LOCUS       sequence-416368-        7257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strep-TEV-PaCas1 + PaCas2/3 (Cas2 K11D, R12E).
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-461
FEATURES             Location/Qualifiers
     source          1..7257
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2470..2488)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2484..2531
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2666..3457)
                     /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      3579..4167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4068..4087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4321..4338
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4353..4492
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4418..4437)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4578..4600
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4594..4785)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5022..5041
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5022..5040
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             5072..5094
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5114..5137
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5138..5158
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     6184..6201
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6185..6201
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             6278..6300
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 actattccag tctccctgcc gactcccggc tgggcgaccc gaacttcccc ttgcacgcca
       61 acaccgaccg ggacagcggc aaactaaagc agcgcctgga cgaacacctg ctcggcgtcg
      121 ccctgcacag tcgcaagctc gccggcaccc tgccacgcct ggagcgacaa ctaccgcgcc
      181 ttgcccggca caagggcttc acccgccggg tcgagcagcc gcgcttccgc tggcaggaca
      241 aggcctacga ctgcgcgatg gcctgccgcg agcaggctat ggagcatgga ttcttcggcc
      301 tcaacctggc gtcgaccggt tgcggtaaga ccctcgccaa cggccgtatc ctgtatgcgc
      361 tggccgatcc gcaacgcggc gcgcgtttca gcatcgctct cggcctgcgc agcttgaccc
      421 tgcaaaccgg gcaggcctac cgcgagcggc tcggcctggg cgacgacgac ctcgctatcc
      481 tggtcggcgg cagcgccgcc cgcgaactgt tcgaaaagca gcaggagcgc ctggagcgca
      541 gcggtagcga gtcagcccag gagctgctgg cggaaaacag ccatgtacac ttcgccggca
      601 cgctcgagga cggccctcta cgcgagtggc tcggcaggaa cagcgcggga aaccgcctac
      661 tccaggcgcc catcctggcc tgcaccatcg accacctgat gcccgccagc gaaagcctgc
      721 gcggcggaca ccagatagcg ccactgctcc gcctgatgac ttccgacctg gtgctcgacg
      781 aggtcgacga cttcgatatc gacgacctgc ccgccctgtc gcggctggtg cactgggccg
      841 gcctgttcgg cagccgcgtg ctgctctcct ccgcgaccct gccgccggcc ttggtgcagg
      901 gcctgttcga ggcctatcgc agcggccggg aaatcttcca gcgccatcgt ggcgctcccg
      961 gacgcgctac ggaaatccgc tgtgcctggt tcgacgagtt ctccagccaa tccagcgccc
     1021 acggcgccgt aacctccttc agcgaagcgc atgcgacctt cgtcgcccag cgtctggcga
     1081 agctcgagca actgccgcca cgtcgccagg cgcagctatg caccgtgcat gccgctggcg
     1141 aggcccgtcc cgcgctgtgc cgcgagttgg ccgggcagat gaatacctgg atggctgacc
     1201 tgcatcgctg ccatcacacc gaacaccaag gacgtcgcat cagtttcggc ctgctacggc
     1261 tggccaacat cgaacccctg atcgaactgg cccaggccat cctcgcccag ggtgcgcccg
     1321 aggggttgca tgtccatctg tgtgtctacc attcgcggca tccccttctg gtccgctcgg
     1381 ccatcgagcg acaactcgat gaactgctga agcgttcgga cgacgacgcc gccgcgctgt
     1441 tcgctcgtcc gacgctggcc aaggcgctcc aggccagcac ggagcgggat catctgttcg
     1501 tcgtactcgc ctcgccggtg gcggaggtcg gtcgcgacca cgattacgac tgggccatcg
     1561 tcgaaccctc ctccatgcgc tcgatcatcc agttggccgg gcgaatccgc cgccatcgct
     1621 ccggcttcag cggcgaggcc aacctatacc tgctatcgcg caatatccgc tcgctggaag
     1681 ggcagaatcc ggcgttccag cggcccggct tcgagacccc cgacttccct cttgacagcc
     1741 acgacctgca cgacctgctc gaccccgccc tactcgcccg catcgacgcc agcccacgaa
     1801 tcgtcgaacc gttcccactg ttcccacgca gccggttggt cgacctggaa caccgacgcc
     1861 tgcgcgcgct gatgcttgcc gacgacccac cgtcgtccct gctcggcgta ccgctctggt
     1921 ggcaaacccc ggcatcgctc agcggcgccc tgcaaaccag ccaaccattt cgcgcaggcg
     1981 ccaaggagcg atgctacgcc ctgctgccgg acgaggacga cgaggagcgc ttgcatttca
     2041 gccgctacga agaagggacc tggagcaacc aggacaacct gttgcgcaac ctcgacctca
     2101 cctatggccc gcgcatccag acatggggca cggtcaacta tcgggaggag ctagtcgcaa
     2161 tggccggccg cgaggacctc gacctgcgtc aatgcgccat gcgctacggc gaggtgagat
     2221 tgcgagaaaa cacccaggga tggagctacc acccttattt ggggttcaag aaatacaact
     2281 gataataaga tcccaactcc ataaggatcc gcgatcgcgg cgcgcccgcc agggttttcc
     2341 cagtcgcggc cgctcgcgca cctggtgcca tcgccctgat agacgttaat taaagtactg
     2401 gccggccacg cgtctcgaga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     2461 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     2521 ggggtttttt gctgaaagga ggaactatat ccgactagta acgtttacaa tttcaggtgg
     2581 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2641 tatgtatccg ctcatgaatt aattcttatt tgccgactac cttggtgatc tcgcctttca
     2701 cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct tcttcttgtc
     2761 caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca
