LOCUS       sequence-469876-        6817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a EGFP-based control sponge in a Cre recombinase-dependent
            manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6817)
  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 6817)
  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-Ctrl-sponge
FEATURES             Location/Qualifiers
     source          1..6817
                     /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    4373..4397
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4726..5439,5440..5442,5443..5445))
                     /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(4729..5439,5440..5442,5443..5445))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4771..4792)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5120..5139
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5379..5400
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    5485..5518
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5529..5562
                     /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    5586..6174
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5639..5659)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6057..6068)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6209..6226)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6215..6422
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6445..6585
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6445..6574
                     /label=AAV2 ITR
     rep_origin      join(6660..6817,1..298)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6747..6766)
                     /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 cttacttgta cgcccacttt
     4381 gtacaagaaa gctgggtgtc gacgcatatc gatggggccc tacgacgctt ttgtacgcca
     4441 tattctacga cgcttttgta cgccgggacc ctacgacgct tttgtacgcc atattctacg
     4501 acgcttttgt acgccgggac cctacgacgc ttttgtacgc catattctac gacgcttttg
     4561 tacgccggga ctctacgacg cttttgtacg ccatattcta cgacgctttt gtacgccggg
     4621 actctacgac gcttttgtac gccatattct acgacgcttt tgtacgccgg gacccagatc
     4681 tccacattcc atagtgagca aatgaccgat taagtcctcc acactttact tgtacagctc
     4741 gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca tgtgatcgcg
     4801 cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag
     4861 cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct
     4921 gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc
     4981 ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt
     5041 gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc
     5101 gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc
     5161 ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg
     5221 gtagcggctg aagcactgca cgccgtaggt cagggtggtc acgagggtgg gccagggcac
     5281 gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc
     5341 ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag
     5401 gatgggcacc accccggtga acagctcctc gcccttgctc accatcatgg tggcctcgag
     5461 cttttttgta caaggccggc cttgataact tcgtataatg tatgctatac gaagttatcc
     5521 gaatcgcaat aacttcgtat aaagtatcct atacgaagtt atcgaagatc atcaagctta
     5581 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     5641 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     5701 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     5761 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     5821 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     5881 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     5941 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     6001 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     6061 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     6121 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     6181 accgagcgct gctcgagaga tcgatctgcc tcgactgtgc cttctagttg ccagccatct
     6241 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     6301 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     6361 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     6421 gacacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     6481 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     6541 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     6601 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     6661 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     6721 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     6781 gttcgccggc tttccccgtc aagctctaaa tcggggg
//
