LOCUS       sequence-472821-        4658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND closed-loop circuit regulating mRuby2 with miR-FF4 design 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4658)
  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 4658)
  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-TS.FF4x1-mRuby2(miR.FF4)-bGH
FEATURES             Location/Qualifiers
     source          1..4658
                     /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      3326..3335
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           3418..3511
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           3558..3572
                     /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           3600..3728
                     /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    4434..4658
                     /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 tgtaagcttg tggccgcttg
     3301 aagtctttaa ttaaacctag gacaagccac catggtgtct aagggcgaag agctgatcaa
     3361 ggaaaatatg cgtatgaagg tgagtatgtg ctcgcttcgg cagcacatat actatgtttg
     3421 aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca cttgctggga
     3481 ttacttcttc aggttaaccc aacagaaggc tcgagtgctg ttgacagtga gcgccgcttg
     3541 aagtctttaa ttaaatagtg aagccacaga tgtatttaat taaagacttc aagcggtgcc
     3601 tactgcctcg gagaattcaa ggggctactt taggagcaat tatcttgttt actaaaactg
     3661 aataccttgc tatctctttg atacattttt acaaagctga attaaaatgg tataaattaa
     3721 atcacttttt tcaattgttt cctttttttt cctcaggtgg tcatggaagg ttcggtcaac
     3781 ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc cgtacatggg aactcaaacc
     3841 atgaggatca aagtcatcga gggaggaccc ctgccatttg cctttgacat tcttgccacg
     3901 tcgttcatgt atggcagccg tacttttatc aagtacccga aaggcattcc tgatttcttt
     3961 aaacagtcct ttcctgaggg ttttacttgg gaaagagtta cgagatacga agatggtgga
     4021 gtcgtcaccg tcatgcagga caccagcctt gaggatggct gtctcgttta ccacgtccaa
     4081 gtcagagggg taaactttcc ctccaatggt cccgtgatgc agaagaagac caagggttgg
     4141 gagcctaata cagagatgat gtatccagca gatggtggtc tgaggggata cactcatatg
     4201 gcactgaaag ttgatggtgg tggccatctg tcttgctctt tcgtaacaac ttacaggtca
     4261 aaaaagaccg tcgggaacat caagatgccc ggtatccatg ccgttgatca ccgcctggaa
     4321 aggttagagg aaagtgacaa tgaaatgttc gtagtacaac gcgaacacgc agttgccaag
     4381 ttcgccgggc ttggtggtgg gatggacgag ctgtacaagt aacaacgttg cacctgtgcc
     4441 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4501 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4561 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4621 caatagcagg catgctgggg atgcggtggg ctctatgg
//
