LOCUS       Exported                8102 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pNR.CFH, complete sequence.
ACCESSION   KY654504
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8102)
  AUTHORS   Jackson HO, Bibby TS, Moore MC, Terry MJ.
  TITLE     Characterization of constitutive and inducible promoters in 
            Nannochloropsis gaditana (unpublished)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8102)
  AUTHORS   Jackson HO, Bibby TS, Moore MC, Terry MJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-FEB-2017) Ocean and Earth Sciences, National 
            Oceanography Centre, Southampton, European Way, Southampton SO14 
            3ZH, England
REFERENCE   3  (bases 1 to 8102)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8102
                     /organism="Cloning vector pNR.CFH"
                     /mol_type="other DNA"
                     /db_xref="taxon:2079256"
     regulatory      1..1319
                     /regulatory_class="promoter"
                     /note="NR promoter"
     misc_feature    1323..1330
                     /label=NotI restriction site
                     /note="NotI restriction site"
     misc_feature    1342..1349
                     /label=AscI restriction site
                     /note="AscI restriction site"
     misc_feature    1350..1379
                     /label=3C protease site
                     /note="3C protease site"
     CDS             1356..1379
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     misc_feature    1386..1409
                     /label=FLAG tag
                     /note="FLAG tag"
     CDS             1386..1409
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1419..1448
                     /label=His tag
                     /note="His tag"
     CDS             1419..1448
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     regulatory      1452..1959
                     /regulatory_class="terminator"
                     /note="ATPa terminator"
     regulatory      1990..2682
                     /regulatory_class="promoter"
                     /note="UEP promoter"
     CDS             2683..3711
                     /codon_start=1
                     /product="hygromycin phosphotransferase"
                     /label=hygromycin phosphotransferase
                     /note="hph"
                     /protein_id="AUX13698.1"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     regulatory      3712..4149
                     /regulatory_class="terminator"
                     /note="NOS terminator"
     terminator      3743..3995
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(4029..4045)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4053..4069
                     /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(4077..4107)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4122..4143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4162..4790
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      4184..4772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    4850..4897
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     misc_feature    4864..4897
                     /label=FRT site
                     /note="FRT site"
     rep_origin      4878..5860
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     rep_origin      4898..5860
                     /label=2micron
                     /note="2micron"
     CDS             complement(6004..6807)
                     /codon_start=1
                     /product="URA3"
                     /label=URA3
                     /protein_id="AUX13700.1"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6808..7023)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             complement(7147..7941)
                     /codon_start=1
                     /product="KanR"
                     /label=KanR
                     /protein_id="AUX13699.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"