LOCUS       sequence-318784-        6382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP64-ZF10 under the EF1-alpha 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6382)
  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 6382)
  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: pJM520 EF1-alpha 3xFLAG-NLS-VP64-ZF10 in TUPV4
FEATURES             Location/Qualifiers
     source          1..6382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      777..1205
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(864..883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1074..1095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1228..1424
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1275..1410
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1337..1356
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    1544..1665
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1581..1600)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1635..1654
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1714..1730)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1714..1730)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1727..1749)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1738..1754
                     /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(1762..1768,1769..1786,1787..1792))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1807..1828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1945..1962)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2116..2704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2196..2215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2875..3666,3667..3735))
                     /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     3498..3517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3736..3840)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4221..5403
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4452..5394
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5351..5371
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     5405..5429
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      5504..5513
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5513..5578
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5585..5605
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5630..5779
                     /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(6160..6177)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6166..6382,1..8)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ctctatggct tctgaggcag gtcgacactg tttaacaagc ccgtcagtag agcgcatcgc
       61 gataagctct agagggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc
      121 cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc
      181 atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc
      241 tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga
      301 aaaagcttta ggctgaaaga gagatttaga atgacagaat catagaacgg cctgcgtacg
      361 gatggggctc tagctctaga gcttgatatc gaattcctgc agccccgcta gagggacagc
      421 ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga
      481 gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc
      541 ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc
      601 tgctctttga gcctgcagac acctgggggg atacggggaa aaagctttag gctgaaagag
      661 agatttagaa tgacagaatc atagaacggc ctgggcgcgc ctggtggcga acgatcaaca
      721 gacgggagag gcagaaagtc ttcgctgaca gcgaccggac tctagggggt atccccacgc
      781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
      841 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      961 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1021 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1081 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1141 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1201 gaattaattc tgtggaatgt gtgtcagtta ggctcaatta gtcagcaacc atagtcccgc
     1261 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     1321 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     1381 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     1441 gtatatccat tttcggaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     1501 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     1561 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     1621 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     1681 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     1741 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     1801 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1861 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1921 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     1981 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2041 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2101 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2161 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2221 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2281 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2341 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2401 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2461 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2521 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2581 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2641 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2701 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2761 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2821 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     2881 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     2941 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3001 tgcaatgata ccgcgagatc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3061 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3121 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3181 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3241 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3301 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3361 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3421 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3481 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3541 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     3601 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3661 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3721 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     3781 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     3841 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatcgaa gacggttaca
     3901 atatcccgtg aagcttgagt ggaatcaaga tctggggagg tcgctgagta gtgcgcgagc
     3961 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
     4021 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgtga gtaattcata
     4081 caaaaggact cgcccctgcc ttggggaatc ccagggaccg tcgttaaact cccactaacg
     4141 tagaacccag agatcgctgc gttcccgccc cctcacccgc ccgctctcgt catcactgag
     4201 gtggagaaga gcatgcgtga ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     4261 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
     4321 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     4381 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     4441 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
     4501 gcccttgcgt gccttgaatt acttccacgc ccctggctgc agtacgtgat tcttgatccc
     4561 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
     4621 cctcgtgctt gagttgaggc ctggcttggg cgctggggcc gccgcgtgcg aatctggtgg
     4681 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
     4741 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
     4801 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
     4861 tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
     4921 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
     4981 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
     5041 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
     5101 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     5161 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     5221 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     5281 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     5341 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     5401 tgagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
     5461 tccatagaag agaacgggac cgatccaaat tctcgaggct agcgccacca tggattataa
     5521 ggatcacgat ggcgactaca aggaccacga tattgactac aaagacgatg acgataaaat
     5581 ggctcctaag aaaaagcgca aagtcggtat ccatggcgtt cccggatcag acgcattgga
     5641 cgattttgat ctggatatgc tgggaagtga cgccctcgat gattttgacc ttgacatgct
     5701 tggttcggat gcccttgatg actttgacct cgacatgctc ggcagtgacg cccttgatga
     5761 tttcgacctg gacatgctga ttaactctag aagttccgga tcttctggtt ctggttctgg
     5821 ttcaggatcc ggtaccggtg gagctagacc cggggagcgc cccttccagt gtcgcatttg
     5881 catgcggaac ttttcgacta atcaaaaatt ggaagttcat acccgtactc ataccggtga
     5941 aaaaccgttt cagtgtcgga tctgtatgcg aaatttctcc gttagacata atttgcaaag
     6001 acatctacgt acgcacaccg gcgagaagcc attccaatgc cgaatatgca tgcgcaactt
     6061 cagtcaacat ccaaatttga ctagacacct aaaaacccac ctgagaggat cttaagcggc
     6121 cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     6181 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     6241 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     6301 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gtcaatagca
     6361 ggcatgctgg ggatgcggtg gg
//
