LOCUS       sequence-473938-        4716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND base gene regulating mRuby2, CAG-driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4716)
  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 4716)
  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-bGH
FEATURES             Location/Qualifiers
     source          1..4716
                     /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"
     CDS             2246..2956
                     /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    2971..3195
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3232..3255)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(3263..3290)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3382..3468)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(3633..4221)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3713..3732)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4311..4716,1..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 ccrggggggg sggggggggg sggggggsgg ggsggggsgr ggcggagagg kgcggcggca
     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 kgggggtgcc gggcggggcg gggccgcctc gggccggggr gggctcgggg
     1801 grggggcgcg 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 aaggtggtca tggaaggttc ggtcaacggc caccaattca aatgcacagg
     2341 tgaaggagaa ggcaatccgt acatgggaac tcaaaccatg aggatcaaag tcatcgaggg
     2401 aggacccctg ccatttgcct ttgacattct tgccacgtcg ttcatgtatg gcagccgtac
     2461 ttttatcaag tacccgaaag gcattcctga tttctttaaa cagtcctttc ctgagggttt
     2521 tacttgggaa agagttacga gatacgaaga tggtggagtc gtcaccgtca tgcaggacac
     2581 cagccttgag gatggctgtc tcgtttacca cgtccaagtc agaggggtaa actttccctc
     2641 caatggtccc gtgatgcaga agaagaccaa gggttgggag cctaatacag agatgatgta
     2701 tccagcagat ggtggtctga ggggatacac tcatatggca ctgaaagttg atggtggtgg
     2761 ccatctgtct tgctctttcg taacaactta caggtcaaaa aagaccgtcg ggaacatcaa
     2821 gatgcccggt atccatgccg ttgatcaccg cctggaaagg ttagaggaaa gtgacaatga
     2881 aatgttcgta gtacaacgcg aacacgcagt tgccaagttc gccgggcttg gtggtgggat
     2941 ggacgagctg tacaagtaac aacgttgcac ctgtgccttc tagttgccag ccatctgttg
     3001 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3061 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3121 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3181 cggtgggctc tatggcgcta gtagactgca ggtgggcccg agcttaagta actaactaac
     3241 aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg
     3301 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac
     3361 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     3421 gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt
     3481 tccctactct cgcttagtag ttagacgtcc ccgagatcca tgctagcggt aatacggtta
     3541 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3601 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3661 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3721 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3781 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3841 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3901 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3961 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4021 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4081 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4141 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4201 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4261 tggaacgggg cccaatctga ataatgttac aaccaattaa ccaattctga ttagaaaaac
     4321 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     4381 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     4441 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     4501 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     4561 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     4621 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagct
     4681 agacgaaata cgcgatcgct gttaaaagga caatta
//
