LOCUS       sequence-470661-        5470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE0974h 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 5470)
  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 5470)
  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: AiP13458 -
            pAAV-AiE0974h-minBG-SYFP2-P2A-3XFLA...
FEATURES             Location/Qualifiers
     source          1..5470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      428..1016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     917..936
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1078..1207
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      2013..2022
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2019..2021,2022..2024,2025..2735)
                     /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(2325..2344)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2672..2693
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2745..2801
                     /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             2814..2879
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2910..3287
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(3374..3394)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3581..3692
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3810..3950
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3810..3939
                     /label=AAV2 ITR
     rep_origin      4025..4480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4112..4131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4322..4343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4497..4516)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4616..4638
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4676..4694)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4762..4866
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4867..4935,4936..5470,1..257)
                     /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(5085..5104)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
       61 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
      121 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
      181 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
      241 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
      301 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
      361 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
      421 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
      481 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
      541 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
      601 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
      661 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
      721 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
      781 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
      841 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
      901 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
      961 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1021 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct
     1081 gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt
     1141 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
     1201 ggttcctgcg gccgcacgcg ttgtgcgtgt gtgtgtgcat taccctgttc cttccttcct
     1261 cctatgcagc tggagcagta agcttagcac ctgaaggtgt gggaggggta cccttctctt
     1321 tgcattctta tcccctacat gcccacatgg atgtggccag gagctcattg gaaattaggc
     1381 caagctagta ctggttattc atggcaaata taacaaatat atgctcttcc ctggctgtct
     1441 gggctctctc tggctctatt ctcctctttc actccatgta aactagcctc agtatcatgc
     1501 tccagcaggg tagagcagag aactcagcat caggaattaa gcttacagcc catctgccag
     1561 tgctttgcgc atacatcctt ctcttgggaa acctatgagg aagagtatct taggtttggt
     1621 ctcctggtct gttgccatca atgagcctgt gaatcccaag ttttccctca ctctctgctc
     1681 tgttccagca ctgtgccttc ttgtgcatga cataaatgct ctgaatcaca gtttttgcct
     1741 ctatcaaatg gacataatgg cattcatgat catgctcaca gagatttaaa cgcaataact
     1801 tggtatattc tagaagcgta ttcaaaagga acttttttat tgtctcgttc ctcaggctta
     1861 gtatgatttc tcgatggagt ggagataatg aatccttatg ggatggctga ttgaacagag
     1921 agctcgggct gggcataaaa gtcagggcag agccatctat tgcttacatt tgcttctggg
     1981 atccagatct ttcgaagcta gcgctaccgg tcgccaccat ggtctccaag ggcgaggagc
     2041 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgatgtcaac ggccacaagt
     2101 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctga
     2161 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg
     2221 gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2281 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaagacgac ggcaactaca
     2341 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2401 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2461 gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     2521 tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag cagaacaccc
     2581 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccaagc
     2641 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2701 ccgggatcac tctcggcatg gacgagctgt acaagggctc tggtgctacc aacttctcac
     2761 tgttgaaaca ggcaggggat gtagaggaga atccagggcc tggtgctagt ggagactaca
     2821 aagaccatga cggagattat aaagatcatg acatcgatta caaggatgac gatgacaagt
     2881 ccggactcag atctggaggc tccggaggcc cagagccagc gaagtctgct cccgccccga
     2941 aaaagggctc caagaaggcg gtgactaagg cgcagaagaa aggcggcaag aagcgcaagc
     3001 gcagccgcaa ggagagctat tccatctatg tgtacaaggt tctgaagcag gtccaccctg
     3061 acaccggcat ttcgtccaag gccatgggca tcatgaattc gtttgtgaac gacattttcg
     3121 agcgcatcgc aggagaggct tcccgcctgg cgcattacaa caagcgctcg accatcacct
     3181 cccgggagat ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc aagcacgccg
     3241 tgtccgaggg tactaaggcc atcaccaagt acaccagcgc taagtaatga ggcgcgccgc
     3301 ggccgcgaat tcgatatcat aatcaacctc tggattacaa aatttgtgaa agattgactg
     3361 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     3421 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag
     3481 ttcttgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3541 tgttgggcac tgacaattcc gtggctcgag agatcttcga ctgtgccttc tagttgccag
     3601 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3661 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3721 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3781 gagatctcac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc
     3841 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     3901 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     3961 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     4021 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4081 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     4141 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4201 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     4261 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4321 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     4381 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     4441 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     4501 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     4561 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4621 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     4681 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     4741 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4801 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4861 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4921 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4981 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5041 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5101 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5161 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5221 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5281 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5341 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5401 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5461 ctagcttccc
//
