LOCUS       sequence-333509-        2144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 promoter expression plasmid for in vitro transcription of SpCas9 
            sgRNA with spacer #1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2144)
  AUTHORS   Walton RT, Hsu JY, Joung JK, Kleinstiver BP
  TITLE     Scalable characterization of the PAM requirements of CRISPR-Cas 
            enzymes using HT-PAMDA.
  JOURNAL   Nat Protoc. 2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi: 
            10.1038/s41596-020-00465-2.
  PUBMED    33547443
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"; journalName: "Nat Protoc.
            2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi: 
            10.1038/s41596-020-00465-2."
COMMENT     Sequence Label: pT7-SpCas9_sgRNA-site1 (RTW443)
FEATURES             Location/Qualifiers
     source          1..2144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..42
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     26..42
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     171..190
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        171..189
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        208..283
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(465..482)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(465..481)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      522..1109
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1010..1029
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1121..1148)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1172..1987)
                     /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     1895..1914
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1988..2090)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(2091..2122)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 gatttaaatg gtcagtattg agcctcagga aacagctatg acatcaagct gactagataa
       61 tctagctgat cgtggaccga tcatacgtat aatgccgtaa gatcacgggt cgcagcacag
      121 ctcgcggtcc agtagtgatc gacactgctc gatccgctcg caccgctagc taatacgact
      181 cactataggg cacgggcagc ttgccgggtt ttagagctag aaatagcaag ttaaaataag
      241 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttaaa aagcttggat
      301 cgacgagagc agcgcgactg gatctgtcgc ccgtctcaaa cgcaaccctc cggcggtcgc
      361 atatcattca ggacgagcct cagactccag cgtaactgga ctgcaatcaa ctcactggct
      421 caccttccgg tccacgatca gctagaatca agctgactag ataaactggc cgtcgtttta
      481 cacgggtggg cctttcttcg gtagaaaatc aaaggatctt cttgagatcc tttttttctg
      541 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
      601 gatcaagagc taccaactct ttttccgagg taactggctt cagcagagcg cagataccaa
      661 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
      721 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
      781 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
      841 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
      901 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      961 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     1021 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcatcga tttttgtgat
     1081 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcagaaagg cccacccgaa
     1141 ggtgagccag gtgattacat ttaggtcctc attagaaaaa ctcatcgagc atcaagtgaa
     1201 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1261 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1321 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1381 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aagagtttat
     1441 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     1501 caccaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     1561 cgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     1621 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     1681 ctgggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     1741 tcggaagagg cataaattcc gtcagccagt ttagcctgac catctcatct gtaacatcat
     1801 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     1861 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     1921 aatcagcatc catgttggaa tttaatcgcg gcttcaagca agacgtttcc cgttgaatat
     1981 ggctcatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
     2041 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca agtcaaaagc
     2101 ctccggtcgg aggcttttga ctttctgcta tggaggtcag gtat
//
