LOCUS       Exported                8752 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses Target-AID-NG 
            (NLS-nSpCas9-NG-NLS-SH3-3xFLAG-PmCDA1-NLS-UGI) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    pSI-Target-AID-NG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8752)
  AUTHORS   Nishimasu H, Shi X, Ishiguro S, Gao L, Hirano S, Okazaki S, Noda T, 
            Abudayyeh OO, Gootenberg JS, Mori H, Oura S, Holmes B, Tanaka M, 
            Seki M, Hirano H, Aburatani H, Ishitani R, Ikawa M, Yachie N, Zhang 
            F, Nureki O
  TITLE     Engineered CRISPR-Cas9 nuclease with expanded targeting space.
  JOURNAL   Science. 2018 Aug 30. pii: science.aas9129. doi: 
            10.1126/science.aas9129.
  PUBMED    30166441
REFERENCE   2  (bases 1 to 8752)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2018 Aug 30. pii: science.aas9129. doi: 10.1126/science.aas9129."
FEATURES             Location/Qualifiers
     source          1..8752
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(12..31)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        203..582
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        583..786
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     737..757
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     828..847
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        828..846
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      854..863
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             866..886
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3498..3509
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5027..5047
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5255..5320
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5954..5974
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5981..6235
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     primer_bind     complement(6258..6275)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6264..6488
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6559..6575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6559..6575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6572..6594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6583..6599
                     /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)."
     promoter        complement(6607..6637)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6652..6673
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6790..6807)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6961..7549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7041..7060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7720..8580)
                     /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     8343..8362
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8581..8685)
                     /gene="bla"
                     /label=AmpR promoter