LOCUS       sequence-148558-        6019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recruits VP64 to a compatible gRNA for transcriptional activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6019)
  AUTHORS   Chavez A, Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan
            D, Haque SJ, Cecchi RJ, Kowal EJ, Buchthal J, Housden BE, Perrimon 
            N, Collins JJ, Church G
  TITLE     Comparison of Cas9 activators in multiple species.
  JOURNAL   Nat Methods. 2016 May 23. doi: 10.1038/nmeth.3871.
  PUBMED    27214048
REFERENCE   2  (bases 1 to 6019)
  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: "Nat 
            Methods. 2016 May 23. doi: 10.1038/nmeth.3871."
COMMENT     Sequence Label: pcDNA3_3_TOPO_TA-ms2-VP64
FEATURES             Location/Qualifiers
     source          1..6019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      742..751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             754..774
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             784..807
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1189..1338
                     /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(1405..1424)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1449..1497
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1699..2127
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1786..1805)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1996..2017
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2136..2156)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2141..2470
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2321..2456
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2383..2402
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2537..3331
                     /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(2591..2610)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3201..3220
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3507..3628
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3544..3563)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3598..3617
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3677..3693)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3677..3693)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3690..3712)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3701..3717
                     /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(3725..3731,3732..3749,3750..3755))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3770..3791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3908..3925)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4079..4667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4159..4178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4838..5629,5630..5698))
                     /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     5461..5480
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5699..5803)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5878..5897)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tgccaccatg ggacctaaga aaaagaggaa ggtggcggcc
      781 gctgactaca aggatgacga cgataaatct agaatggctt ctaactttac tcagttcgtt
      841 ctcgtcgaca atggcggaac tggcgacgtg actgtcgccc caagcaactt cgctaacggg
      901 atcgctgaat ggatcagctc taactcgcgt tcacaggctt acaaagtaac ctgtagcgtt
      961 cgtcagagct ctgcgcagaa tcgcaaatac accatcaaag tcgaggtgcc taaaggcgcc
     1021 tggcgttcgt acttaaatat ggaactaacc attccaattt tcgccacgaa ttccgactgc
     1081 gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg gaaacccgat tccctcagca
     1141 atcgcagcaa actccggcat ctacgaggcc agcggttccg gacgggctga cgcattggac
     1201 gattttgatc tggatatgct gggaagtgac gccctcgatg attttgacct tgacatgctt
     1261 ggttcggatg cccttgatga ctttgacctc gacatgctcg gcagtgacgc ccttgatgat
     1321 ttcgacctgg acatgctgat taactctaga tgaaagggtt cgatccctac cggttagtaa
     1381 tgagtttaaa cgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     1441 cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata
     1501 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     1561 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     1621 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcagatctg cgcagctggg
     1681 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1741 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     1801 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc
     1861 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1921 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1981 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2041 tctattcttt tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc
     2101 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2161 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2221 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2281 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2341 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2401 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2461 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     2521 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2581 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2641 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2701 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     2761 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     2821 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     2881 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     2941 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3001 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3061 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3121 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3181 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3241 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3301 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcgc gaaatgaccg
     3361 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     3421 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3481 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3541 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3601 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     3661 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3721 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3781 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3841 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3901 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3961 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4021 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4081 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4141 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4201 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4261 tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4321 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4381 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4441 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4501 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4561 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4621 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4681 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4741 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4801 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4861 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4921 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4981 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5041 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5101 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5161 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5221 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5281 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5341 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5401 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5461 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5521 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5581 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5641 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5701 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5761 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5821 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtcga ctctcagtac
     5881 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     5941 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     6001 catgaagaat ctgcttagg
//
