LOCUS       sequence-475645-        5393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND open-loop circuit regulating FXN-P2A-mRuby2 (therapeutically
            relevant gene).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5393)
  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 5393)
  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-FXN-P2A-mRuby2(miRE.FF4)-TS.FF6x1-bGH
FEATURES             Location/Qualifiers
     source          1..5393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(17..40)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(48..75)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(167..253)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(418..1006)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(498..517)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1096..1905)
                     /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     1819..1838
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1897..1916
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2027..3208
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2257..3199
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3156..3176
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     3210..3234
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             3908..3964
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     ncRNA           4203..4217
                     /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           4274..4386
                     /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"
     polyA_signal    5149..5373
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gcccgagctt aagtaactaa ctaacaggaa gagtttgtag aaacgcaaaa aggccatccg
       61 tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
      121 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
      181 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
      241 ttcgttttat ttgatgcctg gcagttccct actctcgctt agtagttaga cgtccccgag
      301 atccatgcta gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      361 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      421 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      481 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      541 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
      601 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
      661 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
      721 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
      781 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
      841 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
      901 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
      961 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1021 cttttctacg gggtctgacg ctcagtggaa cggggcccaa tctgaataat gttacaacca
     1081 attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1141 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1201 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     1261 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1321 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     1381 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     1441 attcattcgt gattgcgcct gagctagacg aaatacgcga tcgctgttaa aaggacaatt
     1501 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     1561 acctgaatca ggatattctt ctaatacctg gaatgctgtt tttccgggga tcgcagtggt
     1621 gagtaaccat gcatcaccag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     1681 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     1741 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaagcgat agattgtcgc
     1801 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     1861 ggaatttaat cgcggcctcg acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     1921 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     1981 gtaacatcag agattttgag acacgggcac ctgcagtctt cgtactgctc cggtgcccgt
     2041 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     2101 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     2161 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     2221 gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc
     2281 gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc cacgcccctg
     2341 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
     2401 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ttgggcgctg
     2461 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
     2521 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
     2581 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga
     2641 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc
     2701 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc
     2761 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
     2821 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc
     2881 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt
     2941 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag
     3001 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     3061 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     3121 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     3181 tttttttctt ccatttcagg tgtcgtgaga gctcgtttag tgaaccgtca gatcgcctgg
     3241 agacgccatc cacgctgtaa tggccaccat gtggactctc gggcgccgcg cagtagccgg
     3301 cctcctggcg tcacccagcc cggcccaggc ccagaccctc acccgggtcc cgcggccggc
     3361 agagttggcc ccactctgcg gccgccgtgg cctgcgcacc gacatcgatg cgacctgcac
     3421 gccccgccgc gcaagttcga accaacgtgg cctcaaccag atttggaatg tcaaaaagca
     3481 gagtgtctat ttgatgaatt tgaggaaatc tggaactttg ggccacccag gctctctaga
     3541 tgagaccacc tatgaaagac tagcagagga aacgctggac tctttagcag agttttttga
     3601 agaccttgca gacaagccat acacgtttga ggactatgat gtctcctttg ggagtggtgt
     3661 cttaactgtc aaactgggtg gagatctagg aacctatgtg atcaacaagc agacgccaaa
     3721 caagcaaatc tggctatctt ctccatccag tggacctaag cgttatgact ggactgggaa
     3781 aaactgggtg tactcccacg acggcgtgtc cctccatgag ctgctggccg cagagctcac
     3841 taaagcctta aaaaccaaac tggacttgtc ttccttggcc tattccggaa aagatgctgg
     3901 aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg
     3961 acctatggtg tctaagggcg aagagctgat caaggaaaat atgcgtatga aggtaagtgt
     4021 gctcgcttcg gcagcacata tactatgttg aatgaggctt cagtacttta cagaatcgtt
     4081 gcctgcacat cttggaaaca cttgctggga ttacttcttc agggacttct taacccaaca
     4141 gaaggctcga gaaggtatat tgctgttgac agtgagcgcc gcttgaagtc tttaattaaa
     4201 tagtgaagcc acagatgtat ttaattaaag acttcaagcg gtgcctactg cctcggactt
     4261 caaggggcta gaattcaagg ggctacttta ggagcaatta tcttgtttac taaaactgaa
     4321 taccttgcta tctctttgat acatttttac aaagctgaat taaaatggta taaattaaat
     4381 cacttttttc aattgtacta acttcgagtc ttcttttttt ttttcacagg tggtcatgga
     4441 aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca atccgtacat
     4501 gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga
     4561 cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc cgaaaggcat
     4621 tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata
     4681 cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt
     4741 ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa
     4801 gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagggg
     4861 atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct ctttcgtaac
     4921 aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc atgccgttga
     4981 tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca
     5041 cgcagttgcc aagttcgccg ggcttggtgg tgggatggac gagctgtaca agtaacaaca
     5101 agcttgtgga accaaagaga ttcctcataa acctaggagg agttgcacct gtgccttcta
     5161 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     5221 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     5281 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     5341 gcaggcatgc tggggatgcg gtgggctcta tggcgctagt agactgcagg tgg
//
