LOCUS       sequence-416370-        7257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strep-TEV-PaCas1 + PaCas2/3 (Cas2 K11D, R12E, R55E, N56D).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7257)
  AUTHORS   Santiago-Frangos A, Henriques WS, Wiegand T, Gauvin CC, Buyukyoruk 
            M, Graham AB, Wilkinson RA, Triem L, Neselu K, Eng ET, Lander GC, 
            Wiedenheft B
  TITLE     Structure reveals why genome folding is necessary for site-specific 
            integration of foreign DNA into CRISPR arrays.
  JOURNAL   Nat Struct Mol Biol. 2023 Nov;30(11):1675-1685. doi: 
            10.1038/s41594-023-01097-2. Epub 2023 Sep 14.
  PUBMED    37710013
REFERENCE   2  (bases 1 to 7257)
  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: 
            "10.1038/s41594-023-01097-2"; journalName: "Nat Struct Mol Biol"; 
            date: "2023-11"; volume: "30"; issue: "11"; pages: "1675-1685"
COMMENT     Sequence Label: pRAW-462
FEATURES             Location/Qualifiers
     source          1..7257
                     /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)"
     CDS             119..142
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             143..163
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     1189..1206
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1190..1206
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             1283..1305
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(4732..4750)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4746..4793
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4928..5719)
                     /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      5841..6429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6330..6349
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6583..6600
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6615..6754
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6680..6699)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6840..6862
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6856..7047)
                     /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 atacatatga aatcttcttg
      121 gagccacccg cagttcgaaa aggaaaacct gtacttccaa tccaatgcag acgacatttc
      181 tcccagcgaa ctgaagacga tccttcactc caagcgtgcc aacctgtact acctgcaaca
      241 ctgccgggta ctggtcaacg gcgggcgggt cgagtacgtc accgacgaag gccggcattc
      301 gcactactgg aacatcccca tcgccaacac caccagcctg ttgctgggca ccggtacctc
      361 cattacccag gcagccatgc gcgaactggc cagggccggg gtattggtag gcttctgcgg
      421 cggcggcgga acgccgctgt tcagcgccaa cgaagtggac gtcgaggtgt cctggctgac
      481 gccgcagagc gaataccgcc ccaccgaata cctgcagcgc tgggtcggct tctggttcga
      541 tgaggaaaaa cgcctggtgg ccgcccggca cttccagcga gccaggctgg agcggattcg
      601 ccacagttgg ctggaagacc gcgtattgcg cgacgccggc ttcgccgtcg atgccaccgc
      661 actggcagtt gccgtggaag attccgcccg agcgttggag caggctccca atcatgagca
      721 tctactgacc gaagaagcgc gcctgagcaa gcgcctgttc aagctcgcag cccaggccac
      781 ccgctacggc gagttcgtcc gcgccaagcg cggcagcggc ggcgacccgg ccaaccgctt
      841 cctcgaccat ggcaactacc tggcctacgg cctcgcggcg accgccacct gggtgctggg
      901 cattccccat ggactggcgg tgctacacgg caagacccgg cgcggaggcc tggtgttcga
      961 tgtggcggac ctgatcaagg actcgctgat cctgccgcag gcatttcttt ccgccatgcg
     1021 cggcgacgag gaacaggact tccgccaggc ctgcctggac aacctgagcc gcgcacaggc
     1081 cctggacttc atgatcgaca cgctgaaaga cgtggcgcag cggagcacgg tgtccgcatg
     1141 ataataacat tggaagtgga taacggatcc gggccctgta caagatcctg taaaacgacg
     1201 gccagtgaat tcactcgagt cccggggcga tcgcagcgga taacaatttc acatcctgca
     1261 ggactcgagt tctagaaata attttgttta actttaagaa ggagatatag atcatgaaca
     1321 tcctgctggt gtcgcaatgc gaagatgaag ccctgagcga aacccgccgc attctcgacc
     1381 agttcgccga gcgccgcggc gaacggacct ggcaaacgcc catcactcaa gccggactgg
     1441 ataccctgcg acgcctgctg aagaaaagcg cacgggagga caccgccgta gcctgtcact
     1501 ggatccgcgg ccgcgaccac agcgaactgc tgtggatcgt cggtgatgcc agccgcttca
     1561 acgcccaggg tgcggtgccg accaacagga cctgccgcga catcctgcgc aaggaagacg
     1621 agaacgactg gcacagcgcc gaggacatcc gcctgctgac ggtgatggca gcgctgttcc
     1681 acgatatcgg caaggccagc caggccttcc aggccaaact gcggaaccgc ggcaaaccga
     1741 tggccgatgc ctatcgtcac gaatgggtat cactgcgcct gttcgaagcc ttcgttggcc
     1801 caggcagcag cgacgaggac tggctgaggc gcctggcgga caagcgagag acgggcgatg
     1861 cctggctgtc gcaactggcc agggacgacc ggcaatccgc gccacccggc ccgttccaga
     1921 aaagccggct accgccgctc gcccaggcgg tcggctggtt gatcgtcagc catcatcgcc
     1981 tgcccaacgg ggaccatcgc ggcagcgcct cgctggcacg cttgccggcc cccatccaga
     2041 gccaatggtg cggcgcacgc gacgcagacg caaaagaaaa ggccgcctgc tggcagttcc
     2101 cccacggcct gcccttcgcc agcgcccatt ggcgcgccag gacagcgcta tgcgcgcaga
     2161 gcatgctcga gcgtcccggc ctgctggctc ggggaccggc cttgttgcat gattcctacg
     2221 tcatgcatgt gtcccgactg atcctgatgc tcgcggacca ccactattcc agtctccctg
     2281 ccgactcccg gctgggcgac ccgaacttcc ccttgcacgc caacaccgac cgggacagcg
     2341 gcaaactaaa gcagcgcctg gacgaacacc tgctcggcgt cgccctgcac agtcgcaagc
     2401 tcgccggcac cctgccacgc ctggagcgac aactaccgcg ccttgcccgg cacaagggct
     2461 tcacccgccg ggtcgagcag ccgcgcttcc gctggcagga caaggcctac gactgcgcga
     2521 tggcctgccg cgagcaggct atggagcatg gattcttcgg cctcaacctg gcgtcgaccg
     2581 gttgcggtaa gaccctcgcc aacggccgta tcctgtatgc gctggccgat ccgcaacgcg
