LOCUS       sequence-325976-        5489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ShadowY-CBD and Clover(T153M/F223R)-Cdc42 in mammalian 
            cells to monitor Cdc42 activity with FLIM imaging..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5489)
  AUTHORS   Shibata ACE, Ueda HH, Eto K, Onda M, Sato A, Ohba T, Nabekura J, 
            Murakoshi H
  TITLE     Photoactivatable CaMKII induces synaptic plasticity in single 
            synapses.
  JOURNAL   Nat Commun. 2021 Feb 2;12(1):751. doi: 10.1038/s41467-021-21025-6.
  PUBMED    33531495
REFERENCE   2  (bases 1 to 5489)
  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"; journalName: "Nat 
            Commun."; date: "2021-02-2"; volume: "12(1):751. doi"; pages: " 
            10.1038/s41467-021-21025-6"
COMMENT     Sequence Label: CMV-ShadowY-CBD-P2A-Clover(T153M/F223R)-Cdc42
FEATURES             Location/Qualifiers
     source          1..5489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        112..415
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        416..619
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     570..590
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      658..667
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(970..989)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1555..1611
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1621..2337
                     /codon_start=1
                     /product="monomeric derivative of the bright green-yellow 
                     fluorescent protein Clover"
                     /label=mClover2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     ERVTAAGITLGMDELSK"
     CDS             2359..2934
                     /codon_start=1
                     /product="human Rho family small GTPase Cdc42"
                     /label=Cdc42
                     /translation="MQTIKCVVVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVM
                     IGGEPYTLGLFDTAGQEDYDRLRPLSYPQTDVFLVCFSVVSPSSFENVKEKWVPEITHH
                     CPKTPFLLVGTQIDLRDDPSTIEKLAKNKQKPITPETAEKLARDLKAVKYVECSALTQK
                     GLKNVFDEAILAALEPPEPKKSRRCVLL"
     polyA_signal    3056..3177
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3093..3112)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3147..3166
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3184..3639
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3271..3290)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3481..3502
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3695..3799
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3800..3868,3869..4660)
                     /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     complement(4018..4037)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4831..5419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5320..5339
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcccctgcgt tatcccctga ttctgtggat aaccgtatta ccaccatgca ttagttatta
       61 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      121 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      181 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      241 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      301 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      361 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      421 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      481 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      541 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      601 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc
      661 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
      721 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
      781 tacggcaagc tgaccctgaa gctcatctgc accaccggca agctgcccgt gccctggccc
      841 accctcgtga ccaccttcgg ctacggcctg atgtgcttcg cgcgctatcc agaccacatg
      901 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
      961 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1021 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1081 cacaagctcg agtacgcttg gcccgtggtg aatgtctata tcatggccga caagcagaag
     1141 aacggcatca aggtgaactt ctctatctat cataacatcg aggacggcag cgtgcagctc
     1201 gccgaccact accagcagaa cacccccatc ggcgacgggc ccgtgctgct gcccgacaac
     1261 cactacctga gctactcggc taagctgagc aaagacccca acgagaagcg cgatcacatg
     1321 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcagatctcg aggaatgaac
     1381 aaaaagaaag agaaagagcg gccagagatt tctctccctg cagattttga acacacaatt
     1441 catgtcggtg ctgatgctgt cacaggggag tttacgggaa taccagagca gtgggcccgc
     1501 ttgcttcaga catcaaatat cactaagttg gagcagaagt gtacaggaag cggagccact
     1561 aacttctccc tgttgaaaca agcaggggat gtcgaagaga atcccgggcc aggatccgga
     1621 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1681 ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac
     1741 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1801 ctcgtgacca ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag
     1861 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct
     1921 ttcaaggacg acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1981 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2041 aagctggagt acaacttcaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2101 ggcatcaagg ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc
     2161 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2221 tacctgagcc atcagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2281 ctgctggagc gcgtgaccgc cgccgggatc actctcggca tggacgagct gtctaagtcc
     2341 ggactcaggt ctcgagggat gcagacaatt aagtgtgttg ttgtgggcga tggtgctgtt
     2401 ggtaaaacat gtctcctgat atcctacaca acaaacaaat ttccatcgga atatgtaccg
     2461 actgtttttg acaactatgc agtcacagtt atgattggtg gagaaccata tactcttgga
     2521 ctttttgata ctgcagggca agaggattat gacagattac gaccgctgag ttatccacaa
     2581 acagatgtat ttctagtctg tttttcagtg gtctctccat cttcatttga aaacgtgaaa
     2641 gaaaagtggg tgcctgagat aactcaccac tgtccaaaga ctcctttctt gcttgttggg
     2701 actcaaattg atctcagaga tgacccctct actattgaga aacttgccaa gaacaaacag
     2761 aagcctatca ctccagagac tgctgaaaag ctggcccgtg acctgaaggc tgtcaagtat
     2821 gtggagtgtt ctgcacttac acagaaaggc ctaaagaatg tatttgacga agcaatattg
     2881 gctgccctgg agcctccaga accgaagaag agccgcaggt gtgtgctgct atgagcggcc
     2941 gcgacactag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     3001 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     3061 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3121 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg
     3181 cgtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3241 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3301 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3361 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3421 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3481 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3541 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3601 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctctagataa taatggtttc
     3661 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3721 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3781 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3841 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3901 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3961 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4021 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4081 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4141 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4201 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4261 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4321 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4381 cgaactactt actctagctt cccggcaaca actgatagac tggatggagg cggataaagt
     4441 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4501 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4561 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4621 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4681 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4741 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4801 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4861 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4921 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4981 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5041 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5101 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5161 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5221 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5281 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5341 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5401 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5461 ctggcctttt gctcacatgg ctcgacaga
//
