LOCUS       sequence-309707-        7481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AMPK biosensor .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7481)
  AUTHORS   Miyamoto T, Rho E, Sample V, Akano H, Magari M, Ueno T, Gorshkov K, 
            Chen M, Tokumitsu H, Zhang J, Inoue T
  TITLE     Compartmentalized AMPK signaling illuminated by genetically encoded 
            molecular sensors and actuators.
  JOURNAL   Cell Rep. 2015 Apr 28;11(4):657-70. doi: 
            10.1016/j.celrep.2015.03.057. Epub 2015 Apr 16.
  PUBMED    25892241
REFERENCE   2  (bases 1 to 7481)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2015.03.057"; journalName: "Cell Rep"; date: 
            "2015-04-28- 28"; volume: "11"; issue: "4"; pages: "657-70"
COMMENT     Sequence Label: Cyto-ABKAR
FEATURES             Location/Qualifiers
     source          1..7481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             688..705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             709..741
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(745..753,754..768)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(775..777,778..780,781..1491)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2014..2748
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     CDS             2755..2781
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(2800..2818)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2801..2818)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2838..2855)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2844..3068
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3114..3542
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3201..3220)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3411..3432
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3551..3571)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3556..3885
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3736..3871
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3798..3817
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3952..4746
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4006..4025)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4616..4635
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4920..5041
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4957..4976)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5011..5030
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5090..5106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5090..5106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5103..5125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5114..5130
                     /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(join(5138..5144,5145..5162,5163..5168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5183..5204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5321..5338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5492..6080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5572..5591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6251..7042,7043..7111))
                     /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     6874..6893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7112..7216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7291..7310)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
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 gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gcggccgcca ccatggtgcg gggttctcat catcatcatc atcatggtat ggctagcatg
      721 actggtggac agcaaatggg tcgggatctg tacgacgatg acgataagga tcccatggtg
      781 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      841 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      901 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      961 accaccctga gctggggcgt gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     1021 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1081 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1141 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1201 gagtacaacg ccatccacgg caacgtctat atcaccgccg acaagcagaa gaacggcatc
     1261 aaggccaact tcggcctcaa ctgcaacatc gaggacggca gcgtgcagct cgccgaccac
     1321 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1381 agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1441 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gcgcatgcat
     1501 aagttttctc aagaacagat cggcgaaaac attgtgtgca gggtcatttg taccacgggt
     1561 caaattccca tccgagattt gtcagctgat atttcacaag tgcttaagga aaaacgatcc
     1621 ataaagaaag tttggacatt tggtagaaac ccagcctgtg actatcattt aggaaacatt
     1681 tcaagactgt caaataagca tttccaaata ctactaggag aagacggtaa ccttttattg
     1741 aatgacattt ccactaatgg gacctggtta aatgggcaaa aagtcgagaa gaacagcaat
     1801 cagttactgt ctcaaggtga tgaaataacc gttggtgtag gcgtggaatc agatatttta
     1861 tctctggtca ttttcataaa cgacaaattt aagcagtgcc tcgagcagaa caaagttgat
     1921 cgctctgcag gtaagccagg cagcggcgag ggcagcacca agatgcgtcg tgtcgccacc
     1981 ctggtggacc tcggcaccgg cggcagcgag ctcatgggcg gcgtgcagct cgccgaccac
     2041 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2101 agctaccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2161 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggaggtacc
     2221 ggtggatcta tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2281 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2341 gccacctacg gcaagctgac cctgaagctg atctgcacca ccggcaagct gcccgtgccc
     2401 tggcccaccc tcgtgaccac cctgggctac ggccttcagt gcttcgcccg ctaccccgac
     2461 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2521 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2581 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2641 ctggggcaca agctggagta caactacaac agccacaacg tctatatcac cgccgacaag
     2701 cagaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga attcctgccc
     2761 cccctggagc gcctgaccct gtaatctaga gggccctatt ctatagtgtc acctaaatgc
     2821 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2881 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2941 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3001 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3061 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3121 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3181 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3241 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3301 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3361 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3421 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3481 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3541 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3601 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3661 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3721 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3781 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3841 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3901 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3961 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4021 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4081 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4141 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4201 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4261 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4321 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4381 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4441 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4501 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4561 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4621 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4681 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4741 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4801 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4861 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4921 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4981 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5041 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5101 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5161 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5221 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6001 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6241 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6301 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6361 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6421 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6481 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6541 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6601 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6661 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6721 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6781 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6841 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6901 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6961 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7021 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7081 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7201 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     7261 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     7321 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     7381 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     7441 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt t
//
