LOCUS       V000236                 9116 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V000236
VERSION     V000236
KEYWORDS    pPBbsr2-4031NES
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9116)
  AUTHORS   Konagaya Y, Terai K, Hirao Y, Takakura K, Imajo M, Kamioka Y,
            Sasaoka N, Kakizuka A, Sumiyama K, Asano T, Matsuda M
  TITLE     A Highly Sensitive FRET Biosensor for AMPK Exhibits Heterogeneous
            AMPK Responses among Cells and Organs.
  JOURNAL   Cell Rep. 2017 Nov 28;21(9):2628-2638. doi:
            10.1016/j.celrep.2017.10.113.
   PUBMED   29186696
REFERENCE   2  (bases 1 to 9116)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9116)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi:
            "10.1016/j.celrep.2017.10"; journalName: "Cell Rep"; date:
            "2017-11-28- 28"; volume: "21"; issue: "9"; pages: "2628-2638"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        626..644
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label="piggyBac left (5') inverted repeat"
                     /note="piggyBac transposon-specific inverted terminal
                     repeat sequence (ITR)"
     enhancer        1011..1390
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1393..1668
                     /label="chicken beta-actin promoter"
     intron          1669..2677
                     /label="chimeric intron"
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     CDS             2735..3448
                     /label="YPet"
                     /note="yellow fluorescent protein optimized for FRET with
                     CyPet (Nguyen and Daugherty, 2005)"
     primer_bind     4975..4996
                     /label="EGFP-C"
                     /note="EGFP, forward primer"
     CDS             5045..5071
                     /label="NES"
                     /note="nuclear export signal from the HIV Rev protein
                     (Fischer et al., 1995)"
     misc_feature    5168..5729
                     /label="IRES"
                     /note="internal ribosome entry site (IRES) of the
                     encephalomyocarditis virus (EMCV)"
     CDS             5738..6157
                     /gene="bsr"
                     /label="Blasticidin-S deaminase"
                     /note="Blasticidin-S deaminase from Bacillus cereus.
                     Accession#: P33967"
     polyA_signal    6206..6430
                     /label="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6477..6493)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    6501..6517
                     /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(6525..6555)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    6570..6591
                     /label="CAP binding site"
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     repeat_region   6825..6887
                     /label="piggyBac right (3') inverted repeat"
                     /note="piggyBac transposon-specific inverted terminal
                     repeat sequence (ITR)"
     promoter        complement(6928..6946)
                     /label="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6967..6983)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(6991..7007)
                     /label="lac operator"
                     /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(7015..7045)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7060..7081)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(7198..7215)
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7369..7957)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(8131..8988)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(8989..9093)
                     /label="AmpR promoter"
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1201 acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1381 tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
     1441 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
     1501 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
     1561 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     1621 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     1681 ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg gctctgactg
     1741 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1801 cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct taaagggctc
     1861 cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
     1921 ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
     1981 gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt
     2041 gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     2101 agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct ccccgagttg
     2161 ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg gggctcgccg
     2221 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg
     2281 gggagggctc gggggagggg cgcggcggcc ccggagcgcc ggcggctgtc gaggcgcggc
     2341 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     2401 ccaaatctgg cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg
     2461 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     2521 gccgccgtcc ccttctccat ctccagcctc ggggctgccg cagggggacg gctgccttcg
     2581 ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc
     2641 tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttgt
     2701 tgtgctgtct catcattttg gcaaagaatt cggcatggtg agcaagggcg aggagctgtt
     2761 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2821 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agcttctatg
     2881 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctgg gctacggcct
     2941 gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     3001 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     3061 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     3121 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     3181 caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggccaact tcaagatccg
     3241 ccacaacatc gaggacggcg gcgtgcagct cgccgaccac taccagcaga acacccccat
     3301 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgtt
     3361 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcctga ccgccgccgg
     3421 gatcactgag ggcatgaacg agctgtacct cgagaagttt tctcaagaac agatcggcga
     3481 aaacattgtg tgcagggtca tttgtaccac gggtcaaatt cccatccgag atttgtcagc
