LOCUS       Exported                7348 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Booster-ROCK, a Red-Shifted Genetically Encoded FRET Biosensor for 
            ROCK activity..
ACCESSION   .
VERSION     .
KEYWORDS    pCAGGS-4591NES
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7348)
  AUTHORS   Watabe T, Terai K, Sumiyama K, Matsuda M
  TITLE     Booster, a Red-Shifted Genetically Encoded Forster Resonance Energy 
            Transfer (FRET) Biosensor Compatible with Cyan Fluorescent 
            Protein/Yellow Fluorescent Protein-Based FRET Biosensors and Blue 
            Light-Responsive Optogenetic Tools.
  JOURNAL   ACS Sens. 2020 Feb 26. doi: 10.1021/acssensors.9b01941.
  PUBMED    32101394
REFERENCE   2  (bases 1 to 7348)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7348
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..665
                     /label=chicken beta-actin promoter
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1735..1744
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1741..2436
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             3610..4263
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             4330..4356
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(4372..4391)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4437..4492
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4491..4510)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4851..4867)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4851..4867)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4864..4886)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4875..4891
                     /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(4899..4929)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4944..4965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5024..5219
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5070..5205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5132..5151
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    5225..5359
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5262..5281)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5316..5335
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5427..5444)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5598..6186)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5678..5697)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6357..7217)
                     /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     6980..6999
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7218..7322)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgatcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cgggggcgag
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
      841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtgcggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctggcgg agccgaaatc tgggaggcgc cgccgcaccc
     1441 cctctagcgg gcgcgggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
     1501 ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg gctgccgcag
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggttg ttgtgctgtc tcatcatttt ggcaagaatt cgccgccacc
     1741 atggtgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     1801 aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     1861 accatgagaa tcaaggcggt cgagggcggc cctctcccct tcgccttcga catcctggct
     1921 accagcttca tgtacggcag caaaaccttc atcaaccaca cccagggcat ccccgacttc
     1981 tttaagcagt ccttccccga gggcttcaca tgggagagag tcaccacata cgaagacggg
     2041 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     2101 aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc
     2161 tgggaggcct ccaccgagac cctgtacccc gctgacggcg gcctggaagg cagagccgac
     2221 atggccctga agctcgtggg cgggggccac ctgatctgca acttgaagac cacatacaga
     2281 tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga cagaagactg
     2341 gaaagaatca aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc
     2401 agatactgcg acctccctag caaactgggg cacagactcg agaagttttc tcaagaacag
     2461 atcggcgaaa acattgtgtg cagggtcatt tgtaccacgg gtcaaattcc catccgagat
     2521 ttgtcagctg atatttcaca agtgcttaag gaaaaacgat ccataaagaa agtttggaca
     2581 tttggtagaa acccagcctg tgactatcat ttaggaaaca tttcaagact gtcaaataag
     2641 catttccaaa tactactagg agaagacggt aaccttttat tgaatgacat ttccactaat
     2701 gggacctggt taaatgggca aaaagtcgag aagaacagca atcagttact gtcccaaggt
     2761 gatgaaataa ccgttggtgt aggcgtggaa tcagatattt tatctctggt cattttcata
     2821 aacgacaaat ttaagcagtg cctggagcag aacaaagttg atcgcggtac cagtgctggt
     2881 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg ttccggcagt
     2941 gctggtggta gtgctggtgg tagtaccagt gctggtggta gtgctggtgg tagtgctggt
     3001 ggtagtgctg gtggtagtgc tggtggttcc ggcagtgctg gtggtagtgc tggtggtagt
     3061 accagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     3121 ggttccggca gtgctggtgg tagtgctggt ggtagtacca gtgctggtgg tagtgctggt
     3181 ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggcagtgc tggtggtagt
     3241 gctggtggta gtaccagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     3301 ggtagtgctg gtggttccgg cagtgctggt ggtagtgctg gtggtagtac cagtgctggt
     3361 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg ttccggcagt
     3421 gctggtggta gtgctggtgg tagtaccagt gctggtggta gtgctggtgg tagtgctggt
     3481 ggtagtgctg gtggtagtgc tggtggttcc ggcagtgctg gtggtagtgc tggtggtagt
     3541 accagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     3601 ggttccggaa tggtgagtgt gattaaacca gagatgaaga tgaggtacta catggacggc
     3661 tccgtcaatg ggcatgagtt cacaattgaa ggtgaaggca caggcagacc ttacgaggga
     3721 catcaagaga tgacactacg cgtcacaatg gccgagggcg ggccaatgcc tttcgcgttt
     3781 gacttagtgt cacacgtgtt ctgttacggc cacagagtat ttactaaata tccagaagag
     3841 ataccagact atttcaaaca agcatttcct gaaggcctgt catgggaaag gtcgttggag
     3901 ttcgaagatg gtgggtccgc ttcagtcagt gcgcatataa gccttagagg aaacaccttc
     3961 taccacaaat ccaaatttac tggggttaac tttcctgccg atggtcctat catgcaaaac
     4021 caaagtgttg attgggagcc atcaaccgag aaaattactg ccagcgacgg agttctgaag
     4081 ggtgatgtta cgatgtacct aaaacttgaa ggaggcggca atcacaaatg ccaattcaag
     4141 actacttaca aggcggcaaa agagattctt gaaatgccag gagaccatta catcggccat
     4201 cgcctcgtca ggaaaaccga aggcaacatt actgagcagg tagaagatgc agtagctcat
     4261 tccgcggccg caaagagaag aaacagaaga aagaccctgg tagatctgcc tctggattct
     4321 agactgcagc tgcctcctct ggaacgcctg actctggatt aagtcgacgg atctttttcc
     4381 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     4441 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     4501 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     4561 aacatatgcc atatgctggc tgccatgaac aaaggtggct ataaagaggt catcagtata
     4621 tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga
     4681 ttttttttta tattttgttt gtgttatttt tttctttaac atccctaaaa ttttccttac
     4741 atgttttact agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc
     4801 ttctcttatg aagatccctc gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg
     4861 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4921 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4981 ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc aattagtcag
     5041 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     5101 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     5161 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     5221 agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5281 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5341 atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5401 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5461 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5521 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5581 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5641 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5701 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5761 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     5821 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5881 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5941 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6001 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     6061 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6121 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6181 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6241 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6301 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6361 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6421 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6481 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6541 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6601 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6661 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6721 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6781 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6841 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6901 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6961 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7021 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7081 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7141 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7201 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7261 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7321 cgcacatttc cccgaaaagt gccacctg
//
