LOCUS       sequence-105906-        7201 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-term SpCas9 piece of inducible split-4 (Cas9(C)-FKBP split-4)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7201)
  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 7201)
  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: PX852
FEATURES             Location/Qualifiers
     source          1..7201
                     /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             1668..4166
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI
                     SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
                     AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH
                     DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE
                     NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL
                     QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
                     VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI
                     LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV
                     GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
                     ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL
                     PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME
                     RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP
                     SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD
                     KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL
                     IHQSITGLYETRIDLSQLGGD"
     CDS             4167..4214
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4242..4259)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4248..4455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4464..4604
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4464..4593
                     /label=AAV2 ITR
     rep_origin      4679..5134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4766..4785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4976..4997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5151..5170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5270..5292
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5330..5348)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5416..5520
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5521..5589,5590..6381)
                     /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(5739..5758)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6552..7140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7041..7060
                     /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 gcgacaagaa 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 ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg
     4201 caaaaaagaa aaagtaagaa ttcctagagc tcgctgatca gcctcgactg tgccttctag
     4261 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4321 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4381 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag
     4441 caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     4501 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     4561 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     4621 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     4681 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4741 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4801 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4861 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4921 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4981 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     5041 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5101 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     5161 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     5221 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     5281 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     5341 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5401 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5461 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5521 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5581 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5641 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5701 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5761 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5821 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5881 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5941 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6001 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6061 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6121 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6181 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc
     6241 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6301 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6361 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6421 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6481 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6541 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6601 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6661 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6721 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6781 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6841 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6901 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6961 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7021 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7081 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7141 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7201 t
//
