LOCUS       Exported                6679 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Mammalian expression of NgAgo (Argonaute from N. gregoryi)..
ACCESSION   .
VERSION     .
KEYWORDS    nls-NgAgo-GK
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  AUTHORS   Gao F, Shen XZ, Jiang F, Wu Y, Han C
  TITLE     DNA-guided genome editing using the Natronobacterium gregoryi 
            Argonaute.
  JOURNAL   Nat Biotechnol. 2016 May 2. doi: 10.1038/nbt.3547.
  PUBMED    27136078
REFERENCE   2  (bases 1 to 6679)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2016 May 2. doi: 10.1038/nbt.3547."
FEATURES             Location/Qualifiers
     source          1..6679
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        16..319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        320..523
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     474..494
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      583..592
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             595..615
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             619..3276
                     /codon_start=1
                     /product="Argonaute protein from Natronobacterium gregoryi 
                     (Gao et al., 2016)"
                     /label=NgAgo
                     /note="reportedly a DNA-guided endonuclease that does not 
                     require a PAM site, although this interpretation has been 
                     challenged (Cyranoski, 2016)"
                     /translation="TVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPRMSLAFE
                     QDNGERRYITLWKNTTPKDVFTYDYATGSTYIFTNIDYEVKDGYENLTATYQTTVENAT
                     AQEVGTTDEDETFAGGEPLDHHLDDALNETPDDAETESDSGHVMTSFASRDQLPEWTLH
                     TYTLTATDGAKTDTEYARRTLAYTVRQELYTDHDAAPVATDGLMLLTPEPLGETPLDLD
                     CGVRVEADETRTLDYTTAKDRLLARELVEEGLKRSLWDDYLVRGIDEVLSKEPVLTCDE
                     FDLHERYDLSVEVGHSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTTYRPRRGH
                     IVWGLRDECATDSLNTLGNQSVVAYHRNNQTPINTDLLDAIEAADRRVVETRRQGHGDD
                     AVSFPQELLAVEPNTHQIKQFASDGFHQQARSKTRLSASRCSEKAQAFAERLDPVRLNG
                     STVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAHPDETFSKGIVNPPESFEVAV
                     VLPEQQADTCKAQWDTMADLLNQAGAPPTRSETVQYDAFSSPESISLNVAGAIDPSEVD
                     AAFVVLPPDQEGFADLASPTETYDELKKALANMGIYSQMAYFDRFRDAKIFYTRNVALG
                     LLAAAGGVAFTTEHAMPGDADMFIGIDVSRSYPEDGASGQINIAATATAVYKDGTILGH
                     SSTRPQLGEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGFMNEDLDPATEFLNE
                     QGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNEPRATVATFGAPEYLATRDG
                     GGLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHNSTARLPITTAYADQASTHATKG
                     YLVQTGAFESNVGFL"
     polyA_signal    3422..3543
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3459..3478)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3513..3532
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3550..4005)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3687..3708)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3899..3918
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4032..4136
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4138..4495
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(4161..4181)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4346..4481
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4408..4427
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4530..5324
                     /codon_start=1
                     /gene="aph(3)-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4584..4603)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5194..5213
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5512..5531)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5556..5603
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5932..6520
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6421..6440
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"