LOCUS       sequence-469891-        6044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the mCherry-based sensor for miR-133. 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-133-MRE
FEATURES             Location/Qualifiers
     source          1..6044
                     /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..791
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   662..791
                     /label=AAV2 ITR
     protein_bind    798..818
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    947..1081
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(984..1003)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1117..1818)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    1832..1856
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(1878..2081)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1907..1927)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2082..2385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    2461..2552
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(2493..2512)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    2566..2614
                     /note="synthetic polyadenylation signal"
     protein_bind    2615..2635
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        2642..2853
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2878..2887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2884..3594
                     /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(3029..3047)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3297..3316
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3736..3753)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3742..3949
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3957..4097
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3957..4086
                     /label=AAV2 ITR
     rep_origin      4172..4627
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4259..4278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4469..4490
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4644..4663)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4763..4785
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4823..4841)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4909..5013
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5014..5082,5083..5874)
                     /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(5232..5251)
                     /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 ccgggcaaag cccgggcgtc gggcgacctt
      721 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      781 taggggttcc ttctagacaa ctttgtatag aaaagttgcc cttaattaat cgagagatct
      841 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
      901 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
      961 agcttataat ggttacaaat aaggcaatag catcacaaat ttcacaaata aggcattttt
     1021 ttcactgcat tctagttttg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     1081 ctcgcggccg cactatgtat aataaagttg ggtacctcaa ttaagcttgt gccccagttt
     1141 gctagggagg tcgcagtatc tggccactgc cacctcgtgc tgctcgacgt aggtctcgtt
     1201 gttggcctcc ttgattcttt ccagtctgta gtccacatag tagacgccag gcatcttgag
     1261 gttcttagcg ggtttcttgg atctatatgt ggtcttggcg tttgcgatca gatggctccc
     1321 gcccacgagc ttcagggcca tgtcgtttct gccttccagg ccgccgtcag cggggtacag
     1381 cgtctcggtg aaggcctccc agccgagtgt tttcttctgc atcacagggc cgttggatgt
     1441 gaagttcacc cctctgatct tgacgttgta gatgaggcag ccgtcctgga ggctggtgtc
     1501 ctgggtagcg gtcagcacgc ccccgtcttc gtatgtggtg actctctccc atgtgaagcc
     1561 ctcagggaag gactgcttga agaagtcggg gatgccctgg gtgtggttga tgaaggtctt
     1621 gctgccgtag aggaagctag tagccaggat gtcgaaggcg aaggggagag ggccgccctc
     1681 gaccaccttg attctcatgg tctgggtgcc ctcgtagggc ttgccttcgc cctcggatgt
     1741 gcacttgaag tgatggttgt ccacggtgcc ctccatgtac agcttcatgt gcatgttctc
     1801 cttaatcagc tcgctcatgg tggcggcgcg ccccactttg tacaagaaag ctgggtgatc
     1861 tgacggttca ctaaaccagc tctgcttata tagacctccc accgtacacg cctaccgccc
     1921 atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc
     1981 caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac
     2041 cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact
     2101 aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc
     2161 caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact
     2221 tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa
     2281 gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg
     2341 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat
     2401 gggctatgaa ctaatgaccc cgtaattgat tactattaat aactactcga gagcctgctt
     2461 agagaaatgt tctggcacct gcacttgcac tggggacagc ctattttgct agtttgtttt
     2521 gtttcgtttt gttttgatgg agagcgtatg ttagtactat cgattcacac aaaaaaccaa
     2581 cacacagatg taatgaaaat aaagatattt tattcaactt tgtatagaaa agttgactag
     2641 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2701 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2761 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2821 ctttttcgca acgggtttgc cgccagaaca caggcgtacg gccaccatga gaccggtgcc
     2881 accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
     2941 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     3001 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     3061 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
     3121 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     3181 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     3241 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     3301 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     3361 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     3421 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     3481 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     3541 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gtaacaactt
     3601 ggatccagtg tggaggactt aatcggtcat ttgctcacta tggaatgtgg catcaaattg
     3661 ctgaggacca aatttttaat ctgttcccct ttgaatacag aatccacgcg tgaattccta
     3721 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3781 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3841 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3901 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag ggccgcagga
     3961 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4021 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4081 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4141 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4201 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     4261 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4321 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4381 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4441 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4501 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     4561 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4621 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4681 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4741 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4801 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4861 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4921 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4981 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5041 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5101 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5161 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5221 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5281 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5341 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5401 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5461 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5521 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5581 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5641 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5701 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     5761 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5821 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5881 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5941 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6001 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
