LOCUS       sequence-472798-        6605 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND EGFP-FMRP closed-loop circuit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6605)
  AUTHORS   Love KS, Johnstone CP, Peterman EL, Gaglione S, Birnbaum ME, 
            Galloway KE
  TITLE     Model-guided design of microRNA-based gene circuits supports precise
            dosage of transgenic cargoes into diverse primary cells.
  JOURNAL   Cell Syst. 2025 Apr 22:101269. doi: 10.1016/j.cels.2025.101269.
  PUBMED    40300600
REFERENCE   2  (bases 1 to 6605)
  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: "Cell Syst. 
            2025 Apr 22:101269. doi: 10.1016/j.cels.2025.101269."
COMMENT     Sequence Label: EF1a-EGFP(miRE.FF4)-FMRP-TS.FF4x1-bGH
FEATURES             Location/Qualifiers
     source          1..6605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..60)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(68..95)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(187..273)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(438..1026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(518..537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1116..1925)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1839..1858
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1917..1936
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2047..3228
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2277..3219
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3176..3196
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     3230..3254
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3283..3292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3334..3355)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3595..3614)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     ncRNA           3815..3829
                     /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"
     ncRNA           3886..3998
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     4359..4380
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4465..4488
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    6381..6605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cgctagtaga ctgcaggtgg gcccgagctt aagtaactaa ctaacaggaa gagtttgtag
       61 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
      121 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
      181 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
      241 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgctt
      301 agtagttaga cgtccccgag atccatgcta gcggtaatac ggttatccac agaatcaggg
      361 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
      421 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
      481 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
      541 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
      601 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
      661 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
      721 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
      781 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
      841 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
      901 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
      961 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1021 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cggggcccaa
     1081 tctgaataat gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
     1141 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     1201 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     1261 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     1321 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     1381 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     1441 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagctagacg aaatacgcga
     1501 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     1561 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1621 tttccgggga tcgcagtggt gagtaaccat gcatcaccag gagtacggat aaaatgcttg
     1681 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1741 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1801 tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     1861 tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg ttgaatatgg
     1921 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
     1981 atatttttat cttgtgcaat gtaacatcag agattttgag acacgggcac ctgcagtctt
     2041 cgtactgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     2101 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
     2161 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2221 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
     2281 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
     2341 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     2401 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2461 gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2521 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2581 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     2641 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     2701 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     2761 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     2821 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     2881 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     2941 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3001 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3061 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3121 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3181 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaga gctcgtttag
     3241 tgaaccgtca gatcgcctgg agacgccatc cacgctgtaa tggccaccat ggtgagcaag
     3301 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     3361 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     3421 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     3481 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     3541 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     3601 ggcaactaca agacccgcgc cgaggtaagt gtgctcgctt cggcagcaca tatactatgt
     3661 tgaatgaggc ttcagtactt tacagaatcg ttgcctgcac atcttggaaa cacttgctgg
     3721 gattacttct tcagggactt cttaacccaa cagaaggctc gagaaggtat attgctgttg
     3781 acagtgagcg ccgcttgaag tctttaatta aatagtgaag ccacagatgt atttaattaa
     3841 agacttcaag cggtgcctac tgcctcggac ttcaaggggc tagaattcaa ggggctactt
     3901 taggagcaat tatcttgttt actaaaactg aataccttgc tatctctttg atacattttt
     3961 acaaagctga attaaaatgg tataaattaa atcacttttt tcaattgtac taacttcgag
     4021 tcttcttttt ttttttcaca ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     4081 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     4141 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     4201 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     4261 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     4321 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     4381 accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact cagatctcga
     4441 gctcaagctt cgaattcgat ggaggactac aaggacgacg atgacaagga gctggtggtg
     4501 gaagtgcggg gctccaatgg cgctttctac aaggcatttg taaaagatgt ccatgaagat
     4561 tctataacag ttgcttttga aaacaactgg caaccagaga gacagattcc attccatgat
     4621 gtgagattcc caccacctgt aggttataat aaagatataa acgaaagtga tgaagttgag
     4681 gtttattcca gagcaaatga aaaagagcct tgctgttggt ggttagctaa agtgaggatg
     4741 ataaagggtg agttttatgt gatagaatat gcagcatgtg atgctacgta taatgaaatt
     4801 gtcacaattg agcgtctacg atctgttaat cccaacaaac ctgctacaaa agatactttc
     4861 cataagatca agctggaggt gccagaagat ttacgacaaa tgtgtgccaa agaatcagca
     4921 cataaggatt ttaaaaaggc agttggtgcc ttctctgtaa cttatgatcc agaaaattat
     4981 cagctggtaa ttttgtccat caatgaagtc acctcaaagc gagcccacat gttgattgac
     5041 atgcactttc gaagtctgcg caccaagttg tctcttatac tgagaaatga agaagccagt
     5101 aaacaactgg agagttcaag gcagcttgcc tcaagatttc atgaacagtt tatcgtacga
     5161 gaagatctga tgggtttagc tattggtact catggtgcta atattcagca agctagaaaa
     5221 gtgcctggcg tcactgctat tgatttagat gaggatacct gcacatttca tatttatgga
     5281 gaggatcaag atgcagtgaa aaaggctaga agctttctgg aatttgctga agatgtcata
     5341 caggttccac gaaacttagt aggcaaagta ataggaaaaa atggaaagct gattcaagag
     5401 atcgtggaca agtcaggagt tgtgagggtg aggattgagg ctgaaaatga gaaaagtgtc
     5461 ccacaagaag aggaaattat gccaccaagt tccctacctt ccaataattc aagggttgga
     5521 cctaactcct ctgaagaaaa gaaacattta gatacaaagg aaaacaccca tttttctcaa
     5581 cctaacagta caaaagtcca gagggtgtta gtggtttcat caattgtagc agggggaccc
     5641 cagaaacctg aacccaaggc ttggcagggt atggtaccat ttgtttttgt gggaacaaaa
     5701 gacagcatcg ctaatgccac tgttcttttg gattatcacc tgaactattt aaaggaagta
     5761 gaccagttgc gtttggagag attacaaatt gatgagcagt tgcgacaaat tggagctagt
     5821 tctagaccac caccaaatcg tacagataag gaaaaaggct atgtgactga tgatggtcaa
     5881 ggaatgggtc gaggtagtag accttacaga aatagggggc acggcagacg cggtcctgga
     5941 tatacttcag gaactaattc tgaagcatca aatgcttctg aaacagaatc tgaccacaga
     6001 gacgaactca gtgattggtc attagctcca acagaggaag agagggagag cttcctgcgc
     6061 agaggagacg gacggcggcg tggaggagga ggaagaggac aaggaggaag aggaagagga
     6121 ggaggcttca aaggaaacga cgatcattcc cgaacagata atcgtccacg taatccaaga
     6181 gaggctaaag gaagaacagc tgatggatcc ctgcagagtg cctccagtga agggagccgg
     6241 ctgcgcacgg gtaaagatcg taaccagaag aaggaaaagc cagacagcgt agatgggctg
     6301 caaccgctgg tgaatggagt accctaacaa caagcttgtg gccgcttgaa gtctttaatt
     6361 aaacctagga ggagttgcac ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     6421 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     6481 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     6541 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     6601 tatgg
//
