LOCUS       sequence-173889-        6541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Cascade subunits Cse2, Cas7, Cas5, Cas6e and pre-crRNA
            containing a shorter version of the wt spacer (-12) truncated by 12 
            nucleotides at the leader-distal end..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6541)
  AUTHORS   Kuznedelov K, Mekler V, Lemak S, Tokmina-Lukaszewska M, Datsenko KA,
            Jain I, Savitskaya E, Mallon J, Shmakov S, Bothner B, Bailey S, 
            Yakunin AF, Severinov K, Semenova E
  TITLE     Altered stoichiometry Escherichia coli Cascade complexes with 
            shortened CRISPR RNA spacers are capable of interference and primed 
            adaptation.
  JOURNAL   Nucleic Acids Res. 2016 Dec 15;44(22):10849-10861. Epub 2016 Oct 13.
  PUBMED    27738137
REFERENCE   2  (bases 1 to 6541)
  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: "Nucleic 
            Acids Res."; date: "2016-12-15"; volume: "44(22)"; pages: 
            "10849-10861. Epub 2016 Oct 13"
COMMENT     Sequence Label: pCDF-casBCDE(-12)
FEATURES             Location/Qualifiers
     source          1..6541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             80..103
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             110..133
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3208..3226)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3222..3269
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3440..4231)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(4232..4323)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4371..5109)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(5319..6401)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6363..6382
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6402..6479)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6525..6541,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6525..6541,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggcaagct ggagccaccc gcagttcgaa aagggtgcac ttgaagtcct
      121 ctttcaggga cccgggtacc agatggctga tgaaattgat gcaatggctt tatatcgagc
      181 ctggcaacaa ctggataatg gatcatgtgc gcaaattaga cgtgtttcag aacctgatga
      241 attacgcgat atccctgcgt tttataggct ggtgcaacct tttggttggg aaaacccacg
      301 tcaccagcag gctcttttgc gcatggtgtt ttgcctgagc gcaggaaaga atgtcatccg
      361 acatcaggac aaaaaatcgg agcaaacaac aggtatctcg ttgggaagag ctttagccaa
      421 tagtggaaga attaacgagc gccgtatctt tcaattaatt cgggctgaca gaacagccga
      481 tatggtccag ttacgtcgat tacttactca cgccgaaccc gtacttgact ggccattaat
      541 ggccaggatg ttgacctggt ggggaaagcg cgaacgccag caacttctgg aagattttgt
      601 attgaccaca aacaaaaatg cgtaaggaaa cctttctatg tctaacttta tcaatattca
      661 tgttctgatc tctcacagcc cttcatgtct gaaccgcgac gatatgaaca tgcagaaaga
      721 cgctattttc ggcggcaaaa gacgagtaag aatttcaagt caaagcctta aacgtgcgat
      781 gcgtaaaagt ggttattacg cacaaaatat tggtgaatcc agtctcagaa ccattcatct
      841 tgcacaatta cgtgatgttc ttcggcaaaa acttggtgaa cgttttgacc aaaaaatcat
      901 cgataagaca ttagcgctgc tctccggtaa atcagttgat gaagccgaaa agatttctgc
      961 cgatgcggtt actccctggg ttgtgggaga aatagcctgg ttctgtgagc aggttgcaaa
     1021 agcagaggct gataatctgg atgataaaaa gctgctcaaa gttcttaagg aagatattgc
     1081 cgccatacgt gtgaatttac agcagggtgt tgatattgcg cttagtggaa gaatggcaac
     1141 cagcggcatg atgactgagt tgggaaaagt tgatggtgca atgtccattg cgcatgcgat
     1201 cactactcat caggttgatt ctgatattga ctggttcacc gctgtagatg atttacagga
     1261 acaaggttct gcacatctgg gaactcagga attttcatcg ggtgtttttt atcgttatgc
     1321 caacattaac ctcgctcaac ttcaggaaaa tttaggtggt gcctccaggg agcaggctct
     1381 ggaaattgca acccatgttg ttcatatgct ggcaacagag gtccctggag caaaacagcg
     1441 tacttatgcc gcttttaacc ctgcggatat ggtaatggtt aatttctccg atatgccact
     1501 ttctatggca aatgcttttg aaaaagcggt taaagcgaaa gatggctttt tgcaaccgtc
     1561 tatacaggcg tttaatcaat attgggatcg cgttgccaat ggatatggtc tgaacggagc
     1621 tgctgcgcaa ttcagcttat ctgatgtaga cccaattact gctcaagtta aacaaatgcc
     1681 tactttagaa cagttaaaat cctgggttcg taataatggc gaggcgtgaa catgagatct
     1741 tatttgatct tgcggcttgc tgggccaatg caagcctggg ggcagccgac ctttgaagga
     1801 acgcgaccta ccggaagatt tccgacccga agcgggttat tagggctact cggggcttgt
     1861 cttgggatcc aacgtgatga tacttcttca ttacaggcgt tatcagagag tgtgcaattt
     1921 gcagtgcgct gcgatgaact cattcttgac gatcgtcgtg tgtctgtaac ggggttgcgt
     1981 gattaccata cagtccttgg agcgcgagaa gattaccgtg gtttgaaaag tcatgaaacg
     2041 attcaaacat ggcgcgaata tttatgtgat gcctccttta ccgtcgctct ctggttaaca
     2101 ccccatgcaa cgatggttat ctcagaactt gaaaaagcag tattaaagcc tcggtataca
     2161 ccttacctgg ggcggagaag ttgcccacta acacacccgc tttttttggg gacatgtcag
     2221 gcatcggatc ctcagaaggc gctattaaat tatgagcccg ttggcggcga tatatatagt
     2281 gaggaatcag ttacagggca tcatttaaaa tttacggcgc gcgacgaacc gatgatcacc
