LOCUS       sequence-309005-        8896 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas12a (D917A) + PA4-mVenus. (removed crRNAs).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8896)
  AUTHORS   Kuo J, Yuan R, Sanchez C, Paulsson J, Silver PA
  TITLE     Toward a translationally independent RNA-based synthetic oscillator 
            using deactivated CRISPR-Cas.
  JOURNAL   Nucleic Acids Res. 2020 Aug 20;48(14):8165-8177. doi: 
            10.1093/nar/gkaa557.
  PUBMED    32609820
REFERENCE   2  (bases 1 to 8896)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2020-08-20- 20"; volume: "48"; issue: 
            "14"; pages: "8165-8177"
COMMENT     Sequence Label: pJK506
FEATURES             Location/Qualifiers
     source          1..8896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..660)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     575..594
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(661..763)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(793..1419)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1324..1343
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        1435..1490
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1471..1489
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1508..1519
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             complement(6045..6995)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      7043..7265
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     misc_RNA        7810..7859
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             7889..7900
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             7909..8622
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             7909..8622
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(7939..7967)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     8562..8582
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      8683..8769
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(8782..8876)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
       61 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
      121 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
      181 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
      241 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
      301 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
      361 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
      421 accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg
      481 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
      541 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
      601 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
      661 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
      721 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
      781 gatatcgacg tcttaagacc cactttcaca tttaagttgt ttttctaatc cgcatatgat
      841 caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata attcgatagc
      901 ttgtcgtaat aatggcggca tactatcagt agtaggtgtt tccctttctt ctttagcgac
      961 ttgatgctct tgatcttcca atacgcaacc taaagtaaaa tgccccacag cgctgagtgc
     1021 atataatgca ttctctagtg aaaaaccttg ttggcataaa aaggctaatt gattttcgag
     1081 agtttcatac tgtttttctg taggccgtgt acctaaatgt acttttgctc catcgcgatg
     1141 acttagtaaa gcacatctaa aacttttagc gttattacgt aaaaaatctt gccagctttc
     1201 cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg ctaaggcgtc
     1261 gagcaaagcc cgcttatttt ttacatgcca atacaatgta ggctgctcta cacctagctt
     1321 ctgggcgagt ttacgggttg ttaaaccttc gattccgacc tcattaagca gctctaatgc
     1381 gctgttaatc actttacttt tatctaatct agacatcatt aattcctaat ttttgttgac
     1441 actctatcgt tgatagagtt attttaccac tccctatcag tgatagagaa aagaattcaa
     1501 aagatctaaa gaggagaaag gatctatgtc aatttatcaa gaatttgtta ataaatatag
     1561 tttaagtaaa actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa
     1621 agcaagaggt ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca
     1681 aataattgat aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag
     1741 cgaagattta ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga
     1801 taatctacaa aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata
     1861 tataaaggac tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa
     1921 agggcaagag tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact
     1981 atttaaagcc aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt
     2041 taaaggttgg acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag
     2101 caatgatatt cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct
     2161 agaaaataaa gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga
     2221 acaaattaaa aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga
     2281 agttaatcaa agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta
     2341 tctaaatcaa agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg
     2401 tgaaaataca aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa
     2461 tgataaaaca ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac
     2521 agaatctaaa tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat
     2581 gcaaagtttt tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga
     2641 aacactatct ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta
     2701 ttttaaaaat gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt
     2761 tattggtaca gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa
     2821 ccctagtaag aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc
     2881 tctagaaact ataaagcttg ccttagaaga atttaataag catagagata tagataaaca
     2941 gtgtaggttt gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat
     3001 agctcaaaac aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa
     3061 agacctactt caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca
     3121 aactaataat ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc
     3181 aaatatttta gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct
     3241 agcgaatata gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag
     3301 tgatgagaaa tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa
     3361 taaagagcct gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt
     3421 gatgaataag aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga
     3481 gggttataaa aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt
     3541 tttcttttct gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag
     3601 aaatcattcc acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt
     3661 taatattgaa gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc
