LOCUS       sequence-466219-        6933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE2066m 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 6933)
  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 6933)
  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: AiP1238 - pAAV-AiE2066m-minBG-FlpO-WPRE-HGHpA
FEATURES             Location/Qualifiers
     source          1..6933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1604..1613
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1610..1615,1616..1636,1637..2908)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     misc_feature    2933..3521
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2986..3006)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3404..3415)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3553..4029
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3752..3771)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4069..4209
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4069..4198
                     /label=AAV2 ITR
     rep_origin      4284..4739
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4371..4390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4581..4602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4756..4775)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4875..4897
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4935..4953)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5021..5125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5126..5194,5195..5986)
                     /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(5344..5363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6157..6745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6646..6665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6807..6933,1..3)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 cctgcggccg cacgcgtcag tggtgttggt taaaggtcaa cagatgaggt tcagagcaca
       61 tcaagtgtat gggaagccac cgataccttt gaagctggag aggccatgat ctgatgatgt
      121 tttaaggctg tcttctgaaa gcctagctgg agaagagatt ttacagccac catgtaacct
      181 atgaagaaac caagcaggaa cttatggcaa tgtgtccctg ggacaggaag gcaccagagg
      241 gagaggagtg gggctggaag cagaccattg caagataatg aagagtcttc ctgaagattg
      301 cagagcttct gatacgaaag gctcaaccat tccagatcac ccattttgca tccagttctg
      361 atgttccacc agtgaacttg acttcattca ggtactgggt ttgttcccaa ccaaatgaac
      421 cacgagcata gatgggaagg ggaacacaat gaaggatgat gttggagccg gtgaagatga
      481 gaaggcccta tggaaactgt caactgccta tgcccagaac ttcttcacag tatggaggtt
      541 tatattacct aatatatgcc ccaactttaa ttaataatgt ccaataggca ctatagatta
      601 tatactctgt atatggaaca agtatatttt caattcaatt aaaatactta agattaaacc
      661 aatgtcatct caaattgaaa ctcagtctcc ctacttcctt gtttccattt tattgccatg
      721 ttttcctccc taccatacaa ctagcatttt ttttaacttg ctgtcagact ttccccattc
      781 agcagtgacc gaaacaaagg gaaaaaatgg tacagaggga aaatcagcag ttctgttcaa
      841 taatggccaa ttgtttccct gacagcatcc agacaaatcg tctaaatcac aaatgcattt
      901 gtgaccctca ttttggaagg gcttctctaa tcttagggat tggaattgaa cttcaaaaat
      961 gggtctcaag gtctttgggg gcccctcaat ttgctgttcc aacaccctag tggctggatg
     1021 gctaccatcc tttttcattc gcttaataaa aataacaaga aaaactataa tagccaaatt
     1081 tcttctctca aatgccaatg tagagagcaa tcattcccaa actctgaggc agtaaagagg
     1141 tgactttgaa caatgccttt attttttcct catttatttc tcaattttta tctccagatt
     1201 agatatgtgc ctataggaag ggaagcagtg ctgggaagtg cagagtagtt tgatgtcaca
     1261 cagcagatta agcaaagaat catgtataat caccactgaa tgcccatgtg catccttcca
     1321 tcttgtggct ttgtgtggga gagtcaaagg tggtctggat ttcaaatgaa cggaaaaatc
     1381 accctgtgac tggatagagg ttccaggaca cactctctct ccctctctct ttccctctct
     1441 ccctctcttt taggaaacac caaaagatgt agctctcatt ggataaaata aaatatgggc
     1501 aaaaacatca tggatactgg catgcattag tattaagtcg agctcgggct gggcataaaa
     1561 gtcagggcag agccatctat tgcttacatt tgcttctggc gtggccacca tggctcctaa
     1621 gaagaagagg aaggtgatga gccagttcga catcctgtgc aagacccccc ccaaggtgct
     1681 ggtgcggcag ttcgtggaga gattcgagag gcccagcggc gagaagatcg ccagctgtgc
     1741 cgccgagctg acctacctgt gctggatgat cacccacaac ggcaccgcca tcaagagggc
     1801 caccttcatg agctacaaca ccatcatcag caacagcctg agcttcgaca tcgtgaacaa
     1861 gagcctgcag ttcaagtaca agacccagaa ggccaccatc ctggaggcca gcctgaagaa
     1921 gctgatcccc gcctgggagt tcaccatcat cccttacaac ggccagaagc accagagcga
     1981 catcaccgac atcgtgtcca gcctgcagct gcagttcgag agcagcgagg aggccgacaa
     2041 gggcaacagc cacagcaaga agatgctgaa ggccctgctg tccgagggcg agagcatctg
     2101 ggagatcacc gagaagatcc tgaacagctt cgagtacacc agcaggttca ccaagaccaa
     2161 gaccctgtac cagttcctgt tcctggccac attcatcaac tgcggcaggt tcagcgacat
     2221 caagaacgtg gaccccaaga gcttcaagct ggtgcagaac aagtacctgg gcgtgatcat
     2281 tcagtgcctg gtgaccgaga ccaagacaag cgtgtccagg cacatctact ttttcagcgc
     2341 cagaggcagg atcgaccccc tggtgtacct ggacgagttc ctgaggaaca gcgagcccgt
     2401 gctgaagaga gtgaacagga ccggcaacag cagcagcaac aagcaggagt accagctgct
     2461 gaaggacaac ctggtgcgca gctacaacaa ggccctgaag aagaacgccc cctaccccat
     2521 cttcgctatc aagaacggcc ctaagagcca catcggcagg cacctgatga ccagctttct
     2581 gagcatgaag ggcctgaccg agctgacaaa cgtggtgggc aactggagcg acaagagggc
     2641 ctccgccgtg gccaggacca cctacaccca ccagatcacc gccatccccg accactactt
     2701 cgccctggtg tccaggtact acgcctacga ccccatcagc aaggagatga tcgccctgaa
     2761 ggacgagacc aaccccatcg aggagtggca gcacatcgag cagctgaagg gcagcgccga
     2821 gggcagcatc agataccccg cctggaacgg catcatcagc caggaggtgc tggactacct
     2881 gagcagctac atcaacaggc ggatctgaga attcgatatc aagcttatcg ataatcaacc
     2941 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3001 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3061 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3121 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3181 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3241 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3301 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt
     3361 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3421 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3481 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct
     3541 cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
     3601 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct
     3661 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa
     3721 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg
     3781 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag
     3841 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
     3901 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg
     3961 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc
     4021 cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag
     4081 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4141 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4201 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4261 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4321 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4381 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4441 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4501 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4561 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     4621 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     4681 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     4741 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4801 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4861 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4921 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     4981 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5041 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5101 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5161 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5221 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5281 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5341 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5401 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5461 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5521 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5581 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5641 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5701 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5761 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5821 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5881 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5941 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6001 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6061 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6121 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6181 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6241 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6301 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6361 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6421 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6481 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6541 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6601 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6661 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6721 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6781 cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga
     6841 ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
     6901 gagagggagt ggccaactcc atcactaggg gtt
//
