LOCUS       sequence-177172-        7134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the genes Csy1, Csy2, Csy3, Csy4, cloned as a 
            polycistronic operon from bacterium Pseudomonas aeruginosa PA14. 
            Csy3 includes an N-terminal 6his tag and TEV protease cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7134)
  AUTHORS   Chowdhury S, Carter J, Rollins MF, Golden SM, Jackson RN, Hoffmann 
            C, Nosaka L, Bondy-Denomy J, Maxwell KL, Davidson AR, Fischer ER, 
            Lander GC, Wiedenheft B
  TITLE     Structure Reveals Mechanisms of Viral Suppressors that Intercept a 
            CRISPR RNA-Guided Surveillance Complex.
  JOURNAL   Cell. 2017 Mar 23;169(1):47-57.e11. doi: 10.1016/j.cell.2017.03.012.
  PUBMED    28340349
REFERENCE   2  (bases 1 to 7134)
  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"; doi: 
            "10.1016/j.cell.2017.03"; journalName: "Cell"; date: "2017-03-23- 
            23"; volume: "169"; issue: "1"; pages: "47-57.e11"
COMMENT     Sequence Label: pCsy_complex
FEATURES             Location/Qualifiers
     source          1..7134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             77..99
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     1459..1476
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1460..1476
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             1503..1525
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             2602..2624
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2644..2661
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2662..2682
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     RBS             3815..3837
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3846..4409
                     /codon_start=1
                     /product="CRISPR-associated endoribonuclease responsible 
                     for pre-crRNA processing in Pseudomonas aeruginosa 
                     (Haurwitz et al., 2010)"
                     /label=Csy4
                     /translation="MDHYLDIRLRPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFP
                     DLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQ
                     AKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGP
                     LQVTAEEGGFTCYGLSKGGFVPWF"
     primer_bind     complement(4564..4582)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4578..4625
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4692..4783
                     /gene="bla"
                     /label=AmpR promoter
     terminator      5648..5687
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      5718..6306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6207..6226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6460..6477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6492..6631
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6557..6576)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6717..6739
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6733..6924)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ggcaccatgc atcgatcccg cgaaattaat acgactcact atagggagac cacaacggtt
       61 tccctctaga aataattttg tttaacttta agaaggagat atagatcatg acctctcccc
      121 tcccaacgcc tacgtggcag gagcttcgcc agttcatcga atccttcatc caggagcgcc
      181 tccagggcaa gctggacaaa ctccaacccg acgaagacga caagcgccag acattgctgg
      241 ccacccaccg gcgggaggcc tggctggccg atgccgcccg gcgggttggc cagttgcagt
      301 tggtgaccca cacgctcaag ccgatccatc ccgacgcccg cggcagcaac ctgcacagcc
      361 tgccgcaagc acccggccaa ccgggcctcg ccggttccca tgagctaggt gacaggctgg
      421 tcagcgatgt ggtgggcaat gccgcggcgc tggacgtatt caagtttctc agtctccagt
      481 atcagggtaa aaatcttctg aactggctga cagaagacag tgccgaggca ttacaggcgc
      541 tgtccgataa cgccgaacag gctcgcgaat ggcggcaagc gttcatcggc atcacgaccg
      601 tcaaaggcgc tcccgcgtcc cacagcctgg ccaagcagtt gtactttccc ctgcccggtt