     3721 ggagtggaaa gattttggat ttagattttc tgatactcaa agatataatt ctatagatga
     3781 attttataga gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag
     3841 ctatattgat agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga
     3901 tttttcagct tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt
     3961 tgatgagaga aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta
     4021 tcgtaaacaa tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa
     4081 aaacaaagat aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg
     4141 ctttactgaa gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg
     4201 agctaataag tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca
     4261 tatattaagt atagctagag gtgaaagaca tttagcttac tatactttgg tagatggtaa
     4321 aggcaatatc atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa
     4381 ctaccatgat aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa
     4441 aaagataaat aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat
     4501 agctaagcta gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt
     4561 taaaagaggg cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat
     4621 tgagaaacta aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct
     4681 tagagcttat cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg
     4741 tattatctac tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt
     4801 aaatcagtta tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt
     4861 tgacaagatt tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa
     4921 ctttggtgac aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat
     4981 taactttaga aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa
     5041 agagttggag aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa
     5101 agcagctatt tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa
     5161 tactatctta caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc
     5221 agtagcagat gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca
     5281 agatgctgat gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag
     5341 gatcaaaaat aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt
     5401 tgagttcgtg cagaatagga ataactaatt gagaagagaa aagaaaaccg ccgatcctgt
     5461 ccaccgcatt actgcaaggt agtggacaag accggcggtc ttaagttttt tggctgaatt
     5521 aattaagtac gggttttgct gcccgcaaac gggctgttct ggtgttgcta gtttgttatc
     5581 agaatcgcag atccggcttc agccggtttg ccggctgaaa gcgctatttc ttccagaatt
     5641 gccatgattt tttccccacg ggaggcgtca ctggctcccg tgttgtcggc agctttgatt
     5701 cgataagcag catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt
     5761 gtctcaggtg ttcaatttca tgttctagtt gctttgtttt actggtttca cctgttctat
     5821 taggtgttac atgctgttca tctgttacat tgtcgatctg ttcatggtga acagctttga
     5881 atgcaccaaa aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg
     5941 tgcatatgga cagttttccc tttgatatgt aacggtgaac agttgttcta cttttgtttg
     6001 ttagtcttga tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat
     6061 ttagccagta tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca
     6121 ttgagatcat acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga
     6181 atttttgcag ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct
     6241 gatgtaatgg ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg
     6301 gttacgagat ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg
     6361 cctcgcttat caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt
     6421 ttcaaaaccc attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg
     6481 aacttaaatt catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt
     6541 tcttttaata accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt
     6601 tccagattat attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta
     6661 aaaactaatt ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca
     6721 aagcctttaa ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct
     6781 ttagctaata caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta
     6841 taagtgaacg ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt
     6901 gccacacagc ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct
     6961 agttcatttg ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt
     7021 taatcactat accaattgag atgggctagt caatgataat tactagtcct tttcctttga
     7081 gttgtgggta tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctaga
     7141 ccctctgtaa attccgctag acctttgtgt gttttttttg tttatattca agtggttata
     7201 atttatagaa taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat
     7261 aactcactac tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct
     7321 cctctacaaa acagacctta aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc
     7381 aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg
     7441 tgacattcag ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag
     7501 gcgcctttta tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg
     7561 cttctcaggg cgttttatgg cgggtctgct atgtggtgct atctgacttt ttgctgttca
     7621 gcagttcctg ccctctgatt ttccagtctg accacttcgg attatcccgt gacaggtcat
     7681 tcagactggc taatgcaccc agtaaggcag cggtatcatc aacaggctta cccgtcttac
     7741 tgtccctagt gcttggccta gtatgcgtct cctttacacc tccacaacta gctggtatta
     7801 tgctagcagc tgtcaccgga tgtgctttcc ggtctgatga gtccgtgagg acgaaacagc
     7861 ctctacaaat aattttgttt aactttacaa agaggagaaa ggtaccgcat gagtaaagga
     7921 gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg
     7981 cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt
     8041 aaattgattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttg
     8101 ggttatggtg ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc
     8161 aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg
     8221 aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag
     8281 ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac
     8341 tataactcac acaatgtata catcacggca gacaaacaaa agaatggaat caaagcgaac
     8401 ttcaaaatta gacacaacat tgaagatgga ggtgttcaac tagcagacca ttatcaacaa
     8461 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtcctaccaa
     8521 tctaagcttt cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta
     8581 acagctgctg ggattacaca tggcatggat gaactataca aataagcttg atatcgaatt
     8641 cctgcagccc gggggatccc attgtacgcg tgctagaggc atcaaataaa acgaaaggct
     8701 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     8761 aggacaaatt ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt
     8821 tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag
     8881 cgagctcgat atcaaa
//
