LOCUS       sequence-317920-        7330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP64-intC-ZF1-DsDed-NLS-HA 
            under the EF1-alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7330)
  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 7330)
  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: pJM588 EF1-alpha 
            3xFLAG-NLS-VP64-intC-ZF1-DsDed-NLS...
FEATURES             Location/Qualifiers
     source          1..7330
                     /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             1400..1549
                     /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"
     CDS             2102..2776
                     /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(2282..2302)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2694..2717
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2786..2806
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2813..2839
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2887..2904)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2893..3117
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3886..4314
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3973..3992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4183..4204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4337..4533
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4384..4519
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4446..4465
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4653..4774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4690..4709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4744..4763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4823..4839)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4823..4839)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4836..4858)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4847..4863
                     /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(4871..4877,4878..4895,4896..4901))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4916..4937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5054..5071)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5225..5813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5305..5324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5984..6775,6776..6844))
                     /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     6607..6626
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6845..6949)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(7330,1..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 ggactataaa gaccatgacg gtgactacaa
     1321 ggatcacgac atagattata aagacgatga cgacaagatg gcacctaaga agaagcgcaa
     1381 agttggctct ggcgaattcg acgcccttga tgactttgac ttggacatgc tggggtcaga
     1441 cgcactcgat gattttgacc ttgatatgct gggaagcgat gcacttgatg actttgactt
     1501 ggacatgctc ggctccgacg ctcttgatga tttcgatctt gacatgctcg gatccggtgg
     1561 tggcgggtct ggtggaggag gtagtggtgg aggtggctct gccaccatga tgctgaagaa
     1621 gatcctgaag atcgaggagc tggacgagag agagctgatc gacatcgagg tgagcggcaa
     1681 ccacctgttc tacgccaacg acatcctgac ccacaacagc aacaggggtg gtggcgggtc
     1741 tggtggagga ggtagtggtg gaggtggctc tggtaccgct agacccgggg agcgcccctt
     1801 ccagtgtcgc atttgcatgc ggaacttttc gcgccaggac aggcttgaca ggcatacccg
     1861 tactcatacc ggtgaaaaac cgtttcagtg tcggatctgt atgcgaaatt tctcccagaa
     1921 ggagcacttg gcggggcatc tacgtacgca caccggcgag aagccattcc aatgccgaat
     1981 atgcatgcgc aacttcagtc gccgcgacaa cctgaaccgg cacctaaaaa cccacctgag
     2041 agaattcggc tctggcggag gcggaagcgg aggcggtgga tcaggctccg gaagtggctc
     2101 catggatagc actgagaacg tcatcaagcc cttcatgcgc ttcaaggtgc acatggaggg
     2161 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggcaagc cctacgaggg
     2221 cacccagacc gccaagctcc aggtgaccaa gggcggcccc ctgcccttcg cctgggacat
     2281 cctgtccccc cagttccagt acggctccaa ggtgtacgtg aagcaccccg ccgacatccc
     2341 cgactacaag aagctgtcct tccccgaggg cttcaagtgg gagaaggtga tgaacttcga
     2401 ggacggcggc gtggtgaccg tgacccagga ctcctccctc caggacggca ccttcatcta
     2461 ccacgtgaag ttcatcggcg tgaacttccc ctccgacggc cccgtaatgc agaagaaaac
     2521 tctgggctgg gagccctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga
     2581 gatccacaag gcgctgaagc tgaagggcgg cggccactac ctggtggagt tcaagtcaat
     2641 ctacatggcc aagaagcccg tgaagctgcc cggctactac tacgtggact ccaagctgga
     2701 catcacctcc cacaacgagg actacaccgt ggtggagcag tacgagcgcg ccgaggcccg
     2761 ccaccacctg ttccagtctg gttctcctaa gaagaagcgc aaagttggca tgtacccata
     2821 cgacgtacca gattacgctt aggcggccgc tcgagtctag agggcccgtt taaacccgct
     2881 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2941 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3001 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3061 agggggagga ttgggaagtc aatagcaggc atgctgggga tgcggtgggc tctatggctt
     3121 ctgaggcagg tcgaagaact atttcctggc tgttacgcga gcgcatcgcg ataagctcta
     3181 gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg
     3241 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc
     3301 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa
     3361 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aaagctttag
     3421 gctgaaagag agatttagaa tgacagaatc atagaacggc ctgcgtacgg atggggctct
     3481 agctctagag cttgatatcg aattcctgca gccccgctag agggacagcc cccccccaaa
     3541 gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg
     3601 ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc
     3661 gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag
     3721 cctgcagaca cctgggggga tacggggaaa aagctttagg ctgaaagaga gatttagaat
     3781 gacagaatca tagaacggcc tgggcgcgcc tggtggcgaa cgatcaacag acgggagagg
     3841 actaaagtct tcgctgacag cgaccggact ctagggggta tccccacgcg ccctgtagcg
     3901 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3961 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4021 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4081 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4141 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4201 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     4261 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     4321 gtggaatgtg tgtcagttag gctcaattag tcagcaacca tagtcccgcc cctaactccg
     4381 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4441 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     4501 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     4561 ttcggaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4621 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4681 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4741 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4801 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4861 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4921 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4981 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5041 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5101 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5161 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5221 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5281 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5341 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5401 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5461 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5521 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5581 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5641 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5701 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5761 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5821 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5881 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5941 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6001 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6061 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6121 cgcgagatcc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6181 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6241 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6301 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6361 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6421 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6481 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6541 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6601 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6661 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6721 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6781 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6841 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6901 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6961 gaaaagtgcc acctgacgtc gacggatcgg gagatcgaag acgggcaaaa ccgatgttga
     7021 cggactaatc ctgacaagat ctggggaggt cgctgagtag tgcgcgagca aaatttaagc
     7081 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     7141 tgcgctgctt cgcgatgtac gggccagata tacgcgtgag taattcatac aaaaggactc
     7201 gcccctgcct tggggaatcc cagggaccgt cgttaaactc ccactaacgt agaacccaga
     7261 gatcgctgcg ttcccgcccc ctcacccgcc cgctctcgtc atcactgagg tggagaagag
     7321 catgcgtgag
//
