LOCUS       sequence-204631-        7567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C terminal (574-1368 aa) R1335K mutant SpCas9 fused to 
            FKBP and ZFP-VEGFA-TS3 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  JOURNAL   Unpublished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            "Unpublished"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-ZFPTS3-3xFLAG-2xNLS
FEATURES             Location/Qualifiers
     source          1..7567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        30..48
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     30..47
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      80..89
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(104..106,107..427)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1308..1319
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2837..2857
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2858..2947
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             2960..2986
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3374..3439
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3473..3499
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3545..3565
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3610..3744
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3634..3653)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3688..3707
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3861..3881)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3861..3879)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3900..3916)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3900..3916)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3913..3935)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3924..3940
                     /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(3948..3954,3955..3972,3973..3978))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3993..4014
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4131..4148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4302..4890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4382..4401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5061..5852,5853..5921))
                     /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     5684..5703
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5922..6026)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6052..6507)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6189..6210)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6401..6420
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6578..7562
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 ttctgcctgg ggacgtcgga gcaagcttga tttaggtgac actatagaat acaagctact
       61 tgttcttttt gcaggatctg ccaccatggg ttcgggccgt acgatgggag tgcaggtgga
      121 aaccatctcc ccaggagacg ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca
      181 ctacaccggg atgcttgaag atggaaagaa atttgattcc tctcgggaca gaaacaagcc
      241 ctttaagttt atgctaggca agcaggaggt gatccgaggc tgggaagaag gggttgccca
      301 gatgagtgtg ggtcagagag ccaaactgac tatatctcca gattatgcct atggtgccac
      361 tgggcaccca ggcatcatcc caccacatgc cactctcgtc ttcgatgtgg agcttctaaa
      421 actggaaggc gccggcggat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt
      481 caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct
      541 ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga
      601 agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt
      661 catgaaacag ctcaagagac gccgatatac aggatggggg cggctgtcaa gaaaactgat
      721 caatggcatc cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg
      781 atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga
      841 catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca tcgctaatct
      901 tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact
      961 cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa
     1021 ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg
     1081 tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca
     1141 gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga
     1201 actggacatc aaccggttgt ccgactacga cgtggatcat atcgtgcccc aaagctttct
     1261 caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata gagggaagag
     1321 tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct
     1381 gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg
     1441 cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat
     1501 caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga
     1561 caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag
     1621 aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc
     1681 ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc tggaatctga
     1741 atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca
     1801 ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa
     1861 gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg
     1921 agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc gcaaggtcct
     1981 gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa
     2041 ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga
     2101 ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc
     2161 caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac
     2221 aatcatggag cgatccagct tcgagaaaaa ccccatcgac tttctcgaag cgaaaggata
     2281 taaagaggtc aaaaaagacc tcatcattaa gctgcccaag tactctctct ttgagcttga
     2341 aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc
     2401 actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg
     2461 gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga
     2521 tgagatcatc gagcaaataa gcgagttctc caaaagagtg atcctcgccg acgctaacct
     2581 cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga
     2641 aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt
     2701 cgacaccacc atagacagaa agaagtacac ctctacaaag gaggtcctgg acgccacact
     2761 gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg
     2821 agacggcacc ggcgggccca agaagaagag gaaggtatac ccatacgatg ttcctgacta
     2881 tgcgggctat ccctatgacg tcccggacta tgcaggatcg tatccttatg acgttccaga
     2941 ttacgctgga tccgccgctc cggcagctaa gaaaaagaaa ctggatttcg aatccggaaa
     3001 gccctataaa tgtcctgaat gtggcaagtc cttctcgcgg agcgacgacc tgacacggca
     3061 ccaacgtacg cacactggtg agaagccata cgcgtgtcct gtcgagtcct gtgaccgccg
     3121 cttcagtcag aagggacacc tgacacggca catccgcatt cacacagggc aaaaaccgtt
     3181 tcaatgccgc atctgcatga ggaacttcag catccgtagc agcctgacac ggcacatccg
     3241 cacccacaca ggagaaaagc ccttcgcctg tgacatctgc ggcaggaagt tcgcgctgag
     3301 ccaccacctg acacggcaca ccaagatcca cctccgtcag aaagaccccg ggggatccgg
     3361 gtcgctcgag ggagactaca aagaccatga cggtgattat aaagatcatg acatcgatta
     3421 caaggatgac gatgacaagc aggggtccga gctcggatcg ggtagagccg cgccggcagc
     3481 taagaaaaag aaactggatc aattgggcgc tagcggtagt tctggaggct caagtggcgc
     3541 cgctccaaag aagaagcgta aggtatctga attctgataa tctagaacta tagtgagtcg
     3601 tattacgtag atccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     3661 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3721 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3781 cagggggagg tgtgggaggt tttttaattc gcggccgcgg cgccaatgca ttgggcccgg
     3841 tacgtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     3901 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3961 gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4021 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4081 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4141 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4201 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4261 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4321 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4381 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4441 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4501 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4561 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4621 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4681 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4741 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4801 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4861 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4921 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4981 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5041 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5101 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5161 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5221 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5281 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5341 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5401 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5461 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5521 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5581 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5641 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5701 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5761 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5821 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5881 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5941 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6001 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     6061 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     6121 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     6181 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     6241 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     6301 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     6361 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     6421 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     6481 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     6541 aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat tcaatatggc
     6601 gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc
     6661 gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat tcaatatggc
     6721 ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac tggggagggg
     6781 tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa gtccgccata
     6841 ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg atcaatatat
     6901 aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca atactatgta
     6961 ttggccctat gccatatagt attccatata tgggttttcc tattgacgta gatagcccct
     7021 cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca tagccactcc
     7081 cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata tgggcggtcc
     7141 tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc ccgcctggct
     7201 caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat ggctcattgc
     7261 ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg gcccacttgg
     7321 cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg aagtacgcca
     7381 gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc gatggctgcc
     7441 aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc gttccattga
     7501 cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct cgtttaggga
     7561 accgcca
//
