LOCUS       Exported                4859 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Genetically encoded H2O2 probe for ratiometric and fluorescence 
            lifetime imaging.
ACCESSION   .
VERSION     .
KEYWORDS    pQE-30-HyPer3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4859)
  AUTHORS   Bilan DS, Pase L, Joosen L, Gorokhovatsky AY, Ermakova YG, Gadella 
            TW, Grabher C, Schultz C, Lukyanov S, Belousov VV
  TITLE     HyPer-3: a genetically encoded H2O2 probe with improved performance 
            for ratiometric and fluorescence lifetime imaging.
  JOURNAL   ACS Chem Biol. 2012 Dec 20.
  PUBMED    23256573
REFERENCE   2  (bases 1 to 4859)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Chem 
            Biol. 2012 Dec 20."
FEATURES             Location/Qualifiers
     source          1..4859
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        10..54
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             151..1584
                     /codon_start=1
                     /product="improved fluorescent indicator for intracellular 
                     H2O2 (Bilan et al., 2013)"
                     /label=HyPer-3
                     /translation="EMASQQGETMSGPLHIGLIPTVGPYLLPHIIPMLYQTFPKLEMYL
                     HEAQTHQLLAQLDSGKLDCVILALVKESEAFIEVPLFDEPMLLAIYEDHPWANRECVPM
                     ADLAGEKLLMLEDGHCLRDQAMSAGYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQL
                     ADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELY
                     NVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTT
                     GKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTR
                     AEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGTGFCFEAGADEDTHFRATSLETL
                     RNMVAAGSGITLLPALAVPPERKRDGVVYLPCIKPEPRRTIGLVYRPGSPLRSRYEQLA
                     EAIRARMDGHFDKVLKQAV"
     primer_bind     756..777
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(886..907)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1147..1166)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     terminator      1606..1700
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1744..2403
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1810..1829)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2468..2554
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2601..2623)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     misc_feature    2709..2849
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2764..2783
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2864..2881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3035..3623)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3115..3134)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3794..4654)
                     /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     4417..4436
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4655..4759)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4827..4845
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"