LOCUS       sequence-318791-        7435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-DsDed-ZF10-intN-VP64 under the
            EF1-alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7435)
  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 7435)
  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: pJM592 EF1-alpha 3xFLAG-NLS-DsDed-ZF10-intN-VP64 
            in...
FEATURES             Location/Qualifiers
     source          1..7435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          231..1173
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1130..1150
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1184..1208
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1283..1292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1292..1357
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1364..1384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1427..2101
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWEKVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(1607..1627)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2019..2042
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2795..2944
                     /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(2992..3009)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2998..3222
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3991..4419
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4078..4097)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4288..4309
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4442..4638
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4489..4624
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4551..4570
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4758..4879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4795..4814)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4849..4868
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4928..4944)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4928..4944)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4941..4963)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4952..4968
                     /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(4976..4982,4983..5000,5001..5006))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5021..5042
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5159..5176)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5330..5918)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5410..5429)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6089..6880,6881..6949))
                     /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     6712..6731
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6950..7054)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(7435,1..1182)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
ORIGIN
        1 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
       61 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
      121 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
      181 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
      241 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
      301 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
      361 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
      421 tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
      481 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
      541 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
      601 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
      661 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
      721 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
      781 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
      841 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
      901 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
      961 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     1021 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     1081 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     1141 acagtggttc aaagtttttt tcttccattt caggtgtcgt gagagctcgt ttagtgaacc
     1201 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga gaacgggacc
     1261 gatccaaatt ctcgaggcta gcgccaccat ggattataag gatcacgatg gcgactacaa
     1321 ggaccacgat attgactaca aagacgatga cgataaaatg gctcctaaga aaaagcgcaa
     1381 agtcggtatc catggcgttc ccggatcagg ctccggaagt ggctccatgg atagcactga
     1441 gaacgtcatc aagcccttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1501 cgagttcgag atcgagggcg agggcgaggg caagccctac gagggcaccc agaccgccaa
     1561 gctccaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt
     1621 ccagtacggc tccaaggtgt acgtgaagca ccccgccgac atccccgact acaagaagct
     1681 gtccttcccc gagggcttca agtgggagaa ggtgatgaac ttcgaggacg gcggcgtggt
     1741 gaccgtgacc caggactcct ccctccagga cggcaccttc atctaccacg tgaagttcat
     1801 cggcgtgaac ttcccctccg acggccccgt aatgcagaag aaaactctgg gctgggagcc
     1861 ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc acaaggcgct
     1921 gaagctgaag ggcggcggcc actacctggt ggagttcaag tcaatctaca tggccaagaa
     1981 gcccgtgaag ctgcccggct actactacgt ggactccaag ctggacatca cctcccacaa
     2041 cgaggactac accgtggtgg agcagtacga gcgcgccgag gcccgccacc acctgttcca
     2101 gtctggttct ggttctggtt caggatccgg taccggtgga gctagacccg gggagcgccc
     2161 cttccagtgt cgcatttgca tgcggaactt ttcgactaat caaaaattgg aagttcatac
     2221 ccgtactcat accggtgaaa aaccgtttca gtgtcggatc tgtatgcgaa atttctccgt
     2281 tagacataat ttgcaaagac atctacgtac gcacaccggc gagaagccat tccaatgccg
     2341 aatatgcatg cgcaacttca gtcaacatcc aaatttgact agacacctaa aaacccacct
     2401 gagaggatct acttctggtg gtggcgggtc tggaggagga ggtactggtg gaggtggcac
     2461 tggtaccaga agcggatact gcctggacct gaagacccag gtgcagaccc ctcagggcat
     2521 gaaggagatc agcaacatcc aggtgggcga cctggtgctg agcaacaccg gctacaacga
     2581 ggtgctgaac gtgttcccca agagcaagaa gaagagctac aagatcaccc tggaggacgg
     2641 caaggagatc atctgcagcg aggagcacct gttccccacc cagaccggcg agatgaacat
     2701 cagcggcggc ctgaaggagg gcatgtgcct gtacgtgaag gagggaggcg gaagcggagg
     2761 cggtggatca ggctccggaa gtggctccga attcgacgcc cttgatgact ttgacttgga
     2821 catgctgggg tcagacgcac tcgatgattt tgaccttgat atgctgggaa gcgatgcact
     2881 tgatgacttt gacttggaca tgctcggctc cgacgctctt gatgatttcg atcttgacat
     2941 gctctaggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     3001 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3061 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3121 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3181 aagtcaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcaggtcgaa
     3241 gaactatttc ctggctgtta cgcgagcgca tcgcgataag ctctagaggg acagcccccc
     3301 cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc
     3361 ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga
     3421 cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc
     3481 tttgagcctg cagacacctg gggggatacg gggaaaaagc tttaggctga aagagagatt
     3541 tagaatgaca gaatcataga acggcctgcg tacggatggg gctctagctc tagagcttga
     3601 tatcgaattc ctgcagcccc gctagaggga cagccccccc ccaaagcccc cagggatgta
     3661 attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg
     3721 gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt
     3781 ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg
     3841 ggggatacgg ggaaaaagct ttaggctgaa agagagattt agaatgacag aatcatagaa
     3901 cggcctgggc gcgcctggtg gcgaacgatc aacagacggg agaggactaa agtcttcgct
     3961 gacagcgacc ggactctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     4021 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4081 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4141 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4201 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4261 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4321 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4381 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     4441 gttaggctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4501 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4561 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4621 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg aggttgggct
     4681 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4741 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4801 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4861 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     4921 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4981 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5041 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5101 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5161 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5221 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5281 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5341 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5401 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5461 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5521 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5581 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5641 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5701 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5761 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5821 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5881 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5941 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6001 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6061 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6121 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6181 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct
     6241 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6301 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6361 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6421 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6481 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6541 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6601 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6661 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6721 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6781 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6841 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6901 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6961 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7021 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7081 acgtcgacgg atcgggagat cgaagacggg caaaaccgat gttgacggac taatcctgac
     7141 aagatctggg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     7201 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     7261 tgtacgggcc agatatacgc gtgagtaatt catacaaaag gactcgcccc tgccttgggg
     7321 aatcccaggg accgtcgtta aactcccact aacgtagaac ccagagatcg ctgcgttccc
     7381 gccccctcac ccgcccgctc tcgtcatcac tgaggtggag aagagcatgc gtgag
//
