LOCUS       sequence-304516-        2154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA in vitro transcription plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2154)
  AUTHORS   Lazzarotto CR, Nguyen NT, Tang X, Malagon-Lopez J, Guo JA, Aryee MJ,
            Joung JK, Tsai SQ
  TITLE     Defining CRISPR-Cas9 genome-wide nuclease activities with 
            CIRCLE-seq.
  JOURNAL   Nat Protoc. 2018 Nov;13(11):2615-2642. doi: 
            10.1038/s41596-018-0055-0.
  PUBMED    30341435
REFERENCE   2  (bases 1 to 2154)
  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"; journalName: 
            "Nat Protoc"; date: "2018-11"; volume: "13"; issue: "11"; pages: 
            "2615-2642"
COMMENT     Sequence Label: pCRL1
FEATURES             Location/Qualifiers
     source          1..2154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(58..89)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     primer_bind     137..153
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     137..153
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        329..404
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(586..603)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(586..602)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      643..1230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1131..1150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1242..1269)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1293..2108)
                     /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     2016..2035
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(join(2109..2154,1..57))
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaagt
       61 caaaagcctc cggtcggagg cttttgactt tctgctatgg aggtcaggta tgatttaaat
      121 ggtcagtatt gagcctcagg aaacagctat gacatcaagc tgactagata atctagctga
      181 tcgtggaccg atcatacgta taatgccgta agatcacggg tcgcagcaca gctcgcggtc
      241 cagtagtgat cgacactgct cgatccgctc gcaccgctag ctaatacgac tcactataga
      301 gagacctgcg acagagaccg tggtctcagt tttagagcta gaaatagcaa gttaaaataa
      361 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgcttttaa aaagcttgga
      421 tcgacgagag cagcgcgact ggatctgtcg cccgtctcaa acgcaaccct ccggcggtcg
      481 catatcattc aggacgagcc tcagactcca gcgtaactgg actgcaatca actcactggc
      541 tcaccttccg gtccacgatc agctagaatc aagctgacta gataaactgg ccgtcgtttt
      601 acacgggtgg gcctttcttc ggtagaaaat caaaggatct tcttgagatc ctttttttct
      661 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
      721 ggatcaagag ctaccaactc tttttccgag gtaactggct tcagcagagc gcagatacca
      781 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
      841 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
      901 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      961 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1021 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1081 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1141 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcatcg atttttgtga
     1201 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcagaaag gcccacccga
     1261 aggtgagcca ggtgattaca tttaggtcct cattagaaaa actcatcgag catcaagtga
     1321 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     1381 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     1441 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     1501 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caagagttta
     1561 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     1621 gcaccaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     1681 ccgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     1741 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     1801 cctgggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     1861 gtcggaagag gcataaattc cgtcagccag tttagcctga ccatctcatc tgtaacatca
     1921 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     1981 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     2041 aaatcagcat ccatgttgga atttaatcgc ggcttcaagc aagacgtttc ccgttgaata
     2101 tggctcattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacg
//
