LOCUS       sequence-313076-        6379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP64-ZF10 under the EF1-alpha 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  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 6379)
  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: pJM520 EF1-alpha 3xFLAG-NLS-VP64-ZF10 in TUPV4
FEATURES             Location/Qualifiers
     source          1..6379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        350..1532
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          581..1523
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1480..1500
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1534..1558
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1633..1642
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1642..1707
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1714..1734
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1759..1908
                     /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(2289..2306)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2295..2519
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3288..3716
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3375..3394)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3585..3606
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3739..3935
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3786..3921
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3848..3867
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4055..4176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4092..4111)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4146..4165
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4225..4241)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4225..4241)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4238..4260)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4249..4265
                     /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(4273..4279,4280..4297,4298..4303))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4318..4339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4456..4473)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4627..5212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4707..4726)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5383..6174,6175..6243))
                     /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     6006..6025
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6244..6348)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcgaag acggttacaa tatcccgtga agcttgagtg gaatcaagat
       61 ctggggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
      121 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
      181 gggccagata tacgcgtgag taattcatac aaaaggactc gcccctgcct tggggaatcc
      241 cagggaccgt cgttaaactc ccactaacgt agaacccaga gatcgctgcg ttcccgcccc
      301 ctcacccgcc cgctctcgtc atcactgagg tggagaagag catgcgtgag gctccggtgc
      361 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
      421 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
      481 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
      541 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
      601 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacgcc
      661 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
      721 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcttgggc
      781 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
      841 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata
      901 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
      961 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     1021 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     1081 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     1141 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc
     1201 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     1261 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     1321 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     1381 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     1441 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     1501 aaagtttttt tcttccattt caggtgtcgt gagagctcgt ttagtgaacc gtcagatcgc
     1561 ctggagacgc catccacgct gttttgacct ccatagaaga gaacgggacc gatccaaatt
     1621 ctcgaggcta gcgccaccat ggattataag gatcacgatg gcgactacaa ggaccacgat
     1681 attgactaca aagacgatga cgataaaatg gctcctaaga aaaagcgcaa agtcggtatc
     1741 catggcgttc ccggatcaga cgcattggac gattttgatc tggatatgct gggaagtgac
     1801 gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc
     1861 gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactctaga
     1921 agttccggat cttctggttc tggttctggt tcaggatccg gtaccggtgg agctagaccc
     1981 ggggagcgcc ccttccagtg tcgcatttgc atgcggaact tttcgactaa tcaaaaattg
     2041 gaagttcata cccgtactca taccggtgaa aaaccgtttc agtgtcggat ctgtatgcga
     2101 aatttctccg ttagacataa tttgcaaaga catctacgta cgcacaccgg cgagaagcca
     2161 ttccaatgcc gaatatgcat gcgcaacttc agtcaacatc caaatttgac tagacaccta
     2221 aaaacccacc tgagaggatc ttaagcggcc gctcgagtct agagggcccg tttaaacccg
     2281 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2341 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2401 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2461 caagggggag gattgggaag tcaatagcag gcatgctggg gatgcggtgg gctctatggc
     2521 ttctgaggca ggtcgacact gtttaacaag cccgtcagta gagcgcatcg cgataagctc
     2581 tagagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
     2641 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
     2701 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
     2761 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt
     2821 aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgcgtac ggatggggct
     2881 ctagctctag agcttgatat cgaattcctg cagccccgct agagggacag ccccccccca
     2941 aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg
     3001 ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc
     3061 ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg
     3121 agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgaaaga gagatttaga
     3181 atgacagaat catagaacgg cctgggcgcg cctggtggcg aacgatcaac agacgggaga
     3241 ggcagaaagt cttcgctgac agcgaccgga ctctaggggg tatccccacg cgccctgtag
     3301 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3361 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3421 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3481 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3541 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3601 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3661 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3721 ctgtggaatg tgtgtcagtt aggctcaatt agtcagcaac catagtcccg cccctaactc
     3781 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3841 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3901 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3961 ttttcggagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4021 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4081 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4141 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4201 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4261 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4321 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4381 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4441 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4501 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4561 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4621 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4681 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4741 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4801 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4861 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4921 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4981 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5041 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5101 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5161 cggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5221 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5281 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5341 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5401 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5461 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5521 gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5581 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5641 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5701 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5761 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5821 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5881 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5941 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6001 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6061 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6121 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6181 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6241 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6301 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6361 aaaagtgcca cctgacgtc
//
