LOCUS       sequence-469872-        6875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a EGFP-based sponge for miR-133 in a Cre 
            recombinase-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6875)
  AUTHORS   Zolboot N, Xiao Y, Du JX, Ghanem MM, Choi SY, Junn MJ, Zampa F, 
            Huang Z, MacRae IJ, Lippi G
  TITLE     MicroRNA mechanisms instructing Purkinje cell specification.
  JOURNAL   Neuron. 2025 Apr 1:S0896-6273(25)00181-3. doi: 
            10.1016/j.neuron.2025.03.009.
  PUBMED    40179877
REFERENCE   2  (bases 1 to 6875)
  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. 
            2025 Apr 1:S0896-6273(25)00181-3. doi: 
            10.1016/j.neuron.2025.03.009."
COMMENT     Sequence Label: pAAV[FLEX]-CAG-EGFP-miR-133-sponge
FEATURES             Location/Qualifiers
     source          1..6875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     140..161
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(315..334)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     434..456
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(494..512)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        580..684
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(685..753,754..1545)
                     /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(903..922)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1716..2304
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2205..2224
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2366..2495
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        2513..2892
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2894..3171
                     /label=chicken beta-actin promoter
     intron          3172..4188
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4112..4134
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4196..4215
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(4274..4307)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(4318..4351)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4362..4386
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4784..5497,5498..5500,5501..5503))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(4787..5497,5498..5500,5501..5503))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4829..4850)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5178..5197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5437..5458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    5543..5576
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5587..5620
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    5644..6232
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5697..5717)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6115..6126)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6267..6284)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6273..6480
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6503..6643
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6503..6632
                     /label=AAV2 ITR
     rep_origin      join(6718..6875,1..298)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6805..6824)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
       61 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
      121 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
      181 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
      241 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
      301 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
      361 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
      421 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
      481 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
      541 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
      601 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
      661 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
      721 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
      781 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
      841 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
      901 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
      961 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     1021 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     1081 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     1141 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     1201 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     1261 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     1321 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     1381 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     1441 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     1501 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     1561 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     1621 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     1681 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1741 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1801 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1861 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1921 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1981 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2041 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2101 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2161 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2221 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2281 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2341 ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag
     2401 gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
     2461 agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgtg tcgacattga
     2521 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     2581 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2641 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2701 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2761 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2821 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2881 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     2941 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     3001 gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg
     3061 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     3121 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     3181 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     3241 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     3301 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
     3361 gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
     3421 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     3481 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
     3541 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
     3601 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc
     3661 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
     3721 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     3781 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
     3841 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
     3901 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     3961 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     4021 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     4081 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     4141 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     4201 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tccgtacggc cggtaccctt
     4261 caggtcgacg tcgataactt cgtatagcat acattatacg aagttatccg aatcgcaata
     4321 acttcgtata ggatacttta tacgaagtta tttggcgcgc ccccactttg tacaagaaag
     4381 ctgggtgtcg acgcatatcg atgggaccct ttggtccagg caaccagcta atattctttg
     4441 gtccaggcaa ccagctagga ccctttggtc caggcaacca gctaatattc tttggtccag
     4501 gcaaccagct aggacccttt ggtccaggca accagctaat attctttggt ccaggcaacc
     4561 agctaggacc ctttggtcca ggcaaccagc taatattctt tggtccaggc aaccagctag
     4621 gaccctttgg tccaggcaac cagctaatat tctttggtcc aggcaaccag ctaggaccct
     4681 ttggtccagg caaccagcta atattctttg gtccaggcaa ccagctagga cccagatctc
     4741 cacattccat agtgagcaaa tgaccgatta agtcctccac actttacttg tacagctcgt
     4801 ccatgccgag agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct
     4861 tctcgttggg gtctttgctc agggcggact gggtgctcag gtagtggttg tcgggcagca
     4921 gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc
     4981 cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg
     5041 ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc
     5101 cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga
     5161 acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct
     5221 ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt
     5281 agcggctgaa gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg
     5341 gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct
     5401 cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga
     5461 tgggcaccac cccggtgaac agctcctcgc ccttgctcac catcatggtg gcctcgagct
     5521 tttttgtaca aggccggcct tgataacttc gtataatgta tgctatacga agttatccga
     5581 atcgcaataa cttcgtataa agtatcctat acgaagttat cgaagatcat caagcttatc
     5641 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     5701 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     5761 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     5821 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     5881 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     5941 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     6001 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     6061 ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     6121 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     6181 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     6241 cgagcgctgc tcgagagatc gatctgcctc gactgtgcct tctagttgcc agccatctgt
     6301 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     6361 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     6421 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     6481 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     6541 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     6601 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     6661 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     6721 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     6781 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     6841 tcgccggctt tccccgtcaa gctctaaatc ggggg
//
