LOCUS       sequence-202430-        6180 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 6180)
  AUTHORS   Scot Wolfe
  TITLE     Inducible Cas9-DNA-binding-domain fusions for precise genome editing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6180)
  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            "Unpublished"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..6180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..55
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     37..54
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    110..134
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      138..147
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             144..1862
                     /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             1875..1895
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1911..2189
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     polyA_signal    2231..2365
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2255..2274)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2309..2328
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2478..2498)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2478..2496)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2517..2533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2517..2533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2530..2552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2541..2557
                     /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(2565..2571,2572..2589,2590..2595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2610..2631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2748..2765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2919..3507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2999..3018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3678..4469,4470..4538))
                     /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     4301..4320
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4539..4643)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4669..5124)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4806..4827)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5018..5037
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(5198..6180,1..2)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 accgccattc tgcctgggga cgtcggagca agcttgattt aggtgacact atagaataca
       61 agctacttgt tctttttgca ggatcccatc gattcgaatt caaggatcaa caagtttgta
      121 caaaaaagca ggctggcgcc accatggaca agaagtactc cattgggctc gatatcggta
      181 ccaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca
      241 aagttctggg caataccgat cgccacagca taaagaagaa cctcattgga gccctcctgt
      301 tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata
      361 cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg
      421 tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc
      481 acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc
      541 caaccatata tcatctgagg aagaagctgg tagacagtac tgataaggct gacttgcggt
      601 tgatctatct cgcgctggcg cacatgatca aatttcgggg acacttcctc atcgaggggg
      661 acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt cagacttaca
      721 atcagctttt cgaggagaac ccgatcaacg catccggcgt tgacgccaaa gcaatcctga
      781 gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga
      841 agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta
      901 aatctaactt cgacctggcc gaagatgcca agctgcaact gagcaaagac acctacgatg
      961 atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg
     1021 caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca
     1081 ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga
     1141 ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg
     1201 atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt
     1261 acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc
     1321 tgaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc
     1381 agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt
     1441 tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag
     1501 gccccctcgc tcggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca
     1561 tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg
     1621 aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc
     1681 tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag
     1741 ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct
     1801 tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg
     1861 aaggcaccgg cgggcccaag aagaagagga aggtaatcgg atcctccgga atcctctggc
     1921 atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg
     1981 tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga
     2041 ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt
     2101 ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cacccaagcc tgggacctct
     2161 attatcatgt gttccgacga atctcaaagc tcgagtaata atctagaact atagtgagtc
     2221 gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     2281 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     2341 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     2401 tcagggggag gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc attgggcccg
     2461 gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     2521 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2581 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2641 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2701 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2761 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2821 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2881 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2941 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3001 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3061 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3121 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3181 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3241 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3301 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3361 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3421 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3481 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3541 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3601 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3661 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3721 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3781 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3841 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3901 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3961 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4021 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4081 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4141 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4201 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4261 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4321 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4381 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4441 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4501 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4561 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4621 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     4681 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     4741 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     4801 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     4861 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     4921 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4981 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     5041 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5101 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
     5161 ggcgatcggt gcgggcctct tcgctattac gccagtcgac catagccaat tcaatatggc
     5221 gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc
     5281 gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat tcaatatggc
     5341 ggacttggca ccatgccaat tcaatatggc ggacttggca ctgtgccaac tggggagggg
     5401 tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa gtccgccata
     5461 ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg atcaatatat
     5521 aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca atactatgta
     5581 ttggccctat gccatatagt attccatata tgggttttcc tattgacgta gatagcccct
     5641 cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca tagccactcc
     5701 cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata tgggcggtcc
     5761 tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc ccgcctggct
     5821 caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat ggctcattgc
     5881 ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg gcccacttgg
     5941 cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg aaggacgcca
     6001 gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc gatggctgcc
     6061 aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc gttccattga
     6121 cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct cgtttaggga
//
