LOCUS       sequence-469860-        6875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP and miR-137 primary transcript 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-pri-miR-137
FEATURES             Location/Qualifiers
     source          1..6875
                     /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..1455
                     /label=chicken beta-actin promoter
     intron          1456..2472
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2396..2418
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2480..2499
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(2558..2591)
                     /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(2602..2635)
                     /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)."
     CDS             complement(join(3069..3782,3783..3785,3786..3788))
                     /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(3072..3782,3783..3785,3786..3788))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3114..3135)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3463..3482
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3722..3743
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3828..3861
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3872..3905
                     /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    3929..4517
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3982..4002)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4400..4411)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4552..4569)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4558..4765
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4788..4928
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4788..4917
                     /label=AAV2 ITR
     rep_origin      5003..5458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5090..5109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5300..5321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5475..5494)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5594..5616
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5654..5672)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5740..5844
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5845..5913,5914..6705)
                     /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(6063..6082)
                     /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 gggggggggc gcgcggcggg gggggcgggg
     1321 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
     1381 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     1441 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
     1501 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
     1561 gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt
     1621 ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc
     1681 tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc
     1741 ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag
     1801 gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc
     1861 tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg
     1921 caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg
     1981 ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg
     2041 gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc
     2101 ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa
     2161 tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg
     2221 aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag
     2281 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag
     2341 cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
     2401 ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc
     2461 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa
     2521 gaattccgta cggccggtac ccttcaggtc gacgtcgata acttcgtata gcatacatta
     2581 tacgaagtta tccgaatcgc aataacttcg tataggatac tttatacgaa gttatttggc
     2641 gcgcctctgg gtcaccagag gagccaggaa gatccagaac gaaaccaccc gaggaaaaga
     2701 aaacataaaa agtgctacct tggcaaccgg gagcttttag tggccagctg gtgggctggg
     2761 aggggcggcc gctgcccgcc tgccgctggt actctcctcg actacgcgta ttcttaagca
     2821 ataacaacgt aatccgtatt atccacccaa gaatacccgt caccgaagag agtcagaggg
     2881 ccaagctgcc gctgcctctg cctctgctac cgctgtcact gctgccgctg ccgctgccgc
     2941 caccgccgcc gccgccagaa agaactcttg ctgcggagcc cgcccctggc tagggctggg
     3001 ctgagcttga ccgggaccag atctccacat tccatagtga gcaaatgacc gattaagtcc
     3061 tccacacttt acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac
     3121 tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg
     3181 ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg
     3241 tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc
     3301 ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac
     3361 tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg
     3421 cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg
     3481 tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag
     3541 tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg
     3601 gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc
     3661 agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt
     3721 acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg
     3781 ctcaccatca tggtggcctc gagctttttt gtacaaggcc ggccttgata acttcgtata
     3841 atgtatgcta tacgaagtta tccgaatcgc aataacttcg tataaagtat cctatacgaa
     3901 gttatcgaag atcatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     3961 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     4021 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     4081 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     4141 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     4201 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     4261 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     4321 gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac
     4381 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     4441 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     4501 ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatcgatct gcctcgactg
     4561 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4621 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     4681 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     4741 aagacaatag caggcatgct ggggacacgt gcggaccgag cggccgcagg aacccctagt
     4801 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     4861 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     4921 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4981 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5041 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     5101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     5221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     5341 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     5401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     5461 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5521 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5581 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5641 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5701 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5761 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5821 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5881 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5941 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6001 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6061 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6121 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6181 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6241 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6301 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6361 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6421 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6481 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6541 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6601 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6661 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6721 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6781 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6841 gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