      661 ccggctacca cctgctagca ccgctgtttc ccaccagtct ggtgcatcac gtccacgctc
      721 tgctccgcga agcgcgcttc ggcgacgcgg ccaaggcggc acgcgaagcg cgcagccgcc
      781 aggagtcatg gccccacgga ttcagcgagt accccaacct ggcgatccag aagttcggcg
      841 gtaccaagcc gcagaacatc agtcagttga acaacgagcg ccgtggcgag aactggttgc
      901 tgccatcgct tccgccgaac tggcaaaggc agaatgtaaa cgcgccgatg cggcactctt
      961 cggtcttcga gcatgacttc ggaagaactc ctgaagtatc ccgcctaacc cgtaccctgc
     1021 agcgttttct tgccaagaca gtccataaca acctcgccat ccgccagcgg cgtgcccaat
     1081 tggtggcgca aatctgcgac gaagccctgc aatacgccgc ccgtctgcgc gaactggagc
     1141 ccggctggag cgcaaccccc gggtgccaac tgcatgacgc ggagcagctc tggctcgatc
     1201 cgttgcgcgc acagaccgat gagacgttct tgcagcgccg actacgaggt gactggcctg
     1261 ccgaggtcgg aaatcgcttc gccaactggc tgaaccgggc ggtcagcagc gacagtcaga
     1321 tactgggtag cccggaagcc gcccaatgga gccaggagct gagcaaggaa ctgacgatgt
     1381 tcaaggagat actcgaagat gagcgtgact gataataaga tcccaactcc ataaggatcc
     1441 gggccctgta caagatcctg taaaacgacg gccagtgaat tcactcgagt tctagaaata
     1501 attttgttta actttaagaa ggagatatag atcatgagcg tgactgatcc cgaggcgctg
     1561 ctgttgctgc cacgcctgtc catccagaac gccaacgcca tttccagccc cttgacctgg
     1621 ggtttccctt cgcccggcgc tttcaccgga ttcgtccatg ccttgcagcg tcgagtcgga
     1681 atctccctcg atatcgaact ggacggtgtc ggcatcgtct gtcaccgctt cgaagcgcag
     1741 atatcgcaac cggccggcaa gcgcaccaag gtcttcaacc tgacccgcaa tccgctgaac
     1801 cgcgacggct ccaccgcagc catcgtcgaa gaaggtcgcg cccatctgga ggtcagtctg
     1861 ctgctcggag tgcatggcga tggtctggac gatcaccccg cacaggaaat cgccaggcag
     1921 gtacaggagc aggctggcgc catgcgcctc gccggtggca gcatcctgcc ctggtgcaat
     1981 gagcgcttcc cggctccgaa cgccgaactg ctgatgctgg gtggcagcga cgaacagcgg
     2041 cgcaagaacc agcgccggct tacccgccgc ctgctccccg gcttcgccct ggtgagtcgc
     2101 gaggcgttgt tgcaacaaca tctggaaacc ttgcgcacca cgctccccga agccaccacg
     2161 ctcgatgcac tgctcgatct ttgtcgaatc aacttcgagc ctcctgcaac ctcgtccgaa
     2221 gaagaagcat cgccgcccga tgcagcctgg caagtgcgcg acaagcccgg ctggctggtg
     2281 ccgatcccgg ctggctacaa cgccctgtca cccctgtacc tccccggcga agtgcgaaat
     2341 gcccgtgata gagaaacacc gctgcgcttc gtcgaaaacc tcttcggtct gggcgaatgg
     2401 ctcagccccc atcgcgtcgc cgcgttatcg gacctgcttt ggtatcacca cgccgagcct
     2461 gacaaagggc tctaccgctg gagcacaccc cgtttcgtcg aacacgccat cgcataataa
     2521 taagatccca actccataag gatccgcgat cgcagcggat aacaatttca catcctgcag
     2581 gactcgagtt ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgaaatct
     2641 tctcaccatc accatcacca tgaaaacctg tacttccaat ccaatgcatc caagccaata
     2701 ctgagcaccg cttccgtcct cgccttcgaa cgtaagctcg acccttccga cgcccttatg
     2761 agcgctggcg cctgggcgca acgcgacgcc tcgcaggaat ggccggccgt gaccgtgcgc
     2821 gagaagtccg tacgcggcac catctccaac cgcctcaaga ccaaggaccg tgacccggcc
     2881 aagctggacg cctcgatcca gtcgcccaac ctgcagacgg tggacgtggc caacctgccg
     2941 agcgacgccg acaccctcaa ggtccgcttc accctccgtg tgctcggcgg cgccggaaca
     3001 ccgtctgcct gcaacgacgc ggcctaccgt gacaagctgc tgcaaacggt cgccacctac
     3061 gtgaacgatc agggcttcgc cgagctggct cgtcgttatg cgcacaacct ggccaacgcc
     3121 cgcttcctgt ggcgcaaccg ggtgggcgcg gaagcggtgg aagtccgtat caaccatatc
     3181 cgccagggcg aggtggctcg cgcctggcgc ttcgacgccc tggccatcgg cttgcgcgac
     3241 ttcaaggccg acgccgaact ggacgcgctc gccgaactga tcgccagcgg tctctcaggc
     3301 agtgggcatg tcctgctcga agtggtcgcc ttcgcccgta tcggcgacgg ccaggaagtc
     3361 ttcccctccc aggaactgat cctcgacaaa ggcgacaaga aaggccagaa gagcaagacc
     3421 ctgtacagcg tacgcgatgc cgcggccatc cactcgcaga agatcggcaa tgccctgcgc
     3481 accatcgata cgtggtatcc cgacgaagat ggcctcggcc ccatcgccgt ggagccctac
     3541 ggctccgtca catcccaggg caaagcctat cgccagccca agcagaagct ggacttctat
     3601 acgctgctcg acaactgggt actgcgcgac gaggcgcccg ccgtggagca acagcattat
     3661 gtgatcgcca acctgatccg tggcggcgtg ttcggtgaag ccgaagagaa gtaataataa
