LOCUS       sequence-317644-        6373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP64-intN under the EF1-alpha 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6373)
  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 6373)
  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: pJM554 EF1-alpha 3xFLAG-NLS-VP64-intN in TUPV2
FEATURES             Location/Qualifiers
     source          1..6373
                     /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"
     primer_bind     complement(1930..1947)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1936..2160
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2929..3357
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3016..3035)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3226..3247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3380..3576
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3427..3562
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3489..3508
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3696..3817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3733..3752)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3787..3806
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3866..3882)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3866..3882)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3879..3901)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3890..3906
                     /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(3914..3920,3921..3938,3939..3944))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3959..3980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4097..4114)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4268..4856)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4348..4367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5027..5818,5819..5887))
                     /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     5650..5669
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5888..5992)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6373,1..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 ggaggaggag gtactggtgg aggtggcact ggtaccagaa gcggatactg
     1621 cctggacctg aagacccagg tgcagacccc tcagggcatg aaggagatca gcaacatcca
     1681 ggtgggcgac ctggtgctga gcaacaccgg ctacaacgag gtgctgaacg tgttccccaa
     1741 gagcaagaag aagagctaca agatcaccct ggaggacggc aaggagatca tctgcagcga
     1801 ggagcacctg ttccccaccc agaccggcga gatgaacatc agcggcggcc tgaaggaggg
     1861 catgtgcctg tacgtgaagg agtgagcggc cgctcgagtc tagagggccc gtttaaaccc
     1921 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1981 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2041 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2101 gcaaggggga ggattgggaa gtcaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2161 cttctgaggc aggtcgaaga actatttcct ggctgttacg cgagcgcatc gcgataagct
     2221 ctagagggac agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag
     2281 ggggcagcag cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga
     2341 gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct
     2401 gaacgcttct cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaagctt
     2461 taggctgaaa gagagattta gaatgacaga atcatagaac ggcctgcgta cggatggggc
     2521 tctagctcta gagcttgata tcgaattcct gcagccccgc tagagggaca gccccccccc
     2581 aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg
     2641 gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag
     2701 cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt
     2761 gagcctgcag acacctgggg ggatacgggg aaaaagcttt aggctgaaag agagatttag
     2821 aatgacagaa tcatagaacg gcctgggcgc gcctggtggc gaacgatcaa cagacgggag
     2881 aggactaaag tcttcgctga cagcgaccgg actctagggg gtatccccac gcgccctgta
     2941 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3001 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3061 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3121 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3181 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3241 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3301 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3361 tctgtggaat gtgtgtcagt taggctcaat tagtcagcaa ccatagtccc gcccctaact
     3421 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3481 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3541 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3601 attttcggag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3661 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3721 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3781 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     3841 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3901 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3961 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4021 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4081 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4141 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4201 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4261 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4321 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4381 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4441 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4501 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4561 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4621 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4681 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4741 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4801 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4861 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4921 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4981 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5041 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5101 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5161 taccgcgaga tccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5221 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5281 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5341 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5401 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5461 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5521 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5581 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5641 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5701 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5761 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5821 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5881 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5941 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6001 cccgaaaagt gccacctgac gtcgacggat cgggagatcg aagacgggca aaaccgatgt
     6061 tgacggacta atcctgacaa gatctgggga ggtcgctgag tagtgcgcga gcaaaattta
     6121 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     6181 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt gagtaattca tacaaaagga
     6241 ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa ctcccactaa cgtagaaccc
     6301 agagatcgct gcgttcccgc cccctcaccc gcccgctctc gtcatcactg aggtggagaa
     6361 gagcatgcgt gag
//
