LOCUS       sequence-470721-        5111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE0516h is an enhancer sequence, designed to drive AAV-mediated 
            transgene expression in specific populations of brain cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5111)
  AUTHORS   Ben-Simon Y, Hooper M, Narayan S, Daigle TL, Dwivedi D, Way SW, 
            Oster A, Stafford DA, Mich JK, Taormina MJ, Martinez RA, Opitz-Araya
            X, Roth JR, Alexander JR, Allen S, Amster A, Arbuckle J, Ayala A, 
            Baker PM, Bakken TE, Barcelli T, Barta S, Bendrick J, Bertagnolli D,
            Bielstein C, Bishwakarma P, Bowlus J, Boyer G, Brouner K, Casian B, 
            Casper T, Chakka AB, Chakrabarty R, Chance RK, Chavan S, Clark M, 
            Colbert K, Collman F, Daniel S, Departee M, DiValentin P, Donadio N,
            Dotson N, Egdorf T, Fliss T, Gabitto M, Garcia J, Gary A, Gasperini 
            M, Gloe J, Goldy J, Gore BB, Graybuck L, Greisman N, Haeseleer F, 
            Halterman C, Haradon Z, Hastings SD, Helback O, Ho W, Hockemeyer D, 
            Huang C, Huff S, Hunker A, Johansen N, Jones D, Juneau Z, Kalmbach 
            B, Kannan M, Khem S, Kussick E, Kutsal R, Larsen R, Lee C, Lee AY, 
            Leibly M, Lenz GH, Li S, Liang E, Lusk N, Madigan Z, Malloy J, 
            Malone J, McCue R, Melchor J, Mollenkopf T, Moosman S, Morin E, 
            Newman D, Ng L, Ngo K, Omstead V, Otto S, Oyama A, Pena N, Pham T, 
            Phillips E, Pom CA, Potekhina L, Ransford S, Ray PL, Rette D, 
            Reynoldson C, Rimorin C, Rocha D, Ruiz A, Sanchez REA, Sawyer L, 
            Sedeno-Cortes A, Sevigny JP, Shapovalova N, Shepard N, Shulga L, 
            Sigler AR, Siverts L, Soliman S, Somasundaram S, Staats B, Stewart 
            K, Szelenyi E, Tieu M, Trader C, Tran A, van Velthoven CTJ, Walker 
            M, Wang Y, Weed N, Wirthlin M, Wood T, Wynalda B, Yao Z, Zhou T, 
            Ariza J, Dee N, Reding M, Ronellenfitch K, Mufti S, Sunkin SM, Smith
            KA, Esposito L, Waters J, Thyagarajan B, Yao S, Lein ES, Zeng H, 
            Levi BP, Ngai J, Ting JT, Tasic B
  TITLE     A suite of enhancer AAVs and transgenic mouse lines for genetic 
            access to cortical cell types.
  JOURNAL   Cell. 2025 May 16:S0092-8674(25)00513-6. doi: 
            10.1016/j.cell.2025.05.002.
  PUBMED    40403729
REFERENCE   2  (bases 1 to 5111)
  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"; journalName: "Cell. 2025 
            May 16:S0092-8674(25)00513-6. doi: 10.1016/j.cell.2025.05.002."
COMMENT     Sequence Label: AiP13650 -
            pAAV-AiE0516h-minBG-SYFP2-P2A-3XFLA...
