LOCUS       sequence-202450-        7570 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 7570)
  AUTHORS   Scot Wolfe
  TITLE     Inducible Cas9-DNA-binding-domain fusions for precise genome editing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7570)
  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            "Unpublished"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..7570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(109..111,112..432)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1313..1324
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2842..2862
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2863..2952
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             2965..2991
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3379..3444
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3478..3504
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3550..3570
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3615..3749
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3639..3658)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3693..3712
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3866..3886)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3866..3884)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3905..3921)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3905..3921)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3918..3940)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3929..3945
                     /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(3953..3959,3960..3977,3978..3983))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3998..4019
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4136..4153)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4307..4895)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4387..4406)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5066..5857,5858..5926))
                     /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     5689..5708
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5927..6031)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6057..6512)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6194..6215)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6406..6425
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6586..7570
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atctgccacc atgggttcgg gccgtacgat gggagtgcag
      121 gtggaaacca tctccccagg agacgggcgc accttcccca agcgcggcca gacctgcgtg
      181 gtgcactaca ccgggatgct tgaagatgga aagaaatttg attcctctcg ggacagaaac
      241 aagcccttta agtttatgct aggcaagcag gaggtgatcc gaggctggga agaaggggtt
      301 gcccagatga gtgtgggtca gagagccaaa ctgactatat ctccagatta tgcctatggt
      361 gccactgggc acccaggcat catcccacca catgccactc tcgtcttcga tgtggagctt
      421 ctaaaactgg aaggcgccgg cggatgtttc gactctgttg aaatcagcgg agtggaggat
      481 cgcttcaacg catccctggg aacgtatcac gatctcctga aaatcattaa agacaaggac
      541 ttcctggaca atgaggagaa cgaggacatt cttgaggaca ttgtcctcac ccttacgttg
      601 tttgaagata gggagatgat tgaagaacgc ttgaaaactt acgctcatct cttcgacgac
      661 aaagtcatga aacagctcaa gagacgccga tatacaggat gggggcggct gtcaagaaaa
      721 ctgatcaatg gcatccgaga caagcagagt ggaaagacaa tcctggattt tcttaagtcc
      781 gatggatttg ccaaccggaa cttcatgcag ttgatccatg atgactctct cacctttaag
      841 gaggacatcc agaaagcaca agtttctggc cagggggaca gtcttcacga gcacatcgct
      901 aatcttgcag gtagcccagc tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat
      961 gaactcgtca aagtaatggg aaggcataag cccgagaata tcgttatcga gatggcccga
     1021 gagaaccaaa ctacccagaa gggacagaag aacagtaggg aaaggatgaa gaggattgaa
     1081 gagggtataa aagaactggg gtcccaaatc cttaaggaac acccagttga aaacacccag
     1141 cttcagaatg agaagctcta cctgtactac ctgcagaacg gcagggacat gtacgtggat
     1201 caggaactgg acatcaaccg gttgtccgac tacgacgtgg atcatatcgt gccccaaagc
     1261 tttctcaaag atgattctat tgataataaa gtgttgacaa gatccgataa aaatagaggg
     1321 aagagtgata acgtcccctc agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag
     1381 ctgctgaacg ccaaactgat cacacaacgg aagttcgata atctgactaa ggctgaacga
     1441 ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc
     1501 cagatcacca agcacgtggc ccaaattctc gattcacgca tgaacaccaa gtacgatgaa
     1561 aatgacaaac tgattcgaga ggtgaaagtt attactctga agtctaagct ggtctcagat
     1621 ttcagaaagg actttcagtt ttataaggtg agagagatca acaattacca ccatgcgcat
     1681 gatgcctacc tgaatgcagt ggtaggcact gcacttatca aaaaatatcc caagctggaa
     1741 tctgaatttg tttacggaga ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct
     1801 gagcaggaaa taggcaaggc caccgctaag tacttctttt acagcaatat tatgaatttt
     1861 ttcaagaccg agattacact ggccaatgga gagattcgga agcgaccact tatcgaaaca
     1921 aacggagaaa caggagaaat cgtgtgggac aagggtaggg atttcgcgac agtccgcaag
     1981 gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg aagtacagac cggaggcttc
     2041 tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat
     2101 tgggacccca agaaatacgg cggattcgat tctcctacag tcgcttacag tgtactggtt
     2161 gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc
     2221 atcacaatca tggagcgatc cagcttcgag aaaaacccca tcgactttct cgaagcgaaa
     2281 ggatataaag aggtcaaaaa agacctcatc attaagctgc ccaagtactc tctctttgag
     2341 cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg agctgcagaa aggtaacgag
     2401 ctggcactgc cctctaaata cgttaatttc ttgtatctgg ccagccacta tgaaaagctc
     2461 aaagggtctc ccgaagataa tgagcagaag cagctgttcg tggaacaaca caaacactac
     2521 cttgatgaga tcatcgagca aataagcgag ttctccaaaa gagtgatcct cgccgacgct
