LOCUS       sequence-429179-        5453 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses T7-RNAP, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5453)
  AUTHORS   Qi T, Wang Y, Yang Y, Gao S, Liu J, Huang Q, Tian Y, Tang J, Zheng 
            WV, Wang Y
  TITLE     Phage-assisted evolution of compact Cas9 variants targeting a simple
            NNG PAM.
  JOURNAL   Nat Chem Biol. 2023 Dec 5. doi: 10.1038/s41589-023-01481-5.
  PUBMED    38052959
REFERENCE   2  (bases 1 to 5453)
  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: "Nat Chem 
            Biol. 2023 Dec 5. doi: 10.1038/s41589-023-01481-5."
COMMENT     Sequence Label: pMR-A-3-Sasg
FEATURES             Location/Qualifiers
     source          1..5453
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(804..1595)
                     /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(1596..1667)
                     /gene="bla"
                     /label=AmpR promoter
     misc_RNA        complement(1727..1802)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(1824..1858)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     rep_origin      1960..2548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2449..2468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2702..2719
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2734..2874
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2800..2819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2960..2982
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2976..3167)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ggtccaaaga gttcggcttc cgtcaacaag tgctggaaga taccattcag ccagctattg
       61 attccggcaa gggtctgatg ttcactcagc cgaatcaggc tgctggatac atggctaagc
      121 tgatttggga atctgtgagc gtgacggtgg tagctgcggt tgaagcaatg aactggctta
      181 agtctgctgc taagctgctg gctgctgagg tcaaagataa gaagactgga gagattcttc
      241 gcaagcgttg cgctgtgcat tgggtaactc ctgatggttt ccctgtgtgg caggaataca
      301 agaagcctat tcagacgcgc ttgaacctga tgttcctcgg tcagttccgc ttacagccta
      361 ccattaacac caacaaagat agcgagattg atgcacacaa acaggagtct ggtatcgctc
      421 ctaactttgt acacagccaa gacggtagcc accttcgtaa gactgtagtg tgggcacacg
      481 agaagtacgg tatcgaatct tttgcactga ttcacgactc cttcggtacc attccggctg
      541 acgctgcgaa cctgttcaaa gcagtgcgcg aaactatggt tgacacatat gagtcttgtg
      601 atgtactggc tgatttctac gaccagttcg ctgaccagtt gcacgagtct caattggaca
      661 aaatgccagc acttccggct aaaggtaact tgaacctccg tgacatctta gagtccgact
      721 tcgcgttcgc ctaatggcac tcctcagcaa atataatgac cctcttgata acccaagagg
      781 gcatttttta atgcccatgg cgtttatttg ccgactacct tggtgatctc gcctttcacg
      841 tagtggacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc ttcttgtcca
      901 agataagcct gtctagcttc aagtatgacg ggctgatact gggccggcag gcgctccatt
      961 gcccagtcgg cagcgacatc cttcggcgcg attttgccgg ttactgcgct gtaccaaatg
     1021 cgggacaacg taagcactac atttcgctca tcgccagccc agtcgggcgg cgagttccat
     1081 agcgttaagg tttcatttag cgcctcaaat agatcctgtt caggaaccgg atcaaagagt
     1141 tcctccgccg ctggacctac caaggcaacg ctatgttctc ttgcttttgt cagcaagata
     1201 gccagatcaa tgtcgatcgt ggctggctcg aagatacctg caagaatgtc attgcgctgc
     1261 cattctccaa attgcagttc gcgcttagct ggataacgcc acggaatgat gtcgtcgtgc
     1321 acaacaatgg tgacttctac agcgcggaga atctcgctct ctccagggga agccgaagtt
     1381 tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat caagccttac ggtcaccgta
     1441 accagcaaat caatatcact gtgtggcttc aggccgccat ccactgcgga gccgtacaaa
     1501 tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac ctctgatagt
     1561 tgagtcgata cttcggcgat caccgcttcc ctcatactct tcctttttca atattattga
     1621 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgagg aagaggacat
     1681 ccggtgcgaa aaaaccccgc cgaagcgggg ttttttgcga aaaaaatctc gccaacaagt
     1741 tgacgagata aacacggcat tttgccttgt tttagtagat tctgtttcca gagtactaaa
     1801 acaagatgtt tgagtgataa cttactagta ttatacctag gactgagcta gctgtcaaac
     1861 tctgctagca agtaaggccg actagcgaaa aaaccccgcc gaagcggggt tttttgcgac
     1921 atgcccatgg cgtatttaga aaaataggcc aaatagggtt tgagatcctt tttttctgcg
