LOCUS       sequence-474138-        5175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND closed-loop circuit regulating mRuby2, CAG-driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5175)
  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 5175)
  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: CAG-mRuby2(miRE.FF4)-TS.FF4x1-bGH
FEATURES             Location/Qualifiers
     source          1..5175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     318..337
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     396..415
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     enhancer        588..891
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        893..1170
                     /label=chicken beta-actin promoter
     intron          1171..2187
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2111..2133
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2195..2214
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2240..2249
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           2484..2498
                     /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           2555..2667
                     /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    3430..3654
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3691..3714)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(3722..3749)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3841..3927)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(4092..4680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4172..4191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4770..5175,1..404))
                     /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"
ORIGIN
        1 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
       61 cctgaatcag gatattcttc taatacctgg aatgctgttt ttccggggat cgcagtggtg
      121 agtaaccatg catcaccagg agtacggata aaatgcttga tggtcggaag aggcataaat
      181 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
      241 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaagcgata gattgtcgca
      301 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
      361 gaatttaatc gcggcctcga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
      421 ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg
      481 taacatcaga gattttgaga cacgggcacc tgcagtcttc gtactactag ttattaatag
      541 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      601 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      661 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      721 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      781 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      841 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg
      901 agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat
      961 ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg
     1021 ccgggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca
     1081 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
     1141 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
     1201 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
     1261 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga
     1321 cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga gggccctttg
     1381 tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc
     1441 ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc
     1501 cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc
     1561 gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc
     1621 gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc gagttgctga gcacggcccg
     1681 gcttcgggtg cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg
     1741 tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg
     1801 gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat
     1861 tgccttttat ggtaatcgtg cgagagggcg cagggacttc ctttgtccca aatctgtgcg
     1921 gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc
     1981 ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc
     2041 cttctccctc tccagcctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg
     2101 ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc tgctaaccat
     2161 gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct
     2221 catcattttg gcaaaaatgg ccaccatggt gtctaagggc gaagagctga tcaaggaaaa
     2281 tatgcgtatg aaggtaagtg tgctcgcttc ggcagcacat atactatgtt gaatgaggct
     2341 tcagtacttt acagaatcgt tgcctgcaca tcttggaaac acttgctggg attacttctt
     2401 cagggacttc ttaacccaac agaaggctcg agaaggtata ttgctgttga cagtgagcgc
     2461 cgcttgaagt ctttaattaa atagtgaagc cacagatgta tttaattaaa gacttcaagc
     2521 ggtgcctact gcctcggact tcaaggggct agaattcaag gggctacttt aggagcaatt
     2581 atcttgttta ctaaaactga ataccttgct atctctttga tacattttta caaagctgaa
     2641 ttaaaatggt ataaattaaa tcactttttt caattgtact aacttcgagt cttctttttt
     2701 tttttcacag gtggtcatgg aaggttcggt caacggccac caattcaaat gcacaggtga
     2761 aggagaaggc aatccgtaca tgggaactca aaccatgagg atcaaagtca tcgagggagg
     2821 acccctgcca tttgcctttg acattcttgc cacgtcgttc atgtatggca gccgtacttt
     2881 tatcaagtac ccgaaaggca ttcctgattt ctttaaacag tcctttcctg agggttttac
     2941 ttgggaaaga gttacgagat acgaagatgg tggagtcgtc accgtcatgc aggacaccag
     3001 ccttgaggat ggctgtctcg tttaccacgt ccaagtcaga ggggtaaact ttccctccaa
     3061 tggtcccgtg atgcagaaga agaccaaggg ttgggagcct aatacagaga tgatgtatcc
     3121 agcagatggt ggtctgaggg gatacactca tatggcactg aaagttgatg gtggtggcca
     3181 tctgtcttgc tctttcgtaa caacttacag gtcaaaaaag accgtcggga acatcaagat
     3241 gcccggtatc catgccgttg atcaccgcct ggaaaggtta gaggaaagtg acaatgaaat
     3301 gttcgtagta caacgcgaac acgcagttgc caagttcgcc gggcttggtg gtgggatgga
     3361 cgagctgtac aagtaacaac aagcttgtgg ccgcttgaag tctttaatta aacctaggag
     3421 gagttgcacc tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3481 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3541 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3601 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcgctag
     3661 tagactgcag gtgggcccga gcttaagtaa ctaactaaca ggaagagttt gtagaaacgc
     3721 aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg
     3781 gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg
     3841 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     3901 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcttagtagt
     3961 tagacgtccc cgagatccat gctagcggta atacggttat ccacagaatc aggggataac
     4021 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4081 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4141 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4201 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4261 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4321 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4381 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4441 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4501 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4561 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4621 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4681 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacggggc ccaatctgaa
     4741 taatgttaca accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac
     4801 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     4861 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     4921 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     4981 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     5041 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     5101 tcaaccaaac cgttattcat tcgtgattgc gcctgagcta gacgaaatac gcgatcgctg
     5161 ttaaaaggac aatta
//
