LOCUS       Exported                3771 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Can use a PCR template to assemble new desired guide RNA. Contains 
            an Arabidopsis U6 promoter to drive guide RNA (targeting AtPDS3 gene
            target site 1) expression with a TTTTTT as terminator. .
ACCESSION   .
VERSION     .
KEYWORDS    pUC119-gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3771)
  AUTHORS   Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM,
            Sheen J
  TITLE     Multiplex and homologous recombination-mediated genome editing in 
            Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.
  JOURNAL   Nat Biotechnol. 2013 Aug;31(8):688-91. doi: 10.1038/nbt.2654.
  PUBMED    23929339
REFERENCE   2  (bases 1 to 3771)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2013-08"; volume: "31"; issue:
            "8"; pages: "688-91"
FEATURES             Location/Qualifiers
     source          1..3771
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        407..710
                     /label=AtU6-1 promoter
                     /note="promoter for the Arabidopsis thaliana U6-1 snRNA 
                     gene (Waibel and Filipowicz, 1990)"
     misc_RNA        731..806
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(818..842)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(900..917)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(900..916)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(909..931)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1129..1584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1216..1235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1426..1447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1601..1620)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1720..1742
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1780..1798)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1866..1970
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1971..2831
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2189..2208)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3002..3590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3491..3510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3744..3761
                     /label=L4440
                     /note="L4440 vector, forward primer"