     3721 cattggaagt ggataacgga tccgcgatcg cggcgcgccc gccagggttt tcccagtcgc
     3781 ggccgccctg caggactcga gttctagaaa taattttgtt taactttaag aaggagatat
     3841 agatcatgga ccactacctc gacattcgct tgcgaccgga cccggaattt cccccggcgc
     3901 aactcatgag cgtgctcttc ggcaagctcc accaggccct ggtggcacag ggcggggaca
     3961 ggatcggcgt gagcttcccc gacctcgacg aaagccgctc ccggctgggc gagcgcctgc
     4021 gcattcatgc ctcggcggac gaccttcgtg ccctgctcgc ccggccctgg ctggaagggt
     4081 tgcgggacca tctgcaattc ggagaaccgg cagtcgtgcc tcaccccaca ccgtaccgtc
     4141 aggtcagtcg ggttcaggcg aaaagcaatc cggaacgcct gcggcggcgg ctcatgcgcc
     4201 ggcacgatct gagtgaggag gaggctcgga aacgcattcc cgatacggtc gcgagagcct
     4261 tggacctgcc cttcgtcacg ctacgcagcc agagcaccgg acagcacttc cgtctcttca
     4321 tccgccacgg gccgttgcag gtgacggcag aggaaggagg attcacctgt tacgggttga
     4381 gcaaaggagg tttcgttccc tggttctgat aataagatcc caactccata aggatccgcg
     4441 atcgcggcgc gccacctggt gccatcgccc tgatagacgt taattaaagt actggccggc
     4501 cacgcgtctc gagagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     4561 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     4621 ttttgctgaa aggaggaact atatccgact agtagcccgc ctaatgagcg ggcttttttt
     4681 taattcccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4741 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4801 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4861 aacgctcgtg aaagtaaaag acgcagagga ccaattgggg gcacgagtgg gatacataga
     4921 actggacttg aatagcggta aaatccttga gagttttcgc cctgaagagc gttttccaat
     4981 gatgagcact ttcaaagttc tgctatgtgg agcagtatta tcccgtgtag atgcggggca
     5041 agagcaactc ggacgacgaa tacactattc gcagaatgac ttggttgaat actccccagt
     5101 gacagaaaag caccttacgg acggaatgac ggtaagagaa ttatgtagtg ccgccataac
     5161 gatgagtgat aacactgcgg cgaacttact tctgacaacc atcggtggac cgaaggaatt
     5221 aaccgctttt ttgcacaata tgggagacca tgtaactcgc cttgaccgtt gggaaccaga
     5281 actgaatgaa gccataccaa acgacgagcg agacaccaca atgcctgcgg caatggcaac
     5341 aacattacgc aaactattaa ctggcgaact acttactctg gcttcacggc aacaattaat
     5401 agactggctt gaagcggata aagttgcagg accactactg cgttcggcac ttcctgctgg
     5461 ctggtttatt gctgataaat ctggggcagg agagcgtggt tcacggggta tcattgccgc
     5521 acttggacca gatggtaagc cttcccgtat cgtagttatc tacacgacgg gtagtcaggc
     5581 aactatggac gaacgaaata gacagattgc tgaaataggg gcttcactga ttaagcattg
     5641 gtaaaccgat acaattaaag gctccttttg gagccttttt ttttggacgg accggtagaa
     5701 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5761 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5821 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     5881 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5941 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6001 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6061 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6121 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6181 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6241 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6301 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6361 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     6421 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     6481 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     6541 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     6601 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     6661 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     6721 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     6781 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     6841 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     6901 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     6961 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     7021 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     7081 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgtt
//
