LOCUS       sequence-147557-        6841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MCP:VP64 under actin promoter for ""Scaffold"" CRISPRa in 
            Drosophila cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6841)
  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 6841)
  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: pAct:MCP-VP64
FEATURES             Location/Qualifiers
     source          1..6841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        140..2649
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     protein_bind    2667..2691
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2697..2706
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2709..2729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2739..2762
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3144..3293
                     /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"
     protein_bind    complement(3310..3334)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      3342..3351
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3348..3350,3351..3353,3354..4064)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3348..3350,3351..3353,3354..4064)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3393..3414)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3654..3673)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4001..4022
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          4169..4234
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4364..4384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4809..4943
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4846..4865)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4900..4919
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(5040..5108,5109..5900)
                     /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     complement(5258..5277)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6071..6659
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6560..6579
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6813..6830
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcaggcaact
       61 cgtgaaaggt aggcggatca gcggccgcag catgcaattc tatattctaa aaacacaaat
      121 gatacttcta aaaaaaaatc atgaatggca tcaactctga atcaaatctt tgcagatgca
      181 cctacttctc atttccactg tcacatcatt tttccagatc tcgctgcctg ttatgtggcc
      241 cacaaaccaa gacacgtttt atggccatta aagctggctg atcgtcgcca aacaccaaat
      301 acataatgaa tatgtacaca ttcgagaaag aagcgatcaa agaagcgtct tcgggcggag
      361 taggagaatg cggaggagaa ggagaacgag ctgatctagt atctctccac aatccaatgc
      421 caactgacca actggccata ttcggagcaa tttgaagcca atttccatcg cctggcgatc
      481 gctccattct tggctatatg tttttcaccg ttacccgggg ccattttcaa agactcgtcg
      541 gcaagataag attgtgtcac tcgctgtctc tcttcatttg tcgaagaatg ctgaggaatt
      601 tcgcgatgac gtcggcgagt attttgaaga atgagaataa tttgtattta tacgaaaatc
      661 agttagtgga attttctaca aaaacatgtt atctatagat aattttgttg caaaatatgt
      721 tgactatgac aaagattgta tgtatatacc tttaatgtat tctcattttc ttatgtattt
      781 ataatggcaa tgatgatact gatgatattt taagatgatg ccagaccaaa aggcttgaat
      841 ttctgcgtct tttgccgaac gcagtgcatg tgcaattgtt gttttttgga atattcaatt
      901 ttcggactgt ccgctttgat ttcagtttct tggcttattc aaaaagcaaa gtaaagccaa
      961 aaaagcgaga tggcaatacc aaatgcggca aaacggtagt ggaaggaaag gggtgcgggg
     1021 cagcggaagg aagggtgggg cggggcgtgg cggggtctgt ggctgggcgc gacgtcaccg
     1081 acgttggagc cactcctttg accatgtgtg cgtgtgtgta ttattcgtgt ctcgccactc
     1141 gccggttgtt tttttctttt tatgctgcgc tctctctagc gccatctcgc ttacgcatgc
     1201 tcaacgcacc gcatgttgcc gtttcctttt atgcgtcatt ttggctcgaa ataggcaatt
     1261 atttaaacaa agattagtca acgaaaacgc taaaataaat aagtctacaa tatggttact
     1321 tattgccatg tgtgtgcagc caacgatagc aacaaaagca acaacacagg tggctttccc
     1381 tctttcactt tttgtttgca agccgcgtgc gagcaagacg gcacgaccgg caaacgcaat
     1441 tacgctgaca aagagcagac gaagttttgg cgaaaaacat caaggcgcct gatacgaatg
     1501 catttgcaat aacaattgcg atatttaata ttgtttatga agctgtttga cttcaaaaca
     1561 cacaaaaaaa aaaataaaac aaattatttg aaagagaatt aggaatcgga cgcttatcgt
     1621 tagggtaaca acaagaaatg cttactgagt cacagcctct ggaaaactgc cgcaagccag
     1681 agagagagag aaaaagaggg agagcagctt agaccgcatg tgcttgtgtg tgaggcgtct
     1741 ctctcttcgt ctctgttgcg caaacgcata gactgcactg agaaaatcga ttacctattt
     1801 tttatgaatg aatatttgca ctattactat tcaaaactat taagatagca atcacattca
     1861 atagccaaat actataccac ctgagcgatg caacgaaatg atcaatttga gcaaaaatgc
     1921 tgcatattta ggacggcatc attatagaaa tgcttcttgc tgtgtacttt tctctcgtct
     1981 ggcagctgtt tcgccgttat tgttaaaacc ggcttaagtt aggtgtgttt tctacgacta
     2041 gtgaatgccc tactagaaga tgtgtgttgc acaaaatgtc cctggaataa ccaatttgaa
     2101 gtgcagatag cagtaaacgt aagctaatat gaatattatt taactgtaat gttttaatat
     2161 cgctggacat tactaataaa cccactataa acacatgtac atatgtatgt tttggcatac
     2221 aatgagtagt tggggaaaaa atgtgtaaaa gcaccgtgac catcacagca taaagataac
     2281 cagctgaagt atcgaatatg agtaaccccc aaattgaatc acatgccgca actgatagga
