LOCUS       sequence-379141-        6737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Non-phosphorylatable negative-control mutant of ExRai-AMPKAR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6737)
  AUTHORS   Schmitt DL, Curtis SD, Lyons AC, Zhang JF, Chen M, He CY, Mehta S, 
            Shaw RJ, Zhang J
  TITLE     Spatial regulation of AMPK signaling revealed by a sensitive kinase 
            activity reporter.
  JOURNAL   Nat Commun. 2022 Jul 5;13(1):3856. doi: 10.1038/s41467-022-31190-x.
  PUBMED    35790710
REFERENCE   2  (bases 1 to 6737)
  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: "Nat 
            Commun."; date: "2022-07-5"; volume: "13(1):3856. doi"; pages: " 
            10.1038/s41467-022-31190-x"
COMMENT     Sequence Label: pcDNA3-ExRai-AMPKAR(T/A)
FEATURES             Location/Qualifiers
     source          1..6737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /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(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1278..1299
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1405..1426)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1666..1685)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(2290..2308)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2291..2308)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2328..2345)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2334..2558
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2604..3032
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2691..2710)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2901..2922
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3041..3061)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3046..3375
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3226..3361
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3288..3307
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3442..4236
                     /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(3496..3515)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4106..4125
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4410..4531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4447..4466)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4501..4520
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4580..4596)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4580..4596)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4593..4615)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4604..4620
                     /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(4628..4634,4635..4652,4653..4658))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4673..4694
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4811..4828)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4982..5570)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5062..5081)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5741..6532,6533..6601))
                     /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     6364..6383
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6602..6706)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat gaggagagtg
     1021 gctgctctgg tggatctggg cggcaccggc ggcagcgagc tcagcttctg caacgtctat
     1081 atcaaggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc
     1141 gaggacggcg gcgtgcagct cgcctaccac taccagcaga acacccccat cggcgacggc
     1201 cccgtgctgc tgcccgacaa ccactacctg agcgtgcagt ccaaactttc gaaagacccc
     1261 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1321 ggcatggacg agctgtacaa gggcggtacc ggagggagca tggtgagcaa gggcgaggag
     1381 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1441 ttcagcgtgt ccggcgaggg tgagggcgat gccacctacg gcaagctgac cctgaagttc
     1501 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1561 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1621 gccatgcccg aaggctacat ccaggagcgc accatcttct tcaaggacga cggcaactac
     1681 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1741 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacttgctg
     1801 cataagtttt ctcaagaaca gatcggcgaa aacattgtgt gcagggtcat ttgtaccacg
     1861 ggtcaaattc ccatccgaga tttgtcagct gatatttcac aagtgcttaa ggaaaaacga
     1921 tccataaaga aagtttggac atttggtaga aacccagcct gtgactatca tttaggaaac
     1981 atttcaagac tgtcaaataa gcatttccaa atactactag gagaagacgg taacctttta
     2041 ttgaatgaca tttccactaa tgggacctgg ttaaatgggc aaaaagtcga gaagaacagc
     2101 aatcagttac tgtctcaagg tgatgaaata accgttggtg taggcgtgga atcagatatt
     2161 ttatctctgg tcattttcat aaacgacaaa tttaagcagt gcctcgagca gaacaaagtt
     2221 gatcgctagg aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
     2281 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     2341 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2401 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2461 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2521 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2581 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2641 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2701 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2761 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2821 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2881 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2941 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3001 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3061 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3121 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3181 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3241 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3301 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3361 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     3421 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3481 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3541 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3601 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3661 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3721 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3781 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3841 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3901 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3961 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4021 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4081 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4141 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4201 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4261 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4321 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4381 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4441 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4501 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4561 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4621 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4681 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4741 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4801 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4861 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4921 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4981 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5041 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5101 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5161 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5221 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5281 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5341 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5401 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5461 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5521 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5581 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5641 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5701 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5761 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5821 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5881 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5941 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6001 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6061 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6121 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6181 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6241 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6301 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6361 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6421 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6481 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6541 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6601 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6661 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6721 aagtgccacc tgacgtc
//
