LOCUS       sequence-321725-        4927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of VP64-ZF43 under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4927)
  AUTHORS   Donahue PS, Draut JW, Muldoon JJ, Edelstein HI, Bagheri N, Leonard 
            JN
  TITLE     The COMET toolkit for composing customizable genetic programs in 
            mammalian cells.
  JOURNAL   Nat Commun. 2020 Feb 7;11(1):779. doi: 10.1038/s41467-019-14147-5.
  PUBMED    32034124
REFERENCE   2  (bases 1 to 4927)
  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 
            Commun."; date: "2020-02-7"; volume: "11(1):779. doi"; pages: " 
            10.1038/s41467-019-14147-5"
COMMENT     Sequence Label: pPD303 VP64-ZF43
FEATURES             Location/Qualifiers
     source          1..4927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..975
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             982..1002
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1027..1176
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     primer_bind     complement(1557..1574)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1563..1787
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1833..2261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1920..1939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2130..2151
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2284..2480
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2331..2466
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2393..2412
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2600..2721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2637..2656)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2691..2710
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2770..2786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2770..2786)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2783..2805)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2794..2810
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2818..2824,2825..2842,2843..2848))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2863..2884
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3001..3018)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3172..3760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3252..3271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3931..4722,4723..4791))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4554..4573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4792..4896)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagtcccaa gctggctagc
      901 gccaccatgg attataagga tcacgatggc gactacaagg accacgatat tgactacaaa
      961 gacgatgacg ataaaatggc tcctaagaaa aagcgcaaag tcggtatcca tggcgttccc
     1021 ggatcagacg cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat
     1081 tttgaccttg acatgcttgg ttcggatgcc cttgatgact ttgacctcga catgctcggc
     1141 agtgacgccc ttgatgattt cgacctggac atgctgatta actctagaag ttccggatct
     1201 tctggttctg gttctggttc aggatccggt accggtggag ctagacccgg ggagcgcccc
     1261 ttccagtgtc gcatttgcat gcggaacttt tcgcgccagg acaggcttga caggcatacc
     1321 cgtactcata ccggtgaaaa accgtttcag tgtcggatct gtatgcgaaa tttctcccag
     1381 aaggagcact tggcggggca tctacgtacg cacaccggcg agaagccatt ccaatgccga
     1441 atatgcatgc gcaacttcag tcgccgcgac aacctgaacc ggcacctaaa aacccacctg
     1501 agaggatctt aagcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     1561 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1621 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1681 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1741 ttgggaagtc aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1801 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     1861 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1921 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1981 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2041 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2101 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2161 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2221 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2281 cagttaggct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2341 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2401 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2461 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggaggttggg
     2521 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     2581 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     2641 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2701 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     2761 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2821 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2881 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2941 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3001 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3061 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3121 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3181 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3241 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3301 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3361 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3421 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3481 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3541 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3601 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3661 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3721 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3781 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3841 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3901 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3961 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4021 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagatccacg
     4081 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4141 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4201 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4261 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4321 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4381 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4441 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4501 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4561 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4621 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4681 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4741 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4801 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4861 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4921 tgacgtc
//
