LOCUS       sequence-492424-        4984 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  ZFa Plasmid expressing VP16-ZF37-2A-mCherry from the EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4984)
  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 4984)
  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-mCherry-BGH_pKG1501
FEATURES             Location/Qualifiers
     source          1..4984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(111..130)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             122..931
                     /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(189..208)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1021..1608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1509..1528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      1773..1859
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1951..1978
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     1985..2008
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     promoter        2045..3226
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2275..3217
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3174..3194
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     3228..3252
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3288..3297
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3297..3362
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3369..3389
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3942..3998
                     /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             4011..4718
                     /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(4156..4174)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4424..4443
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4725..4745
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4760..4984
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cgctcgctga ctgcaggtgc ccgtgtctca aaatctctga tgttacattg cacaagataa
       61 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
      121 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
      181 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
      241 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
      301 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
      361 gaccatcaag cattttatcc gtactcctgg tgatgcatgg ttactcacca ctgcgatccc
      421 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
      481 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
      541 cagcgatcgc gtatttcgtc tagctcaggc gcaatcacga atgaataacg gtttggttga
      601 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
      661 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
      721 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
      781 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
      841 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
      901 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacatta
      961 ttcagattgg gccccgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1021 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1081 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1141 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     1201 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1261 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1321 ggcgcagcgt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1381 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1441 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1501 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     1561 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     1621 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     1681 ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg ggacgtctaa
     1741 ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcggaag
     1801 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
     1861 cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg gcaggacgcc
     1921 cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat ggcctttttg
     1981 ctttctacaa actcttcctg ttagttagtt acttaagctc gggcccacct gcagtctact
     2041 tactgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     2101 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     2161 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     2221 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt
     2281 gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga
     2341 attacttcca cgcccctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag
     2401 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga
     2461 ggcctggctt gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct
     2521 cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt
     2581 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt
     2641 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg
     2701 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct
     2761 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc
     2821 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa
     2881 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc
     2941 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca
     3001 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta
     3061 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt
     3121 gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc
     3181 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgagagc tcgtttagtg
     3241 aaccgtcaga tcgcctggag acgccatcca cgctgtaatg agccaccgcc accatggatt
     3301 ataaggatca cgatggcgac tacaaggacc acgatattga ctacaaagac gatgacgata
     3361 aaatggctcc taagaaaaag cgcaaagtcg gtatccatgg cgttcccgtc gacggtggac
     3421 tgcaggcccc accaaccgat gtttctttag gtgatgagtt acatttggac ggtgaggacg
     3481 tagctatggc acatgcagat gccttagacg attttgactt ggatatgtta ggagatggag
     3541 attcaccagg acctggtttc acaccacacg attcagctcc atacggtgct ttggacatgg
     3601 ccgatttcga atttgaacag atgtttactg atgccttggg tatagacgaa tacggtggtg
     3661 gagctagacc cggggagcgc cccttccagt gtcgcatttg catgcggaac ttttcgagaa
     3721 atttcatttt gcaaagacat acccgtactc ataccggtga aaaaccgttt cagtgtcgga
     3781 tctgtatgcg aaatttctcc gatagagcta atttgaggag gcatctgcgt acgcacaccg
     3841 gcgagaagcc attccaatgc cgaatatgca tgcgcaactt cagtagacat gatcaattga
     3901 ctagacacct aaaaacccac ctgagaggat ctggaagcgg agctactaac ttcagcctgc
     3961 tgaagcaggc tggagacgtg gaggagaacc ctggacctgc accaggaagt atggtgagca
     4021 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     4081 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     4141 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     4201 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     4261 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     4321 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     4381 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     4441 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     4501 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     4561 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     4621 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     4681 gccgccactc caccggcggc atggacgagc tgtacaagca attgccaaaa aagaagagaa
     4741 aggtataaca acgttgcacc tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4801 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4861 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     4921 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     4981 atgg
//
