LOCUS       40924_22690        2584 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expression of customizable guide RNA (gRNA) for bacterial gene 
            knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2584)
  AUTHORS   Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim 
            WA
  TITLE     Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific 
            Control of Gene Expression.
  JOURNAL   Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.
  PUBMED    23452860
REFERENCE   2  (bases 1 to 2584)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2584)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2013.02"; journalName: "Cell"; date: "2013-02-28-
            28"; volume: "152"; issue: "5"; pages: "1173-83"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..39
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        60..135
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             156..185
                     /codon_start=1
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
                     /translation="EQKLISEEDL"
     CDS             201..218
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     primer_bind     complement(274..291)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse
                     primer"
     terminator      444..490
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     complement(543..560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(714..1302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1476..2333)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2334..2438)
                     /label=AmpR promoter
     primer_bind     2506..2524
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     terminator      complement(2528..2571)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 atctttgaca gctagctcag tcctaggtat aatactagta actttcagtt tagcggtctg
       61 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
      121 gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc tcagaagagg
      181 atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg gtctccagct
      241 tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag
      301 aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc
      361 catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc
      421 gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
      481 ttcgttttat ctgttgtttg tcggtgaact ggatccttac tcgagtctag actgcaggct
      541 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      601 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      661 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccac
      721 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      781 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      841 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      901 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      961 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1021 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1081 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1141 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1201 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1261 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1321 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1381 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1441 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1501 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1561 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1621 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1681 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1741 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1801 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1861 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1921 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1981 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     2041 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     2101 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2161 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2221 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2281 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2341 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     2401 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     2461 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     2521 aggcagaatt tcagataaaa aaaatcctta gctttcgcta aggatgattt ctggaattct
     2581 aaag
//
