LOCUS       sequence-318787-        6715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP64-ZF1-intN under the 
            EF1-alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6715)
  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 6715)
  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: pJM591 EF1-alpha 3xFLAG-NLS-VP64-ZF1-intN in TUPV2
FEATURES             Location/Qualifiers
     source          1..6715
                     /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(2622..2639)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2628..2852
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3621..4049
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3708..3727)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3918..3939
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4072..4268
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4119..4254
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4181..4200
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4388..4509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4425..4444)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4479..4498
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4558..4574)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4558..4574)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4571..4593)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4582..4598
                     /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(4606..4612,4613..4630,4631..4636))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4651..4672
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4789..4806)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4960..5548)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5040..5059)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5719..6510,6511..6579))
                     /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     6342..6361
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6580..6684)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcgaag acgggcaaaa ccgatgttga cggactaatc ctgacaagat
       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 tttcgcgcca ggacaggctt
     2041 gacaggcata cccgtactca taccggtgaa aaaccgtttc agtgtcggat ctgtatgcga
     2101 aatttctccc agaaggagca cttggcgggg catctacgta cgcacaccgg cgagaagcca
     2161 ttccaatgcc gaatatgcat gcgcaacttc agtcgccgcg acaacctgaa ccggcaccta
     2221 aaaacccacc tgagaggatc tactggtggt ggtggcgggt ctggaggagg aggtactggt
     2281 ggaggtggca ctggtaccag aagcggatac tgcctggacc tgaagaccca ggtgcagacc
     2341 cctcagggca tgaaggagat cagcaacatc caggtgggcg acctggtgct gagcaacacc
     2401 ggctacaacg aggtgctgaa cgtgttcccc aagagcaaga agaagagcta caagatcacc
     2461 ctggaggacg gcaaggagat catctgcagc gaggagcacc tgttccccac ccagaccggc
     2521 gagatgaaca tcagcggcgg cctgaaggag ggcatgtgcc tgtacgtgaa ggagtgagcg
     2581 gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     2641 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2701 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2761 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagtcaatag
     2821 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcaggtcgaa gaactatttc
     2881 ctggctgtta cgcgagcgca tcgcgataag ctctagaggg acagcccccc cccaaagccc
     2941 ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc
     3001 gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc
     3061 acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg
     3121 cagacacctg gggggatacg gggaaaaagc tttaggctga aagagagatt tagaatgaca
     3181 gaatcataga acggcctgcg tacggatggg gctctagctc tagagcttga tatcgaattc
     3241 ctgcagcccc gctagaggga cagccccccc ccaaagcccc cagggatgta attacgtccc
     3301 tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc
     3361 cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat
     3421 cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg
     3481 ggaaaaagct ttaggctgaa agagagattt agaatgacag aatcatagaa cggcctgggc
     3541 gcgcctggtg gcgaacgatc aacagacggg agaggactaa agtcttcgct gacagcgacc
     3601 ggactctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3661 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3721 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3781 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3841 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3901 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3961 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4021 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttaggctca
     4081 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     4141 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     4201 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     4261 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg aggttgggct tcggaatcgt
     4321 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4381 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4441 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4501 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     4561 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4621 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4681 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4741 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4801 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4861 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4921 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4981 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5041 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5101 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5161 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5221 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5281 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5341 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5401 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5461 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5521 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5581 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5641 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5701 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5761 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5821 gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct caccggctcc
     5881 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5941 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6001 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6061 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6121 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6181 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6241 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6301 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6361 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6421 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6481 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6541 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6601 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6661 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
