LOCUS       sequence-317681-        6943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP64-ZF1-intC-ZF10-NLS-HA 
            under the EF1-alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6943)
  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 6943)
  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: pJM590 EF1-alpha 
            3xFLAG-NLS-VP64-ZF1-intC-ZF10-NLS-...
FEATURES             Location/Qualifiers
     source          1..6943
                     /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             1409..1558
                     /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             2393..2413
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2426..2452
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2500..2517)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2506..2730
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3499..3927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3586..3605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3796..3817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3950..4146
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3997..4132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4059..4078
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4266..4387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4303..4322)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4357..4376
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4436..4452)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4436..4452)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4449..4471)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4460..4476
                     /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(4484..4490,4491..4508,4509..4514))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4529..4550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4667..4684)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4838..5426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4918..4937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5597..6388,6389..6457))
                     /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     6220..6239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6458..6562)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6943,1..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 ccggatcaga cgcattggac gattttgatc tggatatgct
     1441 gggaagtgac gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga
     1501 ctttgacctc gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat
     1561 taactctaga agttccggat cttctggttc tggttctggt tcaggatccg gtaccggtgg
     1621 agctagaccc ggggagcgcc ccttccagtg tcgcatttgc atgcggaact tttcgcgcca
     1681 ggacaggctt gacaggcata cccgtactca taccggtgaa aaaccgtttc agtgtcggat
     1741 ctgtatgcga aatttctccc agaaggagca cttggcgggg catctacgta cgcacaccgg
     1801 cgagaagcca ttccaatgcc gaatatgcat gcgcaacttc agtcgccgcg acaacctgaa
     1861 ccggcaccta aaaacccacc tgagaggatc tggaggcgga agcggaggcg gtggatcagg
     1921 ctccggaagt ggctccatga tgctgaagaa gatcctgaag atcgaggagc tggacgagag
     1981 agagctgatc gacatcgagg tgagcggcaa ccacctgttc tacgccaacg acatcctgac
     2041 ccacaacagc ggatccggtg gtggcgggtc tggaggagga ggtagtggtg gaggtggctc
     2101 tggtaccggt ggagctagac ccggggagcg ccccttccag tgtcgcattt gcatgcggaa
     2161 cttttcgact aatcaaaaat tggaagttca tacccgtact cataccggtg aaaaaccgtt
     2221 tcagtgtcgg atctgtatgc gaaatttctc cgttagacat aatttgcaaa gacatctacg
     2281 tacgcacacc ggcgagaagc cattccaatg ccgaatatgc atgcgcaact tcagtcaaca
     2341 tccaaatttg actagacacc taaaaaccca cctgagagga tctggctctg gccctaagaa
     2401 gaagcgcaaa gttggctctg gcatgtaccc atacgacgta ccagattacg cttaggcggc
     2461 cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2521 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2581 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2641 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gtcaatagca
     2701 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc aggtcgaaga actatttcct
     2761 ggctgttacg cgagcgcatc gcgataagct ctagagggac agcccccccc caaagccccc
     2821 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc
     2881 tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac
     2941 ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca
     3001 gacacctggg gggatacggg gaaaaagctt taggctgaaa gagagattta gaatgacaga
     3061 atcatagaac ggcctgcgta cggatggggc tctagctcta gagcttgata tcgaattcct
     3121 gcagccccgc tagagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc
     3181 ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg
     3241 catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg
     3301 ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg
     3361 aaaaagcttt aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgggcgc
     3421 gcctggtggc gaacgatcaa cagacgggag aggactaaag tcttcgctga cagcgaccgg
     3481 actctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3541 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3601 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3661 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3721 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3781 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3841 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3901 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt taggctcaat
     3961 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4021 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4081 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4141 tgcaaaaagc tcccgggagc ttgtatatcc attttcggag gttgggcttc ggaatcgttt
     4201 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4261 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4321 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4381 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4441 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4501 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4561 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4621 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4681 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4741 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4801 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4861 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4921 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4981 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5041 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5101 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5161 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5221 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5281 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5341 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5401 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5461 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5521 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5581 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5641 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5701 gcttaccatc tggccccagt gctgcaatga taccgcgaga tccacgctca ccggctccag
     5761 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5821 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5881 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5941 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6001 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6061 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6121 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6181 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6241 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6301 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6361 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6421 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6481 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6541 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     6601 cgggagatcg aagacgggca aaaccgatgt tgacggacta atcctgacaa gatctgggga
     6661 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     6721 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     6781 atatacgcgt gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac
     6841 cgtcgttaaa ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc
     6901 gcccgctctc gtcatcactg aggtggagaa gagcatgcgt gag
//
