LOCUS       sequence-469866-        6044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the mCherry-based sensor for miR-206. TagBFP2 is expressed
            as a transduction reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6044)
  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 6044)
  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-rev(CMV-TagBFP2)-EFS-mCherry-206-MRE
FEATURES             Location/Qualifiers
     source          1..6044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    297..431
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(334..353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(467..1168)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    1182..1206
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(1228..1431)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1257..1277)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1432..1735
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    1811..1902
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(1843..1862)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    1916..1964
                     /note="synthetic polyadenylation signal"
     protein_bind    1965..1985
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        1992..2203
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2228..2237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2234..2944
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2379..2397)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2647..2666
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3086..3103)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3092..3299
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3307..3447
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3307..3436
                     /label=AAV2 ITR
     rep_origin      3522..3977
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3609..3628)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3819..3840
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3994..4013)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4113..4135
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4173..4191)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4259..4363
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4364..4432,4433..5224)
                     /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(4582..4601)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5395..5983
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5884..5903
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgcc cttaattaat
      181 cgagagatct gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
      241 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
      301 tgtttattgc agcttataat ggttacaaat aaggcaatag catcacaaat ttcacaaata
      361 aggcattttt ttcactgcat tctagttttg gtttgtccaa actcatcaat gtatcttatc
      421 atgtctggat ctcgcggccg cactatgtat aataaagttg ggtacctcaa ttaagcttgt
      481 gccccagttt gctagggagg tcgcagtatc tggccactgc cacctcgtgc tgctcgacgt
      541 aggtctcgtt gttggcctcc ttgattcttt ccagtctgta gtccacatag tagacgccag
      601 gcatcttgag gttcttagcg ggtttcttgg atctatatgt ggtcttggcg tttgcgatca
      661 gatggctccc gcccacgagc ttcagggcca tgtcgtttct gccttccagg ccgccgtcag
      721 cggggtacag cgtctcggtg aaggcctccc agccgagtgt tttcttctgc atcacagggc
      781 cgttggatgt gaagttcacc cctctgatct tgacgttgta gatgaggcag ccgtcctgga
      841 ggctggtgtc ctgggtagcg gtcagcacgc ccccgtcttc gtatgtggtg actctctccc
      901 atgtgaagcc ctcagggaag gactgcttga agaagtcggg gatgccctgg gtgtggttga
      961 tgaaggtctt gctgccgtag aggaagctag tagccaggat gtcgaaggcg aaggggagag
     1021 ggccgccctc gaccaccttg attctcatgg tctgggtgcc ctcgtagggc ttgccttcgc
     1081 cctcggatgt gcacttgaag tgatggttgt ccacggtgcc ctccatgtac agcttcatgt
     1141 gcatgttctc cttaatcagc tcgctcatgg tggcggcgcg ccccactttg tacaagaaag
     1201 ctgggtgatc tgacggttca ctaaaccagc tctgcttata tagacctccc accgtacacg
     1261 cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg
     1321 attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg
     1381 tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat
     1441 agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg
     1501 ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat
     1561 atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg
     1621 tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg
     1681 gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa
     1741 ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat aactactcga
     1801 gagcctgctt agagaaatgt tctggcacct gcacttgcac tggggacagc ctattttgct
     1861 agtttgtttt gtttcgtttt gttttgatgg agagcgtatg ttagtactat cgattcacac
     1921 aaaaaaccaa cacacagatg taatgaaaat aaagatattt tattcaactt tgtatagaaa
     1981 agttgactag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     2041 cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     2101 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     2161 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggcgtacg gccaccatga
     2221 gaccggtgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     2281 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     2341 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     2401 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     2461 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     2521 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     2581 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     2641 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     2701 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     2761 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     2821 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     2881 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     2941 gtaacaactt ggatccagtg tggaggactt aatcggtcat ttgctcacta tggaatgtgg
     3001 tctcacactg aaacattcca catttttaat ctgttcccct ttgaatacag aatccacgcg
     3061 tgaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3121 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3181 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     3241 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     3301 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     3361 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     3421 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     3481 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     3541 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     3601 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3661 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3721 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3781 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3841 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     3901 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     3961 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4021 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4081 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4141 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     4201 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4261 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4321 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4381 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4441 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4501 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4561 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4621 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4681 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4741 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4801 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4861 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4921 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4981 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5041 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     5101 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5161 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5221 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5281 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5341 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5401 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5461 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5521 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5581 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5641 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5701 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5761 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5821 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5881 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5941 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6001 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