     2581 aacctcgata aggtgctttc tgcttacaat aagcacaggg ataagcccat cagggagcag
     2641 gcagaaaaca ttatccactt gtttactctg accaacttgg gcgcgcctgc agccttcaag
     2701 tacttcgaca ccaccataga cagaaagaag tacacctcta caaaggaggt cctggacgcc
     2761 acactgattc atcagtcaat tacggggctc tatgaaacaa gaatcgacct ctctcagctc
     2821 ggtggagacg gcaccggcgg gcccaagaag aagaggaagg tatacccata cgatgttcct
     2881 gactatgcgg gctatcccta tgacgtcccg gactatgcag gatcgtatcc ttatgacgtt
     2941 ccagattacg ctggatccgc cgctccggca gctaagaaaa agaaactgga tttcgaatcc
     3001 ggaaagccct ataaatgtcc tgaatgtggc aagtccttct cgcggagcga cgacctgaca
     3061 cggcaccaac gtacgcacac tggtgagaag ccatacgcgt gtcctgtcga gtcctgtgac
     3121 cgccgcttca gtcagaaggg acacctgaca cggcacatcc gcattcacac agggcaaaaa
     3181 ccgtttcaat gccgcatctg catgaggaac ttcagcatcc gtagcagcct gacacggcac
     3241 atccgcaccc acacaggaga aaagcccttc gcctgtgaca tctgcggcag gaagttcgcg
     3301 ctgagccacc acctgacacg gcacaccaag atccacctcc gtcagaaaga ccccggggga
     3361 tccgggtcgc tcgagggaga ctacaaagac catgacggtg attataaaga tcatgacatc
     3421 gattacaagg atgacgatga caagcagggg tccgagctcg gatcgggtag agccgcgccg
     3481 gcagctaaga aaaagaaact ggatcaattg ggcgctagcg gtagttctgg aggctcaagt
     3541 ggcgccgctc caaagaagaa gcgtaaggta tctgaattct gataatctag aactatagtg
     3601 agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga caaaccacaa
     3661 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     3721 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     3781 aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc cgcggcgcca atgcattggg
     3841 cccggtacgt acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     3901 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3961 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4021 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4081 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4141 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4201 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4261 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4321 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4381 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4441 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4501 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4561 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4621 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4681 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4741 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4801 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4861 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4921 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4981 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5041 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5101 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5161 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5221 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5281 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5341 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5401 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5461 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5521 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5581 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5641 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5701 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5761 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5821 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5881 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5941 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6001 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     6061 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     6121 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     6181 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     6241 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     6301 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     6361 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     6421 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     6481 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
     6541 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagtcgacca tagccaattc
     6601 aatatggcgt atatggactc atgccaattc aatatggtgg atctggacct gtgccaattc
     6661 aatatggcgt atatggactc gtgccaattc aatatggtgg atctggaccc cagccaattc
     6721 aatatggcgg acttggcacc atgccaattc aatatggcgg acttggcact gtgccaactg
     6781 gggaggggtc tacttggcac ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt
     6841 ccgccatatt gaattggcat ggtgccaata atggcggcca tattggctat atgccaggat
     6901 caatatatag gcaatatcca atatggccct atgccaatat ggctattggc caggttcaat
     6961 actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga
     7021 tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata
     7081 gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg acgtcatatg
     7141 ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc
     7201 gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg
     7261 ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc
     7321 ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt actattggaa
     7381 ggacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa tggcccgcga
     7441 tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc aaatgggcgt
     7501 tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg
     7561 tttagggaac
//