     2821 ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa
     2881 tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc
     2941 atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga
     3001 gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga
     3061 tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct
     3121 gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt
     3181 gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag
     3241 tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg
     3301 taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca
     3361 aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata
     3421 gttgagtcga tacttcggcg atcaccgctt ccctcataac accccttgta ttactgttta
     3481 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3541 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3601 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3661 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3721 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3781 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3841 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3901 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3961 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4021 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4081 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4141 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4201 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4261 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4321 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4381 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     4441 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4501 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4561 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4621 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     4681 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     4741 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     4801 agggggattt ctgttcatgg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4861 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4921 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4981 acagatgtag gtgttggcac catgcatcga tcccgcgaaa ttaatacgac tcactatagg
     5041 gagaccacaa cggtttccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
     5101 tatgaaatct tcttggagcc acccgcagtt cgaaaaggaa aacctgtact tccaatccaa
     5161 tgcagacgac atttctccca gcgaactgaa gacgatcctt cactccaagc gtgccaacct
     5221 gtactacctg caacactgcc gggtactggt caacggcggg cgggtcgagt acgtcaccga
     5281 cgaaggccgg cattcgcact actggaacat ccccatcgcc aacaccacca gcctgttgct
     5341 gggcaccggt acctccatta cccaggcagc catgcgcgaa ctggccaggg ccggggtatt
     5401 ggtaggcttc tgcggcggcg gcggaacgcc gctgttcagc gccaacgaag tggacgtcga
     5461 ggtgtcctgg ctgacgccgc agagcgaata ccgccccacc gaatacctgc agcgctgggt
     5521 cggcttctgg ttcgatgagg aaaaacgcct ggtggccgcc cggcacttcc agcgagccag
     5581 gctggagcgg attcgccaca gttggctgga agaccgcgta ttgcgcgacg ccggcttcgc
     5641 cgtcgatgcc accgcactgg cagttgccgt ggaagattcc gcccgagcgt tggagcaggc
     5701 tcccaatcat gagcatctac tgaccgaaga agcgcgcctg agcaagcgcc tgttcaagct
     5761 cgcagcccag gccacccgct acggcgagtt cgtccgcgcc aagcgcggca gcggcggcga
     5821 cccggccaac cgcttcctcg accatggcaa ctacctggcc tacggcctcg cggcgaccgc
     5881 cacctgggtg ctgggcattc cccatggact ggcggtgcta cacggcaaga cccggcgcgg
     5941 aggcctggtg ttcgatgtgg cggacctgat caaggactcg ctgatcctgc cgcaggcatt
     6001 tctttccgcc atgcgcggcg acgaggaaca ggacttccgc caggcctgcc tggacaacct
     6061 gagccgcgca caggccctgg acttcatgat cgacacgctg aaagacgtgg cgcagcggag
     6121 cacggtgtcc gcatgataat aacattggaa gtggataacg gatccgggcc ctgtacaaga
     6181 tcctgtaaaa cgacggccag tgaattcact cgagtcccgg ggcgatcgca gcggataaca
     6241 atttcacatc ctgcaggact cgagttctag aaataatttt gtttaacttt aagaaggaga
     6301 tatagatcat gaacatcctg ctggtgtcgc aatgcgaaga tgaagccctg agcgaaaccc
     6361 gccgcattct cgaccagttc gccgagcgcc gcggcgaacg gacctggcaa acgcccatca
     6421 ctcaagccgg actggatacc ctgcgacgcc tgctgaagaa aagcgcacgg cgcaacaccg
     6481 ccgtagcctg tcactggatc cgcggccgcg accacagcga actgctgtgg atcgtcggtg
     6541 atgccagccg cttcaacgcc cagggtgcgg tgccgaccaa caggacctgc cgcgacatcc
     6601 tgcgcaagga agacgagaac gactggcaca gcgccgagga catccgcctg ctgacggtga
     6661 tggcagcgct gttccacgat atcggcaagg ccagccaggc cttccaggcc aaactgcgga
     6721 accgcggcaa accgatggcc gatgcctatc gtcacgaatg ggtatcactg cgcctgttcg
     6781 aagccttcgt tggcccaggc agcagcgacg aggactggct gaggcgcctg gcggacaagc
     6841 gagagacggg cgatgcctgg ctgtcgcaac tggccaggga cgaccggcaa tccgcgccac
     6901 ccggcccgtt ccagaaaagc cggctaccgc cgctcgccca ggcggtcggc tggttgatcg
     6961 tcagccatca tcgcctgccc aacggggacc atcgcggcag cgcctcgctg gcacgcttgc
     7021 cggcccccat ccagagccaa tggtgcggcg cacgcgacgc agacgcaaaa gaaaaggccg
     7081 cctgctggca gttcccccac ggcctgccct tcgccagcgc ccattggcgc gccaggacag
     7141 cgctatgcgc gcagagcatg ctcgagcgtc ccggcctgct ggctcgggga ccggccttgt
     7201 tgcatgattc ctacgtcatg catgtgtccc gactgatcct gatgctcgcg gaccacc
//
