LOCUS       sequence-320208-        4981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of VP16-ZF62 under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4981)
  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 4981)
  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: pPD533 VP16-ZF62
FEATURES             Location/Qualifiers
     source          1..4981
                     /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"
     primer_bind     complement(1611..1628)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1617..1841
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1887..2315
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1974..1993)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2184..2205
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2338..2534
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2385..2520
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2447..2466
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2654..2775
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2691..2710)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2745..2764
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2824..2840)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2824..2840)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2837..2859)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2848..2864
                     /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(2872..2878,2879..2896,2897..2902))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2917..2938
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3055..3072)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3226..3814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3306..3325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3985..4776,4777..4845))
                     /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     4608..4627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4846..4950)
                     /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 ggatccgtcg acggtggact gcaggcccca ccaaccgatg tttctttagg tgatgagtta
     1081 catttggacg gtgaggacgt agctatggca catgcagatg ccttagacga ttttgacttg
     1141 gatatgttag gagatggaga ttcaccagga cctggtttca caccacacga ttcagctcca
     1201 tacggtgctt tggacatggc cgatttcgaa tttgaacaga tgtttactga tgccttgggt
     1261 atagacgaat acggtggtgg aggtaccggt ggagctagac ccggggagcg ccccttccag
     1321 tgtcgcattt gcatgcggaa cttttcgact ggtcaaagat tgagaattca tacccgtact
     1381 cataccggtg aaaaaccgtt tcagtgtcgg atctgtatgc gaaatttctc ccaaaatcaa
     1441 aatttggcta gacatctacg tacgcacacc ggcgagaagc cattccaatg ccgaatatgc
     1501 atgcgcaact tcagtgataa atctgttttg gctagacacc taaaaaccca cctgagagga
     1561 tcttaagcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     1621 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1681 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1741 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1801 agtcaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     1861 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     1921 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1981 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2041 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2101 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2161 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2221 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2281 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     2341 ggctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2401 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2461 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2521 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggaggt tgggcttcgg
     2581 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     2641 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2701 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2761 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     2821 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2881 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2941 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3001 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3061 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3181 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3241 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3301 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3361 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3421 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3481 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3541 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3601 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3661 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3721 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3781 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3841 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3901 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3961 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4021 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4081 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc
     4141 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4201 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4261 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4321 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4381 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4441 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4501 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4561 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4621 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4681 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4741 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4801 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4861 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4921 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4981 c
//
