LOCUS       sequence-484449-        4764 bp    DNA     circular SYN 22-AUG-2025
DEFINITION  ComMAND closed-loop circuit regulating mRuby2 with 4 copies of 
            microRNA target sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4764)
  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 4764)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 22, 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.FF4x4-bGH
FEATURES             Location/Qualifiers
     source          1..4764
                     /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"
     promoter        526..1707
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          756..1698
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1655..1675
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1709..1733
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1762..1771
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           2006..2020
                     /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           2077..2189
                     /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    3019..3243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3280..3303)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(3311..3338)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3430..3516)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(3681..4269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3761..3780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4359..4764,1..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
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMLLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
       61 cctgaatcag gatattcttc taatacctgg aatgctgttt ttccggggat cgcagtggtg
      121 agtaaccatg catcaccagg agtacggata aaatgcttga tggtcggaag aagcataaat
      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 gtactgctcc ggtgcccgtc
      541 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
      601 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
      661 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
      721 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
      781 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acgcccctgg
      841 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
      901 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggct tgggcgctgg
      961 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
     1021 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     1081 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     1141 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     1201 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     1261 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     1321 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     1381 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     1441 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     1501 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     1561 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     1621 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     1681 ttttttcttc catttcaggt gtcgtgagag ctcgtttagt gaaccgtcag atcgcctgga
     1741 gacgccatcc acgctgtaat ggccaccatg gtgtctaagg gcgaagagct gatcaaggaa
     1801 aatatgcgta tgaaggtaag tgtgctcgct tcggcagcac atatactatg ttgaatgagg
     1861 cttcagtact ttacagaatc gttgcctgca catcttggaa acacttgctg ggattacttc
     1921 ttcagggact tcttaaccca acagaaggct cgagaaggta tattgctgtt gacagtgagc
     1981 gccgcttgaa gtctttaatt aaatagtgaa gccacagatg tatttaatta aagacttcaa
     2041 gcggtgccta ctgcctcgga cttcaagggg ctagaattca aggggctact ttaggagcaa
     2101 ttatcttgtt tactaaaact gaataccttg ctatctcttt gatacatttt tacaaagctg
     2161 aattaaaatg gtataaatta aatcactttt ttcaattgta ctaacttcga gtcttctttt
     2221 tttttttcac aggtggtcat ggaaggttcg gtcaacggcc accaattcaa atgcacaggt
     2281 gaaggagaag gcaatccgta catgggaact caaaccatga ggatcaaagt catcgaggga
     2341 ggacccctgc catttgcctt tgacattctt gccacgtcgt tcatgtatgg cagccgtact
     2401 tttatcaagt acccgaaagg cattcctgat ttctttaaac agtcctttcc tgagggtttt
     2461 acttgggaaa gagttacgag atacgaagat ggtggagtcg tcaccgtcat gcaggacacc
     2521 agccttgagg atggctgtct cgtttaccac gtccaagtca gaggggtaaa ctttccctcc
     2581 aatggtcccg tgatgcagaa gaagaccaag ggttgggagc ctaatacaga gatgatgtat
     2641 ccagcagatg gtggtctgag gggatacact catatggcac tgaaagttga tggtggtggc
     2701 catctgtctt gctctttcgt aacaacttac aggtcaaaaa agaccgtcgg gaacatcaag
     2761 atgcccggta tccatgccgt tgatcaccgc ctggaaaggt tagaggaaag tgacaatgaa
     2821 atgttcgtag tacaacgcga acacgcagtt gccaagttcg ccgggcttgg tggtgggatg
     2881 gacgagctgt acaagtaaca acaagcttgt ggccgcttga agtctttaat taaaccgctt
     2941 gaagtcttta attaaacccg cttgaagtct ttaattaaac cgcttgaagt ctttaattaa
     3001 acctaggagg agttgcacct gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3061 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3121 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3181 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3241 tggcgctagt agactgcagg tgggcccgag cttaagtaac taactaacag gaagagtttg
     3301 tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt
     3361 ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc
     3421 tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa
     3481 ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg
     3541 cttagtagtt agacgtcccc gagatccatg ctagcggtaa tacggttatc cacagaatca
     3601 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3661 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3721 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3781 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3841 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3901 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3961 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4021 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4081 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4141 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4201 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4261 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacggggcc
     4321 caatctgaat aatgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc
     4381 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     4441 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     4501 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     4561 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     4621 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     4681 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagctag acgaaatacg
     4741 cgatcgctgt taaaaggaca atta
//
