LOCUS       sequence-325980-        5495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ShadowY-CBD(H83L/H86L) and Clover(T153M/F223R)-RhoA in 
            mammalian cells as a negative control for FLIM imaging..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5495)
  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 5495)
  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(H83L/H86L)-P2A-Clover(T153M/F223R)-RhoA
FEATURES             Location/Qualifiers
     source          1..5495
                     /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..2940
                     /codon_start=1
                     /product="human Rho family small GTPase RhoA"
                     /label=RhoA
                     /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     polyA_signal    3062..3183
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3099..3118)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3153..3172
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3190..3645
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3277..3296)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3487..3508
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3701..3805
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3806..3874,3875..4666)
                     /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(4024..4043)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4837..5425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5326..5345
                     /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 tggaatgaac
     1381 aaaaagaaag agaaagagcg gccagagatt tctctccctt cagattttga actcacaatt
     1441 cttgtcggtt ttgatgctgt cacaggggag tttacgggaa taccagagca gtgggcccgc
     1501 ttgcttctga 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 ggctgccatc cggaagaaac tggtgattgt tggtgatgga
     2401 gcctgtggaa agacatgctt gctcatagtc ttcagcaagg accagttccc agaggtgtat
     2461 gtgcccacag tgtttgagaa ctatgtggca gatatcgagg tggatggaaa gcaggtagag
     2521 ttggctttgt gggacacagc tgggcaggaa gattatgatc gcctgaggcc cctctcctac
     2581 ccagataccg atgttatact gatgtgtttt tccatcgaca gccctgatag tttagaaaac
     2641 atcccagaaa agtggacccc agaagtcaag catttctgtc ccaacgtgcc catcatcctg
     2701 gttgggaaca agaaggatct tcggaatgat gagcacacaa ggcgggagct agccaagatg
     2761 aagcaggagc cggtgaaacc tgaagaaggc agagatatgg caaacaggat tggcgctttt
     2821 gggtacatgg agtgttcagc aaagaccaaa gatggagtga gagaggtttt tgaaatggct
     2881 acgagagctg ctctgcaagc tagacgtggg aagaaaaaat ctgggtgcct tgtcttgtga
     2941 gcggccgcga cactagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     3001 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     3061 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3121 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3181 ttaacgcgta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3241 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3301 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3361 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3421 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3481 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3541 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3601 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctct agataataat
     3661 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3721 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3781 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3841 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3901 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3961 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     4021 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     4081 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     4141 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     4201 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     4261 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     4321 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     4381 aactggcgaa ctacttactc tagcttcccg gcaacaactg atagactgga tggaggcgga
     4441 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     4501 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     4561 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4621 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4681 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4741 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4801 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4861 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4921 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4981 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5041 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5101 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5161 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5221 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5281 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5341 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5401 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5461 cttttgctgg ccttttgctc acatggctcg acaga
//