     2341 cccatggaag tacactcttc atggcgatat acaagacaca cacaagcacg aacacccagt
     2401 tgcggaggaa attctccgta aatgaaaacc caatcggcga acaattcata cccatatatg
     2461 gtaaaagttt tgaacgcgac ttgagagcgg agagcattgc ggctgataag gttttagcgc
     2521 taagcgggct ttataaaacg ggctgcggga ccagttttca tatcactacc gtttgagttc
     2581 ttgtgctgtg tggatactcc tcccgacaca aagccgctcc atcagccagc agtcgtctaa
     2641 tccagagacc ccggatccag atatcgacaa gtttgtacaa aaaagcaggc tacaaagcca
     2701 ccatgggacc taagaaaaag aggaaggtgg cggccgctga ctacaaggat gacgacgata
     2761 aatctagaat ggcttctaac tttactcagt tcgttctcgt cgacaatggc ggaactggcg
     2821 acgtgactgt cgccccaagc aacttcgcta acgggatcgc tgaatggatc agctctaact
     2881 cgcgttcaca ggcttacaaa gtaacctgta gcgttcgtca gagctctgcg cagaatcgca
     2941 aatacaccat caaagtcgag gtgcctaaag gcgcctggcg ttcgtactta aatatggaac
     3001 taaccattcc aattttcgcc acgaattccg actgcgagct tattgttaag gcaatgcaag
     3061 gtctcctaaa agatggaaac ccgattccct cagcaatcgc agcaaactcc ggcatctacg
     3121 aggccagcgg ttccggacgg gctgacgcat tggacgattt tgatctggat atgctgggaa
     3181 gtgacgccct cgatgatttt gaccttgaca tgcttggttc ggatgccctt gatgactttg
     3241 acctcgacat gctcggcagt gacgcccttg atgatttcga cctggacatg ctgattaact
     3301 ctagatgaaa cccagctttc ttgtacaaag tggtgagctc cgccaccatg gtgagcaagg
     3361 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     3421 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     3481 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     3541 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     3601 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     3661 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3721 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3781 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     3841 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     3901 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     3961 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     4021 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagcaccgg tccacgtgac
     4081 gtaagctagc aggatctttg tgaaggaacc ttacttctgt ggtgtgacat aattggacaa
     4141 actacctaca gagatttaaa gctctaaggt aaatataaaa tttttaagtg tataatgtgt
     4201 taaactactg attctaattg tttgtgtatt ttagattcca acctatggaa ctgatgaatg
     4261 ggagcagtgg tggaatgcct ttaatgagga aaacctgttt tgctcagaag aaatgccatc
     4321 tagtgatgat gaggctactg ctgactctca acattctact cctccaaaaa agaagagaaa
     4381 ggtagaagac cccaaggact ttccttcaga attgctaagt tttttgagtc atgctgtgtt
     4441 tagtaataga actcttgctt gctttgctat ttacaccaca aaggaaaaag ctgcactgct
     4501 atacaagaaa attatggaaa aatattctgt aacctttata agtaggcata acagttataa
     4561 tcataacata ctgttttttc ttactccaca caggcataga gtgtctgcta ttaataacta
     4621 tgctcaaaaa ttgtgtacct ttagcttttt aatttgtaaa ggggttaata aggaatattt
     4681 gatgtatagt gccttgacta gagatcataa tcagccatac cacatttgta gaggttttac
     4741 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     4801 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4861 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4921 atgtatctta tcatgtctgg atcccgttta aactacgcgt aattcaaaca gggttctggc
     4981 gtcgttctcg tactgttttc cccaggccag tgctttagcg ttattgaaaa aggaagagta
     5041 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5101 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5161 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5221 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5281 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5341 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5401 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5461 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5521 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5581 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5641 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5701 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5761 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5821 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5881 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5941 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6001 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6061 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6121 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6181 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     6241 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6301 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6361 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6421 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6481 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6541 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6601 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6661 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6721 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     6781 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     6841 a
//
