LOCUS       sequence-239492-        5559 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for catalytically inactivated 
            deltaproteobacteria CasX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5559)
  AUTHORS   Liu JJ, Orlova N, Oakes BL, Ma E, Spinner HB, Baney KLM, Chuck J, 
            Tan D, Knott GJ, Harrington LB, Al-Shayeb B, Wagner A, Brotzmann J, 
            Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA
  TITLE     CasX enzymes comprise a distinct family of RNA-guided genome 
            editors.
  JOURNAL   Nature. 2019 Feb 4. pii: 10.1038/s41586-019-0908-x. doi: 
            10.1038/s41586-019-0908-x.
  PUBMED    30718774
REFERENCE   2  (bases 1 to 5559)
  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: "Nature. 
            2019 Feb 4. pii: 10.1038/s41586-019-0908-x. doi: 
            10.1038/s41586-019-0908-x."
COMMENT     Sequence Label: pBLO ddd64.1
FEATURES             Location/Qualifiers
     source          1..5559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2668..2739
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2755..2782
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2810..2827)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2944..3489)
                     /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(3024..3043)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3603..3697)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3718..4377)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4292..4311
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4378..4480)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(4510..5136)
                     /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     5041..5060
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        5152..5207
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5188..5206
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             5225..5236
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 cgaccagaac aagctgaagc ccgagatgga cgagaagggc aatctgacca cagccggctt
       61 tgcctgctct cagtgtggcc agcctctgtt cgtgtacaag ctggaacagg tgtccgagaa
      121 aggcaaggcc tacaccaact acttcggcag atgtaacgtg gccgagcacg agaagctgat
      181 tctgctggcc cagctgaaac ctgagaagga ctctgatgag gccgtgacct acagcctggg
      241 caagtttgga cagagagccc tggacttcta cagcatccac gtgaccaaag aaagcacaca
      301 ccccgtgaag cccctggctc agatcgccgg caatagatac gcctctggac ctgtgggcaa
      361 agccctgtcc gatgcctgca tgggaacaat cgccagcttc ctgagcaagt accaggacat
      421 catcatcgag caccagaagg tggtcaaggg caaccagaag agactggaaa gcctgaggga
      481 gctggccggc aaagagaacc tggaataccc cagcgtgacc ctgcctcctc agcctcacac
      541 aaaagaaggc gtggacgcct acaacgaagt gatcgccaga gtgagaatgt gggtcaacct
      601 gaacctgtgg cagaagctga aactgtccag ggacgacgcc aagcctctgc tgagactgaa
      661 gggcttccct agcttccccg tggtggaaag aagggagaat gaggtggact ggtggaacac
      721 catcaacgaa gtcaagaaac tgatcgatgc caagagagac atgggcagag tgttttggag
      781 cggagtgacc gccgagaaga gaaacaccat cctggaaggc tacaactacc tgcctaacga
      841 gaacgaccac aagaagagag agggcagcct ggaaaaccct aagaagcccg ccaagagaca
      901 gttcggcgac ctgctgctgt acctggagaa gaagtatgcc ggcgactggg gcaaagtgtt
      961 cgatgaggcc tgggagagaa tcgacaagaa gattgccggc ctgaccagcc acatcgagag
     1021 agaggaagcc agaaacgccg aggacgccca gtctaaagcc gtgctgacag attggctgag
     1081 agccaaggcc agcttcgtgc tggaaaggct gaaagaaatg gatgagaaag agttctacgc
     1141 ctgcgagatc cagctgcaga agtggtacgg ggacctgaga ggcaatccct ttgccgtgga
     1201 agccgagaac agagtggtgg acatcagcgg ctttagcatc ggctctgacg gccactccat
     1261 ccagtacagg aacctgctgg cctggaagta cctggaaaat ggcaagagag agttctacct
     1321 gctgatgaac tacggcaaga agggcagaat cagattcacc gacggcaccg acatcaagaa
     1381 aagcggcaag tggcagggcc tgctgtatgg cggcggaaag gccaaagtga tcgacctgac
     1441 cttcgatccc gacgacgagc agctgatcat tctgcctctg gccttcggca ccagacaggg
     1501 cagagagttc atctggaacg atctgctgtc cctggaaacc ggcctgatca agctggccaa
     1561 cggcagggtg atcgagaaaa ccatctacaa caagaaaatc ggcagagatg agcccgcact
     1621 gtttgtggcc ctgacctttg agaggagaga agtggtcgat cccagcaaca tcaagcccgt
     1681 gaacctgatc ggagtggcca gaggcgagaa tatccctgcc gtgattgccc tgacagaccc
     1741 tgagggatgt cccctgcctg agtttaagga tagctctggc ggccctaccg acatcctgag
     1801 aatcggcgag ggctacaaag agaagcagag agccatccag gccgccaaag aggtggaaca
