LOCUS       sequence-447012-        5624 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE0961h_3xC2 is an enhancer sequence, designed to drive 
            AAV-mediated transgene expression in striatal MSNs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5624)
  AUTHORS   Hunker AC, Wirthlin ME, Gill G, Johansen NJ, Hooper M, Omstead V, 
            Vargas S, Lerma MN, Taskin N, Weed N, Laird WD, Bishaw YM, Bendrick 
            JL, Gore BB, Ben-Simon Y, Opitz-Araya X, Martinez RA, Way SW, 
            Thyagarajan B, Otto S, Sanchez REA, Alexander JR, Amaya A, Amster A,
            Arbuckle J, Ayala A, Baker PM, Barcelli T, Barta S, Bertagnolli D, 
            Bielstein C, Bishwakarma P, Bowlus J, Boyer G, Brouner K, Casian B, 
            Casper T, Chakka AB, Chakrabarty R, Chong P, Clark M, Colbert K, 
            Daniel S, Dawe T, Departee M, DiValentin P, Donadio NP, Dotson NI, 
            Dwivedi D, Egdorf T, Fliss T, Gary A, Goldy J, Grasso C, Groce EL, 
            Gudsnuk K, Han W, Haradon Z, Hastings S, Helback O, Ho WV, Huang C, 
            Johnson T, Jones DL, Juneau Z, Kenney J, Leibly M, Li S, Liang E, 
            Loeffler H, Lusk NA, Madigan Z, Malloy J, Malone J, McCue R, Melchor
            J, Mich JK, Moosman S, Morin E, Naidoo R, Newman D, Ngo K, Nguyen K,
            Oster AL, Ouellette B, Oyama AA, Pena N, Pham T, Phillips E, Pom C, 
            Potekhina L, Ransford S, Ray PL, Reding M, Rette DF, Reynoldson C, 
            Rimorin C, Sigler AR, Rocha DB, Ronellenfitch K, Ruiz A, Sawyer L, 
            Sevigny JP, Shapovalova NV, Shepard N, Shulga L, Soliman S, Staats 
            B, Taormina MJ, Tieu M, Wang Y, Wilkes J, Wood T, Zhou T, Williford 
            A, Dee N, Mollenkopf T, Ng L, Esposito L, Kalmbach BE, Yao S, Ariza 
            J, Collman F, Mufti S, Smith K, Waters J, Ersing I, Patrick M, Zeng 
            H, Lein ES, Kojima Y, Horwitz G, Owen SF, Levi BP, Daigle TL, Tasic 
            B, Bakken TE, Ting JT
  TITLE     Enhancer AAV toolbox for accessing and perturbing striatal cell 
            types and circuits.
  JOURNAL   Neuron. 2025 May 21;113(10):1507-1524.e17. doi: 
            10.1016/j.neuron.2025.04.035.
  PUBMED    40403704
REFERENCE   2  (bases 1 to 5624)
  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.2025.04"; journalName: "Neuron"; date: 
            "2025-05-21- 21"; volume: "113"; issue: "10"; pages: "1507-1524.e17"
COMMENT     Sequence Label: AiP14497 -
            pAAV-AiE0961h_3xC2-minBG-SYFP2-P2A-...