FEATURES             Location/Qualifiers
     source          1..5111
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   290..419
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(481..1069)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(561..580)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1240..2031,2032..2100))
                     /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     1863..1882
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2101..2205)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2273..2291
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2329..2351)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2451..2470
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2487..2942)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2624..2645)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2836..2855
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3017..3157
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3028..3157
                     /label=AAV2 ITR
     polyA_signal    3275..3386
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3573..3593
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3680..4057)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     CDS             complement(4088..4153)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(4166..4222)
                     /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             complement(join(4232..4942,4943..4945,4946..4948))
                     /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(4274..4295)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4623..4642
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      4945..4954
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ctacatgttg tgtggaatgt ctgggggtgt gtgctgtact tcttttttca tttcctgtcc
       61 ttagaattct acaggcaatt tgcccaaaat gattgtaaat cagactcttt tatgttttct
      121 gaggcaccga agagcctgaa gaacaggaga tgacataaaa aaatagttta catccagttc
      181 ttttgattta aaggagtgag taaacagtaa aagcagcttt tcataacttt tagttcaatg
      241 tgaattttgt tatactaagt tgtcttggaa ttttaacgcg tgcggccgca ggaaccccta
      301 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
      361 aaggtcgccc gacgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcagga
      421 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      481 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      541 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      601 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      661 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      721 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      781 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      841 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      901 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      961 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1021 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1081 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1141 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1201 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1261 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1321 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1381 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1441 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1501 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1561 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1621 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1681 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1741 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1801 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1861 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1921 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1981 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2041 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2101 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2161 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2221 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     2281 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     2341 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     2401 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     2461 gcagattgta ctgagagtgc accataaaat tgtaaacgtt aatattttgt taaaattcgc
     2521 gttaaatttt tgttaaatca gctcattttt taaccaatag accgaaatcg gcaaaatccc
     2581 ttataaatca aaagaatagc ccgagataga gttgagtgtt gttccagttt ggaacaagag
     2641 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2701 tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc
     2761 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2821 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctaaggcgc tggcaagtgt
     2881 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2941 gtactatggt tgctttgacg tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     3001 taccgcatca ggcgcccctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
     3061 gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
     3121 agagagggag tggccaactc catcactagg ggttcctgcg gccgctcggt ccgcacgtga
     3181 gatctcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc
     3241 ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga
     3301 aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac
     3361 agatggctgg caactagaag gcacagtcga agatctctcg agccacggaa ttgtcagtgc
     3421 ccaacagccg agcccctgtc cagcagcggg caaggcaggc ggcgatgagt tccgccgtgg
     3481 caagaactaa ccaggattta tacaaggagg agaaaatgaa agccatacgg gaagcaatag
     3541 catgatacaa aggcattaaa gcagcgtatc cacatagcgt aaaaggagca acatagttaa
     3601 gaataccagt caatctttca caaattttgt aatccagagg ttgattatga tatcgaattc
     3661 gcggccgcgg cgcgcctcat tacttagcgc tggtgtactt ggtgatggcc ttagtaccct
     3721 cggacacggc gtgcttggcc aactccccag gcagcagcag gcgcacggcc gtctggatct
     3781 cccgggaggt gatggtcgag cgcttgttgt aatgcgccag gcgggaagcc tctcctgcga
     3841 tgcgctcgaa aatgtcgttc acaaacgaat tcatgatgcc catggccttg gacgaaatgc
     3901 cggtgtcagg gtggacctgc ttcagaacct tgtacacata gatggaatag ctctccttgc
     3961 ggctgcgctt gcgcttcttg ccgcctttct tctgcgcctt agtcaccgcc ttcttggagc
     4021 cctttttcgg ggcgggagca gacttcgctg gctctgggcc tccggagcct ccagatctga
     4081 gtccggactt gtcatcgtca tccttgtaat cgatgtcatg atctttataa tctccgtcat
     4141 ggtctttgta gtctccacta gcaccaggcc ctggattctc ctctacatcc cctgcctgtt
     4201 tcaacagtga gaagttggta gcaccagagc ccttgtacag ctcgtccatg ccgagagtga
     4261 tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt
     4321 tgctcagctt ggactggtag ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc
     4381 cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gccgccgtcc tcgatgttgt
     4441 ggcggatctt gaagttggcc ttgatgccgt tcttctgctt gtcggcggtg atatagacgt
     4501 tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt
     4561 cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc
     4621 gggtcttgta gttgccgtcg tctttgaaga agatggtgcg ctcctggacg tagccttcgg
     4681 gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg gcgaagcact
     4741 gcacgccgta gcccagggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg
     4801 tgcagatcag cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca
     4861 cgctgaactt gtggccgttg acatcgccgt ccagctcgac caggatgggc accaccccgg
     4921 tgaacagctc ctcgcccttg gagaccatgg tggcgaccgg tagcgctagc ttcgaaagat
     4981 ctggatccca gaagcaaatg taagcaatag atggctctgc cctgactttt atgcccagcc
     5041 cgagctcggt accccctcct caacataaga ccaagcagtt ttggtttcct ttttggggct
     5101 gtttatttat t
//
