LOCUS       sequence-472784-        3219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND base gene regulating mRuby2, EFS-driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3219)
  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 3219)
  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: EFS-mRuby2-bGH
FEATURES             Location/Qualifiers
     source          1..3219
                     /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..2258
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2264..2273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2270..2980
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     polyA_signal    2995..3219
                     /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 cgtactgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     2101 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     2161 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     2221 acgttctttt tcgcaacggg tttgccgcca gaacacagta atggccacca tggtgtctaa
     2281 gggcgaagag ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag gttcggtcaa
     2341 cggccaccaa ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg gaactcaaac
     2401 catgaggatc aaagtcatcg agggaggacc cctgccattt gcctttgaca ttcttgccac
     2461 gtcgttcatg tatggcagcc gtacttttat caagtacccg aaaggcattc ctgatttctt
     2521 taaacagtcc tttcctgagg gttttacttg ggaaagagtt acgagatacg aagatggtgg
     2581 agtcgtcacc gtcatgcagg acaccagcct tgaggatggc tgtctcgttt accacgtcca
     2641 agtcagaggg gtaaactttc cctccaatgg tcccgtgatg cagaagaaga ccaagggttg
     2701 ggagcctaat acagagatga tgtatccagc agatggtggt ctgaggggat acactcatat
     2761 ggcactgaaa gttgatggtg gtggccatct gtcttgctct ttcgtaacaa cttacaggtc
     2821 aaaaaagacc gtcgggaaca tcaagatgcc cggtatccat gccgttgatc accgcctgga
     2881 aaggttagag gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg cagttgccaa
     2941 gttcgccggg cttggtggtg ggatggacga gctgtacaag taacaacgtt gcacctgtgc
     3001 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3061 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3121 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3181 acaatagcag gcatgctggg gatgcggtgg gctctatgg
//
