LOCUS       sequence-98933-e        2144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for generation of a eGFP specific sgRNA from a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2144)
  AUTHORS   Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F
  TITLE     Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by 
            homology-independent DNA repair.
  JOURNAL   Genome Res. 2014 Jan;24(1):142-53. doi: 10.1101/gr.161638.113. Epub 
            2013 Oct 31.
  PUBMED    24179142
REFERENCE   2  (bases 1 to 2144)
  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: 
            "10.1101/gr.161638.113"; journalName: "Genome Res"; date: "2014-01";
            volume: "24"; issue: "1"; pages: "142-53"
COMMENT     Sequence Label: DR274-eGFP sgRNA
FEATURES             Location/Qualifiers
     source          1..2144
                     /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(298..316)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(445..461)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(445..461)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      509..540
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        541..643
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             644..1459
                     /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(717..736)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1483..1510
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(1522..2109)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1602..1621)
                     /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 aggtggcatc gccctcgcct
      301 atagtgagtc gtattagcta gcggtgcgag cggatcgagc agtgtcgatc actactggac
      361 cgcgagctgt gctgcgaccc gtgatcttac ggcattatac gtatgatcgg tccacgatca
      421 gctagattat ctagtcagct tgatgtcata gctgtttcct gaggctcaat actgaccatt
      481 taaatcatac ctgacctcca tagcagaaag tcaaaagcct ccgaccggag gcttttgact
      541 tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt
      601 attttttgag ttatcgagat tttcaggagc taaggaagct aaaatgagcc atattcaacg
      661 ggaaacgtct tgcttgaagc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
      721 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
      781 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
      841 agatgagatg gtcaggctaa actggctgac ggaatttatg cctcttccga ccatcaagca
      901 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatcccag ggaaaacagc
      961 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     1021 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaacg gcgatcgcgt
     1081 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttggtg cgagtgattt
     1141 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactctt
     1201 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     1261 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     1321 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     1381 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcacttgat
     1441 gctcgatgag tttttctaat gaggacctaa atgtaatcac ctggctcacc ttcgggtggg
     1501 cctttctgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1561 tcgatgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1621 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1681 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1741 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1801 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1861 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1921 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     1981 cgctctgctg aagccagtta cctcggaaaa agagttggta gctcttgatc cggcaaacaa
     2041 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2101 ggatctcaag aagatccttt gattttctac cgaagaaagg ccca
//