FEATURES             Location/Qualifiers
     source          1..5624
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      1090..1099
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1096..1098,1099..1101,1102..1812)
                     /codon_start=1
                     /product="monomeric optimized variant of EYFP, identical to
                     mVenus but with L68V (Kremers et al., 2006)"
                     /label=SYFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1402..1421)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1749..1770
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1822..1878
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1891..1956
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1987..2364
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(2451..2471)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    2658..2769
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2887..3027
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2887..3016
                     /label=AAV2 ITR
     rep_origin      3102..3557
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3189..3208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3399..3420
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3574..3593)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3693..3715
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3753..3771)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3839..3943
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3944..4012,4013..4804)
                     /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(4162..4181)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4975..5563
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5464..5483
                     /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 gcgtcgtaag taaaactttc ttgaatagga acttacagta
      181 ctgatgagaa agacccacag gactgttcat gaataaacat gaatgtgcct attgtttcgt
      241 gctgcacaca atcctattat agaaaaatat tcaaattata tagatactct attataaaag
      301 aaaaccttat gtgacatggg cttatttcag ggctctgtca aaacagggct gtcactttaa
      361 tgtcaaaaaa gggagtgaat tttctgcatc tacctacccc actcttcccg gcaatggctc
      421 ctgtcatccg taagtaaaac tttcttgaat aggaacttac agtactgatg agaaagaccc
      481 acaggactgt tcatgaataa acatgaatgt gcctattgtt tcgtgctgca cacaatccta
      541 ttatagaaaa atattcaaat tatatagata ctctattata aaagaaaacc ttatgtgaca
      601 tgggcttatt tcagggctct gtcaaaacag ggctgtcact ttaatgtcaa aaaagggagt
      661 gaattttctg catctaccta ccccactctt cccggcaatg gctcctgtca tccgtaagta
      721 aaactttctt gaataggaac ttacagtact gatgagaaag acccacagga ctgttcatga
      781 ataaacatga atgtgcctat tgtttcgtgc tgcacacaat cctattatag aaaaatattc
      841 aaattatata gatactctat tataaaagaa aaccttatgt gacatgggct tatttcaggg
      901 ctctgtcaaa acagggctgt cactttaatg tcaaaaaagg gagtgaattt tctgcatcta
      961 cctaccccac tcttcccggc aatggctcct gtcatcgagc tcgggctggg cataaaagtc
     1021 agggcagagc catctattgc ttacatttgc ttctgggatc cagatctttc gaagctagcg
     1081 ctaccggtcg ccaccatggt ctccaagggc gaggagctgt tcaccggggt ggtgcccatc
     1141 ctggtcgagc tggacggcga tgtcaacggc cacaagttca gcgtgtccgg cgagggcgag
     1201 ggcgatgcca cctacggcaa gctgaccctg aagctgatct gcaccaccgg caagctgccc
     1261 gtgccctggc ccaccctcgt gaccaccctg ggctacggcg tgcagtgctt cgcccgctac
     1321 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1381 gagcgcacca tcttcttcaa agacgacggc aactacaaga cccgcgccga ggtgaagttc
     1441 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1501 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcaccgcc
     1561 gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc
     1621 ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1681 ctgcccgaca accactacct gagctaccag tccaagctga gcaaagaccc caacgagaag
     1741 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1801 gagctgtaca agggctctgg tgctaccaac ttctcactgt tgaaacaggc aggggatgta
     1861 gaggagaatc cagggcctgg tgctagtgga gactacaaag accatgacgg agattataaa
     1921 gatcatgaca tcgattacaa ggatgacgat gacaagtccg gactcagatc tggaggctcc
     1981 ggaggcccag agccagcgaa gtctgctccc gccccgaaaa agggctccaa gaaggcggtg
     2041 actaaggcgc agaagaaagg cggcaagaag cgcaagcgca gccgcaagga gagctattcc
     2101 atctatgtgt ataaggttct gaagcaggtc caccctgaca ccggcatttc gtccaaggcc
     2161 atgggcatca tgaactcgtt tgtgaacgac attttcgagc gcatcgcagg tgaggcttcc
     2221 cgcctggcgc attacaacaa gcgctcgacc atcacctcca gggagatcca gacggccgtg
     2281 cgcctgctgc tgcctgggga gttggccaag cacgccgtgt ccgagggtac taaggccatc
     2341 accaagtaca ccagcgctaa gtaatgaggc gcgccgcggc cgcgaattcg atatcataat
     2401 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2461 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     2521 gctttcattt tctcctcctt gtataaatcc tggttagttc ttgccacggc ggaactcatc
     2581 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     2641 gctcgagaga tcttcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2701 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2761 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2821 cagcaagggg gaggattggg aagacaatag caggcatgag atctcacgtg cggaccgagc
     2881 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     2941 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     3001 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     3061 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     3121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     3181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3301 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3361 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3421 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     3481 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     3541 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3601 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3661 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3721 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     3781 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     3841 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3901 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     3961 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4021 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4081 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4141 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4201 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4261 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4321 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4381 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4441 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4501 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4561 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     4621 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     4681 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     4741 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     4801 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     4861 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4921 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4981 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5041 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5101 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5161 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5221 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5281 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5341 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5401 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5461 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5521 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5581 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
