LOCUS       sequence-224298-        6262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control of AKARet-cyto.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6262)
  AUTHORS   Tang S, Yasuda R
  TITLE     Imaging ERK and PKA Activation in Single Dendritic Spines during 
            Structural Plasticity.
  JOURNAL   Neuron. 2017 Mar 22;93(6):1315-1324.e3. doi: 
            10.1016/j.neuron.2017.02.032. Epub 2017 Mar 9.
  PUBMED    28285819
REFERENCE   2  (bases 1 to 6262)
  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"; doi: 
            "10.1016/j.neuron.2017.02.032"; journalName: "Neuron"; date: 
            "2017-03-22- 22"; volume: "93"; issue: "6"; pages: "1315-1324.e3"
COMMENT     Sequence Label: negAKARet-cyto
FEATURES             Location/Qualifiers
     source          1..6262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..742
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1058..1060,1061..1063,1064..1774)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITLGMDELYK"
     CDS             join(2582..2584,2585..2587,2588..3298)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2582..2584,2585..2587,2588..3298)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    3377..3498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3388..3407)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3442..3461
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3679..4134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3766..3785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3976..3997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4201..4220)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4320..4342
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4380..4398)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4466..4570
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4571..4639,4640..5431)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDVGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4789..4808)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5602..6189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6090..6109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggcg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt acttaatacg actcactata ggctagcatg gtgagcaagg gcgaggagct
     1081 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1141 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctcat
     1201 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg
     1261 cctgatgtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1321 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1381 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1441 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actggaacag
     1501 cgtcaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1561 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1621 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct
     1681 gttcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcc tgaccgccgc
     1741 cgggatcact ctcggcatgg acgagctgta caagctcgag aagttttctc aagaacagat
     1801 cggcgaaaac attgtgtgca gggtcatttg taccacgggt caaattccca tccgagattt
     1861 gtcagctgat atttcacaag tgcttaagga aaaacgatcc ataaagaaag tttggacatt
     1921 tggtagaaac ccagcctgtg actatcattt aggaaacatt tcaagactgt caaataagca
     1981 tttccaaata ctactaggag aagacggtaa ccttttattg aatgacattt ccactaatgg
     2041 gacctggtta aatgggcaaa aagtcgagaa gaacagcaat cagttactgt cccaaggtga
     2101 tgaaataacc gttggtgtag gcgtggaatc agatatttta tctctggtca ttttcataaa
     2161 cgacaaattt aagcagtgcc tggagcagaa caaagttgat cgcggtacca gtgctggtgg
     2221 tagtgctggt ggtagtgctg gtggtagtgc tggtggttcc ggcagtgctg gtggtagtgc
     2281 tggtggtagt accagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg
     2341 tagtgctggt ggttccggca gtgctggtgg tagtgctggt ggtagtacca gtgctggtgg
     2401 tagtgctggt ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggcagtgc
     2461 tggtggtagt gctggtggta gtaccagtgc tggtggtagt gctggtggta gtgctggtgg
     2521 tagtgctggt ggtagtgctg gtggttccgg attgaggcgc gcggcgctgg ttgacacgcg
     2581 tatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2641 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2701 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2761 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2821 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2881 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2941 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     3001 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     3061 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     3121 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     3181 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     3241 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga
     3301 tgctcttcaa aaaaaacttg aagaacttga acttgacgaa tgaactagta tctgcggccg
     3361 cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     3421 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     3481 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     3541 gagatgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag
     3601 gatccgggct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     3661 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3721 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3781 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3841 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3901 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3961 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4021 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4081 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
     4141 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     4201 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4261 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4321 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4381 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4441 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4501 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4561 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4621 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4681 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4741 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagtgctgc tatgtggcgc
     4801 ggtattatcc cgtattgacg tcgggcaaga gcaactcggt cgccgcatac actattctca
     4861 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4921 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4981 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5041 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5101 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5161 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5221 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5281 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5341 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5401 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5461 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5521 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5581 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5641 aacaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5701 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     5761 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5821 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5881 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5941 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6001 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6061 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6121 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6181 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6241 gctcacatgg ctcgacagat ct
//
