LOCUS       sequence-67465-e        6699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for TALE proteins fused to VP64 activation domain
            compatible with GoldenGate cloning system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6699)
  AUTHORS   Perez-Pinera P, Ousterout DG, Brunger JM, Farin AM, Glass KA, Guilak
            F, Crawford GE, Hartemink AJ, Gersbach CA
  TITLE     Synergistic and tunable human gene activation by combinations of 
            synthetic transcription factors.
  JOURNAL   Nat Methods. 2013 Mar;10(3):239-42. doi: 10.1038/nmeth.2361. Epub 
            2013 Feb 3.
  PUBMED    23377379
REFERENCE   2  (bases 1 to 6699)
  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"; doi: 
            "10.1038/nmeth.2361"; journalName: "Nat Methods"; date: "2013-03"; 
            volume: "10"; issue: "3"; pages: "239-42"
COMMENT     Sequence Label: pcDNA3.1-GoldenGate-VP64
FEATURES             Location/Qualifiers
     source          1..6699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             907..972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             979..999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1312..1719)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     1569..1591
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1583..1600
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1584..1600
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1610..1628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1676..1696)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1676..1694)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1715..1731)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1739..1755
                     /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(1763..1769,1770..1787,1788..1793))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1808..1829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             2024..2044
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2054..2203
                     /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             2210..2236
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2293..2310)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2299..2523
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2569..2997
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2656..2675)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2866..2887
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3006..3026)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3011..3340
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3191..3326
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3253..3272
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3407..4201
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3461..3480)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4071..4090
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4375..4496
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4412..4431)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4466..4485
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4545..4561)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4569..4585
                     /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(4593..4599,4600..4617,4618..4623))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4638..4659
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4776..4793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4947..5532)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5027..5046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5703..6494,6495..6563))
                     /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     6326..6345
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6564..6668)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
      961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gccgcggatc tgtggatcta
     1021 cgcacgctcg gctacagtca gcagcagcaa gagaagatca aaccgaaggt gcgttcgaca
     1081 gtggcgcagc accacgaggc actggtgggc catgggttta cacacgcgca catcgttgcg
     1141 ctcagccaac acccggcagc gttagggacc gtcgctgtca cgtatcagca cataatcacg
     1201 gcgttgccag aggcgacaca cgaagacatc gttggcgtcg gcaaacagtg gtccggcgca
     1261 cgcgccctgg aggccttgct cacggatgcg ggggagttga gaggtccgcc gttacagttg
     1321 gacacaggcc aacttgtgaa gattgcaaaa cgtggcggcg tgaccgcaat ggaggcagtg
     1381 catgcatcgc gcaatgcact gacgggtgcc cccctggaga cgggcgccgc tacagggcgc
     1441 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     1501 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     1561 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     1621 actatagggc gaattgggta ccgggccccc cctcgaggtc ctccagcttt tgttcccttt
     1681 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1741 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1801 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccacc
     1861 ggtcgtctcc aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga
     1921 tgcagtgaaa aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat
     1981 tggcgaacgc acgtcccatc gcgttgccgg atccaaggct agcccgaaaa agaaacgcaa
     2041 agttgggcgc gccgacgcgc tggacgattt cgatctcgac atgctgggtt ctgatgccct
     2101 cgatgacttt gacctggata tgttgggaag cgacgcattg gatgactttg atctggacat
     2161 gctcggctcc gatgctctgg acgatttcga tctcgatatg ttaattaact acccgtacga
     2221 cgttccggac tacgcttctt gaatcggtag gaattcgcgg ccgtctagac ttaagtttaa
     2281 accgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2341 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2401 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2461 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2521 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2581 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2641 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2701 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2761 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2821 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2881 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2941 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3001 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3061 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3181 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3241 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3301 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3361 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3421 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3481 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3541 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3601 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3661 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3721 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3781 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3841 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3901 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3961 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4021 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4081 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4141 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4201 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4261 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4321 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4381 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4441 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4501 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4561 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4621 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4681 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4741 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4801 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4861 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4921 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4981 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5041 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5101 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5161 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5221 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5281 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5341 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5401 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5461 aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5521 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5581 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5641 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5701 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5761 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5821 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5881 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5941 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6001 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6061 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6121 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6181 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6241 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6301 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6361 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6421 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6481 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6541 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6601 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6661 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
