LOCUS       sequence-318378-        6382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP64-ZF1 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: pJM465 EF1-alpha 3xFLAG-NLS-VP64-ZF1 in TUPV2
FEATURES             Location/Qualifiers
     source          1..6382
                     /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"
     primer_bind     complement(1939..1956)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1945..2169
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2938..3366
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3025..3044)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3235..3256
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3389..3585
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3436..3571
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3498..3517
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3705..3826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3742..3761)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3796..3815
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3875..3891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3875..3891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3888..3910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3899..3915
                     /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(3923..3929,3930..3947,3948..3953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3968..3989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4106..4123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4277..4865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4357..4376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5036..5827,5828..5896))
                     /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     5659..5678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5897..6001)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6382,1..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 gaccgggacc
     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 ttaagcggcc gctcgagtct agagggcccg
     1921 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     1981 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2041 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2101 ggcaggacag caagggggag gattgggaag tcaatagcag gcatgctggg gatgcggtgg
     2161 gctctatggc ttctgaggca ggtcgaagaa ctatttcctg gctgttacgc gagcgcatcg
     2221 cgataagctc tagagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc
     2281 ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg
     2341 catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg
     2401 ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg
     2461 aaaaagcttt aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgcgtac
     2521 ggatggggct ctagctctag agcttgatat cgaattcctg cagccccgct agagggacag
     2581 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     2641 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     2701 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     2761 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgaaaga
     2821 gagatttaga atgacagaat catagaacgg cctgggcgcg cctggtggcg aacgatcaac
     2881 agacgggaga ggactaaagt cttcgctgac agcgaccgga ctctaggggg tatccccacg
     2941 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3001 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3061 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3121 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3181 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3241 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3301 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3361 cgaattaatt ctgtggaatg tgtgtcagtt aggctcaatt agtcagcaac catagtcccg
     3421 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3481 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3541 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3601 tgtatatcca ttttcggagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     3661 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     3721 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3781 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     3841 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3901 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3961 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4021 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4081 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4141 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4201 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4261 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4321 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4681 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4861 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4921 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4981 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5041 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5101 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5161 ctgcaatgat accgcgagat ccacgctcac cggctccaga tttatcagca ataaaccagc
     5221 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5281 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5341 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5401 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5461 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5521 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5581 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5641 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5701 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5761 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5821 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5881 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6001 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga agacgggcaa
     6061 aaccgatgtt gacggactaa tcctgacaag atctggggag gtcgctgagt agtgcgcgag
     6121 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     6181 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtg agtaattcat
     6241 acaaaaggac tcgcccctgc cttggggaat cccagggacc gtcgttaaac tcccactaac
     6301 gtagaaccca gagatcgctg cgttcccgcc ccctcacccg cccgctctcg tcatcactga
     6361 ggtggagaag agcatgcgtg ag
//
