LOCUS       sequence-470622-        5235 bp    DNA     circular SYN 30-APR-2025
DEFINITION  AiE0966m 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 5235)
  AUTHORS   Ben-Simon Y, Hooper M, Narayan S, Daigle T, Dwivedi D, Way SW, Oster
            A, Stafford DA, Mich JK, Taormina MJ, Martinez RA, Opitz-Araya X, 
            Roth JR, Allen S, Ayala A, Bakken TE, Barcelli T, Barta S, Bendrick 
            J, Bertagnolli D, Bowlus J, Boyer G, Brouner K, Casian B, Casper T, 
            Chakka AB, Chakrabarty R, Chance RK, Chavan S, Departee M, Donadio 
            N, Dotson N, Egdorf T, Gabitto M, Garcia J, Gary A, Gasperini M, 
            Goldy J, Gore BB, Graybuck L, Greisman N, Haeseleer F, Halterman C, 
            Helback O, Hockemeyer D, Huang C, Huff S, Hunker A, Johansen N, 
            Juneau Z, Kalmbach B, Khem S, Kussick E, Kutsal R, Larsen R, Lee C, 
            Lee AY, Leibly M, Lenz GH, Liang E, Lusk N, Malone J, Mollenkopf T, 
            Morin E, Newman D, Ng L, Ngo K, Omstead V, Oyama A, Pham T, Pom CA, 
            Potekhina L, Ransford S, Rette D, Rimorin C, Rocha D, Ruiz A, 
            Sanchez REA, Sedeno-Cortes A, Sevigny JP, Shapovalova N, Shulga L, 
            Sigler AR, Siverts LA, Somasundaram S, Stewart K, Szelenyi E, Tieu 
            M, Trader C, van Velthoven CTJ, Walker M, 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 J, Tasic B
  TITLE     A suite of enhancer AAVs and transgenic mouse lines for genetic 
            access to cortical cell types.
  JOURNAL   bioRxiv [Preprint]. 2024 Sep 26:2024.06.10.597244. doi: 
            10.1101/2024.06.10.597244.
  PUBMED    38915722
REFERENCE   2  (bases 1 to 5235)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Sep 26:2024.06.10.597244. doi: 
            10.1101/2024.06.10.597244."
COMMENT     Sequence Label: AiP13303 -
            pAAV-AiE0966m-minBG-SYFP2-P2A-3XFLA...
FEATURES             Location/Qualifiers
     source          1..5235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   352..481
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(543..1131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(623..642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1302..2093,2094..2162))
                     /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     1925..1944
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2163..2267)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2335..2353
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2391..2413)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2513..2532
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2549..3004)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2686..2707)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2898..2917
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3079..3219
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3090..3219
                     /label=AAV2 ITR
     polyA_signal    3337..3448
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3635..3655
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3742..4119)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     CDS             complement(4150..4215)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(4228..4284)
                     /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             complement(join(4294..5004,5005..5007,5008..5010))
                     /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(4336..4357)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4685..4704
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      5007..5016
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 tcctcatgcc caaactcgac tgcctgtcct tcctacaccc ctaggagtcg cctgaaccac
       61 accagtataa ctcaactgca gtatggggag ggctcacatt gcctcccctt ggtgccaagg
      121 aaccaattac agttgtacac atgtgacgtg ccaccctcct gttcctggct ggggacccgg
      181 actcatccag ggatggattg gcttttgtgc agaggtcccc ttggaaaggc agagagccag
      241 ctagctgcat agctggtttg ctccagaact agggagatgc ctctacactc ccacctacct
      301 gtacccaggc tatcttttta gaaacacctg ggccagtacg cgtgcggccg caggaacccc
      361 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
      421 caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
