LOCUS       sequence-217143-        4670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 10xHis-Cas14a1 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 4670)
  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 4670)
  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: pLBH559_Tet-HisCas14a1Locus
FEATURES             Location/Qualifiers
     source          1..4670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..71
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    52..70
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             89..100
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             116..133
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     terminator      2289..2316
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2344..2361)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2478..3023)
                     /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(2558..2577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3137..3231)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3252..3911)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3826..3845
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3912..4014)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(4044..4667)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGSKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     4575..4594
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 taattcctaa tttttgttga cactctatcg ttgatagagt tattttacca ctccctatca
       61 gtgatagaga aaagaattca aaagatctaa agaggagaaa ggatctatga aatctcacca
      121 tcaccatcac catgaaaacc tgtacttcca atccaatatt ggaagtggaa tggccaaaaa
      181 caccattacc aaaacactga aactgcgtat tgtgcgtccg tataatagcg cagaagtgga
      241 aaaaattgtt gccgacgaaa aaaacaaccg cgaaaaaatc gcactggaaa agaacaaaga
      301 caaagtgaaa gaagcctgca gcaaacatct gaaagttgca gcatattgta ccacacaggt
      361 tgaacgtaat gcatgcctgt tttgtaaagc acgtaaactg gatgacaaat tctaccaaaa
      421 actgcgtggt cagtttccgg atgcagtttt ttggcaagaa atcagcgaaa tttttcgcca
      481 gctgcagaaa caggcagcag aaatctataa tcagagcctg atcgaactgt actacgagat
      541 ttttatcaaa ggcaaaggta ttgcaaatgc cagcagcgtt gaacattatc tgagtgatgt
      601 ttgttatacc cgtgcagcag aactgtttaa aaacgcagca attgcaagcg gtctgcgtag
      661 caaaatcaaa agcaattttc gtctgaaaga actgaaaaac atgaaaagtg gtctgccgac
      721 caccaaaagc gataattttc cgattccgct ggttaaacag aaaggtggtc agtataccgg
      781 ttttgaaatt agcaatcata atagcgactt catcatcaag attccgtttg gtcgttggca
      841 ggtcaaaaaa gagattgata aatatcgtcc gtgggagaaa tttgactttg aacaggttca
      901 gaaaagcccg aaaccgatta gcctgctgct gagcacccag cgtcgtaaac gtaataaagg
      961 ttggagcaaa gatgaaggca ccgaagccga aatcaaaaaa gttatgaatg gcgattatca
     1021 gaccagctac attgaagtta aacgtggcag caaaatcggt gaaaaaagcg catggatgct
     1081 gaatctgagc attgatgttc cgaaaattga taaaggtgtg gatccgagca ttattggtgg
     1141 tattgatgtt ggtgttaaat caccgctggt ttgcgcaatt aacaatgcat ttagccgtta
     1201 tagcatcagc gataacgacc tgtttcactt caacaagaaa atgtttgcac gtcgtcgtat
     1261 cctgctgaaa aaaaaccgtc ataaacgtgc aggtcatggt gcaaaaaaca aactgaaacc
     1321 gatcaccatt ctgaccgaaa aaagtgaacg ttttcgcaaa aagctgattg aacgttgggc
     1381 atgtgaaatc gcggatttct tcattaaaaa caaagttggc accgtgcaga tggaaaatct
     1441 ggaaagcatg aaacgtaaag aggacagcta ttttaacatt cgcctgcgtg gcttttggcc
     1501 gtatgcagaa atgcagaaca aaatcgaatt caaactgaag cagtatggca tcgaaattcg
     1561 taaagttgca ccgaataata ccagcaaaac ctgtagcaaa tgtggccatc tgaacaacta
     1621 tttcaacttc gagtaccgca agaaaaacaa attcccgcac tttaaatgcg aaaaatgcaa
     1681 cttcaaagaa aacgccgatt ataatgcagc cctgaatatt tcaaacccga aactgaaaag
     1741 caccaaagag gaaccgtaaa tatttatact ttattatcct tcattgacaa aaatgagaat
     1801 gttatcccag ataacatttg atgtacacag attcacactt cactgataaa gtggagaacc
     1861 gcttcaccaa aagctgtccc ttaggggatt agaacttgag tgaaggtggg ctgcttgcat
     1921 cagcctaatg tcgagaagtg ctttcttcgg aaagtaaccc tcgaaacaaa ttcatttttc
     1981 ctctccaatt ctgcacaaaa aaaggtgagt ccttataaac cggcgtgcag aacgccggct
     2041 cacctttttt cttcattcga ttttatgctt aaaagccgta aaaacgcgga attcggcgcc
     2101 gttgcagaac ccgaatagac gaatgaagga atgcaactac cttacaccgc ttgcgaaagt
     2161 tgtcagaaga taatcttgca gaacccgaat agacgaatga aggaatgcaa tcttgacaga
     2221 gcccgattgc gttatctcca ggagaaacat ataaaagcat caaccgctga tcggactaga
     2281 gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacctaggga tatattccgc
     2341 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta
     2401 cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg
     2461 gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga
     2521 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2581 ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg
     2641 ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc
     2701 caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa
     2761 ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg
     2821 taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga
     2881 caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc
     2941 agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta
     3001 cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga
     3061 tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt
     3121 tgttactagt gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga
     3181 acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc
     3241 tcgatatcaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     3301 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc
     3361 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
     3421 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
     3481 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     3541 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     3601 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
     3661 atcaccagct caccgtcttt cattgccata cgaaattccg gatgagcatt catcaggcgg
     3721 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
     3781 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
     3841 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
     3901 tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc
     3961 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct
     4021 cattttcgcc agatatcgac gtcttaagac ccactttcac atttaagttg tttttctaat
     4081 ccgcatatga tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat
     4141 aattcgatag cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct
     4201 tctttagcga cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca
     4261 gcgctgagtg catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat
     4321 tgattttcga gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgat
     4381 ccatcgcgat gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct
     4441 tgccagcttt ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg
     4501 gctaaggcgt cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct
     4561 acacctagct tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc
     4621 agctctaatg cgctgttaat cactttactt ttatctaatc tagacatcat
//