     1981 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2041 tcaagagcta ccacctcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2101 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2161 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2221 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2281 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2341 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2401 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2461 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2521 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2581 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2641 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2701 cagcgagtca gtgagcgagg aagcggatga gcgcctgatg cggtattttc tccttacgca
     2761 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     2821 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     2881 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2941 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3001 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3061 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3121 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3181 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3241 atacgggtta ctgatgatga acaagaggac atccggtcaa ataaaacgaa aggctcagtc
     3301 gaaagactgg gcctttcgtt ttgctgagga gacttaggga ctctcacagc taacaccacg
     3361 tcgtccctat ctgctgccct aggtctatga gtggttgctg gataacttta cgggcatgca
     3421 taaggctcgt ataatatatt cagggagacc acaacggttt ccctctacaa ataattttgt
     3481 ttaactttta ctagagtggg accctacctg caggtgcagt aaggaggaaa aaaaaatgaa
     3541 cacgattaac atcgctaaga acgacttctc tgacatcgaa ctggctgcta tcccgttcaa
     3601 cactctggct gaccattacg gtgagcgttt agctcgcgaa cagttggccc ttgagcatga
     3661 gtcttacgag atgggtgaag cacgcttccg caagatgttt gagtgataac ttaaagctgg
     3721 tgaggttgcg gataacgctg ccgccaagcc tctcatcact accctactcc ctaagatgat
     3781 tgcacgcatc aacgactggt ttgaggaagt gaaagctaag cgcggcaagc gcccgacagc
     3841 cttccagttc ctgcaagaaa tcaagccgga agccgtagcg tacatcacca ttaagaccac
     3901 tctggcttgc ctaaccagtg ctgacaatac aaccgttcag gctgtagcaa gcgcaatcgg
     3961 tcgggccatt gaggacgagg ctcgcttcgg tcgtatccgt gaccttaaag ctaagcactt
     4021 caagaaaaac gttgaggaac aactcaacaa gcgcgtaggg cacgtctaca agaaagcatt
     4081 tatgcaagtt gtcgaggctg acatgctctc taagggtcta ctcggtggcg aggcgtggtc
     4141 ttcgtggcat aaggaagact ctattcatgt aggagtacgc tgcatcgaga tgctcattga
     4201 gtcaaccgga atggttagct tacaccgcca aaatgctggc gtagtaggtc aagactctga
     4261 gactatcgaa ctcgcacctg aatacgctga ggctatcgca acccgtgcag gtgcgctggc
     4321 tggcatctct ccgatgttcc aaccttgcgt agttcctcct aagccgtgga ctggcattac
     4381 tggtggtggc tattgggcta acggtcgtcg tcctctggcg ctggtgcgta ctcacagtaa
     4441 gaaagcactg atgcgctacg aagacgttta catgcctgag gtgtacaaag cgattaacat
     4501 tgcgcaaaac accgcatgga aaatcaacaa gaaagtccta gcggtcgcca acgtaatcac
     4561 caagtggaag cattgtccgg tcgaggacat ccctgcgatt gagcgtgaag aactcccgat
     4621 gaaaccggaa gacatcgaca tgaatcctga ggctctcacc gcgtggaaac gtgctgccgc
     4681 tgctgtgtac cgcaaggaca aggctcgcaa gtctcgccgt atcagccttg agttcatgct
     4741 tgagcaagcc aataagtttg ctaaccataa ggccatctgg ttcccttaca acatggactg
     4801 gcgcggtcgt gtttacgctg tgtcaatgtt caacccgcaa ggtaacgata tgaccaaagg
     4861 actgcttacg ctggcgaaag gtaaaccaat cggtaaggaa ggttactact ggctgaaaat
     4921 ccacggtgca aactgtgcgg gtgtcgataa ggttccgttc cctgagcgca tcaagttcat
     4981 tgaggaaaac cacgagaaca tcatggcttg cgctaagtct ccactggaga acacttggtg
     5041 ggctgagcaa gattctccgt tctgcttcct tgcgttctgc tttgagtacg ctggggtaca
     5101 gcaccacggc ctgagctata actgctccct tccgctggcg tttgacgggt cttgctctgg
     5161 catccagcac ttctccgcga tgctccgaga tgaggtaggt ggtcgcgcgg ttaacttgct
     5221 tcctagtgaa accgttcagg acatctacgg gattgttgct aagaaagtca acgagattct
     5281 acaagcagac gcaatcaatg ggaccgataa cgaagtagtt accgtgaccg atgagaacac
     5341 tggtgaaatc tctgagaaag tcaagctggg cactaaggca ctggctggtc aatggctggc
     5401 ttacggtgtt actcgcagtg tgactaagcg ttcagtcatg acgctggctt acg
//
