LOCUS       sequence-250433-        6933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for mutant Ca2+/calmodulin-dependent protein 
            kinase II alpha (T286D/T305A/T306A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6933)
  AUTHORS   Chang JY, Nakahata Y, Hayano Y, Yasuda R
  TITLE     Mechanisms of Ca(2+)/calmodulin-dependent kinase II activation in 
            single dendritic spines.
  JOURNAL   Nat Commun. 2019 Jun 25;10(1):2784. doi: 10.1038/s41467-019-10694-z.
  PUBMED    31239443
REFERENCE   2  (bases 1 to 6933)
  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: "2019-06-25"; volume: "10(1):2784. doi"; pages: " 
            10.1038/s41467-019-10694-z"
COMMENT     Sequence Label: pCAG-mEGFP-CaMKIIa (T286D/T305A/T306A)
FEATURES             Location/Qualifiers
     source          1..6933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     624..646
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     712..731
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      765..774
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(771..773,774..776,777..1487)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(816..837)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1077..1096)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1424..1445
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2972..2991)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3037..3092
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3091..3110)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3453..3469)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3453..3469)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3466..3488)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3477..3493
                     /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(3501..3507,3508..3525,3526..3531))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3546..3567
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3626..3821
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3672..3807
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3827..3961
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3864..3883)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3918..3937
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4029..4046)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4200..4788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4280..4299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4959..5750,5751..5819))
                     /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     5582..5601
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5820..5924)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5959..6338
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6340..6616
                     /label=chicken beta-actin promoter
     intron          join(6617..6933,1..704)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
ORIGIN
        1 gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg
       61 cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
      121 agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc
      181 tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt
      241 gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg
      301 ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc
      361 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
      421 ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg
      481 ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc
      541 gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct
      601 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagcta
      661 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
      721 tggttattgt gctgtctcat cattttggca aagaattcgc tagcgccacc atggtgagca
      781 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      841 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
      901 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
      961 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1021 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     1081 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1141 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1201 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     1261 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1321 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     1381 cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1441 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc ggactcagat
     1501 ctcgagctat ggctaccatc acctgcaccc gattcacgga agagtaccag ctcttcgagg
     1561 aactgggaaa gggagccttc tccgtggtgc gcaggtgtgt gaaggtgctg gctggccagg
     1621 agtatgctgc caagattatc aacaccaaga agctctcagc cagagatcac cagaagttgg
     1681 aacgcgaggc ccgcatctgc cgcttgttga agcaccccaa tatcgtccga ctccatgaca
     1741 gcatctccga ggaggggcac cactacctta tcttcgatct ggtcactggt ggggagctgt
     1801 tcgaagacat tgtggcccgg gagtattaca gtgaggctga tgccagccac tgtatccagc
     1861 agatcctgga ggctgtgcta cactgtcacc agatgggggt ggtgcatcgc gacctgaagc
     1921 ctgagaatct gttgctggct tcgaagctca agggtgctgc ggtgaagctg gcagactttg
     1981 gcctggccat agaggttgag ggagagcagc aggcatggtt tgggttcgca gggacacctg
     2041 gatacctctc cccagaagtg ctgcggaagg acccatacgg gaagcctgtg gacctgtggg
     2101 cctgtggcgt catcctgtat atcttgctgg ttgggtatcc cccattctgg gatgaggacc
     2161 agcaccgcct gtaccagcag atcaaagctg gtgcctacga tttcccatca ccagaatggg
     2221 acaccgtcac cccggaagcc aaggatctga tcaataagat gctgaccatc aacccgtcca
     2281 aacgcatcac ggccgctgag gctctcaagc acccctggat ctcgcaccgc tccactgtgg
     2341 cctcctgcat gcacagacag gaggacgtgg actgcctgaa gaagttcaat gccaggagga
     2401 aactgaaggg agccatcctc gccgctatgc tggccaccag gaacttctcc ggagggaaga
     2461 gtggaggaaa caagaagaat gatggcgtga aggaatcctc tgagagcacc aacaccacca
     2521 tcgaggatga agacaccaaa gtgcgcaaac aggaaattat caaagtgaca gagcagctga
     2581 tcgaagccat aagcaatgga gactttgaat cctacacgaa gatgtgcgac cctggaatga
     2641 cagcctttga accggaggcc ctggggaacc tggtcgaggg cctggacttt catcgattct
     2701 attttgaaaa cctgtggtcc cggaacagca agcccgtgca caccaccatc ctgaaccctc
     2761 acatccacct gatgggtgac gagtcagcct gcatcgccta catccgcatc actcagtacc
     2821 tggatgcggg tggcatcccc cgcacggccc agtcagagga gacccgtgtc tggcaccgca
     2881 gggatggaaa atggcagatc gtccacttcc acagatctgg ggcgccctcc gtcctgcccc
     2941 attgagcggc cgcgactcta gagtcgacgg atctttttcc ctctgccaaa aattatgggg
     3001 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     3061 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     3121 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg
     3181 ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg
     3241 tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt
     3301 ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat
     3361 ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc
     3421 tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3481 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3541 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3601 ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc
     3661 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3721 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3781 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc
     3841 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3901 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     3961 ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4021 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4081 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4141 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4201 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4261 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4321 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4381 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4441 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4501 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4561 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4621 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4681 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4741 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4801 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4861 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4921 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4981 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5041 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5101 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5161 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5221 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5281 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5341 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5401 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5461 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5521 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5581 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5641 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5701 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5761 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5821 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5881 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5941 gtgccacctg ggtcgatcga cattgattat tgactagtta ttaatagtaa tcaattacgg
     6001 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     6061 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     6121 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     6181 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     6241 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     6301 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
     6361 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     6421 aattattttg tgcagcgatg ggggcggggg gggggggggg cgcgcgccag gcggggcggg
     6481 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
     6541 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
     6601 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
     6661 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     6721 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
     6781 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
     6841 ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc
     6901 cggcggctgt gagcgctgcg ggcgcggcgc ggg
//
