LOCUS       sequence-248670-        8175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes rapamycin and tamoxifen activated split-dCAS9 (C-terminal) 
            as a Type 3 part to be used in the MTK system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8175)
  AUTHORS   Fonseca JP, Bonny AR, Kumar GR, Ng AH, Town J, Wu QC, Aslankoohi E, 
            Chen SY, Dods G, Harrigan P, Osimiri LC, Kistler AL, El-Samad H
  TITLE     A Toolkit for Rapid Modular Construction of Biological Circuits in 
            Mammalian Cells.
  JOURNAL   ACS Synth Biol. 2019 Nov 15;8(11):2593-2606. doi: 
            10.1021/acssynbio.9b00322. Epub 2019 Nov 5.
  PUBMED    31686495
REFERENCE   2  (bases 1 to 8175)
  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"; doi: 
            "10.1021/acssynbio.9b00322"; journalName: "ACS Synth Biol"; date: 
            "2019-11-15- 15"; volume: "8"; issue: "11"; pages: "2593-2606"
COMMENT     Sequence Label: MTK3_020
FEATURES             Location/Qualifiers
     source          1..8175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             783..1718
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             1752..2072
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             5607..5627
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5664..5688
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5718..5867
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             5892..5912
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6342..6698
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             6717..7286
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     terminator      complement(7306..7340)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(7411..8070)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7985..8004
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtagtcgg tctcatatgt
      781 ctgctggaga catgagagct gccaaccttt ggccaagccc gctcatgatc aaacgctcta
      841 agaagaacag cctggccttg tccctgacgg ccgaccagat ggtcagtgcc ttgttggatg
      901 ctgagccccc catactctat tccgagtatg atcctaccag acccttcagt gaagcttcga
      961 tgatgggctt actgaccaac ctggcagaca gggagctggt tcacatgatc aactgggcga
     1021 agagggtgcc aggctttgtg gatttgaccc tccatgatca ggtccacctt ctagaatgtg
     1081 cctggctaga gatcctgatg attggcctcg tctggcgctc catggagcac ccagtgaagc
     1141 tactgtttgc tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta gagggcatgg
     1201 tggagatctt cgacatgctg ctggctacat catctcggtt ccgcatgatg aatctgcagg
     1261 gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg tacacatttc
     1321 tgtccagcac cctgaagtct ctggaagaga aggaccatat ccaccgagtc ctggacaaga
     1381 tcacagacac tttgatccac ctgatggcca aggcaggcct gaccctgcag cagcagcacc
     1441 agcggctggc ccagctcctc ctcatcctct cccacatcag gcacatgagt aacaaaggca
     1501 tggagcatct gtacagcatg aagtgcaaga acgtggtgcc cctctatgac ctgctgctgg
     1561 aggcggcgga cgcccaccgc ctacatgcgc ccactagccg tggaggggca tccgtggagg
     1621 agacagacca aagccacttg gccactgcgg gctctacttc atcgcattcc ttgcaaaagt
     1681 attacatcac gggggaggca gagggtttcc ctgccacagc tggcggtggt ggcagcggcg
     1741 gtggtggcag cggtgtgcag gtagaaacaa tctccccggg agatggccgc acgttcccca
     1801 agaggggaca gacctgtgtg gtgcactaca ccggtatgct cgaagacggc aagaagttcg
     1861 atagctcccg agatcgaaac aagcccttca agttcatgct gggcaagcaa gaggtcatac
     1921 gcggttggga agaaggcgtg gcccagatga gcgtagggca gcgcgccaag ctgaccatta
     1981 gccccgacta cgcctacggg gccaccgggc accccggcat cattccaccc catgcgacac
     2041 tcgtctttga tgtggagctg ctcaagctgg aaggcggtgg tggcagcggg ggaggtggtt
     2101 ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa
     2161 acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc
     2221 tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc
     2281 ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg
     2341 accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt ctgctgagtg
     2401 atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc
     2461 gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc
     2521 ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg
     2581 acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg
     2641 acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca
     2701 ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca
     2761 ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc
     2821 tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt
     2881 ggatgactcg caaatcagaa gaaaccatca ctccctggaa cttcgaggaa gtcgtggata
     2941 agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta
     3001 acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc
     3061 tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc
     3121 agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc
     3181 tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg
     3241 aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca
     3301 aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta
     3361 cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg
     3421 acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa
     3481 gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta
     3541 agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct
     3601 ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca
