LOCUS       sequence-325690-        4713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE2004m_3xC2 is an optimized enhancer sequence designed to drive 
            AAV-mediated transgene expression in L4/5_IT cortical neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4713)
  AUTHORS   Graybuck LT, Daigle TL, Sedeno-Cortes AE, Walker M, Kalmbach B, Lenz
            GH, Morin E, Nguyen TN, Garren E, Bendrick JL, Kim TK, Zhou T, 
            Mortrud M, Yao S, Siverts LA, Larsen R, Gore BB, Szelenyi ER, Trader
            C, Balaram P, van Velthoven CTJ, Chiang M, Mich JK, Dee N, Goldy J, 
            Cetin AH, Smith K, Way SW, Esposito L, Yao Z, Gradinaru V, Sunkin 
            SM, Lein E, Levi BP, Ting JT, Zeng H, Tasic B
  TITLE     Enhancer viruses for combinatorial cell-subclass-specific labeling.
  JOURNAL   Neuron. 2021 Mar 23. pii: S0896-6273(21)00159-8. doi: 
            10.1016/j.neuron.2021.03.011.
  PUBMED    33789083
REFERENCE   2  (bases 1 to 4713)
  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: "Neuron. 
            2021 Mar 23. pii: S0896-6273(21)00159-8. doi: 
            10.1016/j.neuron.2021.03.011."
COMMENT     Sequence Label: AiP11818-pAAV-AiE2004m_3xC2-minCMV-SYFP2-WPRE3...
FEATURES             Location/Qualifiers
     source          1..4713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        625..663
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      725..734
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(731..733,734..736,737..1447)
                     /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(776..797)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1037..1056)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1384..1405
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1540..1560)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    1747..1858
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1976..2116
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1976..2105
                     /label=AAV2 ITR
     rep_origin      2191..2646
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2278..2297)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2488..2509
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2663..2682)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2782..2804
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2842..2860)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2928..3032
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3033..3101,3102..3893)
                     /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(3251..3270)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4064..4652
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4553..4572
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtttaccc tgttgaatat cactgactca ctaacttgca
      181 ttgccatgct aacttgcttt cagagagatc tcagaacaca tcatcttctg ctatttcaat
      241 acatgcacat taatttccta tcaacgtgtg ctgatcagga actctgtaat ctggcaccgt
      301 taccctgttg aatatcactg actcactaac ttgcattgcc atgctaactt gctttcagag
      361 agatctcaga acacatcatc ttctgctatt tcaatacatg cacattaatt tcctatcaac
      421 gtgtgctgat caggaactct gtaatctggc accgttaccc tgttgaatat cactgactca
      481 ctaacttgca ttgccatgct aacttgcttt cagagagatc tcagaacaca tcatcttctg
      541 ctatttcaat acatgcacat taatttccta tcaacgtgtg ctgatcagga actctgtaat
      601 ctggcaccgc ttaaggagct cagaggtagg cgtgtacggt gggaggccta tataagcaga
      661 gctggtttag tgaaccgtca gatcgcctgg ggatccagat ctttcgaagc tagcgctacc
      721 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      781 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      841 tgccacctac ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc
      901 ctggcccacc ctcgtgacca ccctgggcta cggcgtgcag tgcttcgccc gctaccccga
      961 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1021 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1081 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1141 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa
     1201 gcagaagaac ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt
     1261 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1321 cgacaaccac tacctgagct accagtccaa gctgagcaaa gaccccaacg agaagcgcga
     1381 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1441 gtacaagtaa gtcgacggcg cgccgcggcc gcgaattcga tatcataatc aacctctgga
     1501 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     1561 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     1621 ctcctccttg tataaatcct ggttagttct tgccacggcg gaactcatcg ccgcctgcct
     1681 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg ctcgagagat
     1741 cttcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1801 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1861 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     1921 aggattggga agacaatagc aggcatgaga tctcacgtgc ggaccgagcg gccgcaggaa
     1981 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     2041 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     2101 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     2161 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     2221 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     2281 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2341 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2401 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2461 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2521 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     2581 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     2641 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     2701 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2761 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2821 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2881 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2941 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3001 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     3061 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     3121 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     3181 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     3241 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     3301 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3361 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3421 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3481 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3541 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3601 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3661 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3721 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3781 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3841 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3901 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3961 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4021 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4081 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4141 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4201 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4261 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4321 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4381 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4441 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4501 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4561 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4621 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4681 tcctggcctt ttgctggcct tttgctcaca tgt
//
