LOCUS       sequence-494319-        5991 bp    DNA     circular SYN 02-DEC-2025
DEFINITION  Expresses EGFP in mammalian cells regulated by a synthetic 
            microRNA-based dosage compensation circuit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5991)
  TITLE     miRNA circuit modules for precise control of gene expression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5991)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-synmiR-4-mRuby-Pro-EGFP-8×9
FEATURES             Location/Qualifiers
     source          1..5991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      653..1241
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1142..1161
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1303..1432
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1447..1495
                     /note="synthetic polyadenylation signal"
     ncRNA           complement(1699..1713)
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     CDS             complement(1875..2585)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     regulatory      2582..2591
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2607..2818)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        2863..3166
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     regulatory      3404..3413
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3410..3412,3413..3415,3416..4129)
                     /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             join(3410..3412,3413..3415,3416..4126)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3455..3476)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3716..3735)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4063..4084
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4409..4530
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4446..4465)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4500..4519
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4556..4696
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4556..4685
                     /label=AAV2 ITR
     rep_origin      4771..5226
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4858..4877)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5068..5089
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5243..5262)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5362..5384
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5422..5440)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5508..5612
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5613..5681,5682..5991,1..482)
                     /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(5831..5850)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
       61 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
      121 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
      181 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
      241 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
      301 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
      361 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
      421 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
      481 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
      541 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
      601 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      661 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      721 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      781 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
      841 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      901 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      961 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     1021 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     1081 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     1141 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     1201 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     1261 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg
     1321 ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     1381 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
     1441 acgcgtcaca caaaaaacca acacacagat ctaatgaaaa taaagatctt ttattatttc
     1501 ttccacaagc ctgcaggaaa aagaaagagg atgaatggtc aggaaaagtg atttaattta
     1561 taccatttta attcagcttt gtaaaaatgt atcaaagaga tagcaaggta ttcagtttta
     1621 gtaaacaaga taattgctcg aattctagcc ccttgaagtc cgaggcagta ggcatcgtaa
     1681 agatcatgaa attgaataca tctgtggctt cactattcaa tttcatgatc tttacggcgc
     1741 tcactgtcaa cagcaatata ccttctcgag ccttctgttg ggttaagaag tcgaagtaat
     1801 cccagcaagt gtttccaaga tgtgcaggca acgattctgt aaagtactga agcctcattc
     1861 aaacttacct gctacttata tagctcgtcc atgccaccac caaggttgct gtatttggca
     1921 actgccactt ctctttgcac tacgtaggtt tcattgtcac tctcctcgat cctttccagg
     1981 cggtgatcaa cggcatggac accgggcatc ttgatgttcc cgacggtctt ttttgacctg
     2041 taagttgtca cgaagttgca gtgcagatgg ccaccaccat caactttcag tgcgatgtca
     2101 gtgtatcctc tcagaccacc atctgctgga tacatcatct ctgtattagg ctcccaaccc
     2161 ttggtcttct tctgcatcac gggaccattg gagggaaagt ttacccctct gaccttgacg
     2221 ttgtagacga gctcgccatc ctcaaggctg gtgtcctgcg tgacggtgac gactccacca
     2281 tcttcgtatc tcgtaactct ttcccaagta aaaccctcag gaaaggactg tttaaagaaa
     2341 tcagggatgt cggccgggta cttgataaag gtacggctgc catacatgaa cgacgtggca
     2401 agaatgtcaa aggcaaatgg caggggtcct ccctcgatga ctttgatcct catggtttgc
     2461 actccctcgt acggtctgcc ttctccttca cctgtgcatt tgaattggtg gccgttgacc
     2521 gaaccttcca tgaccacctt catacgcata ttttccttga tcagctcttc gcccttagac
     2581 accatggtgg cgtcgactca cgacacctgt gttctggcgg caaacccgtt gcgaaaaaga
     2641 acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg aaaaaggcgg
     2701 agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta ggcaccggtt
     2761 caattgccga cccctccccc caacttctcg gggactgtgg gcgatgtgcg ctctgcccac
     2821 tgacgggcac cggagcccta tgacttgcat agctgcgtat ggcgttacat aacttacggt
     2881 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     2941 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3001 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3061 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3121 tcctacttgg cagtacatct acgtattagt catcgctatt accatgaatt gagggcgtca
     3181 ccgctaaggc tccgccccag cctgggctcc acaaccaatg aagggtaatc tcgacaaaga
     3241 gcaaggggtg gggcgcgggc gcgcaggtgc agcagcacac aggctggtcg ggagggcggg
     3301 gcgcgacgtc tgccgtgcgg ggtcccggca tcggttgcgc gcgcgctccc tcctctcgga
     3361 gagagggctg tggtaaaacc cgtccggaaa ccacttaagc ttcgccacca tggtgagcaa
     3421 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3481 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3541 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3601 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     3661 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     3721 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3781 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3841 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     3901 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3961 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     4021 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     4081 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtagt tctttgagta
     4141 ctctgatcag aaattgaata caacaattgt actctgatca gaaattgaat aatcacttgg
     4201 tactctgatc agaaattgaa tacaactgga gtactctgat cagaaattga ataactagtt
     4261 ctttgagtac tctgatcaga aattgaatac aacaattgta ctctgatcag aaattgaata
     4321 atcacttggt actctgatca gaaattgaat acaactggag tactctgatc agaaattgaa
     4381 taagggccca tcgataccga gcgctagcaa cttgtttatt gcagcttata atggttacaa
     4441 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4501 tggtttgtcc aaactcatca atgtatctta tcatgtctgc ggaccgagcg gccgcaggaa
     4561 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     4621 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     4681 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4741 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     4801 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     4861 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4921 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4981 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5041 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5101 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     5161 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     5221 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     5281 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5341 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5401 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5461 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5521 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5581 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5641 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5701 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5761 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5821 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5881 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5941 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc a
//