      481 gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      541 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      601 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      661 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      721 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      781 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      841 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      901 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      961 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     1021 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1081 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1141 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1201 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1261 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1321 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1381 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1441 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1501 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1561 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1621 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1681 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1741 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1801 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1861 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1921 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1981 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2041 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2101 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2161 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2221 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     2281 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     2341 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2401 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     2461 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     2521 gagcagattg tactgagagt gcaccataaa attgtaaacg ttaatatttt gttaaaattc
     2581 gcgttaaatt tttgttaaat cagctcattt tttaaccaat agaccgaaat cggcaaaatc
     2641 ccttataaat caaaagaata gcccgagata gagttgagtg ttgttccagt ttggaacaag
     2701 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     2761 gatggcccac tacgtgaacc atcacccaaa tcaagttttt tggggtcgag gtgccgtaaa
     2821 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     2881 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctaaggc gctggcaagt
     2941 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3001 gcgtactatg gttgctttga cgtatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     3061 aataccgcat caggcgcccc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc
     3121 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
     3181 gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgctcg gtccgcacgt
     3241 gagatctcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca
     3301 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
     3361 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
     3421 acagatggct ggcaactaga aggcacagtc gaagatctct cgagccacgg aattgtcagt
     3481 gcccaacagc cgagcccctg tccagcagcg ggcaaggcag gcggcgatga gttccgccgt
     3541 ggcaagaact aaccaggatt tatacaagga ggagaaaatg aaagccatac gggaagcaat
     3601 agcatgatac aaaggcatta aagcagcgta tccacatagc gtaaaaggag caacatagtt
     3661 aagaatacca gtcaatcttt cacaaatttt gtaatccaga ggttgattat gatatcgaat
     3721 tcgcggccgc ggcgcgcctc attacttagc gctggtgtac ttggtgatgg ccttagtacc
     3781 ctcggacacg gcgtgcttgg ccaactcccc aggcagcagc aggcgcacgg ccgtctggat
     3841 ctcccgggag gtgatggtcg agcgcttgtt gtaatgcgcc aggcgggaag cctctcctgc
     3901 gatgcgctcg aaaatgtcgt tcacaaacga attcatgatg cccatggcct tggacgaaat
     3961 gccggtgtca gggtggacct gcttcagaac cttgtacaca tagatggaat agctctcctt
     4021 gcggctgcgc ttgcgcttct tgccgccttt cttctgcgcc ttagtcaccg ccttcttgga
     4081 gccctttttc ggggcgggag cagacttcgc tggctctggg cctccggagc ctccagatct
     4141 gagtccggac ttgtcatcgt catccttgta atcgatgtca tgatctttat aatctccgtc
     4201 atggtctttg tagtctccac tagcaccagg ccctggattc tcctctacat cccctgcctg
     4261 tttcaacagt gagaagttgg tagcaccaga gcccttgtac agctcgtcca tgccgagagt
     4321 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     4381 tttgctcagc ttggactggt agctcaggta gtggttgtcg ggcagcagca cggggccgtc
     4441 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgccgccgt cctcgatgtt
     4501 gtggcggatc ttgaagttgg ccttgatgcc gttcttctgc ttgtcggcgg tgatatagac
     4561 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     4621 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     4681 gcgggtcttg tagttgccgt cgtctttgaa gaagatggtg cgctcctgga cgtagccttc
     4741 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc gggcgaagca
     4801 ctgcacgccg tagcccaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     4861 ggtgcagatc agcttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     4921 cacgctgaac ttgtggccgt tgacatcgcc gtccagctcg accaggatgg gcaccacccc
     4981 ggtgaacagc tcctcgccct tggagaccat ggtggcgacc ggtagcgcta gcttcgaaag
     5041 atctggatcc cagaagcaaa tgtaagcaat agatggctct gccctgactt ttatgcccag
     5101 cccgagctct cgcaggaaaa caggatggac ttgtagaatg ttgtcgattg cagctttgca
     5161 cacggatctg agagtgtcta ccagcctgcc agcctgttca ccagcgcttc tcttagcctt
     5221 atagccactc gtggc
//
