LOCUS       sequence-250748-        6935 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for mutant Ca2+/calmodulin-dependent protein 
            kinase II alpha (T305D/T306D).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6935)
  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 6935)
  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 (T305D/T306D)
FEATURES             Location/Qualifiers
     source          1..6935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          261..1281
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1201..1223
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1289..1308
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1342..1351
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1348..1350,1351..1353,1354..2064)
                     /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(1393..1414)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1654..1673)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2001..2022
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3549..3568)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3614..3669
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3668..3687)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4030..4046)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4030..4046)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4043..4065)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4054..4070
                     /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(4078..4084,4085..4102,4103..4108))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4123..4144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4203..4398
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4249..4384
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4404..4538
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4441..4460)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4495..4514
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4606..4623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4777..5365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4857..4876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5536..6327,6328..6396))
                     /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     6159..6178
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6397..6501)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6536..6915
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(6917..6935,1..260)
                     /label=chicken beta-actin promoter
ORIGIN
        1 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt
       61 ttaattattt tgtgcagcga tgggggcggg gggggggggg ggggcgcgcg ccgggggggg
      121 cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga
      181 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
      241 agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg
      301 ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg
      361 ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga cggcttgttt
      421 cttttctgtg gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga
      481 gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc
      541 tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc
      601 gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca
      661 aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg
      721 ggctgcaacc ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg
      781 cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg
      841 tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg
      901 gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat
      961 ggtaatcgtg cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat
     1021 ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca
     1081 ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc
     1141 tccagcctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg
     1201 ggttcggctt ctggcgtgtg accggcggct ctagctagag cctctgctaa ccatgttcat
     1261 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     1321 tttggcaaag aattcgctag cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
     1381 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1441 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1501 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1561 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1621 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1681 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1741 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1801 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1861 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1921 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac
     1981 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2041 ctcggcatgg acgagctgta caagtccgga ctcagatctc gagctatggc taccatcacc
     2101 tgcacccgat tcacggaaga gtaccagctc ttcgaggaac tgggaaaggg agccttctcc
     2161 gtggtgcgca ggtgtgtgaa ggtgctggct ggccaggagt atgctgccaa gattatcaac
     2221 accaagaagc tctcagccag agatcaccag aagttggaac gcgaggcccg catctgccgc
     2281 ttgttgaagc accccaatat cgtccgactc catgacagca tctccgagga ggggcaccac
     2341 taccttatct tcgatctggt cactggtggg gagctgttcg aagacattgt ggcccgggag
     2401 tattacagtg aggctgatgc cagccactgt atccagcaga tcctggaggc tgtgctacac
     2461 tgtcaccaga tgggggtggt gcatcgcgac ctgaagcctg agaatctgtt gctggcttcg
     2521 aagctcaagg gtgctgcggt gaagctggca gactttggcc tggccataga ggttgaggga
     2581 gagcagcagg catggtttgg gttcgcaggg acacctggat acctctcccc agaagtgctg
     2641 cggaaggacc catacgggaa gcctgtggac ctgtgggcct gtggcgtcat cctgtatatc
     2701 ttgctggttg ggtatccccc attctgggat gaggaccagc accgcctgta ccagcagatc
     2761 aaagctggtg cctacgattt cccatcacca gaatgggaca ccgtcacccc ggaagccaag
     2821 gatctgatca ataagatgct gaccatcaac ccgtccaaac gcatcacggc cgctgaggct
     2881 ctcaagcacc cctggatctc gcaccgctcc actgtggcct cctgcatgca cagacaggag
     2941 accgtggact gcctgaagaa gttcaatgcc aggaggaaac tgaagggagc catcctcgac
     3001 gatatgctgg ccaccaggaa cttctccgga gggaagagtg gaggaaacaa gaagaatgat
     3061 ggcgtgaagg aatcctctga gagcaccaac accaccatcg aggatgaaga caccaaagtg
     3121 cgcaaacagg aaattatcaa agtgacagag cagctgatcg aagccataag caatggagac
     3181 tttgaatcct acacgaagat gtgcgaccct ggaatgacag cctttgaacc ggaggccctg
     3241 gggaacctgg tcgagggcct ggactttcat cgattctatt ttgaaaacct gtggtcccgg
     3301 aacagcaagc ccgtgcacac caccatcctg aaccctcaca tccacctgat gggtgacgag
     3361 tcagcctgca tcgcctacat ccgcatcact cagtacctgg atgcgggtgg catcccccgc
     3421 acggcccagt cagaggagac ccgtgtctgg caccgcaggg atggaaaatg gcagatcgtc
     3481 cacttccaca gatctggggc gccctccgtc ctgccccatt gagcggccgc gactctagag
     3541 tcgacggatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat
     3601 ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg
     3661 tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat
     3721 ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta
     3781 taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa
     3841 gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca
     3901 tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc
     3961 ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttggc
     4021 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4081 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4141 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
     4201 ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4261 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4321 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4381 ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa
     4441 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4501 tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca
     4561 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4621 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4681 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4741 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4801 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4861 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4921 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4981 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5041 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5101 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5161 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5221 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5281 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5341 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5401 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5461 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5521 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5581 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5641 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5701 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5761 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5821 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5881 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5941 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6001 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6061 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6121 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6181 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6241 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6301 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6361 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6421 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6481 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt cgatcgacat
     6541 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     6601 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     6661 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     6721 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     6781 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     6841 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     6901 atcgctatta ccatggtcga ggtgagcccc acgtt
//
