtatga acacaataag gctcatgcac aattggttcg agaggttgat gtggagaagg
     3481 tgtctgcttt tgagaatcca tatgtagatg caataaagag cttgtggaat gatcctggaa
     3541 tccaggagtg ctacgacaga cgacgggaat atcagttatc tgactctacc aaatactatc
     3601 tgaatgactt ggaccgtgta gccgaccctt cctatctgcc tacacaacaa gacgtgctta
     3661 gagttcgagt ccccactaca gggatcatcg aatacccctt tgacttacaa agtgtcattt
     3721 tcagaatggt cgatgtaggg ggcctgaggt cagagagaag aaaatggata cactgctttg
     3781 aaaatgtcac ctccatcatg tttctagtag cgcttagcga atatgatcaa gttcttgtgg
     3841 agtcagacaa tgagaaccgc atggaggaga gcaaagcagc ctttagaaca attatcacct
     3901 acccctggtt ccagaactcc tctgtgattc tgttcttaaa caagaaagat cttctagagg
     3961 agaaaatcat gtattcccac ctagtcgact acttcccaga atatgatgga ccccagagag
     4021 atgcccaggc agctcgagaa ttcatcctga aaatgttcgt ggacctgaac cccgacagtg
     4081 acaaaatcat ctactcccac ttcacgtgcg ccacagatac cgagaacatc cgcttcgtct
     4141 ttgcagccgt caaggacacc atcctgcagc tgaacctgaa ggagtacaat ctggtctaac
     4201 agttgctcga gagtacccgg gatactcgcg gccgccaccg cggtggagct ccagcttttg
     4261 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     4321 gtgaaattgt tatccgctaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg
     4381 ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa
     4441 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     4501 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     4561 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc
     4621 atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc
     4681 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     4741 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     4801 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     4861 ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4921 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4981 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5041 ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag
     5101 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     5161 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     5221 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt
     5281 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5341 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5401 tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5461 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5521 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5581 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5641 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5701 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5761 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5821 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     5881 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5941 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6001 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6061 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     6121 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6181 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6241 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6301 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6361 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6421 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6481 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6541 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6601 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6661 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6721 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6781 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6841 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6901 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6961 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7021 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7081 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7141 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7201 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7261 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7321 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7381 ccccgaaaag tgccacctg
//
