LOCUS       sequence-217146-        4715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas14b2 locus (including crRNA and tracrRNA) under control
            of a tetracycline inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4715)
  AUTHORS   Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, 
            Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA
  TITLE     Programmed DNA destruction by miniature CRISPR-Cas14 enzymes.
  JOURNAL   Science. 2018 Oct 18. pii: science.aav4294. doi: 
            10.1126/science.aav4294.
  PUBMED    30337455
REFERENCE   2  (bases 1 to 4715)
  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: "Science. 
            2018 Oct 18. pii: science.aav4294. doi: 10.1126/science.aav4294."
COMMENT     Sequence Label: pLBH547_Tet-Cas14b2Locus
FEATURES             Location/Qualifiers
     source          1..4715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             63..74
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2308..2335
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2363..2380)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2497..3042)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2577..2596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3156..3250)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3271..3930)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3845..3864
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3931..4033)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(4063..4689)
                     /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     4594..4613
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        join(4705..4715,1..45)
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
ORIGIN
        1 atcgttgata gagttatttt accactccct atcagtgata gagaaaagaa ttcaaaagat
       61 ctaaagagga gaaaggatct atggaagaaa gcattattac cggtgtgaaa ttcaaactgc
      121 gcatcgataa agaaaccacc aaaaaactga acgagtactt cgatgaatat ggcaaagcaa
      181 ttaacttcgc cgtgaagatc attcagaaag aactggcaga tgatcgtttt gcaggtaaag
      241 caaaactgga ccagaataaa aacccgatcc tggatgaaaa cggcaaaaaa atctatgaat
      301 tcccggatga attttgcagc tgtggtaaac aggttaacaa gtacgttaac aacaaaccgt
      361 tttgccaaga gtgctataaa atccgcttta ccgaaaatgg tattcgcaaa cgtatgtata
      421 gcgccaaagg tcgtaaagcc gaacataaaa tcaatatcct gaacagcacc aacaagatca
      481 gcaaaaccca ttttaactat gccattcgcg aagccttcat tctggataaa agcatcaaaa
      541 agcagcgcaa aaaacgtaat gaacgtctgc gtgaaagtaa aaaacgtctg cagcagttta
      601 tcgatatgcg tgatggtaaa cgtgaaattt gcccgaccat taaaggtcag aaagtggatc
      661 gttttattca tccgagctgg atcaccaaag ataaaaagct ggaagatttt cgcggttata
      721 ccctgagcat tatcaacagc aaaattaaga ttctggatcg caacatcaaa cgcgaagaaa
      781 aaagcctgaa agaaaaaggc cagatcatct ttaaagccaa acgtctgatg ctggataaat
      841 ccattcgttt tgttggtgat cgcaaagtgc tgtttacaat tagtaaaacc ctgccgaaag
      901 agtatgaact ggatctgccg agcaaagaaa aacggctgaa ttggctgaaa gagaagatcg
      961 agattatcaa gaaccagaaa ccgaaatatg cctatctgct gcgcaaaaac attgagagcg
     1021 aaaaaaaacc gaactatgag tactatctgc agtacaccct ggaaattaaa ccggaactga
     1081 aagattttta tgatggtgcc attggtattg accgtggcat taatcatatt gccgtttgca
     1141 cctttattag caacgatggt aaagttaccc ctccgaaatt tttcagcagc ggtgaaattc
     1201 tgcgtctgaa aaatctgcag aaagagcgtg atcgctttct gctgcgtaaa cacaacaaaa
     1261 atcgcaaaaa aggcaacatg cgcgtgatcg aaaacaaaat caatctgatc ctgcaccgtt
     1321 atagcaagca gattgttgat atggccaaaa agctgaatgc cagcattgtt tttgaagaac
     1381 tgggtcgtat tggtaaaagc cgcaccaaaa tgaaaaaaag ccagcgttat aaactgagcc
     1441 tgttcatctt caagaaactg agcgatctgg ttgattacaa aagccgtcgt gaaggtattc
     1501 gtgttaccta tgttccgcct gaatatacca gcaaagaatg tagccattgc ggtgaaaaag
     1561 ttaataccca gcgtccgttt aatggcaact atagcctgtt taaatgcaac aaatgtggca
     1621 tccagctgaa cagcgattat aatgcaagca tcaacattgc gaaaaagggc ctgaaaattc
     1681 cgaatagcac ctaataatgt tggttaagcc acaatatgga atattgttct tatggacagt
     1741 attgacttaa attaataatc ttcgaaggct atatgcggaa gatttggcgt tgttgtaacg
     1801 caataagggg taaccctgaa aaggtttgaa atcatataaa cctagtttta tttgagttta
     1861 ggctcagata aaatgaacag accaatcttt aattccgttc tgatttaaaa aatcagaatc
     1921 tctttataaa tagtattaca aaaagtgtac attccaaaat ccgaaagcag aattgacctt
     1981 tttaagccta aaaaagccaa atttcaaggc tctttcatac tcagaacaaa gggattaagg
     2041 aatgcaacta ccttacaccg cttgcgaaag ttgtcagaag ataatctttc atactcagaa
     2101 caaagggatt aaggaatgca actatcttat ccatttcttg acatcaaatt ttcttgcagc
     2161 atctgaattg cttaattgct ttccttgctt cagcaggaaa tagccaagat tttccagttc
     2221 tgctggcgtt attgcaagca cggggatttt gtgcttgctg tagattttgt tcttcaggac
     2281 cttctttttc catgctagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     2341 acctagggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     2401 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     2461 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     2521 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     2581 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     2641 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     2701 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     2761 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     2821 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     2881 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     2941 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     3001 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
     3061 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     3121 agccccatac gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg
     3181 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
     3241 aacaggagtc caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta
     3301 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
     3361 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa
     3421 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     3481 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     3541 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     3601 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     3661 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg
     3721 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     3781 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     3841 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     3901 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     3961 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     4021 tacgtgccga tcaacgtctc attttcgcca gatatcgacg tcttaagacc cactttcaca
     4081 tttaagttgt ttttctaatc cgcatatgat caattcaagg ccgaataaga aggctggctc
     4141 tgcaccttgg tgatcaaata attcgatagc ttgtcgtaat aatggcggca tactatcagt
     4201 agtaggtgtt tccctttctt ctttagcgac ttgatgctct tgatcttcca atacgcaacc
     4261 taaagtaaaa tgccccacag cgctgagtgc atataatgca ttctctagtg aaaaaccttg
     4321 ttggcataaa aaggctaatt gattttcgag agtttcatac tgtttttctg taggccgtgt
     4381 acctaaatgt acttttgctc catcgcgatg acttagtaaa gcacatctaa aacttttagc
     4441 gttattacgt aaaaaatctt gccagctttc cccttctaaa gggcaaaagt gagtatggtg
     4501 cctatctaac atctcaatgg ctaaggcgtc gagcaaagcc cgcttatttt ttacatgcca
     4561 atacaatgta ggctgctcta cacctagctt ctgggcgagt ttacgggttg ttaaaccttc
     4621 gattccgacc tcattaagca gctctaatgc gctgttaatc actttacttt tatctaatct
     4681 agacatcatt aattcctaat ttttgttgac actct
//
