LOCUS       sequence-492550-        4030 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for rtTA-2A-mRuby2 driven from the EFS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4030)
  AUTHORS   Kabaria SR, Bae Y, Ehmann ME, Lende-Dorn BA, Beitz AM, Peterman EL, 
            Love KS, Ploessl DS, Galloway KE
  TITLE     Programmable promoter editing for precise control of transgene 
            expression.
  JOURNAL   Nat Biotechnol. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y.
  PUBMED    41083901
REFERENCE   2  (bases 1 to 4030)
  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: "Nat 
            Biotechnol. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y."
COMMENT     Sequence Label: EFS-rtTA-2A-mRuby2-bGH_pKG2064
FEATURES             Location/Qualifiers
     source          1..4030
                     /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"
     CDS             2271..3014
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             3024..3080
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3081..3791
                     /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    3806..4030
                     /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 atgtgccacc atgagccggc
     2281 tggacaagag caaagtgatc aacagcgccc tggaactgct gaacggcgtg ggcatcgagg
     2341 gcctgaccac ccggaagctg gcccagaaac tgggcgtgga acagcccacc ctgtactggc
     2401 acgtgaagaa caagcgggcc ctgctggacg ccctgcccat cgagatgctg gaccggcacc
     2461 acacccacag ctgccctctg gaaggcgaga gctggcagga cttcctgcgg aacaacgcca
     2521 agagctacag atgcgccctg ctgagccacc gggacggcgc caaagtgcac ctgggcacca
     2581 gacccaccga gaagcagtac gagacactgg aaaaccagct ggccttcctg tgccagcagg
     2641 gcttcagcct ggaaaacgcc ctgtacgccc tgagcgccgt gggccacttc accctgggct
     2701 gcgtgctgga agaacaggaa caccaggtcg ccaaagagga aagagagaca cccaccaccg
     2761 acagcatgcc ccccctgctg aagcaggcca tcgagctgtt cgacagacag ggcgccgagc
     2821 ccgccttcct gttcggcctg gaactgatca tctgcggcct cgagaagcag ctgaagtgcg
     2881 agagcggcgg acccaccgac gccctggacg acttcgacct ggacatgctg cccgccgatg
     2941 ccctggatga ttttgatctg gatatgctcc ctgctgacgc tctcgatgac ttcgatctcg
     3001 atatgctgcc aggcggaagc ggagctacta acttcagcct gctgaagcag gctggagacg
     3061 tggaggagaa ccctggacct atggtgtcta agggcgaaga gctgatcaag gaaaatatgc
     3121 gtatgaaggt ggtcatggaa ggttcggtca acggccacca attcaaatgc acaggtgaag
     3181 gagaaggcaa tccgtacatg ggaactcaaa ccatgaggat caaagtcatc gagggaggac
     3241 ccctgccatt tgcctttgac attcttgcca cgtcgttcat gtatggcagc cgtactttta
     3301 tcaagtaccc gaaaggcatt cctgatttct ttaaacagtc ctttcctgag ggttttactt
     3361 gggaaagagt tacgagatac gaagatggtg gagtcgtcac cgtcatgcag gacaccagcc
     3421 ttgaggatgg ctgtctcgtt taccacgtcc aagtcagagg ggtaaacttt ccctccaatg
     3481 gtcccgtgat gcagaagaag accaagggtt gggagcctaa tacagagatg atgtatccag
     3541 cagatggtgg tctgagggga tacactcata tggcactgaa agttgatggt ggtggccatc
     3601 tgtcttgctc tttcgtaaca acttacaggt caaaaaagac cgtcgggaac atcaagatgc
     3661 ccggtatcca tgccgttgat caccgcctgg aaaggttaga ggaaagtgac aatgaaatgt
     3721 tcgtagtaca acgcgaacac gcagttgcca agttcgccgg gcttggtggt gggatggacg
     3781 agctgtacaa gtaacaacgt tgcacctgtg ccttctagtt gccagccatc tgttgtttgc
     3841 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3901 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3961 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     4021 ggctctatgg
//
