LOCUS       sequence-177602-        6697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Cascade subunits Cse2, Cas7, Cas5, Cas6e and pre-crRNA
            containing a longer version of the wt spacer (+6) extended by 6 
            nucleotides at the leader-distal end..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6697)
  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 6697)
  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(+6)
FEATURES             Location/Qualifiers
     source          1..6697
                     /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(3299..3317)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3313..3360
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3531..4322)
                     /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(4323..4414)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(5475..6557)
                     /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     6519..6538
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6558..6635)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6681..6697,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6681..6697,1..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 cgctgctgag ggtgacgatc ccgcatgtat gagttccccg cgccagcggg
     3241 gataaaccga gctcccattt tcaaacccat caagacgcct tcgcctaggc tgctgccacc
     3301 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     3361 ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt caataaaccg
     3421 gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc gacgaccggg
     3481 tcatcgtggc cggatcttgc ggcccctcgg cttgaacgaa ttgttagaca ttatttgccg
     3541 actaccttgg tgatctcgcc tttcacgtag tggacaaatt cttccaactg atctgcgcgc
     3601 gaggccaagc gatcttcttc ttgtccaaga taagcctgtc tagcttcaag tatgacgggc
     3661 tgatactggg ccggcaggcg ctccattgcc cagtcggcag cgacatcctt cggcgcgatt
     3721 ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg
     3781 ccagcccagt cgggcggcga gttccatagc gttaaggttt catttagcgc ctcaaataga
     3841 tcctgttcag gaaccggatc aaagagttcc tccgccgctg gacctaccaa ggcaacgcta
     3901 tgttctcttg cttttgtcag caagatagcc agatcaatgt cgatcgtggc tggctcgaag
     3961 atacctgcaa gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg cttagctgga
     4021 taacgccacg gaatgatgtc gtcgtgcaca acaatggtga cttctacagc gcggagaatc
     4081 tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt
     4141 gtttcatcaa gccttacggt caccgtaacc agcaaatcaa tatcactgtg tggcttcagg
     4201 ccgccatcca ctgcggagcc gtacaaatgt acggccagca acgtcggttc gagatggcgc
     4261 tcgatgacgc caactacctc tgatagttga gtcgatactt cggcgatcac cgcttccctc
     4321 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4381 tacatatttg aatgtattta gaaaaataaa caaatagcta gctcactcgg tcgctacgct
     4441 ccgggcgtga gactgcggcg ggcgctgcgg acacatacaa agttacccac agattccgtg
     4501 gataagcagg ggactaacat gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc
     4561 ataggctccg ccctcctgcc agagttcaca taaacagacg cttttccggt gcatctgtgg
     4621 gagccgtgag gctcaaccat gaatctgaca gtacgggcga aacccgacag gacttaaaga
     4681 tccccaccgt ttccggcggg tcgctccctc ttgcgctctc ctgttccgac cctgccgttt
     4741 accggatacc tgttccgcct ttctccctta cgggaagtgt ggcgctttct catagctcac
     4801 acactggtat ctcggctcgg tgtaggtcgt tcgctccaag ctgggctgta agcaagaact
     4861 ccccgttcag cccgactgct gcgccttatc cggtaactgt tcacttgagt ccaacccgga
     4921 aaagcacggt aaaacgccac tggcagcagc cattggtaac tgggagttcg cagaggattt
     4981 gtttagctaa acacgcggtt gctcttgaag tgtgcgccaa agtccggcta cactggaagg
     5041 acagatttgg ttgctgtgct ctgcgaaagc cagttaccac ggttaagcag ttccccaact
     5101 gacttaacct tcgatcaaac cacctcccca ggtggttttt tcgaaggtta agtcagttgg
     5161 ggaactgctt aaccttcgat caaaccacct ccccaggtgg ttttttcgtt tacagggcaa
     5221 aagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tactgaaccg
     5281 ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     5341 tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5401 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5461 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5521 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5581 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5641 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5701 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     5761 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5821 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5881 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5941 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6001 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     6061 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6121 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6181 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6241 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6301 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6361 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6421 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6481 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6541 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6601 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     6661 cgactcctgc attaggaaat taatacgact cactata
//
