LOCUS       sequence-470619-        5067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE0964m 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 5067)
  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 5067)
  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: AiP13302 -
            pAAV-AiE0964m-minBG-SYFP2-P2A-3XFLA...
FEATURES             Location/Qualifiers
     source          1..5067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      630..1218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1119..1138
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1280..1409
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      1812..1821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1818..1820,1821..1823,1824..2534)
                     /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(2124..2143)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2471..2492
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2544..2600
                     /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             2613..2678
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2709..3086
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(3173..3193)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3380..3491
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3609..3749
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3609..3738
                     /label=AAV2 ITR
     rep_origin      3824..4279
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3911..3930)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4121..4142
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4296..4315)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4415..4437
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4475..4493)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4561..4665
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4666..4734,4735..5067,1..459)
                     /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(4884..4903)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
       61 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
      121 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
      181 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
      241 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
      301 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
      361 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
      421 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
      481 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
      541 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
      601 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
      661 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      721 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
      781 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      841 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      901 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      961 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1021 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     1081 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     1141 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     1201 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     1261 tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc
     1321 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
     1381 agtggccaac tccatcacta ggggttcctg cggccgcacg cgttgggaga catcttgtgg
     1441 cattctgtag gtggaagtgt cgaggaagga aggaagtggg ggatccaaaa gcccgcgtcc
     1501 ctgcagacac gctgggtgac attgggcacg gaagtttcta ttttgaggcc ctacctttct
     1561 agtgtctaaa gtgaggggag tgttttgaga ctcccaagac cagtgaaggt gcatagggga
     1621 ggggggagga cagtcacctc cagctcgtcc gagggtgaag gagcatgggg cttctctaag
     1681 ccctctgagg aagctgcctg ctcagtcttc acatgagaga gctcgggctg ggcataaaag
     1741 tcagggcaga gccatctatt gcttacattt gcttctggga tccagatctt tcgaagctag
     1801 cgctaccggt cgccaccatg gtctccaagg gcgaggagct gttcaccggg gtggtgccca
     1861 tcctggtcga gctggacggc gatgtcaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1921 agggcgatgc cacctacggc aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc
     1981 ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg cgtgcagtgc ttcgcccgct
     2041 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2101 aggagcgcac catcttcttc aaagacgacg gcaactacaa gacccgcgcc gaggtgaagt
     2161 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2221 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg
     2281 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg
     2341 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2401 tgctgcccga caaccactac ctgagctacc agtccaagct gagcaaagac cccaacgaga
     2461 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2521 acgagctgta caagggctct ggtgctacca acttctcact gttgaaacag gcaggggatg
     2581 tagaggagaa tccagggcct ggtgctagtg gagactacaa agaccatgac ggagattata
     2641 aagatcatga catcgattac aaggatgacg atgacaagtc cggactcaga tctggaggct
     2701 ccggaggccc agagccagcg aagtctgctc ccgccccgaa aaagggctcc aagaaggcgg
     2761 tgactaaggc gcagaagaaa ggcggcaaga agcgcaagcg cagccgcaag gagagctatt
     2821 ccatctatgt gtacaaggtt ctgaagcagg tccaccctga caccggcatt tcgtccaagg
     2881 ccatgggcat catgaattcg tttgtgaacg acattttcga gcgcatcgca ggagaggctt
     2941 cccgcctggc gcattacaac aagcgctcga ccatcacctc ccgggagatc cagacggccg
     3001 tgcgcctgct gctgcctggg gagttggcca agcacgccgt gtccgagggt actaaggcca
     3061 tcaccaagta caccagcgct aagtaatgag gcgcgccgcg gccgcgaatt cgatatcata
     3121 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     3181 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     3241 tggctttcat tttctcctcc ttgtataaat cctggttagt tcttgccacg gcggaactca
     3301 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3361 tggctcgaga gatcttcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3421 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3481 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3541 gacagcaagg gggaggattg ggaagacaat agcaggcatg agatctcacg tgcggaccga
     3601 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     3661 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     3721 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     3781 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     3841 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3901 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3961 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4021 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4081 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4141 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     4201 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4261 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4321 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4381 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4441 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     4501 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4561 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4621 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4681 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4741 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4801 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4861 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4921 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4981 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5041 accatgagtg ataacactgc ggccaac
//