     3541 tgatatttca caagtgctta aggaaaaacg atccataaag aaagtttgga catttggtag
     3601 aaacccagcc tgtgactatc atttaggaaa catttcaaga ctgtcaaata agcatttcca
     3661 aatactacta ggagaagacg gtaacctttt attgaatgac atttccacta atgggacctg
     3721 gttaaatggg caaaaagtcg agaagaacag caatcagtta ctgtcccaag gtgatgaaat
     3781 aaccgttggt gtaggcgtgg aatcagatat tttatctctg gtcattttca taaacgacaa
     3841 atttaagcag tgcctggagc agaacaaagt tgatcgcggt accagtgctg gtggtagtgc
     3901 tggtggtagt gctggtggta gtgctggtgg tagtgctggt ggttccggca gtgctggtgg
     3961 tagtgctggt ggtagtacca gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc
     4021 tggtggtagt gctggtggtt ccggcagtgc tggtggtagt gctggtggta gtaccagtgc
     4081 tggtggtagt gctggtggta gtgctggtgg tagtgctggt ggtagtgctg gtggttccgg
     4141 cagtgctggt ggtagtgctg gtggtagtac cagtgctggt ggtagtgctg gtggtagtgc
     4201 tggtggtagt gctggtggta gtgctggtgg ttccggaggc agcggcgagg gcagcaccaa
     4261 gatgaggagg gtggccaccc tggtggacct gggcaccggc ggcagcgagc tgggcggccg
     4321 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     4381 cggcgacgta aacggccaca ggttcagcgt gtccggcgag ggcgagggcg atgccaccta
     4441 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     4501 cctcgtgacc accctgacct ggggcgtgca gtgcttcagc cgctaccccg accacatgaa
     4561 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gtaccatctt
     4621 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     4681 ggtgaaccgc atcgagctga agggcatcgg cttcaaggag gacggcaaca tcctggggca
     4741 caagctagag tacaactaca tcagccacaa cgtctatatc accgccgaca agcagaagaa
     4801 cggcatcaag gcccacttca agatccgcca caacatcgag gacggcggcg tgcagctcgc
     4861 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     4921 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     4981 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtctagact
     5041 gcagctgcct cctctggaac gcctgactct ggattaagtc gagggccggc gcgccgcggc
     5101 cggccgcggt aacaattgtt aactaactta agctagcaac ggtttccctc tagcgggatc
     5161 aattccgccc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     5221 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     5281 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     5341 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     5401 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     5461 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     5521 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     5581 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     5641 atgctttaca tgtgtttagt cgaggttaaa aaacgtctag gccccccgaa ccacggggac
     5701 gtggttttcc tttgaaaaac acgataatac catggtcatg aaaacattta acatttctca
     5761 acaagatcta gaattagtag aagtagcgac agagaagatt acaatgcttt atgaggataa
     5821 taaacatcat gtgggagcgg caattcgtac gaaaacagga gaaatcattt cggcagtaca
     5881 tattgaagcg tatataggac gagtaactgt ttgtgcagaa gccattgcga ttggtagtgc
     5941 agtttcgaat ggacaaaagg attttgacac gattgtagct gttagacacc cttattctga
     6001 cgaagtagat agaagtattc gagtggtaag tccttgtggt atgtgtaggg agttgatttc
     6061 agactatgca ccagattgtt ttgtgttaat agaaatgaat ggcaagttag tcaaaactac
     6121 gattgaagaa ctcattccac tcaaatatac ccgaaattaa aagtcgagat gcatcgatga
     6181 tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     6241 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     6301 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     6361 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     6421 ggctctatgg cttctgaggc ggaaagaacc tgcagcccaa gcttggcgta atcatggtca
     6481 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6541 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6601 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatctt aaaagttttg
     6661 ttactttata gaagaaattt tgagtttttg ttttttttta ataaataaat aaacataaat
     6721 aaattgtttg ttgaatttat tattagtatg taagtgtaaa tataataaaa cttaatatct
     6781 attcaaatta ataaataaac ctcgatatac agaccgataa aacacatgcg tcaattttac
     6841 gcatgattat ctttaacgta cgtcacaata tgattatctt tctagggtta atctagtata
     6901 cgcgtttaat taatccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     6961 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     7021 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     7081 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7141 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     7201 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7261 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7321 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     7381 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     7441 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7501 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7561 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7621 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7681 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7741 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7801 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7861 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7921 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     7981 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     8041 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     8101 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     8161 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     8221 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     8281 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     8341 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     8401 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     8461 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     8521 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     8581 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     8641 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     8701 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     8761 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     8821 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     8881 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     8941 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     9001 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     9061 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
