LOCUS       sequence-421851-        6356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV construct expressing GCaMP6f driven by cholinergic 
            neuron-targeting enhancer. Alias: pAAV_S9E27_GCaMP6f.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6356)
  AUTHORS   Furlanis E, Dai M, Leyva Garcia B, Tran T, Vergara J, Pereira A, 
            Gorissen BL, Wills S, Vlachos A, Hairston A, Dwivedi D, Du S, 
            McMahon J, Huang S, Morabito A, Vazquez A, Kim S, Lee AT, Chang EF, 
            Razzaq T, Qazi A, Vargish G, Yuan X, Caccavano A, Hunt S, 
            Chittajallu R, McLean N, Hewitt L, Paranzino E, Rice H, Cummins AC, 
            Plotnikova A, Mohanty A, Tangen AC, Shin JH, Azadi R, Eldridge MAG, 
            Alvarez VA, Averbeck BB, Alyahyay M, Vallejo TR, Soheib M, Vattino 
            LG, MacGregor CP, Chatain CP, Banks E, Olah VJ, Naskar S, Hill S, 
            Liebergall S, Badiani R, Hyde L, Hanley E, Xu Q, Allaway KC, 
            Goldberg EM, Rowan MJM, Nowakowski TJ, Lee S, Favuzzi E, Kaeser PS, 
            Sjulson L, Batista-Brito R, Takesian AE, Ibrahim LA, Iqbal A, Pelkey
            KA, McBain CJ, Dimidschstein J, Fishell G, Wang Y
  TITLE     An enhancer-AAV toolbox to target and manipulate distinct 
            interneuron subtypes.
  JOURNAL   Neuron. 2025 May 21;113(10):1525-1547.e15. doi: 
            10.1016/j.neuron.2025.05.002.
  PUBMED    40403705
REFERENCE   2  (bases 1 to 6356)
  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.2025.05"; journalName: "Neuron"; date: 
            "2025-05-21- 21"; volume: "113"; issue: "10"; pages: "1525-1547.e15"
COMMENT     Sequence Label: pAAV_BiCHATe27_GCaMP6f
FEATURES             Location/Qualifiers
     source          1..6356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     intron          1150..1282
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1303..1312
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1309..1317,1318..1335,1336..1338,1339..1371,1372..1374,
                     1375..1398,1399..1425,1426..1482,1483..1488,1489..1758,
                     1759..1776,1777..2211,2212..2217,2218..2661)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1405..1414
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1695..1716
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1822..1843)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2083..2102)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2702..3290
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2755..2775)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3173..3184)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3459..3540
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3496..3515)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3550..3569
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3619..3759
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3619..3748
                     /label=AAV2 ITR
     rep_origin      3834..4289
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3921..3940)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4131..4152
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4306..4325)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4425..4447
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4485..4503)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4571..4675
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4676..4744,4745..5536)
                     /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(4894..4913)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5707..6295
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6196..6215
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtatcgat cctcgggtaa tttgtaaaga caatttttct
      181 tttccccctc tgaacctagg ccttaggctg gctaggcaag tctccactac agccccgccc
      241 actccatctc ggctgtggct cgcgggaaag gcagccccct acagttagtc tccccagcca
      301 tcttctccgg ttcccctgat gctctcatgg ccccagccat cctagagctg gtgcgccatc
      361 gctcaccttt agcattcggc ctgctggggg tgagcttttg ggttagaagg aacaaggcat
      421 cagatggggt cgtgtttttt tacattccaa tcagttttcg gaggggaaga acagacattt
      481 ttgtcagcta attgcattta attacagaga caatcgctgt ttccgaacgt tgttgtgtcc
      541 actccctcat ccttcaatca gttcaagcac atccggcccc agctgggagc cttttaattg
      601 cataatttgg atttcacttc tggaaagaaa cttgagaaag actccgtgag ggtctccatt
      661 aacacaatca gctccccagg cacccggggc tggccctaat tgacccatct cagctaatgc
      721 ctctttccaa agaacgcccg cgtcatttct ttggcttgtt tgtgctattg tatcctgttt
      781 tagtgtcttg atcttaaaca tctttaaaga gggcactcag tggcttgttt aactggaagt
      841 ggtccagctg gtttggaagg acagtgcatc aggtgatgta agtccaaggg taggaatttc
      901 tggcctctcc ttcccctgcc cattgcaccg ctctccccaa caggaagtag ctacacatga
      961 taaggtgcac tgtggcttct aggacttatc gatgtttaaa cttcctgcag cccgggctgg
     1021 gcataaaagt cagggcagag ccatctattg cttacatttg cttctagcct gcaagtcgag
     1081 gagcgcagcc ttccagaagc agagcgcggc gccttaagct gcagaagttg gtcgtgaggc
     1141 actgggcacg taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc
     1201 ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc
     1261 cactttgcct ttctctccac agcgagctcg ctagcgcccg gtgccaccat gggttctcat
     1321 catcatcatc atcatggtat ggctagcatg actggtggac agcaaatggg tcgggatctg
     1381 tacgacgatg acgataagga tctcgccacc atggtcgact catcacgtcg taagtggaat
     1441 aagacaggtc acgcagtcag agctataggt cggctgagct cactcgagaa cgtctatatc
     1501 aaggccgaca agcagaagaa cggcatcaag gcgaacttca agatccgcca caacatcgag
     1561 gacggcggcg tgcagctcgc ctaccactac cagcagaaca cccccatcgg cgacggcccc
     1621 gtgctgctgc ccgacaacca ctacctgagc gtgcagtcca aactttcgaa agaccccaac
     1681 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1741 atggacgagc tgtacaaggg cggtaccgga gggagcatgg tgagcaaggg cgaggagctg
     1801 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1861 agcgtgtccg gcgagggtga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1921 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     1981 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2041 atgcccgaag gctacatcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2101 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2161 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa cctgccggac
     2221 caactgactg aagagcagat cgcagaattt aaagaggaat tctccctatt tgacaaggac
     2281 ggggatggga caataacaac caaggagctg gggacggtga tgcggtctct ggggcagaac
     2341 cccacagaag cagagctgca ggacatgatc aatgaagtag atgccgacgg tgacggcaca
     2401 atcgacttcc ctgagttcct gacaatgatg gcaagaaaaa tgaaatacag ggacacggaa
     2461 gaagaaatta gagaagcgtt cggtgtgttt gataaggatg gcaatggcta catcagtgca
     2521 gcagagcttc gccacgtgat gacaaacctt ggagagaagt taacagatga agaggttgat
     2581 gaaatgatca gggaagcaga catcgatggg gatggtcagg taaactacga agagtttgta
     2641 caaatgatga cagcgaagtg aggcgcgccc ctgcagggaa ttcgatatca agcttatcga
     2701 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2761 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2821 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2881 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2941 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     3001 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     3061 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3121 cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3181 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3241 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     3301 agcgctgctc gagagatctg tgatagcggc catcaagctg gccgcgactc tagatcataa
     3361 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     3421 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     3481 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3541 attctagttg tggtttgtcc aaactcatca atgtatcagc ttatcgatac cgcatgcacg
     3601 tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     3661 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     3721 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     3781 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     3841 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3901 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3961 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4021 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     4081 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4141 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     4201 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4261 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     4321 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4381 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4441 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     4501 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     4561 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4621 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4681 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4741 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4801 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4861 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4921 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4981 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5041 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5101 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5161 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5221 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5281 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5341 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5401 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5461 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5521 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5581 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5641 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5701 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5761 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5821 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5881 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5941 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6001 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6061 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6121 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6181 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6241 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6301 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
