LOCUS       Exported                6708 bp ds-DNA     circular SYN 23-AUG-2022
DEFINITION  Booster-PKA, a Red-Shifted Genetically Encoded FRET Biosensor for 
            monitoring PKA activity..
ACCESSION   .
VERSION     .
KEYWORDS    pCAGGS-4493NES
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6708)
  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 6708)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 23, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6708
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1545..1567
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1633..1652
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1682..1691
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1688..2383
                     /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             2981..3634
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             3680..3706
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(3722..3741)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3787..3842
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3841..3860)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4203..4219)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4203..4219)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4216..4238)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4227..4243
                     /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(4251..4281)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4296..4317
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4376..4571
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4422..4557
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4484..4503
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4577..4711
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4614..4633)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4668..4687
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4779..4796)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4950..5538)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5030..5049)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5709..6569)
                     /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     6332..6351
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6570..6674)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      541 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      601 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      661 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      721 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      781 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      841 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      901 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
      961 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1021 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1081 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1141 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1201 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1261 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1321 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1381 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1441 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1501 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1561 tgtgaccggc ggctctagct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     1621 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aagaattcgc
     1681 cgccaccatg gtgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     1741 caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     1801 cacccagacc atgagaatca aggcggtcga gggcggccct ctccccttcg ccttcgacat
     1861 cctggctacc agcttcatgt acggcagcaa aaccttcatc aaccacaccc agggcatccc
     1921 cgacttcttt aagcagtcct tccccgaggg cttcacatgg gagagagtca ccacatacga
     1981 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     2041 caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac
     2101 actcggctgg gaggcctcca ccgagaccct gtaccccgct gacggcggcc tggaaggcag
     2161 agccgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact tgaagaccac
     2221 atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacag
     2281 aagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc
     2341 tgtggccaga tactgcgacc tccctagcaa actggggcac agactcgaga agttttctca
     2401 agaacagatc ggcgaaaaca ttgtgtgcag ggtcatttgt accacgggtc aaattcccat
     2461 ccgagatttg tcagctgata tttcacaagt gcttaaggaa aaacgatcca taaagaaagt
     2521 ttggacattt ggtagaaacc cagcctgtga ctatcattta ggaaacattt caagactgtc
     2581 aaataagcat ttccaaatac tactaggaga agacggtaac cttttattga atgacatttc
     2641 cactaatggg acctggttaa atgggcaaaa agtcgagaag aacagcaatc agttactgtc
     2701 ccaaggtgat gaaataaccg ttggtgtagg cgtggaatca gatattttat ctctggtcat
     2761 tttcataaac gacaaattta agcagtgcct ggagcagaac aaagttgatc gcggtaccag
     2821 tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggttc
     2881 cggcagtgct ggtggtagtg ctggtggtag taccagtgct ggtggtagtg ctggtggtag
     2941 tgctggtggt agtgctggtg gtagtgctgg tggttccgga atggtgagtg tgattaaacc
     3001 agagatgaag atgaggtact acatggacgg ctccgtcaat gggcatgagt tcacaattga
     3061 aggtgaaggc acaggcagac cttacgaggg acatcaagag atgacactac gcgtcacaat
     3121 ggccgagggc gggccaatgc ctttcgcgtt tgacttagtg tcacacgtgt tctgttacgg
     3181 ccacagagta tttactaaat atccagaaga gataccagac tatttcaaac aagcatttcc
     3241 tgaaggcctg tcatgggaaa ggtcgttgga gttcgaagat ggtgggtccg cttcagtcag
     3301 tgcgcatata agccttagag gaaacacctt ctaccacaaa tccaaattta ctggggttaa
     3361 ctttcctgcc gatggtccta tcatgcaaaa ccaaagtgtt gattgggagc catcaaccga
     3421 gaaaattact gccagcgacg gagttctgaa gggtgatgtt acgatgtacc taaaacttga
     3481 aggaggcggc aatcacaaat gccaattcaa gactacttac aaggcggcaa aagagattct
     3541 tgaaatgcca ggagaccatt acatcggcca tcgcctcgtc aggaaaaccg aaggcaacat
     3601 tactgagcag gtagaagatg cagtagctca ttccgcggcc gcattgaggc gcgcgacgct
     3661 ggttgactct agactgcagc tgcctcctct ggaacgcctg actctggatt aagtcgacgg
     3721 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt
     3781 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct
     3841 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt
     3901 agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag
     3961 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     4021 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     4081 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     4141 tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca
     4201 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4261 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4321 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     4381 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4441 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4501 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4561 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4621 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4681 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     4741 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4801 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4861 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4921 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4981 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5041 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5101 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5161 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5221 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5281 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5341 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5401 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5461 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5521 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5581 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5641 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5701 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5761 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5821 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5881 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5941 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6001 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6061 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6121 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6181 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6241 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6301 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6361 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6421 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6481 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6541 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6601 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6661 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ggtcgatc
//
