LOCUS       sequence-491728-        6128 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Negative control viral expression vecotor for plasma 
            membrane-tethered ibARK in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6128)
  AUTHORS   Dewa KI, Kaseda K, Kuwahara A, Kubotera H, Yamasaki A, Awata N, 
            Komori A, Holtz MA, Kasai A, Skibbe H, Takata N, Yokoyama T, Tsuda 
            M, Numata G, Nakamura S, Takimoto E, Sakamoto M, Ito M, Masuda T, 
            Nagai J
  TITLE     The astrocytic ensemble acts as a multiday trace to stabilize 
            memory.
  JOURNAL   Nature. 2025 Oct 15. doi: 10.1038/s41586-025-09619-2.
  PUBMED    41094146
REFERENCE   2  (bases 1 to 6128)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2025 Oct 15. doi: 10.1038/s41586-025-09619-2."
COMMENT     Sequence Label: GfaABC1D-Lck-mCherry-ibARK(D110A)
FEATURES             Location/Qualifiers
     source          1..6128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     protein_bind    137..157
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     intron          1008..1140
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1123..1142
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1185..1203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1219..1243
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1358..1367
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1364..2071
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1509..1527)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1777..1796
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2507..2531)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2541..3129
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2594..2614)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3012..3023)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3159..3176)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3165..3372
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3380..3520
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3380..3509
                     /label=AAV2 ITR
     rep_origin      3595..4050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3682..3701)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3892..3913
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4067..4086)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4186..4208
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4246..4264)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4332..4436
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4437..4505,4506..5297)
                     /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"
     rep_origin      5468..6056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5957..5976
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6118..6128,1..130)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tctagacaac tttgtataga aaagttgtgt agttaatgat taacccgcca
      181 tgctacttat ctacgtagcc atgctctagg aagatctaac atatcctggt gtggagtagg
      241 ggacgctgct ctgacagagg ctcgggggcc tgagctggct ctgtgagctg gggaggaggc
      301 agacagccag gccttgtctg caagcagacc tggcagcatt gggctggccg ccccccaggg
      361 cctcctcttc atgcccagtg aatgactcac cttggcacag acacaatgtt cggggtgggc
      421 acagtgcctg cttcccgccg caccccagcc cccctcaaat gccttccgag aagcccattg
      481 agcagggggc ttgcattgca ccccagcctg acagcctggc atcttgggat aaaagcagca
      541 cagcccccta ggggctgccc ttgctgtgtg gcgccaccgg cggtggagaa caaggctcta
      601 ttcagcctgt gcccaggaaa ggggatcagg ggatgcccag gcatggacag tgggtggcag
      661 ggggggagag gagggctgtc tgcttcccag aagtccaagg acacaaatgg gtgaggggag
      721 agctctcccc atagctgggc tgcggcccaa ccccaccccc tcaggctatg ccagggggtg
      781 ttgccagggg cacccgggca tcgccagtct agcccactcc ttcataaagc cctcgcatcc
      841 caggagcgag cagagccaga gcaggttgga gaggagacgc atcacctccg ctgctcgcaa
      901 gctttattgc ggtagtttat cacagttaaa ttgctaacgc agtcagtgct tctgacacaa
      961 cagtctcgaa cttaagctgc agaagttggt cgtgaggcac tgggcaggta agtatcaagg
     1021 ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag agaagactct
     1081 tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacag
     1141 gtgtccactc ccagttcaat tacagctctt aaggctagag tacttaatac gactcactat
     1201 aggctagcct cgagaattcc aagtttgtac aaaaaagcag gctgccacca tgggctgtgg
     1261 ctgcagctca aaccctgaag atgactggat ggagaacatt gacgtgtgtg agaactgcca
     1321 ttatcccccg ccgaagcttc gaattgatcc accggtcgcc accatggtga gcaagggcga
     1381 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     1441 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1501 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     1561 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     1621 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1681 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     1741 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     1801 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     1861 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     1921 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     1981 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     2041 ctccaccggc ggcatggacg agctgtacaa gaagtacctg gaggaccggg gcgaggtgac
     2101 ttttgagaag atcttctccc agaagctggg gtacctgctt ttccgagact tctgcctgaa
     2161 gcacctggag gaggccaagc ccttggtaga gttctacgag gagatcaaga aatacgagaa
     2221 gctggagaca gaggaggagc gcctggtctg cagccgagag atcttcgcca cgtacatcat
     2281 gaaggagctg ctggcctgct cacatccttt ctcgaagagc gccattgagc acgtccaggg
     2341 ccatctggtg aagaagcagg tgcctccgga tctcttccag ccatatattg aagaaatttg
     2401 ccagaacctc cgaggagacg tgttccagaa attcatcgag agcgataaat tcacacgatt
     2461 ttgccagtgg aagaatgtag agctcaacat ccacggtagt ggctagaccc agctttcttg
     2521 tacaaagtgg gaattccgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     2581 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     2641 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     2701 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     2761 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     2821 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     2881 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga
     2941 cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     3001 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     3061 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     3121 cctccccgca tcgggaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     3181 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3241 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3301 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag
     3361 gcatgctggg gagggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     3421 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     3481 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     3541 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     3601 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3661 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3721 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3781 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     3841 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3901 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     3961 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4021 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     4081 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4141 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4201 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     4261 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4321 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4381 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4441 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4501 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4561 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4621 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4681 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4741 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4801 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4861 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4921 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4981 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5041 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5101 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg
     5161 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5221 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5281 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5341 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5401 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5461 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5521 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5581 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5641 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5701 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5761 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5821 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5881 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5941 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6001 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6061 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct
     6121 gcaggcag
//
