LOCUS       sequence-105910-        6205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-term SpCas9 piece of inducible transcriptional activator 
            (dCas9(N)-FRB-2xNES).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6205)
  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 6205)
  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: PX855
FEATURES             Location/Qualifiers
     source          1..6205
                     /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             join(1278..1301,1302..1304,1305..2879)
                     /codon_start=1
                     /product="N-terminal portion of catalytically inactive 
                     Streptococcus pyogenes dCas9 carrying the D10A mutation 
                     (Zetsche et al., 2015)"
                     /label=dCas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMR"
     CDS             2925..3197
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQL"
     primer_bind     complement(3246..3263)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3252..3459
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3468..3608
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3468..3597
                     /label=AAV2 ITR
     rep_origin      3683..4138
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3770..3789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3980..4001
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4155..4174)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4274..4296
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4334..4352)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4420..4524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4525..4593,4594..5385)
                     /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(4743..4762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5556..6144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6045..6064
                     /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 ggccatcggc 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 gcagctagat ttagctagct taatcctcta agaattccta gagctcgctg
     3241 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     3301 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3361 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     3421 gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt
     3481 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3541 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     3601 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3661 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3721 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3781 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3841 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     3901 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3961 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4021 tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4081 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     4141 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     4201 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4261 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4321 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4381 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4441 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4501 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4561 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4621 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4681 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4741 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4801 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4861 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4921 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4981 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5041 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5101 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5161 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5221 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     5281 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5341 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5401 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5461 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5521 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5581 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5641 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5701 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5761 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5821 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5881 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5941 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6001 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6061 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6121 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6181 ttttgctggc cttttgctca catgt
//
