LOCUS       sequence-462035-        5455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AiE2135m_3xC2 is an optimized enhancer sequence, designed to induce 
            Flp-dependent recombination in specific populations of brain cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5455)
  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 5455)
  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: AiP1537 - pAAV-AiE2135m_3xC2-minBG-FlpO-HGHpA
FEATURES             Location/Qualifiers
     source          1..5455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      890..899
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(896..901,902..922,923..2194)
                     /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"
     polyA_signal    2202..2678
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2401..2420)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2718..2858
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2718..2847
                     /label=AAV2 ITR
     rep_origin      2933..3388
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3020..3039)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3230..3251
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3405..3424)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3524..3546
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3584..3602)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3670..3774
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3775..3843,3844..4635)
                     /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(3993..4012)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4806..5394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5295..5314
                     /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 gcgttccaag gccccctttc ctgccaaaca aagcccccag
      181 ccacccgact gccgcccaac aggcacctgg ggcctctggg cctgggaatc aaagcctgtt
      241 ttgcaaagga ggcccagtgt ttggagaaca cccatcccct gctgcccggt ccttcaaaga
      301 accggcattg ttctgcgctg agaaggcgcc tcttgacgcg ctgggcaggg tcggcttgac
      361 gctggtggac ctccaaggcc ccctttcctg ccaaacaaag cccccagcca cccgactgcc
      421 gcccaacagg cacctggggc ctctgggcct gggaatcaaa gcctgttttg caaaggaggc
      481 ccagtgtttg gagaacaccc atcccctgct gcccggtcct tcaaagaacc ggcattgttc
      541 tgcgctgaga aggcgcctct tgacgcgctg ggcagggtcg gcttgacgct ggtggacctc
      601 caaggccccc tttcctgcca aacaaagccc ccagccaccc gactgccgcc caacaggcac
      661 ctggggcctc tgggcctggg aatcaaagcc tgttttgcaa aggaggccca gtgtttggag
      721 aacacccatc ccctgctgcc cggtccttca aagaaccggc attgttctgc gctgagaagg
      781 cgcctcttga cgcgctgggc agggtcggct tgacgctggt ggaccgagct cgggctgggc
      841 ataaaagtca gggcagagcc atctattgct tacatttgct tctggcgtgg ccaccatggc
      901 tcctaagaag aagaggaagg tgatgagcca gttcgacatc ctgtgcaaga ccccccccaa
      961 ggtgctggtg cggcagttcg tggagagatt cgagaggccc agcggcgaga agatcgccag
     1021 ctgtgccgcc gagctgacct acctgtgctg gatgatcacc cacaacggca ccgccatcaa
     1081 gagggccacc ttcatgagct acaacaccat catcagcaac agcctgagct tcgacatcgt
     1141 gaacaagagc ctgcagttca agtacaagac ccagaaggcc accatcctgg aggccagcct
     1201 gaagaagctg atccccgcct gggagttcac catcatccct tacaacggcc agaagcacca
     1261 gagcgacatc accgacatcg tgtccagcct gcagctgcag ttcgagagca gcgaggaggc
     1321 cgacaagggc aacagccaca gcaagaagat gctgaaggcc ctgctgtccg agggcgagag
     1381 catctgggag atcaccgaga agatcctgaa cagcttcgag tacaccagca ggttcaccaa
     1441 gaccaagacc ctgtaccagt tcctgttcct ggccacattc atcaactgcg gcaggttcag
     1501 cgacatcaag aacgtggacc ccaagagctt caagctggtg cagaacaagt acctgggcgt
     1561 gatcattcag tgcctggtga ccgagaccaa gacaagcgtg tccaggcaca tctacttttt
     1621 cagcgccaga ggcaggatcg accccctggt gtacctggac gagttcctga ggaacagcga
     1681 gcccgtgctg aagagagtga acaggaccgg caacagcagc agcaacaagc aggagtacca
     1741 gctgctgaag gacaacctgg tgcgcagcta caacaaggcc ctgaagaaga acgcccccta
     1801 ccccatcttc gctatcaaga acggccctaa gagccacatc ggcaggcacc tgatgaccag
     1861 ctttctgagc atgaagggcc tgaccgagct gacaaacgtg gtgggcaact ggagcgacaa
     1921 gagggcctcc gccgtggcca ggaccaccta cacccaccag atcaccgcca tccccgacca
     1981 ctacttcgcc ctggtgtcca ggtactacgc ctacgacccc atcagcaagg agatgatcgc
     2041 cctgaaggac gagaccaacc ccatcgagga gtggcagcac atcgagcagc tgaagggcag
     2101 cgccgagggc agcatcagat accccgcctg gaacggcatc atcagccagg aggtgctgga
     2161 ctacctgagc agctacatca acaggcggat ctgagaatta cgggtggcat ccctgtgacc
     2221 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     2281 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     2341 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     2401 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     2461 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     2521 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     2581 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     2641 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
     2701 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     2761 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     2821 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     2881 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     2941 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3001 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3061 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3121 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     3181 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3241 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     3301 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3361 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     3421 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3481 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3541 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     3601 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     3661 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3721 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3781 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3841 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3901 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3961 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4021 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4081 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4141 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4201 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4261 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4321 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4381 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4441 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4501 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4561 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4621 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4681 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4741 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4801 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4861 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4921 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4981 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5041 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5101 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5161 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5221 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5281 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5341 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5401 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
