LOCUS       sequence-472817-        4719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND closed-loop circuit regulating mRuby2 with 2 copies of 
            microRNA target sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4719)
  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 4719)
  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-mRuby2(miRE.FF4)-TS.FF4x2-bGH
FEATURES             Location/Qualifiers
     source          1..4719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..56)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(64..91)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(183..269)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(434..1022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(514..533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1112..1921)
                     /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     1835..1854
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1913..1932
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2043..3224
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2273..3215
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3172..3192
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     3226..3250
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3279..3288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           3523..3537
                     /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           3594..3706
                     /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    4491..4715
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 agtagactgc aggtgggccc gagcttaagt aactaactaa caggaagagt ttgtagaaac
       61 gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc
      121 gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc
      181 ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag
      241 tcttccgact gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcttagta
      301 gttagacgtc cccgagatcc atgctagcgg taatacggtt atccacagaa tcaggggata
      361 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      421 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      481 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      541 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      601 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      661 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      721 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      781 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      841 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      901 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      961 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1021 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacggg gcccaatctg
     1081 aataatgtta caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa
     1141 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1201 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1261 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1321 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     1381 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     1441 catcaaccaa accgttattc attcgtgatt gcgcctgagc tagacgaaat acgcgatcgc
     1501 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     1561 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgtttttc
     1621 cggggatcgc agtggtgagt aaccatgcat caccaggagt acggataaaa tgcttgatgg
     1681 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     1741 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     1801 agcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     1861 aatcagcatc catgttggaa tttaatcgcg gcctcgacgt ttcccgttga atatggctca
     1921 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat
     1981 ttttatcttg tgcaatgtaa catcagagat tttgagacac gggcacctgc agtcttcgta
     2041 ctgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     2101 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     2161 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     2221 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
     2281 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
     2341 tacttccacg cccctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg
     2401 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
     2461 cctggcttgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg
     2521 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt
     2581 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
     2641 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc
     2701 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
     2761 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg
     2821 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat
     2881 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct
     2941 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc
     3001 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
     3061 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga
     3121 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc
     3181 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgagagctc gtttagtgaa
     3241 ccgtcagatc gcctggagac gccatccacg ctgtaatggc caccatggtg tctaagggcg
     3301 aagagctgat caaggaaaat atgcgtatga aggtaagtgt gctcgcttcg gcagcacata
     3361 tactatgttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca
     3421 cttgctggga ttacttcttc agggacttct taacccaaca gaaggctcga gaaggtatat
     3481 tgctgttgac agtgagcgcc gcttgaagtc tttaattaaa tagtgaagcc acagatgtat
     3541 ttaattaaag acttcaagcg gtgcctactg cctcggactt caaggggcta gaattcaagg
     3601 ggctacttta ggagcaatta tcttgtttac taaaactgaa taccttgcta tctctttgat
     3661 acatttttac aaagctgaat taaaatggta taaattaaat cacttttttc aattgtacta
     3721 acttcgagtc ttcttttttt ttttcacagg tggtcatgga aggttcggtc aacggccacc
     3781 aattcaaatg cacaggtgaa ggagaaggca atccgtacat gggaactcaa accatgagga
     3841 tcaaagtcat cgagggagga cccctgccat ttgcctttga cattcttgcc acgtcgttca
     3901 tgtatggcag ccgtactttt atcaagtacc cgaaaggcat tcctgatttc tttaaacagt
     3961 cctttcctga gggttttact tgggaaagag ttacgagata cgaagatggt ggagtcgtca
     4021 ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt ttaccacgtc caagtcagag
     4081 gggtaaactt tccctccaat ggtcccgtga tgcagaagaa gaccaagggt tgggagccta
     4141 atacagagat gatgtatcca gcagatggtg gtctgagggg atacactcat atggcactga
     4201 aagttgatgg tggtggccat ctgtcttgct ctttcgtaac aacttacagg tcaaaaaaga
     4261 ccgtcgggaa catcaagatg cccggtatcc atgccgttga tcaccgcctg gaaaggttag
     4321 aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca cgcagttgcc aagttcgccg
     4381 ggcttggtgg tgggatggac gagctgtaca agtaacaaca agcttgtggc cgcttgaagt
     4441 ctttaattaa accgcttgaa gtctttaatt aaacctagga ggagttgcac ctgtgccttc
     4501 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4561 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     4621 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     4681 tagcaggcat gctggggatg cggtgggctc tatggcgct
//
