LOCUS       sequence-176377-        7394 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the Cas2/3 gene from Pseudomonas aeruginosa PA14, 
            with N-terminal Strep II tag, TEV protease cleavage site, and MBP 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7394)
  AUTHORS   Rollins MF, Chowdhury S, Carter J, Golden SM, Wilkinson RA, 
            Bondy-Denomy J, Lander GC, Wiedenheft B
  TITLE     Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease 
            activity.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Apr 24. pii: 201616395. doi: 
            10.1073/pnas.1616395114.
  PUBMED    28438998
REFERENCE   2  (bases 1 to 7394)
  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: "Proc Natl 
            Acad Sci U S A. 2017 Apr 24. pii: 201616395. doi: 
            10.1073/pnas.1616395114."
COMMENT     Sequence Label: pMBPCas2/3
FEATURES             Location/Qualifiers
     source          1..7394
                     /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"
     primer_bind     102..119
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     103..119
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             196..218
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             238..261
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             271..1371
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1345..1368
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1426..1446
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(4869..4887)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4883..4930
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5065..5856)
                     /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"
     rep_origin      5978..6566
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6467..6486
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6720..6737
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6752..6891
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6817..6836)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6977..6999
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6993..7184)
                     /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 aggcctcgga tccgggccct gtacaagatc ctgtaaaacg acggccagtg
      121 aattcactcg agtcccgggg cgatcgcagc ggataacaat ttcacatcct gcaggactcg
      181 agttctagaa ataattttgt ttaactttaa gaaggagata tacatatgaa atcttcttgg
      241 agccacccgc agttcgaaaa gggttcttct atgaaaatcg aagaaggtaa actggtaatc
      301 tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa
      361 gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag
      421 gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc
      481 tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg
      541 tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct
      601 gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg
      661 gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc
      721 aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc
      781 aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa
      841 gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat
      901 tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg
      961 tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc
     1021 ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc
     1081 agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt
     1141 ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa
     1201 gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc
     1261 atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac
     1321 gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc
     1381 tcgaacaaca acaacaataa caataacaac aacctcggga tcgaggaaaa cctgtacttc
     1441 caatccaatg caaacatcct gctggtgtcg caatgcgaaa agcgcgccct gagcgaaacc
     1501 cgccgcattc tcgaccagtt cgccgagcgc cgcggcgaac ggacctggca aacgcccatc
     1561 actcaagccg gactggatac cctgcgacgc ctgctgaaga aaagcgcacg gcgcaacacc
     1621 gccgtagcct gtcactggat ccgcggccgc gaccacagcg aactgctgtg gatcgtcggt
     1681 gatgccagcc gcttcaacgc ccagggtgcg gtgccgacca acaggacctg ccgcgacatc
     1741 ctgcgcaagg aagacgagaa cgactggcac agcgccgagg acatccgcct gctgacggtg
     1801 atggcagcgc tgttccacga tatcggcaag gccagccagg ccttccaggc caaactgcgg
     1861 aaccgcggca aaccgatggc cgatgcctat cgtcacgaat gggtatcact gcgcctgttc
     1921 gaagccttcg ttggcccagg cagcagcgac gaggactggc tgaggcgcct ggcggacaag
     1981 cgagagacgg gcgatgcctg gctgtcgcaa ctggccaggg acgaccggca atccgcgcca
     2041 cccggcccgt tccagaaaag ccggctaccg ccgctcgccc aggcggtcgg ctggttgatc
     2101 gtcagccatc atcgcctgcc caacggggac catcgcggca gcgcctcgct ggcacgcttg
     2161 ccggccccca tccagagcca atggtgcggc gcacgcgacg cagacgcaaa agaaaaggcc
     2221 gcctgctggc agttccccca cggcctgccc ttcgccagcg cccattggcg cgccaggaca
     2281 gcgctatgcg cgcagagcat gctcgagcgt cccggcctgc tggctcgggg accggccttg
     2341 ttgcatgatt cctacgtcat gcatgtgtcc cgactgatcc tgatgctcgc ggaccaccac
     2401 tattccagtc tccctgccga ctcccggctg ggcgacccga acttcccctt gcacgccaac
     2461 accgaccggg acagcggcaa actaaagcag cgcctggacg aacacctgct cggcgtcgcc
     2521 ctgcacagtc gcaagctcgc cggcaccctg ccacgcctgg agcgacaact accgcgcctt
     2581 gcccggcaca agggcttcac ccgccgggtc gagcagccgc gcttccgctg gcaggacaag
     2641 gcctacgact gcgcgatggc ctgccgcgag caggctatgg agcatggatt cttcggcctc
     2701 aacctggcgt cgaccggttg cggtaagacc ctcgccaacg gccgtatcct gtatgcgctg
