LOCUS       sequence-325982-        5576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ShadowY-Rhotekin(8-89) and Clover(T153M/F223R)-RhoA 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 5576)
  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 5576)
  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-Rhotekin(8-89)-P2A-Clover(T153M/F223R)-RhoA
FEATURES             Location/Qualifiers
     source          1..5576
                     /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             1636..1692
                     /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             1702..2418
                     /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             2440..3021
                     /codon_start=1
                     /product="human Rho family small GTPase RhoA"
                     /label=RhoA
                     /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     polyA_signal    3143..3264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3180..3199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3234..3253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3271..3726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3358..3377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3568..3589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3782..3886
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3887..3955,3956..4747)
                     /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(4105..4124)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4918..5506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5407..5426
                     /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 gcagatctat gggactggag
     1381 gacctcaata tgctctacat ccggcagatg gcactcagcc tggaggacac agagctgcag
     1441 aggaaactag atcatgagat ccggatgagg gatggggcct gcaagctgct ggcagcctgc
     1501 tcccagcgag agcaggctct ggaagccacc aagagcctgc tggtgtgcaa cagccgtatt
     1561 ctcagctaca tgggtgagct gcagcggcga aaggaggccc aggtgctgga gaagacaggc
     1621 tgtacaggaa gcggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag
     1681 aatcccgggc caggatccgg aatggtgagc aagggcgagg agctgttcac cggggtggtg
     1741 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt ccgcggcgag
     1801 ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac caccggcaag
     1861 ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcgtggc ctgcttcagc
     1921 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1981 gtccaggagc gcaccatctc tttcaaggac gacggtacct acaagacccg cgccgaggtg
     2041 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2101 gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa cgtctatatc
     2161 atggccgaca agcagaagaa cggcatcaag gctaacttca agatccgcca caacgttgag
     2221 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     2281 gtgctgctgc ccgacaacca ctacctgagc catcagtccg ccctgagcaa agaccccaac
     2341 gagaagcgcg atcacatggt cctgctggag cgcgtgaccg ccgccgggat cactctcggc
     2401 atggacgagc tgtctaagtc cggactcagg tctcgaggga tggctgccat ccggaagaaa
     2461 ctggtgattg ttggtgatgg agcctgtgga aagacatgct tgctcatagt cttcagcaag
     2521 gaccagttcc cagaggtgta tgtgcccaca gtgtttgaga actatgtggc agatatcgag
     2581 gtggatggaa agcaggtaga gttggctttg tgggacacag ctgggcagga agattatgat
     2641 cgcctgaggc ccctctccta cccagatacc gatgttatac tgatgtgttt ttccatcgac
     2701 agccctgata gtttagaaaa catcccagaa aagtggaccc cagaagtcaa gcatttctgt
     2761 cccaacgtgc ccatcatcct ggttgggaac aagaaggatc ttcggaatga tgagcacaca
     2821 aggcgggagc tagccaagat gaagcaggag ccggtgaaac ctgaagaagg cagagatatg
     2881 gcaaacagga ttggcgcttt tgggtacatg gagtgttcag caaagaccaa agatggagtg
     2941 agagaggttt ttgaaatggc tacgagagct gctctgcaag ctagacgtgg gaagaaaaaa
     3001 tctgggtgcc ttgtcttgtg agcggccgcg acactagatc ataatcagcc ataccacatt
     3061 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     3121 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     3181 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     3241 gtccaaactc atcaatgtat cttaacgcgt acgcgccctg tagcggcgca ttaagcgcgg
     3301 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3361 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3421 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3481 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3541 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3601 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3661 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     3721 caatttcctc tagataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     3781 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3841 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3901 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3961 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4021 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4081 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4141 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4201 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4261 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4321 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4381 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4441 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaact
     4501 gatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4561 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     4621 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4681 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4741 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4801 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4861 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4921 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4981 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5041 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5101 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5161 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5221 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5281 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5341 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5401 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5461 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5521 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatggctc gacaga
//
