LOCUS       sequence-492425-        4948 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  ZFa Plasmid expressing VP16-ZF37-2A-TagBFP from the EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4948)
  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 4948)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 10, 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: EF1a-VP16-ZF37-2A-TagBFP-BGH_pKG3223
FEATURES             Location/Qualifiers
     source          1..4948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     290..309
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      554..640
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      732..759
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     766..789
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     promoter        826..2007
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1056..1998
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1955..1975
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     2009..2033
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2069..2078
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2078..2143
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2150..2170
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2723..2779
                     /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             2792..3493
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    3505..3729
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3840..3859)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3851..4660
                     /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     complement(3918..3937)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(4750..4948,1..389)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
       61 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg tcgggctgaa
      121 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
      181 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      241 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      301 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
      361 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
      421 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
      481 ataaccgtat taccgctagc atggatctcg gggacgtcta actactaagc gagagtaggg
      541 aactgccagg catcaaataa aacgaaaggc tcagtcggaa gactgggcct ttcgttttat
      601 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
      661 cgttgtgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
      721 tcaaactaag cagaaggcca tcctgacgga tggccttttt gctttctaca aactcttcct
      781 gttagttagt tacttaagct cgggcccacc tgcagtctac ttactgctcc ggtgcccgtc
      841 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
      901 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
      961 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     1021 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
     1081 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acgcccctgg
     1141 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
     1201 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggct tgggcgctgg
     1261 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
     1321 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     1381 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     1441 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     1501 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     1561 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     1621 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     1681 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     1741 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     1801 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     1861 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     1921 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     1981 ttttttcttc catttcaggt gtcgtgagag ctcgtttagt gaaccgtcag atcgcctgga
     2041 gacgccatcc acgctgtaat gagccaccgc caccatggat tataaggatc acgatggcga
     2101 ctacaaggac cacgatattg actacaaaga cgatgacgat aaaatggctc ctaagaaaaa
     2161 gcgcaaagtc ggtatccatg gcgttcccgt cgacggtgga ctgcaggccc caccaaccga
     2221 tgtttcttta ggtgatgagt tacatttgga cggtgaggac gtagctatgg cacatgcaga
     2281 tgccttagac gattttgact tggatatgtt aggagatgga gattcaccag gacctggttt
     2341 cacaccacac gattcagctc catacggtgc tttggacatg gccgatttcg aatttgaaca
     2401 gatgtttact gatgccttgg gtatagacga atacggtggt ggagctagac ccggggagcg
     2461 ccccttccag tgtcgcattt gcatgcggaa cttttcgaga aatttcattt tgcaaagaca
     2521 tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg atctgtatgc gaaatttctc
     2581 cgatagagct aatttgagga ggcatctgcg tacgcacacc ggcgagaagc cattccaatg
     2641 ccgaatatgc atgcgcaact tcagtagaca tgatcaattg actagacacc taaaaaccca
     2701 cctgagagga tctggaagcg gagctactaa cttcagcctg ctgaagcagg ctggagacgt
     2761 ggaggagaac cctggacctg caccaggaag tatgagcgag ctgattaagg agaacatgca
     2821 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     2881 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     2941 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     3001 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     3061 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     3121 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     3181 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     3241 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     3301 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     3361 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgaaacgta
     3421 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     3481 caagcttaat taacaacgtt gcacctgtgc cttctagttg ccagccatct gttgtttgcc
     3541 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3601 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3661 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3721 gctctatggc gctcgctgac tgcaggtgcc cgtgtctcaa aatctctgat gttacattgc
     3781 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     3841 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
     3901 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     3961 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     4021 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     4081 gcctcttccg accatcaagc attttatccg tactcctggt gatgcatggt tactcaccac
     4141 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     4201 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     4261 tccttttaac agcgatcgcg tatttcgtct agctcaggcg caatcacgaa tgaataacgg
     4321 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     4381 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     4441 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     4501 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     4561 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     4621 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     4681 gtaacattat tcagattggg ccccgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4741 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4801 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4861 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4921 ccaccacttc aagaactctg tagcaccg
//
