LOCUS       sequence-105905-        6181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-term SpCas9 piece of inducible split-4 (Cas9(N)-FRB-NES split-4)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6181)
  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 6181)
  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: PX851
FEATURES             Location/Qualifiers
     source          1..6181
                     /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)"
     CDS             1278..2879
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMR"
     CDS             2925..3194
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     primer_bind     complement(3222..3239)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3228..3435
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3444..3584
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3444..3573
                     /label=AAV2 ITR
     rep_origin      3659..4114
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3746..3765)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3956..3977
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4131..4150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4250..4272
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4310..4328)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4396..4500
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4501..4569,4570..5361)
                     /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(4719..4738)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5532..6120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6021..6040
                     /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 atgctagatt
     1261 tagctagctt aatcctcgac aagaagtaca gcatcggcct ggacatcggc accaactctg
     1321 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     1381 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     1441 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     1501 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     1561 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     1621 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     1681 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     1741 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     1801 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     1861 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     1921 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     1981 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     2041 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     2101 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     2161 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     2221 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     2281 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     2341 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     2401 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     2461 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     2521 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     2581 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     2641 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     2701 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     2761 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     2821 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagag
     2881 gtggcggtgg ctcgggtggc ggtggctcgg gtggcggtgg ctcggagatg tggcacgagg
     2941 gcctggagga ggccagcaga ctgtacttcg gcgagagaaa cgtgaagggc atgttcgagg
     3001 tgctggagcc cctgcacgcc atgatggaga gaggccccca gaccctgaag gagaccagct
     3061 tcaaccaggc ctacggcaga gacctgatgg aggcccagga gtggtgcaga aagtacatga
     3121 agagcggcaa cgtgaaggac ctgacccagg cctgggacct gtactaccac gtgttcagaa
     3181 gaatcagcaa gcagtaagaa ttcctagagc tcgctgatca gcctcgactg tgccttctag
     3241 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3301 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3361 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag
     3421 caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     3481 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     3541 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     3601 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     3661 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3721 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3781 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3841 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     3901 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3961 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     4021 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4081 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     4141 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4201 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4261 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     4321 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4381 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4441 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4501 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4561 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4621 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4681 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4741 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4801 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     4861 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4921 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4981 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5041 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5101 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5161 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc
     5221 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5281 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5341 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5401 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5461 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5521 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5581 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5641 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     5701 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5761 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5821 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5881 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5941 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6001 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6061 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6121 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6181 t
//