     1861 gagaagagcc ggcggataca gcagaaagtt cgccagcaag tccagaaacc tggccgatga
     1921 catggtcagg aacagcgcca gggacctgtt ctaccacgcc gtgacacatg atgccgtgct
     1981 ggtgttcgcg aacctgtcaa gaggcttcgg cagacaggga aaaagaacct tcatgaccga
     2041 gagacagtac acgaagatgg aagattggct caccgccaaa ctggcctacg agggcctgac
     2101 aagcaagacc tacctgtcca agacactggc ccagtacacc tccaagacct gctccaattg
     2161 cggcttcacc atcaccaccg ccgactacga tggaatgctc gtgagactga agaaaacctc
     2221 cgacggctgg gccaccacac tgaacaacaa agagctgaaa gccgagggcc agatcaccta
     2281 ctacaacaga tacaagaggc agaccgtcga gaaagagctg tccgccgaac tcgacagact
     2341 gagcgaggaa tccggcaaca acgacatcag caagtggacc aagggacgca gagatgaggc
     2401 cctgttcctg ctgaagaaga ggttcagcca cagacctgtg caagagcagt ttgtgtgcct
     2461 ggattgcggc cacgaagtgc acgcagctga acaggccgct ctgaatatcg ccagaagctg
     2521 gctgttcctg aacagcaaca gcaccgagtt caagagctac aagagcggca agcagccttt
     2581 cgttggagcc tggcaggcct tctacaagag aaggctcaaa gaagtgtgga agcccaacgc
     2641 ctgactcgag taaggatctc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     2701 gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca
     2761 ccttcgggtg ggcctttctg cgtttatacc tagggatata ttccgcttcc tcgctcactg
     2821 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag
     2881 atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc
     2941 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg
     3001 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt
     3061 gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt
     3121 gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta
     3181 tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3241 ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag
     3301 gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac
     3361 tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa
     3421 aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa
     3481 cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt
     3541 atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgtt actagtgctt
     3601 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg
     3661 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta
     3721 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg
     3781 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
     3841 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac
     3901 gtttaaatca aaactggtga aactcaccca gggattggct gacacgaaaa acatattctc
     3961 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
     4021 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
     4081 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
     4141 gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat
     4201 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc
     4261 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
     4321 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt
     4381 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
     4441 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtttcatt ttcgccagat
     4501 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
     4561 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
     4621 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
     4681 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
     4741 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
     4801 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
     4861 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
     4921 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
     4981 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
     5041 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
     5101 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt tgttgacact
     5161 ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag aattcaaaag
     5221 atctaaagag gagaaaggat ctatggaaaa gagaatcaac aagatcagga agaagctgag
     5281 cgccgacaac gccaccaagc ctgtgtctag gagtggcccc atgaaaaccc tgctcgtcag
     5341 agtgatgacc gacgacctga agaagagact ggaaaagagg aggaagaaac ccgaagtgat
     5401 gccccaagtg atcagcaaca acgccgccaa caacctgaga atgctgctgg acgactacac
     5461 caagatgaag gaagccatcc tgcaggtcta ctggcaagag ttcaaggacg accacgtggg
     5521 cctgatgtgc aagtttgccc agcctgccag caagaagat
//
