LOCUS       sequence-469879-        6879 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a EGFP-based sponge for miR-206 in a Cre 
            recombinase-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6879)
  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 6879)
  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-206-sponge
FEATURES             Location/Qualifiers
     source          1..6879
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        798..1177
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1179..1456
                     /label=chicken beta-actin promoter
     intron          1457..2473
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2397..2419
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2481..2500
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(2559..2592)
                     /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(2603..2636)
                     /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    2647..2671
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3073..3786,3787..3789,3790..3792))
                     /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(3076..3786,3787..3789,3790..3792))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3118..3139)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3467..3486
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3726..3747
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3832..3865
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3876..3909
                     /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    3933..4521
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3986..4006)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4404..4415)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4556..4573)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4562..4769
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4792..4932
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4792..4921
                     /label=AAV2 ITR
     rep_origin      5007..5462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5094..5113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5304..5325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5479..5498)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5598..5620
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5658..5676)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5744..5848
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5849..5917,5918..6709)
                     /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(6067..6086)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgtgtcgac attgattatt gactagttat taatagtaat caattacggg
      841 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      901 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      961 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1021 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     1081 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1141 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
     1201 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
     1261 attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg
     1321 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
     1381 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
     1441 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
     1501 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     1561 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
     1621 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
     1681 ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc
     1741 cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga
     1801 ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg
     1861 ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct
     1921 gcaacccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg
     1981 gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg
     2041 ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg
     2101 cccccggagc gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta
     2161 atcgtgcgag agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg
     2221 gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa
     2281 ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca
     2341 gcctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
     2401 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
     2461 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
     2521 agaattccgt acggccggta cccttcaggt cgacgtcgat aacttcgtat agcatacatt
     2581 atacgaagtt atccgaatcg caataacttc gtataggata ctttatacga agttatttgg
     2641 cgcgccccca ctttgtacaa gaaagctggg tgtcgacgca tatcgatggg accctggaat
     2701 gtgttaagtg tgtggatatt ctggaatgtg ttaagtgtgt gggggaccct ggaatgtgtt
     2761 aagtgtgtgg atattctgga atgtgttaag tgtgtggggg accctggaat gtgttaagtg
     2821 tgtggatatt ctggaatgtg ttaagtgtgt gggggaccct ggaatgtgtt aagtgtgtgg
     2881 atattctgga atgtgttaag tgtgtgggga ccctggaatg tgttaagtgt gtggatattc
     2941 tggaatgtgt taagtgtgtg ggggaccctg gaatgtgtta agtgtgtgga tattctggaa
     3001 tgtgttaagt gtgtggggac ccagatctcc acattccata gtgagcaaat gaccgattaa
     3061 gtcctccaca ctttacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac
     3121 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg
     3181 ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
     3241 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt
     3301 caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt
     3361 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     3421 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     3481 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
     3541 gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag
     3601 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
     3661 ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc
     3721 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     3781 cttgctcacc atcatggtgg cctcgagctt ttttgtacaa ggccggcctt gataacttcg
     3841 tataatgtat gctatacgaa gttatccgaa tcgcaataac ttcgtataaa gtatcctata
     3901 cgaagttatc gaagatcatc aagcttatcg ataatcaacc tctggattac aaaatttgtg
     3961 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     4021 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     4081 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     4141 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     4201 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     4261 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     4321 cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg attctgcgcg
     4381 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     4441 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     4501 ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatcg atctgcctcg
     4561 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     4621 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     4681 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     4741 tgggaagaca atagcaggca tgctggggac acgtgcggac cgagcggccg caggaacccc
     4801 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     4861 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     4921 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4981 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     5041 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     5101 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5161 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5221 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5281 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     5341 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     5401 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5461 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5521 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5581 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5641 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5701 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5761 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5821 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5881 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5941 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6001 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6061 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6121 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6181 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6241 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6301 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6361 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6421 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6481 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6541 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6601 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6661 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6721 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6781 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6841 ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
