LOCUS       sequence-472820-        3528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ComMAND base gene regulating mRuby2, hPGK-driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3528)
  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 3528)
  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-bGH
FEATURES             Location/Qualifiers
     source          1..3528
                     /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"
     CDS             2579..3289
                     /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    3304..3528
                     /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 atgaaggtgg tcatggaagg
     2641 ttcggtcaac ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc cgtacatggg
     2701 aactcaaacc atgaggatca aagtcatcga gggaggaccc ctgccatttg cctttgacat
     2761 tcttgccacg tcgttcatgt atggcagccg tacttttatc aagtacccga aaggcattcc
     2821 tgatttcttt aaacagtcct ttcctgaggg ttttacttgg gaaagagtta cgagatacga
     2881 agatggtgga gtcgtcaccg tcatgcagga caccagcctt gaggatggct gtctcgttta
     2941 ccacgtccaa gtcagagggg taaactttcc ctccaatggt cccgtgatgc agaagaagac
     3001 caagggttgg gagcctaata cagagatgat gtatccagca gatggtggtc tgaggggata
     3061 cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt tcgtaacaac
     3121 ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg ccgttgatca
     3181 ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac gcgaacacgc
     3241 agttgccaag ttcgccgggc ttggtggtgg gatggacgag ctgtacaagt aacaacgttg
     3301 cacctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3361 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3421 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3481 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg
//
