LOCUS       40924_45193        2792 bp DNA     circular SYN 13-MAY-2021
DEFINITION  For in vitro transcription of sgRNA from the T7 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2792)
  AUTHORS   Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins 
            A, Iyer V, Huang X, Skarnes WC
  TITLE     Efficient genome modification by CRISPR-Cas9 nickase with minimal 
            off-target effects.
  JOURNAL   Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857.
  PUBMED    24584192
REFERENCE   2  (bases 1 to 2792)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2792)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     191..207
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        254..272
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        294..369
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(427..443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(451..467)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(475..505)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(520..541)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(659..676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(830..1418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1592..2404)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(2405..2509)
                     /label=AmpR promoter
     primer_bind     2577..2595
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     primer_bind     complement(2633..2655)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2755..2774
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
ORIGIN
        1 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
       61 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
      121 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      181 tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg gtacctcgcg aatgcatcta
      241 gatatcggat ccctaatacg actcactata ggtgagaccg agagagggtc tcagttttag
      301 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      361 agtcggtgct ttttttaaag ggcccgtcga ctgcagaggc ctgcatgcaa gcttggcgta
      421 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
      481 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
      541 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
      601 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgcggcc gccgcttcct
      661 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
      721 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
      781 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
      841 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
      901 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
      961 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1021 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1081 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1141 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1201 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1261 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1321 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1381 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1441 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1501 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1561 gtatatatga gtaaacttgg tctgacagtt agaaaaactc atcgagcatc aaatgaaact
     1621 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     1681 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     1741 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     1801 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     1861 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     1921 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     1981 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2041 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccag
     2101 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2161 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2221 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     2281 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2341 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     2401 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     2461 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     2521 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     2581 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     2641 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     2701 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     2761 cagagcagat tgtactgaga gtgcaccata tg
//
