LOCUS       Exported                3008 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  sgRNA cloning backbone with MS2 loops at tetraloop and stemloop 2. 
            Contains BbsI sites for insertion of spacer sequences..
ACCESSION   .
VERSION     .
KEYWORDS    sgRNA(MS2) cloning backbone
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3008)
  AUTHORS   Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena 
            C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F
  TITLE     Genome-scale transcriptional activation by an engineered CRISPR-Cas9
            complex.
  JOURNAL   Nature. 2014 Dec 10. doi: 10.1038/nature14136.
  PUBMED    25494202
REFERENCE   2  (bases 1 to 3008)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2014 Dec 10. doi: 10.1038/nature14136."
FEATURES             Location/Qualifiers
     source          1..3008
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        7..247
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5
                     /note="Human U6 promoter, forward primer"
     misc_RNA        291..309
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        361..379
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      491..946
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(578..597)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     788..809
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(963..982)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1082..1104
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1142..1160)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1228..1332
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1333..2193
                     /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(1551..1570)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2364..2952
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2853..2872
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"