LOCUS       sequence-177593-        6608 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 (-18) truncated by 18 
            nucleotides at the leader-distal end..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6608)
  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 6608)
  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(-18)
FEATURES             Location/Qualifiers
     source          1..6608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    7..23
                     /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(3275..3293)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3289..3336
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3507..4298)
                     /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(4299..4390)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4438..5176)
                     /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(5386..6468)
                     /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     6430..6449
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6469..6546)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6592..6608,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6592..6608,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggaaattgt 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 tcttttttac ctgtttgaaa acaaagaatt
     3061 agctgatctt taataataag gaaatgttac attaaggttg gtgggttgtt tttatgggaa
     3121 aaaatgcttt aagaacaaat gtatactttt agagagttcc ccgcgccagc ggggataaac
     3181 cgctgtcttt cgctgcgagt tccccgcgcc agcggggata aaccgagctc ccattttcaa
     3241 acccatcaag acgccttcgc ctaggctgct gccaccgctg agcaataact agcataaccc
     3301 cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag
     3361 cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg
     3421 gctatttaac gaccctgccc tgaaccgacg accgggtcat cgtggccgga tcttgcggcc
     3481 cctcggcttg aacgaattgt tagacattat ttgccgacta ccttggtgat ctcgcctttc
     3541 acgtagtgga caaattcttc caactgatct gcgcgcgagg ccaagcgatc ttcttcttgt
     3601 ccaagataag cctgtctagc ttcaagtatg acgggctgat actgggccgg caggcgctcc
     3661 attgcccagt cggcagcgac atccttcggc gcgattttgc cggttactgc gctgtaccaa
     3721 atgcgggaca acgtaagcac tacatttcgc tcatcgccag cccagtcggg cggcgagttc
     3781 catagcgtta aggtttcatt tagcgcctca aatagatcct gttcaggaac cggatcaaag
     3841 agttcctccg ccgctggacc taccaaggca acgctatgtt ctcttgcttt tgtcagcaag
     3901 atagccagat caatgtcgat cgtggctggc tcgaagatac ctgcaagaat gtcattgcgc
     3961 tgccattctc caaattgcag ttcgcgctta gctggataac gccacggaat gatgtcgtcg
     4021 tgcacaacaa tggtgacttc tacagcgcgg agaatctcgc tctctccagg ggaagccgaa
     4081 gtttccaaaa ggtcgttgat caaagctcgc cgcgttgttt catcaagcct tacggtcacc
     4141 gtaaccagca aatcaatatc actgtgtggc ttcaggccgc catccactgc ggagccgtac
     4201 aaatgtacgg ccagcaacgt cggttcgaga tggcgctcga tgacgccaac tacctctgat
     4261 agttgagtcg atacttcggc gatcaccgct tccctcatac tcttcctttt tcaatattat
     4321 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4381 aataaacaaa tagctagctc actcggtcgc tacgctccgg gcgtgagact gcggcgggcg
     4441 ctgcggacac atacaaagtt acccacagat tccgtggata agcaggggac taacatgtga
     4501 ggcaaaacag cagggccgcg ccggtggcgt ttttccatag gctccgccct cctgccagag
     4561 ttcacataaa cagacgcttt tccggtgcat ctgtgggagc cgtgaggctc aaccatgaat
     4621 ctgacagtac gggcgaaacc cgacaggact taaagatccc caccgtttcc ggcgggtcgc
     4681 tccctcttgc gctctcctgt tccgaccctg ccgtttaccg gatacctgtt ccgcctttct
     4741 cccttacggg aagtgtggcg ctttctcata gctcacacac tggtatctcg gctcggtgta
     4801 ggtcgttcgc tccaagctgg gctgtaagca agaactcccc gttcagcccg actgctgcgc
     4861 cttatccggt aactgttcac ttgagtccaa cccggaaaag cacggtaaaa cgccactggc
     4921 agcagccatt ggtaactggg agttcgcaga ggatttgttt agctaaacac gcggttgctc
     4981 ttgaagtgtg cgccaaagtc cggctacact ggaaggacag atttggttgc tgtgctctgc
     5041 gaaagccagt taccacggtt aagcagttcc ccaactgact taaccttcga tcaaaccacc
     5101 tccccaggtg gttttttcgt ttacagggca aaagattacg cgcagaaaaa aaggatctca
     5161 agaagatcct ttgatctttt ctactgaacc gctctagatt tcagtgcaat ttatctcttc
     5221 aaatgtagca cctgaagtca gccccatacg atataagttg taattctcat gttagtcatg
     5281 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5341 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5401 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5461 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5521 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5581 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5641 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     5701 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5761 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5821 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5881 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5941 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     6001 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     6061 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     6121 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     6181 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     6241 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6301 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6361 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6421 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6481 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6541 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaaa ttaatacgac
     6601 tcactata
//
