LOCUS       sequence-57048-e        2147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  guide RNA (gRNA) expression vector used to create a gRNA to a 
            specific sequence; uses T7 promoter for in vitro transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2147)
  AUTHORS   Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT,
            Yeh JR, Joung JK
  TITLE     Efficient genome editing in zebrafish using a CRISPR-Cas system.
  JOURNAL   Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2501.
  PUBMED    23360964
REFERENCE   2  (bases 1 to 2147)
  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 
            Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2501."
COMMENT     Sequence Label: DR274
FEATURES             Location/Qualifiers
     source          1..2147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6..22
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(204..279)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(301..319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(448..464)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(448..464)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      512..543
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        544..646
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             647..1462
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(720..739)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1486..1513
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(1525..2112)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1605..1624)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccgtgtaaa acgacggcca gtttatctag tcagcttgat tctagctgat cgtggaccgg
       61 aaggtgagcc agtgagttga ttgcagtcca gttacgctgg agtctgaggc tcgtcctgaa
      121 tgatatgcga ccgccggagg gttgcgtttg agacgggcga cagatccagt cgcgctgctc
      181 tcgtcgatcc aagcttttta aaagcaccga ctcggtgcca ctttttcaag ttgataacgg
      241 actagcctta ttttaacttg ctatttctag ctctaaaact gagaccctct ctcggtctct
      301 cctatagtga gtcgtattag ctagcggtgc gagcggatcg agcagtgtcg atcactactg
      361 gaccgcgagc tgtgctgcga cccgtgatct tacggcatta tacgtatgat cggtccacga
      421 tcagctagat tatctagtca gcttgatgtc atagctgttt cctgaggctc aatactgacc
      481 atttaaatca tacctgacct ccatagcaga aagtcaaaag cctccgaccg gaggcttttg
      541 acttgatcgg cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg
      601 cgtatttttt gagttatcga gattttcagg agctaaggaa gctaaaatga gccatattca
      661 acgggaaacg tcttgcttga agccgcgatt aaattccaac atggatgctg atttatatgg
      721 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
      781 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
      841 tacagatgag atggtcaggc taaactggct gacggaattt atgcctcttc cgaccatcaa
      901 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc cagggaaaac
      961 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     1021 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acggcgatcg
     1081 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg gtgcgagtga
     1141 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     1201 cttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     1261 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     1321 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     1381 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcactt
     1441 gatgctcgat gagtttttct aatgaggacc taaatgtaat cacctggctc accttcgggt
     1501 gggcctttct gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1561 aaatcgatgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1621 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1681 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     1741 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1801 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1861 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1921 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     1981 ctgcgctctg ctgaagccag ttacctcgga aaaagagttg gtagctcttg atccggcaaa
     2041 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2101 aaaggatctc aagaagatcc tttgattttc taccgaagaa aggccca
//
