LOCUS       sequence-317636-        6607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP64-intC-ZF1-NLS-HA under the
            EF1-alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6607)
  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 6607)
  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: pJM524 EF1-alpha 3xFLAG-NLS-VP64-intC-ZF1-NLS-HA 
            in...
FEATURES             Location/Qualifiers
     source          1..6607
                     /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             2057..2077
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2090..2116
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2164..2181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2170..2394
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3163..3591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3250..3269)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3460..3481
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3614..3810
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3661..3796
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3723..3742
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3930..4051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3967..3986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4021..4040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4100..4116)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4100..4116)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4113..4135)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4124..4140
                     /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(4148..4154,4155..4172,4173..4178))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4193..4214
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4331..4348)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4502..5090)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4582..4601)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5261..6052,6053..6121))
                     /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     5884..5903
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6122..6226)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6607,1..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 tctggcccta agaagaagcg caaagttggc tctggcatgt acccatacga
     2101 cgtaccagat tacgcttagg cggccgctcg agtctagagg gcccgtttaa acccgctgat
     2161 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2221 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2281 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2341 gggaggattg ggaagtcaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2401 aggcaggtcg aagaactatt tcctggctgt tacgcgagcg catcgcgata agctctagag
     2461 ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca
     2521 gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc
     2581 agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc
     2641 ttctcgctgc tctttgagcc tgcagacacc tggggggata cggggaaaaa gctttaggct
     2701 gaaagagaga tttagaatga cagaatcata gaacggcctg cgtacggatg gggctctagc
     2761 tctagagctt gatatcgaat tcctgcagcc ccgctagagg gacagccccc ccccaaagcc
     2821 cccagggatg taattacgtc cctcccccgc tagggggcag cagcgagccg cccggggctc
     2881 cgctccggtc cggcgctccc cccgcatccc cgagccggca gcgtgcgggg acagcccggg
     2941 cacggggaag gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct
     3001 gcagacacct ggggggatac ggggaaaaag ctttaggctg aaagagagat ttagaatgac
     3061 agaatcatag aacggcctgg gcgcgcctgg tggcgaacga tcaacagacg ggagaggact
     3121 aaagtcttcg ctgacagcga ccggactcta gggggtatcc ccacgcgccc tgtagcggcg
     3181 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3241 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3301 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3361 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3421 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3481 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3541 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     3601 gaatgtgtgt cagttaggct caattagtca gcaaccatag tcccgcccct aactccgccc
     3661 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3721 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3781 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3841 ggaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3901 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     3961 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4021 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4081 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4141 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4201 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4261 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4321 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4381 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4441 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4501 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4561 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4621 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4681 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4741 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4801 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4861 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4921 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4981 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5041 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5101 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5161 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5221 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5281 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5341 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5401 gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5461 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5521 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5581 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5641 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5701 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5761 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5821 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5881 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5941 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6001 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6061 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6121 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6181 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6241 aagtgccacc tgacgtcgac ggatcgggag atcgaagacg ggcaaaaccg atgttgacgg
     6301 actaatcctg acaagatctg gggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     6361 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     6421 gctgcttcgc gatgtacggg ccagatatac gcgtgagtaa ttcatacaaa aggactcgcc
     6481 cctgccttgg ggaatcccag ggaccgtcgt taaactccca ctaacgtaga acccagagat
     6541 cgctgcgttc ccgccccctc acccgcccgc tctcgtcatc actgaggtgg agaagagcat
     6601 gcgtgag
//
