LOCUS       pT7-SpCas9_sgRNA-site2 (RTW448)            2144 bp ds-DNA     circular SYN 25-MAY-2021
DEFINITION  T7 promoter expression plasmid for in vitro transcription of SpCas9 
            sgRNA with spacer #2.
ACCESSION   .
VERSION     .
KEYWORDS    pT7-SpCas9_sgRNA-site2 (RTW448)
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 May 25, 2021 from SnapGene Server 1.1.58
            //www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..2144
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     20..36
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     20..36
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        165..182
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        202..277
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(459..476)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(459..475)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      516..1103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1004..1023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      1115..1142
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1166..1981)
                     /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     1889..1908
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1982..2084)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      2085..2116
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 aatggtcagt attgagcctc aggaaacagc tatgacatca agctgactag ataatctagc
       61 tgatcgtgga ccgatcatac gtataatgcc gtaagatcac gggtcgcagc acagctcgcg
      121 gtccagtagt gatcgacact gctcgatccg ctcgcaccgc tagctaatac gactcactat
      181 agtcgccctc gaacttcacc tgttttagag ctagaaatag caagttaaaa taaggctagt
      241 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt taaaaagctt ggatcgacga
      301 gagcagcgcg actggatctg tcgcccgtct caaacgcaac cctccggcgg tcgcatatca
      361 ttcaggacga gcctcagact ccagcgtaac tggactgcaa tcaactcact ggctcacctt
      421 ccggtccacg atcagctaga atcaagctga ctagataaac tggccgtcgt tttacacggg
      481 tgggcctttc ttcggtagaa aatcaaagga tcttcttgag atcctttttt tctgcgcgta
      541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
      601 gagctaccaa ctctttttcc gaggtaactg gcttcagcag agcgcagata ccaaatactg
      661 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
      721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
      781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
      841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
      901 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
      961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     1021 tttatagtcc tgtcgggttt cgccacctct gacttgagca tcgatttttg tgatgctcgt
     1081 caggggggcg gagcctatgg aaaaacgcca gcaacgcaga aaggcccacc cgaaggtgag
     1141 ccaggtgatt acatttaggt cctcattaga aaaactcatc gagcatcaag tgaaactgca
     1201 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1261 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     1321 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1381 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaagagt ttatgcattt
     1441 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcaccaa
     1501 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgccgttaa
     1561 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1621 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttccctggga
     1681 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1741 gaggcataaa ttccgtcagc cagtttagcc tgaccatctc atctgtaaca tcattggcaa
     1801 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     1861 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     1921 catccatgtt ggaatttaat cgcggcttca agcaagacgt ttcccgttga atatggctca
     1981 ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc
     2041 ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaagtcaa aagcctccgg
     2101 tcggaggctt ttgactttct gctatggagg tcaggtatga ttta
//