     3661 tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg
     3721 tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg
     3781 cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga
     3841 ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca
     3901 cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg
     3961 tggatcagga actggacatc aatcggctct ccgactacga cgtggctgct atcgtgcccc
     4021 agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc gataaagcta
     4081 gatggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc
     4141 ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg
     4201 aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga
     4261 cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg
     4321 atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtat
     4381 cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg
     4441 cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc
     4501 ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa
     4561 agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga
     4621 attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga ccacctatcg
     4681 aaacaaacgg ggaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc
     4741 ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag
     4801 gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa
     4861 aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac
     4921 tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc
     4981 tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg
     5041 cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct
     5101 ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta
     5161 acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa
     5221 agctcaaagg gtcgcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac
     5281 actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg
     5341 acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg
     5401 agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct
     5461 tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg
     5521 acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc
     5581 agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtcg ccagggatcc
     5641 gtcgacttga cgcgttgata tcaacaagtt tgtacaaaaa agcaggctac aaagaggcca
     5701 gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg ggaagtgacg
     5761 ccctcgatga ttttgacctt gacatgcttg gttcggatgc cctcgatgac ttcgatctgg
     5821 acatgctggg atctgacgct cttgatgatt ttgaccttga catgctgatt aactctagaa
     5881 gttccggatc tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc gacaccgacg
     5941 accggcaccg gatcgaggaa aagcggaagc ggacctacga gacattcaag agcatcatga
     6001 agaagtcccc cttttccggg ccaacagatc ctcgacctcc cccccgcaga atagctgtcc
     6061 catcaagatc tagcgcctca gttcccaaac ctgcgccgca accgtatccc tttacctcaa
     6121 gtcttagtac cataaattac gacgagttcc cgacaatggt gttcccctct ggccaaattt
     6181 ctcaagcttc agctttggcc ccagctcctc cacaggtttt gccacaagca cccgcacctg
     6241 caccagctcc ggcgatggta tcagcacttg ctcaggctcc tgctccggtc cccgttctcg
     6301 caccgggtcc cccacaagca gtggcacctc ctgcacctaa gccgacgcaa gcaggtgagg
     6361 ggactctgtc tgaagcactt ctccagttgc agtttgacga cgaagatctt ggagccctgc
     6421 tcgggaactc cacagatcca gccgtcttca cagatctcgc ctcagtggat aactccgagt
     6481 ttcagcaact tcttaatcag ggcattcctg tggcccctca caccaccgag cccatgctga
     6541 tggaataccc cgaggccatc acccggctcg tgacaggcgc tcagaggcct cctgatccag
     6601 ctcctgcccc tctgggagca ccaggcctgc ctaatggact gctgtctggc gacgaggact
     6661 tcagctctat cgccgatatg gatttctcag ccttgctggg ctctggcagc ggcagccggg
     6721 attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt agtgacgtgt
     6781 ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct ccaggaagtc
     6841 catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt ccagtacatg
     6901 agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca gcgcccgcag
     6961 tgactcccga ggccagtcac ctgttggagg atcccgatga agaaacgagc caggctgtca
     7021 aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct gcaatctgtg
     7081 gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg acaaccacac
     7141 ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa ttgaacgaga
     7201 ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc agcacaggac
     7261 tgtccatctt cgacacatct ctgtttggat cctgagacca gaccaataaa aaacgcccgg
     7321 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
     7381 aacaggagtc caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta
     7441 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
     7501 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa
     7561 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     7621 ccagggattg gctgaaacga aaaacatatt ctcaataaac cctttaggga aataggccag
     7681 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     7741 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     7801 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg
     7861 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     7921 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     7981 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     8041 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     8101 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     8161 tacgtgcccg atcaa
//
