LOCUS       sequence-470733-        4906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE0656m 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 4906)
  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 4906)
  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: AiP13658 -
            pAAV-AiE0656m-minBG-SYFP2-P2A-3XFLA...
FEATURES             Location/Qualifiers
     source          1..4906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..95
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(249..268)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     368..390
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(428..446)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        514..618
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(619..687,688..1479)
                     /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(837..856)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1650..2238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2139..2158
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2300..2429
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      2671..2680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2677..2679,2680..2682,2683..3393)
                     /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(2983..3002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3330..3351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3403..3459
                     /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             3472..3537
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3568..3945
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(4032..4052)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4239..4350
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4468..4608
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4468..4597
                     /label=AAV2 ITR
     rep_origin      join(4683..4906,1..232)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4770..4789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
       61 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
      121 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
      181 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
      241 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
      301 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
      361 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
      421 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
      481 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
      541 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
      601 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
      661 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
      721 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
      781 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
      841 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
      901 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
      961 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     1021 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     1081 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     1141 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     1201 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     1261 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     1321 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     1381 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     1441 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     1501 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     1561 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1621 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1681 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1741 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     1801 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1861 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1921 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1981 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2041 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2101 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2161 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2221 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2281 tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc
     2341 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
     2401 agtggccaac tccatcacta ggggttcctg cggccgcacg cgtaaaaaaa aaaagagtgg
     2461 agcagcagga ggacagctgt tccctcctga ctgcagcacg cttgcttcaa ggctttcttc
     2521 acttaggatg gtgttggctg tgcatttctc acatacagac tttgttatgt tgaggtagag
     2581 ctcgggctgg gcataaaagt cagggcagag ccatctattg cttacatttg cttctgggat
     2641 ccagatcttt cgaagctagc gctaccggtc gccaccatgg tctccaaggg cgaggagctg
     2701 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg atgtcaacgg ccacaagttc
     2761 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc
     2821 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gggctacggc
     2881 gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2941 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aagacgacgg caactacaag
     3001 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3061 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3121 cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
     3181 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
     3241 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccaagctg
     3301 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     3361 gggatcactc tcggcatgga cgagctgtac aagggctctg gtgctaccaa cttctcactg
     3421 ttgaaacagg caggggatgt agaggagaat ccagggcctg gtgctagtgg agactacaaa
     3481 gaccatgacg gagattataa agatcatgac atcgattaca aggatgacga tgacaagtcc
     3541 ggactcagat ctggaggctc cggaggccca gagccagcga agtctgctcc cgccccgaaa
     3601 aagggctcca agaaggcggt gactaaggcg cagaagaaag gcggcaagaa gcgcaagcgc
     3661 agccgcaagg agagctattc catctatgtg tacaaggttc tgaagcaggt ccaccctgac
     3721 accggcattt cgtccaaggc catgggcatc atgaattcgt ttgtgaacga cattttcgag
     3781 cgcatcgcag gagaggcttc ccgcctggcg cattacaaca agcgctcgac catcacctcc
     3841 cgggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa gcacgccgtg
     3901 tccgagggta ctaaggccat caccaagtac accagcgcta agtaatgagg cgcgccgcgg
     3961 ccgcgaattc gatatcataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     4021 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     4081 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttagtt
     4141 cttgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     4201 ttgggcactg acaattccgt ggctcgagag atcttcgact gtgccttcta gttgccagcc
     4261 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4321 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4381 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatga
     4441 gatctcacgt gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct
     4501 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     4561 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     4621 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     4681 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4741 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4801 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4861 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgat
//