     2641 gcgcgcgttt cagcatcgct ctcggcctgc gcagcttgac cctgcaaacc gggcaggcct
     2701 accgcgagcg gctcggcctg ggcgacgacg acctcgctat cctggtcggc ggcagcgccg
     2761 cccgcgaact gttcgaaaag cagcaggagc gcctggagcg cagcggtagc gagtcagccc
     2821 aggagctgct ggcggaaaac agccatgtac acttcgccgg cacgctcgag gacggccctc
     2881 tacgcgagtg gctcggcagg aacagcgcgg gaaaccgcct actccaggcg cccatcctgg
     2941 cctgcaccat cgaccacctg atgcccgcca gcgaaagcct gcgcggcgga caccagatag
     3001 cgccactgct ccgcctgatg acttccgacc tggtgctcga cgaggtcgac gacttcgata
     3061 tcgacgacct gcccgccctg tcgcggctgg tgcactgggc cggcctgttc ggcagccgcg
     3121 tgctgctctc ctccgcgacc ctgccgccgg ccttggtgca gggcctgttc gaggcctatc
     3181 gcagcggccg ggaaatcttc cagcgccatc gtggcgctcc cggacgcgct acggaaatcc
     3241 gctgtgcctg gttcgacgag ttctccagcc aatccagcgc ccacggcgcc gtaacctcct
     3301 tcagcgaagc gcatgcgacc ttcgtcgccc agcgtctggc gaagctcgag caactgccgc
     3361 cacgtcgcca ggcgcagcta tgcaccgtgc atgccgctgg cgaggcccgt cccgcgctgt
     3421 gccgcgagtt ggccgggcag atgaatacct ggatggctga cctgcatcgc tgccatcaca
     3481 ccgaacacca aggacgtcgc atcagtttcg gcctgctacg gctggccaac atcgaacccc
     3541 tgatcgaact ggcccaggcc atcctcgccc agggtgcgcc cgaggggttg catgtccatc
     3601 tgtgtgtcta ccattcgcgg catccccttc tggtccgctc ggccatcgag cgacaactcg
     3661 atgaactgct gaagcgttcg gacgacgacg ccgccgcgct gttcgctcgt ccgacgctgg
     3721 ccaaggcgct ccaggccagc acggagcggg atcatctgtt cgtcgtactc gcctcgccgg
     3781 tggcggaggt cggtcgcgac cacgattacg actgggccat cgtcgaaccc tcctccatgc
     3841 gctcgatcat ccagttggcc gggcgaatcc gccgccatcg ctccggcttc agcggcgagg
     3901 ccaacctata cctgctatcg cgcaatatcc gctcgctgga agggcagaat ccggcgttcc
     3961 agcggcccgg cttcgagacc cccgacttcc ctcttgacag ccacgacctg cacgacctgc
     4021 tcgaccccgc cctactcgcc cgcatcgacg ccagcccacg aatcgtcgaa ccgttcccac
     4081 tgttcccacg cagccggttg gtcgacctgg aacaccgacg cctgcgcgcg ctgatgcttg
     4141 ccgacgaccc accgtcgtcc ctgctcggcg taccgctctg gtggcaaacc ccggcatcgc
     4201 tcagcggcgc cctgcaaacc agccaaccat ttcgcgcagg cgccaaggag cgatgctacg
     4261 ccctgctgcc ggacgaggac gacgaggagc gcttgcattt cagccgctac gaagaaggga
     4321 cctggagcaa ccaggacaac ctgttgcgca acctcgacct cacctatggc ccgcgcatcc
     4381 agacatgggg cacggtcaac tatcgggagg agctagtcgc aatggccggc cgcgaggacc
     4441 tcgacctgcg tcaatgcgcc atgcgctacg gcgaggtgag attgcgagaa aacacccagg
     4501 gatggagcta ccacccttat ttggggttca agaaatacaa ctgataataa gatcccaact
     4561 ccataaggat ccgcgatcgc ggcgcgcccg ccagggtttt cccagtcgcg gccgctcgcg
     4621 cacctggtgc catcgccctg atagacgtta attaaagtac tggccggcca cgcgtctcga
     4681 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     4741 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     4801 gaggaactat atccgactag taacgtttac aatttcaggt ggcacttttc ggggaaatgt
     4861 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     4921 ttaattctta tttgccgact accttggtga tctcgccttt cacgtagtgg acaaattctt
     4981 ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa gcctgtctag
     5041 cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga
     5101 catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca
     5161 ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat
     5221 ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac
     5281 ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga
     5341 tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca
     5401 gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt
     5461 ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga
     5521 tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat
     5581 cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg
     5641 tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg
     5701 cgatcaccgc ttccctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     5761 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5821 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5881 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5941 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     6001 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6061 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6121 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6181 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6241 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6301 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6361 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6421 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6481 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     6541 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     6601 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     6661 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     6721 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     6781 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     6841 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     6901 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     6961 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     7021 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     7081 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     7141 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     7201 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgtt
//
