LOCUS       sequence-177596-        6547 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 (-6) truncated by 6 
            nucleotides at the leader-distal end..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6547)
  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 6547)
  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..6547
                     /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(3214..3232)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3228..3275
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3446..4237)
                     /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(4238..4329)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4377..5115)
                     /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(5325..6407)
                     /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     6369..6388
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6408..6485)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6531..6547,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6531..6547,1..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 ggttgttttt atgggaaaaa atgctttaag
     3061 aacaaatgta tacttttaga gagttccccg cgccagcggg gataaaccgc tgtctttcgc
     3121 tgctgagggt gacgagagtt ccccgcgcca gcggggataa accgagctcc cattttcaaa
     3181 cccatcaaga cgccttcgcc taggctgctg ccaccgctga gcaataacta gcataacccc
     3241 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc
     3301 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
     3361 ctatttaacg accctgccct gaaccgacga ccgggtcatc gtggccggat cttgcggccc
     3421 ctcggcttga acgaattgtt agacattatt tgccgactac cttggtgatc tcgcctttca
     3481 cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct tcttcttgtc
     3541 caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca
     3601 ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa
     3661 tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc
     3721 atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga
     3781 gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga
     3841 tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct
     3901 gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt
     3961 gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag
     4021 tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg
     4081 taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca
     4141 aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata
     4201 gttgagtcga tacttcggcg atcaccgctt ccctcatact cttccttttt caatattatt
     4261 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4321 ataaacaaat agctagctca ctcggtcgct acgctccggg cgtgagactg cggcgggcgc
     4381 tgcggacaca tacaaagtta cccacagatt ccgtggataa gcaggggact aacatgtgag
     4441 gcaaaacagc agggccgcgc cggtggcgtt tttccatagg ctccgccctc ctgccagagt
     4501 tcacataaac agacgctttt ccggtgcatc tgtgggagcc gtgaggctca accatgaatc
     4561 tgacagtacg ggcgaaaccc gacaggactt aaagatcccc accgtttccg gcgggtcgct
     4621 ccctcttgcg ctctcctgtt ccgaccctgc cgtttaccgg atacctgttc cgcctttctc
     4681 ccttacggga agtgtggcgc tttctcatag ctcacacact ggtatctcgg ctcggtgtag
     4741 gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg ttcagcccga ctgctgcgcc
     4801 ttatccggta actgttcact tgagtccaac ccggaaaagc acggtaaaac gccactggca
     4861 gcagccattg gtaactggga gttcgcagag gatttgttta gctaaacacg cggttgctct
     4921 tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga tttggttgct gtgctctgcg
     4981 aaagccagtt accacggtta agcagttccc caactgactt aaccttcgat caaaccacct
     5041 ccccaggtgg ttttttcgtt tacagggcaa aagattacgc gcagaaaaaa aggatctcaa
     5101 gaagatcctt tgatcttttc tactgaaccg ctctagattt cagtgcaatt tatctcttca
     5161 aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc
     5221 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5281 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5341 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5401 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     5461 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     5521 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     5581 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     5641 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5701 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5761 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5821 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5881 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5941 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     6001 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     6061 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     6121 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     6181 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     6241 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     6301 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     6361 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     6421 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     6481 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat taatacgact
     6541 cactata
//
