LOCUS       sequence-105911-        7351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-term SpCas9 piece of inducible transcriptional activator 
            (dCas9(C)-FKBP-2xNLS-VP64).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7351)
  AUTHORS   Zetsche B, Volz SE, Zhang F
  TITLE     A split-Cas9 architecture for inducible genome editing and 
            transcription modulation.
  JOURNAL   Nat Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149.
  PUBMED    25643054
REFERENCE   2  (bases 1 to 7351)
  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 
            Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149."
COMMENT     Sequence Label: PX856
FEATURES             Location/Qualifiers
     source          1..7351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1233
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1245..1254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1257..1277
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1302..1622
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             join(1668..2648,2649..2651,2652..4166)
                     /codon_start=1
                     /product="C-terminal portion of catalytically inactive 
                     Streptococcus pyogenes dCas9 carrying the N863A mutation 
                     (Zetsche et al., 2015)"
                     /label=dCas9(C)
                     /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI
                     SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
                     AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH
                     DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE
                     NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL
                     QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEV
                     VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI
                     LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV
                     GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
                     ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL
                     PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME
                     RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP
                     SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD
                     KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL
                     IHQSITGLYETRIDLSQLGGD"
     CDS             4170..4190
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4209..4358
                     /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(4392..4409)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4398..4605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4614..4754
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4614..4743
                     /label=AAV2 ITR
     rep_origin      4829..5284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4916..4935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5126..5147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5301..5320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5420..5442
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5480..5498)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5566..5670
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5671..5739,5740..6531)
                     /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(5889..5908)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6702..7290
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7191..7210
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggccccaa
     1261 agaagaagcg gaaggtcggt atccacggag tcccagcagc cggcgtgcag gtggagacca
     1321 tcagccccgg cgacggcaga accttcccca agagaggcca gacctgcgtg gtgcactaca
     1381 ccggcatgct ggaggacggc aagaagttcg acagcagcag agacagaaac aagcccttca
     1441 agttcatgct gggcaagcag gaggtgatca gaggctggga ggagggcgtg gcccagatga
     1501 gcgtgggcca gagagccaag ctgaccatca gccccgacta cgcctacggc gccaccggcc
     1561 accccggcat catccctcct cacgccaccc tggtgttcga cgtggagctg ctgaagctgg
     1621 agggtggcgg tggctcgggt ggcggtggct cgggtggcgg tggctcgaag cccgccttcc
     1681 tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     1741 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     1801 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     1861 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     1921 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     1981 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     2041 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc
     2101 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg
     2161 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     2221 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga
     2281 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg
     2341 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga
     2401 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     2461 ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc
     2521 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc
     2581 atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa
     2641 gcgacaaggc ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     2701 agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc
     2761 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     2821 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga
     2881 acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt
     2941 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     3001 actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa
     3061 agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga
     3121 agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca
     3181 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc
     3241 ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt
     3301 ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg
     3361 tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga
     3421 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg
     3481 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg
     3541 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg
     3601 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta
     3661 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac
     3721 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca
     3781 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg
     3841 aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag
     3901 tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata
     3961 agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag
     4021 cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca
     4081 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga
     4141 tcgacctgtc tcagctggga ggcgacagcc ccaagaagaa gagaaaggtg gaggccagcg
     4201 gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac gccctcgatg
     4261 attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc gacatgctcg
     4321 gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactaagaa ttcctagagc
     4381 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4441 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4501 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4561 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     4621 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4681 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4741 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4801 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4861 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4921 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4981 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5041 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5101 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5161 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5221 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5281 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     5341 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5401 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5461 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5521 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5581 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5641 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5701 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5761 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5821 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5881 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5941 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6001 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6061 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6121 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6181 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6241 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6301 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6361 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     6421 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6481 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6541 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6601 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6661 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6721 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6781 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6841 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6901 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6961 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7021 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7081 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7141 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7201 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7261 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7321 ctggcctttt gctggccttt tgctcacatg t
//
