LOCUS       sequence-105908-        6295 bp    DNA     circular SYN 14-APR-2025
DEFINITION  N-term SpCas9 piece of inducible split-5 (Cas9(N)-FRB-NES split-5)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6295)
  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 6295)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: PX853
FEATURES             Location/Qualifiers
     source          1..6295
                     /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..2993
                     /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
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             3039..3308
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     primer_bind     complement(3336..3353)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3342..3549
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3558..3698
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3558..3687
                     /label=AAV2 ITR
     rep_origin      3773..4228
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3860..3879)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4070..4091
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4245..4264)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4364..4386
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4424..4442)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4510..4614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4615..4683,4684..5475)
                     /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(4833..4852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5646..6234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6135..6154
                     /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 ggaatgagaa
     2881 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     2941 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagggtggcg
     3001 gtggctcggg tggcggtggc tcgggtggcg gtggctcgga gatgtggcac gagggcctgg
     3061 aggaggccag cagactgtac ttcggcgaga gaaacgtgaa gggcatgttc gaggtgctgg
     3121 agcccctgca cgccatgatg gagagaggcc cccagaccct gaaggagacc agcttcaacc
     3181 aggcctacgg cagagacctg atggaggccc aggagtggtg cagaaagtac atgaagagcg
     3241 gcaacgtgaa ggacctgacc caggcctggg acctgtacta ccacgtgttc agaagaatca
     3301 gcaagcagta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     3361 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3421 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3481 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     3541 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     3601 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     3661 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     3721 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     3781 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3841 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3901 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3961 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4021 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4081 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     4141 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4201 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4261 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4321 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4381 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4441 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4501 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4561 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4621 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4681 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4741 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4801 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4861 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4921 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4981 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5041 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5101 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5161 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5221 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5281 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     5341 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5401 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5461 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5521 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5581 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5641 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5701 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5761 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     5821 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5881 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5941 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6001 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6061 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6121 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6181 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6241 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
