LOCUS       sequence-474144-        3987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND closed-loop circuit regulating mRuby2, hPGK-driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3987)
  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 3987)
  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: hPGK-mRuby2(miRE.FF4)-TS.FF4x1-bGH
FEATURES             Location/Qualifiers
     source          1..3987
                     /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        2057..2567
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2486..2504
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      2573..2582
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           2817..2831
                     /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           2888..3000
                     /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    3763..3987
                     /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 cgtactataa ggatccgggg ttggggttgc gccttttcca aggcagccct gggtttgcgc
     2101 agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctggatctc
     2161 gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc
     2221 ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt
     2281 gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc
     2341 agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag
     2401 ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag
     2461 tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt
     2521 cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagaaa tggccaccat
     2581 ggtgtctaag ggcgaagagc tgatcaagga aaatatgcgt atgaaggtaa gtgtgctcgc
     2641 ttcggcagca catatactat gttgaatgag gcttcagtac tttacagaat cgttgcctgc
     2701 acatcttgga aacacttgct gggattactt cttcagggac ttcttaaccc aacagaaggc
     2761 tcgagaaggt atattgctgt tgacagtgag cgccgcttga agtctttaat taaatagtga
     2821 agccacagat gtatttaatt aaagacttca agcggtgcct actgcctcgg acttcaaggg
     2881 gctagaattc aaggggctac tttaggagca attatcttgt ttactaaaac tgaatacctt
     2941 gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaatt aaatcacttt
     3001 tttcaattgt actaacttcg agtcttcttt ttttttttca caggtggtca tggaaggttc
     3061 ggtcaacggc caccaattca aatgcacagg tgaaggagaa ggcaatccgt acatgggaac
     3121 tcaaaccatg aggatcaaag tcatcgaggg aggacccctg ccatttgcct ttgacattct
     3181 tgccacgtcg ttcatgtatg gcagccgtac ttttatcaag tacccgaaag gcattcctga
     3241 tttctttaaa cagtcctttc ctgagggttt tacttgggaa agagttacga gatacgaaga
     3301 tggtggagtc gtcaccgtca tgcaggacac cagccttgag gatggctgtc tcgtttacca
     3361 cgtccaagtc agaggggtaa actttccctc caatggtccc gtgatgcaga agaagaccaa
     3421 gggttgggag cctaatacag agatgatgta tccagcagat ggtggtctga ggggatacac
     3481 tcatatggca ctgaaagttg atggtggtgg ccatctgtct tgctctttcg taacaactta
     3541 caggtcaaaa aagaccgtcg ggaacatcaa gatgcccggt atccatgccg ttgatcaccg
     3601 cctggaaagg ttagaggaaa gtgacaatga aatgttcgta gtacaacgcg aacacgcagt
     3661 tgccaagttc gccgggcttg gtggtgggat ggacgagctg tacaagtaac aacaagcttg
     3721 tggccgcttg aagtctttaa ttaaacctag gaggagttgc acctgtgcct tctagttgcc
     3781 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3841 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3901 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3961 atgctgggga tgcggtgggc tctatgg
//
