LOCUS       sequence-494311-        5864 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 5864)
  TITLE     miRNA circuit modules for precise control of gene expression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5864)
  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            "Unpublished"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-2-mRuby-Pro-EGFP-4×8
FEATURES             Location/Qualifiers
     source          1..5864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      775..1363
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1264..1283
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1425..1554
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1569..1617
                     /note="synthetic polyadenylation signal"
     ncRNA           complement(1821..1835)
                     /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(1997..2707)
                     /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      2704..2713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2729..2940)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        2985..3288
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     regulatory      3526..3535
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3532..3534,3535..3537,3538..4251)
                     /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(3532..3534,3535..3537,3538..4248)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3577..3598)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3838..3857)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4185..4206
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4404..4525
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4441..4460)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4495..4514
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4551..4691
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4551..4680
                     /label=AAV2 ITR
     rep_origin      4766..5221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4853..4872)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5063..5084
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5238..5257)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5357..5379
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5417..5435)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5503..5607
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5608..5676,5677..5864,1..604)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSLIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5826..5845)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
       61 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
      121 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
      181 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
      241 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
      301 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
      361 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
      421 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
      481 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
      541 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
      601 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
      661 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
      721 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
      781 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
      841 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
      901 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
      961 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1021 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     1081 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1141 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1201 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1261 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     1321 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     1381 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg
     1441 cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag
     1501 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc
     1561 gcacgcgtca cacaaaaaac caacacacag atctaatgaa aataaagatc ttttattatt
     1621 tcttccacaa gcctgcagga aaaagaaaga ggatgaatgg tcaggaaaag tgatttaatt
     1681 tataccattt taattcagct ttgtaaaaat gtatcaaaga gatagcaagg tattcagttt
     1741 tagtaaacaa gataattgct cgaattctag ccccttgaag tccgaggcag taggcattga
     1801 tctacaaagt gacataaata catctgtggc ttcactattt atgtcacttt gtagatcagc
     1861 gctcactgtc aacagcaata taccttctcg agccttctgt tgggttaaga agtcgaagta
     1921 atcccagcaa gtgtttccaa gatgtgcagg caacgattct gtaaagtact gaagcctcat
     1981 tcaaacttac ctgctactta tatagctcgt ccatgccacc accaaggttg ctgtatttgg
     2041 caactgccac ttctctttgc actacgtagg tttcattgtc actctcctcg atcctttcca
     2101 ggcggtgatc aacggcatgg acaccgggca tcttgatgtt cccgacggtc ttttttgacc
     2161 tgtaagttgt cacgaagttg cagtgcagat ggccaccacc atcaactttc agtgcgatgt
     2221 cagtgtatcc tctcagacca ccatctgctg gatacatcat ctctgtatta ggctcccaac
     2281 ccttggtctt cttctgcatc acgggaccat tggagggaaa gtttacccct ctgaccttga
     2341 cgttgtagac gagctcgcca tcctcaaggc tggtgtcctg cgtgacggtg acgactccac
     2401 catcttcgta tctcgtaact ctttcccaag taaaaccctc aggaaaggac tgtttaaaga
     2461 aatcagggat gtcggccggg tacttgataa aggtacggct gccatacatg aacgacgtgg
     2521 caagaatgtc aaaggcaaat ggcaggggtc ctccctcgat gactttgatc ctcatggttt
     2581 gcactccctc gtacggtctg ccttctcctt cacctgtgca tttgaattgg tggccgttga
     2641 ccgaaccttc catgaccacc ttcatacgca tattttcctt gatcagctct tcgcccttag
     2701 acaccatggt ggcgtcgact cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa
     2761 gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc
     2821 ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg
     2881 ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc
     2941 actgacgggc accggagccc tatgacttgc atagctgcgt atggcgttac ataacttacg
     3001 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     3061 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     3121 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     3181 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     3241 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgaa ttgagggcgt
     3301 caccgctaag gctccgcccc agcctgggct ccacaaccaa tgaagggtaa tctcgacaaa
     3361 gagcaagggg tggggcgcgg gcgcgcaggt gcagcagcac acaggctggt cgggagggcg
     3421 gggcgcgacg tctgccgtgc ggggtcccgg catcggttgc gcgcgcgctc cctcctctcg
     3481 gagagagggc tgtggtaaaa cccgtccgga aaccacttaa gcttcgccac catggtgagc
     3541 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3601 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     3661 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3721 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     3781 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3841 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3901 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3961 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     4021 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     4081 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     4141 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     4201 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta gttctttgac
     4261 atgaagtttt cagacataaa tacaacaatt catgaagttt tcagacataa ataatcactt
     4321 gcatgaagtt ttcagacata aatacaactg gacatgaagt tttcagacat aaataactag
     4381 tgaacgggcc catcgatgct agcaacttgt ttattgcagc ttataatggt tacaaataaa
     4441 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4501 tgtccaaact catcaatgta tcttatcatg tctgcggacc gagcggccgc aggaacccct
     4561 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4621 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4681 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4741 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4801 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4861 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4921 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4981 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5041 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     5101 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     5161 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5221 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5281 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5341 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5401 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5461 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5521 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5581 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5641 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5701 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5761 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5821 gttctgctat gtggcgcggt attatccctt attgacgccg ggca
//
