LOCUS       sequence-107709-        2987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses sgRNA(tyr) under U6a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2987)
  AUTHORS   Yin L, Maddison LA, Li M, Kara N, LaFave MC, Varshney GK, Burgess 
            SM, Patton JG, Chen W
  TITLE     Multiplex Conditional Mutagenesis Using Transgenic Expression of 
            Cas9 and sgRNAs.
  JOURNAL   Genetics. 2015 Apr 8. pii: genetics.115.176917.
  PUBMED    25855067
REFERENCE   2  (bases 1 to 2987)
  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: "Genetics. 
            2015 Apr 8. pii: genetics.115.176917."
COMMENT     Sequence Label: pU6a:sgRNA(tyr)
FEATURES             Location/Qualifiers
     source          1..2987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        465..540
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             917..1708
                     /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      1801..2389
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2290..2309
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2543..2560
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2719..2746)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2838..2924)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(2973..2987,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(2987,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc ccggtctctc agttttctcc agcctcggtc
       61 attcagtaag acgagtttcc tgctctcggc gtcttttgtt ctggtcttca aggagggggg
      121 aaatgttctg cgcatgcctg tgggggaggg agaaggacac gtcactgaaa acgtccctgc
      181 atcacaccga gacacccaat cactcaagcc aagaccagat aattttgcat atgctttaca
      241 gtttgaaaat taccacggta atccctcaca caaactctgg atttgagatc tttcaggttt
      301 tatcagtttg caggtttatg tcaccatgat atagggtcag acttgatcta agggagctga
      361 ataagtggtt tagtcactca ccacctccca aaaacatacc cagaagtccc tggtatatat
      421 agctctccct ccagctcttg gctcggactg gaggacttct gggggtttta gagctagaaa
      481 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
      541 ttttttagtc agagacctct agaccagcca ggacagaaat gcctcgactt cgctgctacc
      601 caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc agtggacata
      661 agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag
      721 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
      781 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt
      841 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa
      901 aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga
      961 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg
     1021 ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac
     1081 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc
     1141 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga
     1201 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa
     1261 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct
     1321 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga
     1381 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc
     1441 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta
     1501 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg
     1561 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga
     1621 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg
     1681 cgagatcacc aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa aatcccttaa
     1741 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1801 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1861 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1921 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     1981 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2041 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2101 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2161 ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg
     2221 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2281 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2341 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2401 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2461 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2521 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     2581 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     2641 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctagccagga
     2701 agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc
     2761 ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc
     2821 aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     2881 aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc
     2941 tactctcgcg ttaacgctag catggatgtt ttcccagtca cgacgtt
//
