LOCUS       sequence-233280-        7459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A fluorescent reporter of AMPK activity and cellular energy stress.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7459)
  AUTHORS   Tsou P, Zheng B, Hsu CH, Sasaki AT, Cantley LC
  TITLE     A fluorescent reporter of AMPK activity and cellular energy stress.
  JOURNAL   Cell Metab. 2011 Apr 6;13(4):476-86.
  PUBMED    21459332
REFERENCE   2  (bases 1 to 7459)
  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"; journalName: "Cell 
            Metab."; date: "2011-04-6"; volume: "13(4)"; pages: "476-86"
COMMENT     Sequence Label: ampkar
FEATURES             Location/Qualifiers
     source          1..7459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        29..232
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     277..296
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        277..295
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      315..324
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             336..353
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             357..389
                     /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(393..401,402..416)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(423..425,426..428,429..941)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     CDS             1629..2363
                     /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"
     promoter        complement(2427..2445)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2428..2445)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2465..2482)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2471..2695
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2741..3169
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2828..2847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3038..3059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3178..3198)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3183..3512
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3363..3498
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3425..3444
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3579..4373
                     /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(3633..3652)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4243..4262
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4547..4667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4584..4603)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4637..4656
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4716..4732)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4716..4732)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4729..4751)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4740..4756
                     /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(4764..4770,4771..4788,4789..4794))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4809..4830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4947..4964)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5118..5706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5198..5217)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5877..6668,6669..6737))
                     /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     6500..6519
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6738..6842)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6917..6936)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7108..7459,1..28)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
       61 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      121 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      181 gacgcaaatg ggcggtaggc gtatacggtg ggaggtctat ataagcagag ctctctggct
      241 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
      301 ccaagcttgc ggccgccacc atggtgcggg gttctcatca tcatcatcat catggtatgg
      361 ctagcatgac tggtggacag caaatgggtc gggatctgta cgacgatgac gataaggatc
      421 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      481 acggcgacgt aaacggccac aggttcagcg tgtccggcga gggcgagggc gatgccacct
      541 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      601 ccctcgtgac caccctgacc tggggcgtgc agtgcttcag ccgctacccc gaccacatga
      661 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgtaccatct
      721 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      781 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
      841 acaagctgga gtacaactac atcagccaca acgtctatat caccgccgac aagcagaaga
      901 acggcatcaa ggcccacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
      961 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1021 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     1081 tcctgctgga gttcgtgacc gccgcccgca tgcataagtt ttctcaagaa cagatcggcg
     1141 aaaacattgt gtgcagggtc atttgtacca cgggtcaaat tcccatccga gatttgtcag
     1201 ctgatatttc acaagtgctt aaggaaaaac gatccataaa gaaagtttgg acatttggta
     1261 gaaacccagc ctgtgactat catttaggaa acatttcaag actgtcaaat aagcatttcc
     1321 aaatactact aggagaagac ggtaaccttt tattgaatga catttccact aatgggacct
     1381 ggttaaatgg gcaaaaagtc gagaagaaca gcaatcagtt actgtctcaa ggtgatgaaa
     1441 taaccgttgg tgtaggcgtg gaatcagata ttttatctct ggtcattttc ataaacgaca
     1501 aatttaagca gtgcctcgag cagaacaaag ttgatcgctc tgcaggtaag ccaggcagcg
     1561 gcgagggcag caccaagatg cgtcgtgtcg ccaccctggt ggacctcggc accggcggca
     1621 gcgagctcat gggcggcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1681 acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccaag ctgagcaaag
     1741 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1801 ctctcggcat ggacgagctg tacaagggag gtaccggtgg atctatggtg agcaagggcg
     1861 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1921 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1981 agctgatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctgg
     2041 gctacggcct tcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca
     2101 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     2161 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     2221 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     2281 acaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggccaact
     2341 tcaagatccg ccacaacatc gagtaagaat tctgcagata tccatcacac tggcggccgc
     2401 tcgagcatgc atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga
     2461 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2521 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2581 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2641 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2701 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2761 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2821 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2881 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2941 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3001 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3061 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3121 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     3181 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3241 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3301 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3361 ccatcccgcc cctaactccg cccagttctg cccattctcc gccccatggc tgactaattt
     3421 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3481 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3541 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3601 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3661 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3721 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3781 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3841 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3901 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3961 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4021 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4081 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     4141 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4201 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4261 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4321 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4381 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4441 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4501 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4561 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4621 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4681 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4741 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4801 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4861 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4921 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4981 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5041 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5101 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5161 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5221 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5281 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5341 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5401 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5461 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5521 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5581 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5641 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5701 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5761 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5821 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5881 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5941 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6001 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6061 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6121 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6181 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6241 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6301 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6361 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6421 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6481 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6541 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6601 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6661 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6721 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6781 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6841 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     6901 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     6961 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     7021 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     7081 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
     7141 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     7201 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     7261 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     7321 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     7381 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     7441 ttcctacttg gcagtacat
//
