LOCUS       sequence-472648-        4636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded enhancer AAV vector for multiplexed screening applications;
            AiE0441h_3xC2 is an enhancer active in striatal MSNs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4636)
  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 4636)
  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: AiP14458:
            pAAV-AiE0441h_3xC2-minBG-SYFP2-WPRE3...
FEATURES             Location/Qualifiers
     source          1..4636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(103..124)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     315..334
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   496..636
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   507..636
                     /label=AAV2 ITR
     polyA_signal    754..865
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1064..1084
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(1177..1887,1888..1890,1891..1893))
                     /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(1219..1240)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1568..1587
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1827..1848
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1890..1899
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     repeat_region   2405..2534
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2596..3184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2676..2695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3355..4146,4147..4215))
                     /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     3978..3997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4216..4320)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4388..4406
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4444..4466)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4566..4585
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(join(4602..4636,1..421))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 ttaaattttt gttaaatcag ctcatttttt aaccaataga ccgaaatcgg caaaatccct
       61 tataaatcaa aagaatagcc cgagatagag ttgagtgttg ttccagtttg gaacaagagt
      121 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
      181 ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca
      241 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
      301 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct ggcaagtgta
      361 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
      421 tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat
      481 accgcatcag gcgcccctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg
      541 caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
      601 gagagggagt ggccaactcc atcactaggg gttcctgcgg ccgctcggtc cgcacgtgag
      661 atctcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caccccaccc
      721 cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa
      781 aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca
      841 gatggctggc aactagaagg cacagtcgaa gatctctcga tcggtgactc gagccacgga
      901 attgtcagtg cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg cggcgatgag
      961 ttccgccgtg gcaagaacta accaggattt atacaaggag gagaaaatga aagccatacg
     1021 ggaagcaata gcatgataca aaggcattaa agcagcgtat ccacatagcg taaaaggagc
     1081 aacatagtta agaataccag tcaatctttc acaaattttg taatccagag gttgattatg
     1141 atatcgaatt cgcggccgcg gcgcgccgtc gacttacttg tacagctcgt ccatgccgag
     1201 agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg
     1261 gtctttgctc agcttggact ggtagctcag gtagtggttg tcgggcagca gcacggggcc
     1321 gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgccgc cgtcctcgat
     1381 gttgtggcgg atcttgaagt tggccttgat gccgttcttc tgcttgtcgg cggtgatata
     1441 gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt
     1501 gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc
     1561 ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc
     1621 ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcgggcgaa
     1681 gcactgcacg ccgtagccca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc
     1741 ggtggtgcag atcagcttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc
     1801 ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac
     1861 cccggtgaac agctcctcgc ccttgctcac catggtggcg accggtagcg ctagcttcga
     1921 aagatctgga tcccagaagc aaatgtaagc aatagatggc tctgccctga cttttatgcc
     1981 cagcccgagc tcagaaaggc agaagacaga gatggagaga gagtaaaaat agctcagcat
     2041 taagataatt gcagctcgca gctctgccct ccgttaggaa ttaaatgagg ttttattact
     2101 ttgatttttc tctcctggat cctacgcgac ttctcaagaa cttggggaaa agtttttcag
     2161 gtgggcggcc aggtggtggg ggctgtgtgg gagaaaggca gaagacagag atggagagag
     2221 agtaaaaata gctcagcatt aagataattg cagctcgcag ctctgccctc cgttaggaat
     2281 taaatgaggt tttattactt tgatttttct ctcctggatc ctacgcgact tctcaagaac
     2341 ttggggaaaa gtttttcagg tgggcggcca ggtggtgggg gctgtgtggg acgcgtgcgg
     2401 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     2461 gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg
     2521 cagctgcctg caggacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2581 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2641 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2701 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2761 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2821 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2881 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2941 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3001 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3061 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3121 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3181 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3241 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3301 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3361 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3421 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3481 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3541 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3601 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3661 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3721 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3781 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3841 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3901 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3961 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4021 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4081 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4141 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4201 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4261 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4321 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     4381 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     4441 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     4501 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     4561 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat
     4621 tttgttaaaa ttcgcg
//
