LOCUS       sequence-491513-        4190 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  ZFa Plasmid expressing VP16-ZF37-2A-mCherry from the TET promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4190)
  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 4190)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 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: TET-ZF37-mCherry-BGH_pKG01502
FEATURES             Location/Qualifiers
     source          1..4190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(17..40)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(48..75)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(167..253)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(418..1006)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(498..517)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1096..1905)
                     /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     1819..1838
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1897..1916
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2034..2412
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    2042..2060
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2078..2096
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2114..2132
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2150..2168
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2186..2204
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2222..2240
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2258..2276
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      2474..2483
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2483..2548
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2555..2575
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3128..3184
                     /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             3197..3904
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3342..3360)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3610..3629
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3911..3931
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3946..4170
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gcccgagctt aagtaactaa ctaacaggaa gagtttgtag aaacgcaaaa aggccatccg
       61 tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
      121 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
      181 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
      241 ttcgttttat ttgatgcctg gcagttccct actctcgctt agtagttaga cgtccccgag
      301 atccatgcta gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      361 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      421 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      481 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      541 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
      601 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
      661 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
      721 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
      781 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
      841 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
      901 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
      961 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1021 cttttctacg gggtctgacg ctcagtggaa cggggcccaa tctgaataat gttacaacca
     1081 attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1141 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1201 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     1261 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1321 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     1381 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     1441 attcattcgt gattgcgcct gagctagacg aaatacgcga tcgctgttaa aaggacaatt
     1501 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     1561 acctgaatca ggatattctt ctaatacctg gaatgctgtt tttccgggga tcgcagtggt
     1621 gagtaaccat gcatcaccag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     1681 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     1741 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaagcgat agattgtcgc
     1801 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     1861 ggaatttaat cgcggcctcg acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     1921 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     1981 gtaacatcag agattttgag acacgggcac ctgcagtctt cgtactattc ctcgagttta
     2041 ctccctatca gtgatagaga acgtatgaag agtttactcc ctatcagtga tagagaacgt
     2101 atgcagactt tactccctat cagtgataga gaacgtataa ggagtttact ccctatcagt
     2161 gatagagaac gtatgaccag tttactccct atcagtgata gagaacgtat ctacagttta
     2221 ctccctatca gtgatagaga acgtatatcc agtttactcc ctatcagtga tagagaacgt
     2281 ataagcttta ggcgtgtacg gtgggcgcct ataaaagcag agctcgttta gtgaaccgtc
     2341 agatcgcctg gagcaattcc acaacacttt tgtcttatac caactttccg taccacttcc
     2401 taccctcgta aagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct
     2461 gtaatgagcc accgccacca tggattataa ggatcacgat ggcgactaca aggaccacga
     2521 tattgactac aaagacgatg acgataaaat ggctcctaag aaaaagcgca aagtcggtat
     2581 ccatggcgtt cccgtcgacg gtggactgca ggccccacca accgatgttt ctttaggtga
     2641 tgagttacat ttggacggtg aggacgtagc tatggcacat gcagatgcct tagacgattt
     2701 tgacttggat atgttaggag atggagattc accaggacct ggtttcacac cacacgattc
     2761 agctccatac ggtgctttgg acatggccga tttcgaattt gaacagatgt ttactgatgc
     2821 cttgggtata gacgaatacg gtggtggagc tagacccggg gagcgcccct tccagtgtcg
     2881 catttgcatg cggaactttt cgagaaattt cattttgcaa agacataccc gtactcatac
     2941 cggtgaaaaa ccgtttcagt gtcggatctg tatgcgaaat ttctccgata gagctaattt
     3001 gaggaggcat ctgcgtacgc acaccggcga gaagccattc caatgccgaa tatgcatgcg
     3061 caacttcagt agacatgatc aattgactag acacctaaaa acccacctga gaggatctgg
     3121 aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg
     3181 acctgcacca ggaagtatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
     3241 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     3301 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     3361 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     3421 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     3481 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     3541 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     3601 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     3661 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     3721 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     3781 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     3841 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     3901 caagcaattg ccaaaaaaga agagaaaggt ataacaacgt tgcacctgtg ccttctagtt
     3961 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     4021 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4081 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     4141 ggcatgctgg ggatgcggtg ggctctatgg cgctagtaga ctgcaggtgg
//
