LOCUS       Exported                6083 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector HygR-5 EGFP reporter, complete sequence.
ACCESSION   JQ394982
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6083)
  AUTHORS   Konishi Y, Karnan S, Takahashi M, Ota A, Damdindorj L, Hosokawa Y, 
            Konishi H.
  TITLE     A system for the measurement of gene targeting efficiency in human 
            cell lines using an antibiotic resistance-GFP fusion gene
  JOURNAL   BioTechniques 53 (3), 141-152 (2012)
  PUBMED    22963476
REFERENCE   2  (bases 1 to 6083)
  AUTHORS   Konishi H.
  TITLE     Direct Submission
  JOURNAL   Submitted (07-JAN-2012) Aichi Medical University School of Medicine,
            1-1 Yazako-Karimta, Nagakute, Aichi 480-1195, Japan
REFERENCE   3  (bases 1 to 6083)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6083
                     /organism="Cloning vector HygR-5 EGFP reporter"
                     /mol_type="other DNA"
                     /db_xref="taxon:1249021"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     regulatory      309..810
                     /regulatory_class="promoter"
                     /note="Human cytomegalovirus (CMV) major immediate early 
                     promoter"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      936..945
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             942..2524
                     /codon_start=1
                     /gene="HygR-5 EGFP"
                     /product="HygR-5 EGFP"
                     /label=HygR-5 EGFP
                     /note="fusion protein of hygromycin B phosphotransferase 
                     and 3-truncated enhanced GFP"
                     /protein_id="AFV73966.1"
                     /translation="MDRSGKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVG
                     GRGYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQ
                     DLPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSE
                     AMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSL
                     VDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRP
                     RAKEPPPPPDRSYVSGFEFATMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDA
                     TYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQER
                     TIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMAD
                     KQKNGIKVNF"
     gene            942..2524
                     /gene="HygR-5 EGFP"
                     /label=HygR-5 EGFP
     CDS             957..1973
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     regulatory      2019..2028
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     polyA_signal    2673..2794
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    2704..2754
                     /label=SV40 early mRNA polyA
                     /note="SV40 early mRNA polyA"
     misc_feature    2865..3883
                     /note="partial SV40 largeT; an internal portion of SV40 
                     largeT exon 2"
     primer_bind     complement(3926..3942)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3950..3966
                     /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(3974..4004)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4019..4040
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4328..4916)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5087..5947)
                     /codon_start=1
                     /gene="ampR"
                     /product="beta-lactamase"
                     /label=ampR
                     /note="ampicilin resistance protein"
                     /protein_id="AFV73967.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(5087..5947)
                     /gene="ampR"
                     /label=ampR
     promoter        complement(5948..6052)
                     /gene="bla"
                     /label=AmpR promoter