     2341 ttgcctcgac aatttgcttc ccgagaatgg tatgtgatta aaggaggtat ggatgtatct
     2401 cagtaaagtc atcattgcca gggcctggag cagggatctt taccaacttc accagggatt
     2461 atggcattta tttccaaaca gaccggatgc tgctcgtgat tttctttttc atgttgagaa
     2521 gcgaaacaca ccagaaggct gtcatgtttt attgcagtca gcgcaaatgc ctgtttcaac
     2581 tgccgttgcg acagtcatta aaactaaaca ggttgaattt caacttcagg ttggtgttcc
     2641 actctatttt cggcttcggg caaatccgat caaaactatt ctcgacaatc aaaagcgcct
     2701 ggacagtaaa gggaatatta aacgctgtcg ggttccgtta ataaaagaag cagaacaaat
     2761 cgcgtggttg caacgtaaat tgggcaatgc ggcgcgcgtt gaagatgtgc atcccatatc
     2821 ggaacggcca cagtattttt ctggtgatgg taaaagtgga aagatccaaa cggtttgctt
     2881 tgaaggtgtg ctcaccatca acgacgcgcc agcgttaata gatcttgtac agcaaggtat
     2941 tgggccagct aaatcgatgg gatgtggctt gctatctttg gctccactgt gagcggccgc
     3001 actcgagatt tggatggttt aaggttggtg ggttgttttt atgggaaaaa atgctttaag
     3061 aacaaatgta tacttttaga gagttccccg cgccagcggg gataaaccgc tgtctttcgc
     3121 tgctgagggg agttccccgc gccagcgggg ataaaccgag ctcccatttt caaacccatc
     3181 aagacgcctt cgcctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     3241 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
     3301 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
     3361 aacgaccctg ccctgaaccg acgaccgggt catcgtggcc ggatcttgcg gcccctcggc
     3421 ttgaacgaat tgttagacat tatttgccga ctaccttggt gatctcgcct ttcacgtagt
     3481 ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat
     3541 aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc
     3601 agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg
     3661 acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg
     3721 ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct
     3781 ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca
     3841 gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt
     3901 ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa
     3961 caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca
     4021 aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca
     4081 gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta
     4141 cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag
     4201 tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat tattgaagca
     4261 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4321 aaatagctag ctcactcggt cgctacgctc cgggcgtgag actgcggcgg gcgctgcgga
     4381 cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg tgaggcaaaa
     4441 cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca gagttcacat
     4501 aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg aatctgacag
     4561 tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt cgctccctct
     4621 tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt tctcccttac
     4681 gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt gtaggtcgtt
     4741 cgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg cgccttatcc
     4801 ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact ggcagcagcc
     4861 attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg ctcttgaagt
     4921 gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc tgcgaaagcc
     4981 agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc acctccccag
     5041 gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc tcaagaagat
     5101 cctttgatct tttctactga accgctctag atttcagtgc aatttatctc ttcaaatgta
     5161 gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg
     5221 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     5281 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5341 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5401 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     5461 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5521 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     5581 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     5641 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5701 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5761 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5821 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5881 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5941 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     6001 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     6061 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6121 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     6181 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     6241 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     6301 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     6361 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     6421 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     6481 gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat
     6541 a
//