     2761 gccgatccgc aacgcggcgc gcgtttcagc atcgctctcg gcctgcgcag cttgaccctg
     2821 caaaccgggc aggcctaccg cgagcggctc ggcctgggcg acgacgacct cgctatcctg
     2881 gtcggcggca gcgccgcccg cgaactgttc gaaaagcagc aggagcgcct ggagcgcagc
     2941 ggtagcgagt cagcccagga gctgctggcg gaaaacagcc atgtacactt cgccggcacg
     3001 ctcgaggacg gccctctacg cgagtggctc ggcaggaaca gcgcgggaaa ccgcctactc
     3061 caggcgccca tcctggcctg caccatcgac cacctgatgc ccgccagcga aagcctgcgc
     3121 ggcggacacc agatagcgcc actgctccgc ctgatgactt ccgacctggt gctcgacgag
     3181 gtcgacgact tcgatatcga cgacctgccc gccctgtcgc ggctggtgca ctgggccggc
     3241 ctgttcggca gccgcgtgct gctctcctcc gcgaccctgc cgccggcctt ggtgcagggc
     3301 ctgttcgagg cctatcgcag cggccgggaa atcttccagc gccatcgtgg cgctcccgga
     3361 cgcgctacgg aaatccgctg tgcctggttc gacgagttct ccagccaatc cagcgcccac
     3421 ggcgccgtaa cctccttcag cgaagcgcat gcgaccttcg tcgcccagcg tctggcgaag
     3481 ctcgagcaac tgccgccacg tcgccaggcg cagctatgca ccgtgcatgc cgctggcgag
     3541 gcccgtcccg cgctgtgccg cgagttggcc gggcagatga atacctggat ggctgacctg
     3601 catcgctgcc atcacaccga acaccaagga cgtcgcatca gtttcggcct gctacggctg
     3661 gccaacatcg aacccctgat cgaactggcc caggccatcc tcgcccaggg tgcgcccgag
     3721 gggttgcatg tccatctgtg tgtctaccat tcgcggcatc cccttctggt ccgctcggcc
     3781 atcgagcgac aactcgatga actgctgaag cgttcggacg acgacgccgc cgcgctgttc
     3841 gctcgtccga cgctggccaa ggcgctccag gccagcacgg agcgggatca tctgttcgtc
     3901 gtactcgcct cgccggtggc ggaggtcggt cgcgaccacg attacgactg ggccatcgtc
     3961 gaaccctcct ccatgcgctc gatcatccag ttggccgggc gaatccgccg ccatcgctcc
     4021 ggcttcagcg gcgaggccaa cctatacctg ctatcgcgca atatccgctc gctggaaggg
     4081 cagaatccgg cgttccagcg gcccggcttc gagacccccg acttccctct tgacagccac
     4141 gacctgcacg acctgctcga ccccgcccta ctcgcccgca tcgacgccag cccacgaatc
     4201 gtcgaaccgt tcccactgtt cccacgcagc cggttggtcg acctggaaca ccgacgcctg
     4261 cgcgcgctga tgcttgccga cgacccaccg tcgtccctgc tcggcgtacc gctctggtgg
     4321 caaaccccgg catcgctcag cggcgccctg caaaccagcc aaccatttcg cgcaggcgcc
     4381 aaggagcgat gctacgccct gctgccggac gaggacgacg aggagcgctt gcatttcagc
     4441 cgctacgaag aagggacctg gagcaaccag gacaacctgt tgcgcaacct cgacctcacc
     4501 tatggcccgc gcatccagac atggggcacg gtcaactatc gggaggagct agtcgcaatg
     4561 gccggccgcg aggacctcga cctgcgtcaa tgcgccatgc gctacggcga ggtgagattg
     4621 cgagaaaaca cccagggatg gagctaccac ccttatttgg ggttcaagaa atacaactga
     4681 taataacatt ggaagtggat aacggatccg cgatcgcggc gcgcccgcca gggttttccc
     4741 agtcgcggcc gctcgcgcac ctggtgccat cgccctgata gacgttaatt aaagtactgg
     4801 ccggccacgc gtctcgagag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     4861 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     4921 gggttttttg ctgaaaggag gaactatatc cgactagtaa cgtttacaat ttcaggtggc
     4981 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5041 atgtatccgc tcatgaatta attcttattt gccgactacc ttggtgatct cgcctttcac
     5101 gtagtggaca aattcttcca actgatctgc gcgcgaggcc aagcgatctt cttcttgtcc
     5161 aagataagcc tgtctagctt caagtatgac gggctgatac tgggccggca ggcgctccat
     5221 tgcccagtcg gcagcgacat ccttcggcgc gattttgccg gttactgcgc tgtaccaaat
     5281 gcgggacaac gtaagcacta catttcgctc atcgccagcc cagtcgggcg gcgagttcca
     5341 tagcgttaag gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag
     5401 ttcctccgcc gctggaccta ccaaggcaac gctatgttct cttgcttttg tcagcaagat
     5461 agccagatca atgtcgatcg tggctggctc gaagatacct gcaagaatgt cattgcgctg
     5521 ccattctcca aattgcagtt cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg
     5581 cacaacaatg gtgacttcta cagcgcggag aatctcgctc tctccagggg aagccgaagt
     5641 ttccaaaagg tcgttgatca aagctcgccg cgttgtttca tcaagcctta cggtcaccgt
     5701 aaccagcaaa tcaatatcac tgtgtggctt caggccgcca tccactgcgg agccgtacaa
     5761 atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg acgccaacta cctctgatag
     5821 ttgagtcgat acttcggcga tcaccgcttc cctcataaca ccccttgtat tactgtttat
     5881 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     5941 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6001 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6061 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6121 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6181 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6241 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6301 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6361 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6421 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6481 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6541 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6601 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6661 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     6721 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     6781 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     6841 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     6901 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6961 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     7021 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     7081 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     7141 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     7201 gggggatttc tgttcatggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     7261 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     7321 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     7381 cagatgtagg tgtt
//
