LOCUS       sequence-470604-        5408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE0321m 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 5408)
  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 5408)
  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: AiP14535 -
            pAAV-AiE0321m-minBG-SYFP2-P2A-3XFLA...
FEATURES             Location/Qualifiers
     source          1..5408
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    63..90
                     /gene="telR"
                     /label=telR
                     /note="telomeric sequence from coliphage N15"
     misc_feature    91..118
                     /gene="telL"
                     /label=telL
                     /note="telomeric sequence from coliphage N15"
     rep_origin      187..642
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(274..293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     484..505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(659..678)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     778..800
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(838..856)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        924..1028
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1029..1097,1098..1889)
                     /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(1247..1266)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2060..2648
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2549..2568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    2705..2732
                     /gene="telR"
                     /label=telR
                     /note="telomeric sequence from coliphage N15"
     misc_feature    2733..2760
                     /gene="telL"
                     /label=telL
                     /note="telomeric sequence from coliphage N15"
     repeat_region   2767..2896
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      3527..3536
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3533..3535,3536..3538,3539..4249)
                     /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(3839..3858)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4186..4207
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4259..4315
                     /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             4328..4393
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4424..4801
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(4888..4908)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5095..5206
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   join(5324..5408,1..56)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(5324..5408,1..45)
                     /label=AAV2 ITR
ORIGIN
        1 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
       61 cctatcagca cacaattgcc cattatacgc gcgtataatg gactattgtg tgctgatatg
      121 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
      181 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      241 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
      301 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      361 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
      421 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      481 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
      541 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
      601 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
      661 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
      721 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      781 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
      841 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
      901 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
      961 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1021 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     1081 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     1141 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     1201 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     1261 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     1321 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     1381 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     1441 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     1501 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     1561 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     1621 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     1681 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     1741 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     1801 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     1861 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1921 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1981 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2041 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2101 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2161 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     2221 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2281 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2341 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2401 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2461 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2521 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2581 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2641 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2701 ctcatatcag cacacaattg cccattatac gcgcgtataa tggactattg tgtgctgata
     2761 acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg
     2821 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
     2881 atcactaggg gttcctgcgg ccgcacgcgt agaagagttc atttggtcag gaatgcctta
     2941 ttcttccttc caaagaaagc ccccacatag gcagtgagtg tctagatttt agaagtctag
     3001 aactgagccc tgacttcata taattcatgt gccctggcct taatcttggc actttagtag
     3061 tgctaaaggt ccccaggccc tcccttaggg tcagaacatg gttaataaga gacacagggc
     3121 atgtgattac agactagcaa gagtgttaca ctcacaggtg cctcagccca tgagtcatgg
     3181 cttgtagcct tgcattccaa gaagttctgg atccctgcag ctcagccaat tttggacatt
     3241 aatgtcccca gaacaacaag tagatgctgt actgtggcca aggcaacagc agctgcttct
     3301 gatgctacca tgaggtaaag aagggacaca aaactaaatt cacctcatat aacaaacaaa
     3361 ccagaagaaa aatcaattaa acctccaata ctttttttat ttttataacc taaaaaacta
     3421 tatatacacc tgggagctcg ggctgggcat aaaagtcagg gcagagccat ctattgctta
     3481 catttgcttc tgggatccag atctttcgaa gctagcgcta ccggtcgcca ccatggtctc
     3541 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgatgt
     3601 caacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     3661 gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     3721 caccctgggc tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga
     3781 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaaaga
     3841 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     3901 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     3961 gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga acggcatcaa
     4021 ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg ccgaccacta
     4081 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     4141 ctaccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     4201 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg gctctggtgc
     4261 taccaacttc tcactgttga aacaggcagg ggatgtagag gagaatccag ggcctggtgc
     4321 tagtggagac tacaaagacc atgacggaga ttataaagat catgacatcg attacaagga
     4381 tgacgatgac aagtccggac tcagatctgg aggctccgga ggcccagagc cagcgaagtc
     4441 tgctcccgcc ccgaaaaagg gctccaagaa ggcggtgact aaggcgcaga agaaaggcgg
     4501 caagaagcgc aagcgcagcc gcaaggagag ctattccatc tatgtgtata aggttctgaa
     4561 gcaggtccac cctgacaccg gcatttcgtc caaggccatg ggcatcatga actcgtttgt
     4621 gaacgacatt ttcgagcgca tcgcaggtga ggcttcccgc ctggcgcatt acaacaagcg
     4681 ctcgaccatc acctccaggg agatccagac ggccgtgcgc ctgctgctgc ctggggagtt
     4741 ggccaagcac gccgtgtccg agggtactaa ggccatcacc aagtacacca gcgctaagta
     4801 atgaggcgcg ccgcggccgc gaattcgata tcataatcaa cctctggatt acaaaatttg
     4861 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     4921 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     4981 taaatcctgg ttagttcttg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     5041 gacaggggct cggctgttgg gcactgacaa ttccgtggct cgagagatct tcgactgtgc
     5101 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     5161 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     5221 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     5281 acaatagcag gcatgagatc tcacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     5341 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     5401 gcccgacg
//
