LOCUS       sequence-204614-        6177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N terminal SpCas9 (1-573 aa) fused to FRB in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6177)
  AUTHORS   Scot Wolfe
  TITLE     Inducible Cas9-DNA-binding-domain fusions for precise genome editing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6177)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCSD_N-SpCas9-1-573-FRB-1xNLS
FEATURES             Location/Qualifiers
     source          1..6177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..45
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     27..44
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    100..124
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      128..137
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             134..1852
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             1865..1885
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1901..2179
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     polyA_signal    2221..2355
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2245..2264)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2299..2318
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2468..2488)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2468..2486)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2507..2523)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2507..2523)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2520..2542)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2531..2547
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2555..2561,2562..2579,2580..2585))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2600..2621
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2738..2755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2909..3497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2989..3008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3668..4459,4460..4528))
                     /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     4291..4310
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4529..4633)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4659..5114)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4796..4817)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5008..5027
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5185..6169
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 tgcctgggga cgtcggagca agcttgattt aggtgacact atagaataca agctacttgt
       61 tctttttgca ggatcccatc gattcgaatt caaggatcaa caagtttgta caaaaaagca
      121 ggctggcgcc accatggaca agaagtactc cattgggctc gatatcggta ccaacagcgt
      181 cggctgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca aagttctggg
      241 caataccgat cgccacagca taaagaagaa cctcattgga gccctcctgt tcgactccgg
      301 ggagacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa
      361 gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc
      421 tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc acgagcgcca
      481 cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata
      541 tcatctgagg aagaagctgg tagacagtac tgataaggct gacttgcggt tgatctatct
      601 cgcgctggcg cacatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc
      661 agacaacagc gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt
      721 cgaggagaac ccgatcaacg catccggcgt tgacgccaaa gcaatcctga gcgctaggct
      781 gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg
      841 cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt
      901 cgacctggcc gaagatgcca agctgcaact gagcaaagac acctacgatg atgatctcga
      961 caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct
     1021 gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc
     1081 gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa
     1141 ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa
     1201 aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat
     1261 taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc tgaacagaga
     1321 agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct
     1381 gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa
     1441 cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc
     1501 tcggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca tcactccctg
     1561 gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac
     1621 taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga
     1681 gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa
     1741 gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa
     1801 ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg aaggcaccgg
     1861 cgggcccaag aagaagagga aggtaatcgg atcctccgga atcctctggc atgagatgtg
     1921 gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg tgaaaggcat
     1981 gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga ctctgaagga
     2041 aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt ggtgcaggaa
     2101 gtacatgaaa tcagggaatg tcaaggacct cacccaagcc tgggacctct attatcatgt
     2161 gttccgacga atctcaaagc tcgagtaata atctagaact atagtgagtc gtattacgta
     2221 gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     2281 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     2341 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     2401 gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc attgggcccg gtacccagct
     2461 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     2521 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccggg agcataaagt
     2581 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2641 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2701 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2761 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2821 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2881 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2941 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3001 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3061 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3121 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3181 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3241 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3301 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3361 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3421 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3481 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3541 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3601 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3661 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3721 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3781 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3841 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3901 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3961 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4021 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4081 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4141 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4201 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4261 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4321 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4381 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4441 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4501 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4561 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4621 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     4681 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     4741 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     4801 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     4861 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     4921 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     4981 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     5041 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     5101 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     5161 gcgggcctct tcgctattac gccagtcgac cgccaattca atatggcgta tatggactca
     5221 tgccaattca atatggtgga tctggacctg tgccaattca atatggcgta tatggactcg
     5281 tgccaattca atatggtgga tctggacccc agccaattca atatggcgga cttggcacca
     5341 tgccaattca atatggcgga cctggcactg tgccaactgg ggaggggtct acttggcacg
     5401 gtgccaagtt tgaggagggg tcttggccct gtgccaagtc cgccatattg aattggcatg
     5461 gtgccaataa tggcggccat attggctata tgccaggatc aatatatagg caatatccaa
     5521 tatggcccta tgccaatatg gctattggcc aggttcaata ctatgtattg gccctatgcc
     5581 atatagtatt ccatatatgg gttttcctat tgacgtagat agcccctccc aatgggcggt
     5641 cccatatacc atatatgggg cttcctaata ccgcccatag ccactccccc attgacgtca
     5701 atggtctcta tatatggtct ttcctattga cgtcatatgg gcggtcctat tgacgtatat
     5761 ggcgcctccc ccattgacgt caattacggt aaatggcccg cctggctcaa tgcccattga
     5821 cgtcaatagg accacccacc attgacgtca atgggatggc tcattgccca ttcatatccg
     5881 ttctcacgcc ccctattgac gtcaatgacg gtaaatggcc cacttggcag tacatcaata
     5941 tctattaata gtaacttggc aagtacatta ctattggaag tacgccaggg tacattggca
     6001 gtactcccat tgacgtcaat ggcggtaaat ggcccgcgat ggctgccaag tacatcccca
     6061 ttgacgtcaa tggggagggg caatgacgca aatgggcgtt ccattgacgt aaatgggcgg
     6121 taggcgtgcc taatgggagg tctatataag caatgctcgt ttagggaacc gccattc
//
