LOCUS       sequence-204615-        7567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C terminal (574-1368 aa) R1335K mutant SpCas9 fused to 
            FKBP and ZFP-VEGFA-TS2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7567)
  AUTHORS   Scot Wolfe
  TITLE     Inducible Cas9-DNA-binding-domain fusions for precise genome editing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7567)
  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            "Unpublished"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_FKBP-C-SpCas9MT3-574-1368-NLS-3XHA-NLS-ZFPTS2-3xFLAG-2xNLS
FEATURES             Location/Qualifiers
     source          1..7567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..32
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     14..31
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      64..73
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(88..90,91..411)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1292..1303
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2821..2841
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2842..2931
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             2944..2970
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3358..3423
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3457..3483
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3529..3549
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3594..3728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3618..3637)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3672..3691
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3845..3865)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3845..3863)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3884..3900)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3884..3900)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3897..3919)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3908..3924
                     /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(3932..3938,3939..3956,3957..3962))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3977..3998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4115..4132)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4286..4874)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4366..4385)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5045..5836,5837..5905))
                     /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     5668..5687
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5906..6010)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6036..6491)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6173..6194)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6385..6404
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6562..7546
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cggagcaagc ttgatttagg tgacactata gaatacaagc tacttgttct ttttgcagga
       61 tctgccacca tgggttcggg ccgtacgatg ggagtgcagg tggaaaccat ctccccagga
      121 gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt
      181 gaagatggaa agaaatttga ttcctctcgg gacagaaaca agccctttaa gtttatgcta
      241 ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag
      301 agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc
      361 atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga aggcgccggc
      421 ggatgtttcg actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga
      481 acgtatcacg atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac
      541 gaggacattc ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt
      601 gaagaacgct tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag
      661 agacgccgat atacaggatg ggggcggctg tcaagaaaac tgatcaatgg catccgagac
      721 aagcagagtg gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac
      781 ttcatgcagt tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa
      841 gtttctggcc agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct
      901 atcaaaaagg gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga
      961 aggcataagc ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag
     1021 ggacagaaga acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg
     1081 tcccaaatcc ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac
     1141 ctgtactacc tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaaccgg
     1201 ttgtccgact acgacgtgga tcatatcgtg ccccaaagct ttctcaaaga tgattctatt
     1261 gataataaag tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca
     1321 gaagaagttg tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc
     1381 acacaacgga agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat
     1441 aaagccggct tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc
     1501 caaattctcg attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag
     1561 gtgaaagtta ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt
     1621 tataaggtga gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg
     1681 gtaggcactg cacttatcaa aaaatatccc aagctggaat ctgaatttgt ttacggagac
     1741 tataaagtgt acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc
     1801 accgctaagt acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg
     1861 gccaatggag agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc
     1921 gtgtgggaca agggtaggga tttcgcgaca gtccgcaagg tcctgtccat gccgcaggtg
     1981 aacatcgtta aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg
     2041 aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc
     2101 ggattcgatt ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg
     2161 aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatcc
     2221 agcttcgaga aaaaccccat cgactttctc gaagcgaaag gatataaaga ggtcaaaaaa
     2281 gacctcatca ttaagctgcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga
     2341 atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac
     2401 gttaatttct tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat
     2461 gagcagaagc agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa
     2521 ataagcgagt tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct
     2581 gcttacaata agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg
     2641 tttactctga ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac
     2701 agaaagaagt acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt
     2761 acggggctct atgaaacaag aatcgacctc tctcagctcg gtggagacgg caccggcggg
     2821 cccaagaaga agaggaaggt atacccatac gatgttcctg actatgcggg ctatccctat
     2881 gacgtcccgg actatgcagg atcgtatcct tatgacgttc cagattacgc tggatccgcc
     2941 gctccggcag ctaagaaaaa gaaactggat ttcgaatccg gaaagcccta taaatgtcct
     3001 gaatgtggca agtccttctc ggagaagagc cacctgacac ggcaccaacg cacgcacact
     3061 ggtgagaagc catacgcgtg tcctgtcgag tcctgtgacc gccgcttcag tcggagcgac
     3121 cacctgacac agcacatccg cattcacaca gggcaaaaac cgtttcaatg ccgcatctgc
     3181 atgaggaact tcagcgacaa gggccacctg acccggcaca tccgcaccca cacaggagaa
     3241 aagcccttcg cctgtgacat ctgcggcagg aagttcgcgc ggagcgacga cctgacacgg
     3301 cacaccaaga tccacctccg tcagaaagac cccgggggat ccgggtcgct cgagggagac
     3361 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
     3421 aagcaggggt ccgagctcgg atcgggtaga gccgcgccgg cagctaagaa aaagaaactg
     3481 gatcaattgg gcgctagcgg tagttctgga ggctcaagtg gcgccgctcc aaagaagaag
     3541 cgtaaggtat ctgaattctg ataatctaga actatagtga gtcgtattac gtagatccag
     3601 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     3661 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     3721 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     3781 aggtttttta attcgcggcc gcggcgccaa tgcattgggc ccggtacgta cccagctttt
     3841 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     3901 tgtgaaattg ttatccgctc acaattccac acaacatacg agccgggagc ataaagtgta
     3961 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4021 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4081 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4141 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4201 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4261 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4321 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4381 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4441 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4501 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4561 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4621 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4681 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4741 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4801 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4861 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4921 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4981 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5041 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5101 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5161 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5221 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5281 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5341 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5401 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5461 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5521 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5581 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5641 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5701 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5761 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5821 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5881 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5941 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6001 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     6061 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     6121 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     6181 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     6241 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     6301 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     6361 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     6421 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     6481 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     6541 ggcctcttcg ctattacgcc agtcgaccgc caattcaata tggcgtatat ggactcatgc
     6601 caattcaata tggtggatct ggacctgtgc caattcaata tggcgtatat ggactcgtgc
     6661 caattcaata tggtggatct ggaccccagc caattcaata tggcggactt ggcaccatgc
     6721 caattcaata tggcggacct ggcactgtgc caactgggga ggggtctact tggcacggtg
     6781 ccaagtttga ggaggggtct tggccctgtg ccaagtccgc catattgaat tggcatggtg
     6841 ccaataatgg cggccatatt ggctatatgc caggatcaat atataggcaa tatccaatat
     6901 ggccctatgc caatatggct attggccagg ttcaatacta tgtattggcc ctatgccata
     6961 tagtattcca tatatgggtt ttcctattga cgtagatagc ccctcccaat gggcggtccc
     7021 atataccata tatggggctt cctaataccg cccatagcca ctcccccatt gacgtcaatg
     7081 gtctctatat atggtctttc ctattgacgt catatgggcg gtcctattga cgtatatggc
     7141 gcctccccca ttgacgtcaa ttacggtaaa tggcccgcct ggctcaatgc ccattgacgt
     7201 caataggacc acccaccatt gacgtcaatg ggatggctca ttgcccattc atatccgttc
     7261 tcacgccccc tattgacgtc aatgacggta aatggcccac ttggcagtac atcaatatct
     7321 attaatagta acttggcaag tacattacta ttggaagtac gccagggtac attggcagta
     7381 ctcccattga cgtcaatggc ggtaaatggc ccgcgatggc tgccaagtac atccccattg
     7441 acgtcaatgg ggaggggcaa tgacgcaaat gggcgttcca ttgacgtaaa tgggcggtag
     7501 gcgtgcctaa tgggaggtct atataagcaa tgctcgttta gggaaccgcc attctgcctg
     7561 gggacgt
//
