LOCUS       sequence-318369-        5899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NES-PYL1-VP64 under the CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5899)
  AUTHORS   Muldoon JJ, Kandula V, Hong M, Donahue PS, Boucher JD, Bagheri N, 
            Leonard JN
  TITLE     Model-guided design of mammalian genetic programs.
  JOURNAL   Sci Adv. 2021 Feb 19;7(8). pii: 7/8/eabe9375. doi: 
            10.1126/sciadv.abe9375. Print 2021 Feb.
  PUBMED    33608279
REFERENCE   2  (bases 1 to 5899)
  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: "Sci Adv."; 
            date: "2021-02-19"; volume: "7(8). pii: 7/8/eabe9375. doi"; pages: "
            10.1126/sciadv.abe9375. Print 2021 Feb"
COMMENT     Sequence Label: pPD1122 CMV 3xFLAG-NES-PYL1-VP64 in TUPV3
FEATURES             Location/Qualifiers
     source          1..5899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             676..741
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1405..1554
                     /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(1608..1625)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1614..1838
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2607..3035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2694..2713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2904..2925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3058..3254
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3105..3240
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3167..3186
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3374..3495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3411..3430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3465..3484
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3544..3560)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3544..3560)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3557..3579)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3568..3584
                     /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(3592..3598,3599..3616,3617..3622))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3637..3658
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3775..3792)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3946..4534)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4026..4045)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4705..5496,5497..5565))
                     /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     5328..5347
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5566..5670)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag tcccaagctg
      661 gctagcgcca ccatggacta caaagaccac gacggcgatt acaaggatca tgacatcgat
      721 tacaaagacg atgacgataa gatggaagag ctgagccagg ctctggccag ctcctttagc
      781 gtgtcctccg gaatgggcgg aggagcccct acccaggacg agttcaccca gctgagccag
      841 agcatcgctg agttccacac ctaccagctg ggaaacggac gctgttccag cctgctggca
      901 cagagaatcc acgctcctcc tgagacagtg tggagtgtgg tgcgcagatt cgaccgccct
      961 cagatttaca agcacttcat caagagctgc aacgtgagcg aggacttcga gatgagagtg
     1021 ggatgtacca gagatgtgaa cgtgatcagc ggactgcctg ccaacaccag cagagagaga
     1081 ctggacctgc tggacgatga ccgcagagtg accggcttca gcatcaccgg aggtgagcac
     1141 agactgagaa actacaagag cgtgaccacc gtccaccgct tcgagaagga agaggaagag
     1201 gagcgcatct ggaccgtggt gctggagagc tacgtcgtgg acgtgcccga gggcaacagc
     1261 gaagaggata cccgcctgtt cgctgacacc gtgatcagac tgaacctcca gaagctggcc
     1321 agcatcaccg aggcaatgaa cggatccggt ggtggcgggt ctggaggagg aggtagtggt
     1381 ggaggtggct ctggtaccgc cggagacgct ctggatgact ttgatctgga catgcttggt
     1441 tccgatgcct tggacgattt tgacctcgat atgctgggct ccgacgctct cgacgatttc
     1501 gacctcgaca tgctgggtag cgatgcactg gatgactttg atctggatat gctcggatca
     1561 taggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     1621 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1681 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1741 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaagt
     1801 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcag gtcgaagaga
     1861 ctgcccgaca catcttagtg agcgcatcgc gataagctct agagggacag ccccccccca
     1921 aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg
     1981 ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc
     2041 ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg
     2101 agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgaaaga gagatttaga
     2161 atgacagaat catagaacgg cctgcgtacg gatggggctc tagctctaga gcttgatatc
     2221 gaattcctgc agccccgcta gagggacagc ccccccccaa agcccccagg gatgtaatta
     2281 cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc ggtccggcgc
     2341 tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca
     2401 cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac acctgggggg
     2461 atacggggaa aaagctttag gctgaaagag agatttagaa tgacagaatc atagaacggc
     2521 ctgggcgcgc ctggtggcga acgatcaaca gacgggagag gttacaagtc ttcgctgaca
     2581 gcgaccggac tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2641 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2701 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2761 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2821 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2881 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2941 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3001 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3061 ggctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3121 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3181 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3241 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggaggt tgggcttcgg
     3301 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     3361 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3421 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3481 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     3541 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3601 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3661 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3721 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3781 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3841 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3901 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3961 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4021 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4081 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4141 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4201 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4261 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4321 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4381 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4441 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4501 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4561 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4621 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4681 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4741 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4801 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc
     4861 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4921 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4981 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5041 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5101 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5161 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5221 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5281 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5341 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5401 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5461 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5521 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5581 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5641 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     5701 cgacggttcg ggagatcgaa gacggactaa atagtagttc agacgccgtt aagcgcaaga
     5761 tctggggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     5821 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     5881 cgggccagat atacgcgtt
//
