LOCUS       Exported                7689 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses SaKKH-BE3 in mammalian cells [Edit].
ACCESSION   .
VERSION     .
KEYWORDS    pJL-SaKKH-BE3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7689)
  AUTHORS   Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR
  TITLE     Increasing the genome-targeting scope and precision of base editing 
            with engineered Cas9-cytidine deaminase fusions.
  JOURNAL   Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803.
  PUBMED    28191901
REFERENCE   2  (bases 1 to 7689)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803."
FEATURES             Location/Qualifiers
     source          1..7689
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        179..558
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        559..762
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     713..733
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     804..823
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        804..822
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             836..1522
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             4736..4756
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4763..4828
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4841..5089
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5102..5122
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5131..5148
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5171..5188)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5177..5401
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5472..5488)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5472..5488)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5485..5507)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5496..5512
                     /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(5520..5550)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5565..5586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5703..5720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5874..6462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5954..5973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6633..7493)
                     /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     7256..7275
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7494..7598)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(7677..7689,1..7))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"