LOCUS       sequence-176880-        6278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes negative-control C-terminal (substrate) fragment of 
            bimolecular AMPK/BRSK activity reporter (bimABKAR); cytosol 
            targeted; use in conjunction with pcDNA3-Cerulean-FHA1-NES.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6278)
  AUTHORS   Depry C, Mehta S, Li R, Zhang J
  TITLE     Visualization of Compartmentalized Kinase Activity Dynamics Using 
            Adaptable BimKARs.
  JOURNAL   Chem Biol. 2015 Nov 19;22(11):1470-9. doi: 
            10.1016/j.chembiol.2015.10.004. Epub 2015 Nov 5.
  PUBMED    26548610
REFERENCE   2  (bases 1 to 6278)
  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.chembiol.2015.10.004"; journalName: "Chem Biol"; date: 
            "2015-11-19- 19"; volume: "22"; issue: "11"; pages: "1470-9"
COMMENT     Sequence Label: pcDNA3-AMPKsub(TA)-YPet-NES
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             22..54
                     /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(58..66,67..81)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(448..467)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             865..891
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(910..928)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(911..928)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(948..965)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    954..1178
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1224..1652
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1311..1330)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1521..1542
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1661..1681)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1666..1995
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1846..1981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1908..1927
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2062..2856
                     /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(2116..2135)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2726..2745
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3030..3151
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3067..3086)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3121..3140
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3200..3216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3200..3216)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3213..3235)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3224..3240
                     /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(3248..3254,3255..3272,3273..3278))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3293..3314
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3431..3448)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3602..4190)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3682..3701)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4361..5152,5153..5221))
                     /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     4984..5003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5222..5326)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5401..5420)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5592..5971
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5972..6175
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6126..6146
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6220..6239
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6220..6238
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6258..6267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 catcatcatc atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat
       61 ctgtacgacg atgacgataa ggatcccatg aggagagtgg ctgccctggt ggatctgggc
      121 ggcaccggcg gcagcgagct catggtgagc aaaggcgaag agctgttcac cggcgtggtg
      181 cccatcctgg tggagctgga cggcgacgtg aacggccaca agttcagcgt gagcggcgag
      241 ggcgagggcg acgccaccta cggcaagctg accctgaagc tgctgtgcac caccggcaag
      301 ctgcccgtgc cctggcccac cctggtgacc accctgggct acggcgtgca gtgcttcgcc
      361 cggtaccccg accacatgaa gcagcacgac ttcttcaaga gcgccatgcc cgagggctac
      421 gtgcaggagc ggaccatctt cttcaaggac gacggcaact acaagacccg ggccgaggtg
      481 aagttcgagg gcgacaccct ggtgaaccgg atcgagctga agggcatcga cttcaaggag
      541 gacagcaaca tcctgggcca caagctggag tacaactaca acagccacaa cgtgtacatc
      601 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccggca caacatcgag
      661 gacggcggcg tgcagctggc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      721 gtgctgctgc ccgataacca ctacctgagc taccagagcg ccctgttcaa ggaccccaac
      781 gagaagcggg accacatggt gctgctggag ttcctgaccg ccgccggcat caccgagggt
      841 atgaatgaat tgtacaaaga attcctgccc cccctggagc gcctgaccct gtaatctaga
      901 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
      961 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1021 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1081 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     1141 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     1201 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1261 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1321 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1381 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1441 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1501 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1561 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1621 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     1681 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     1741 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     1801 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     1861 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     1921 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     1981 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     2041 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     2101 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     2161 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     2221 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     2281 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     2341 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     2401 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     2461 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     2521 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     2581 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     2641 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     2701 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     2761 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     2821 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     2881 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     2941 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3001 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     3061 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3121 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     3181 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3241 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3301 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3361 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3421 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3481 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3541 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3601 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3661 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3721 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3781 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3841 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3901 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3961 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4021 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4081 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4141 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4201 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4261 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4321 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4381 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4441 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4501 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4561 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4621 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4681 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4741 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4801 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4861 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4921 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4981 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5041 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5101 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5161 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5221 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5281 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5341 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     5401 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     5461 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     5521 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     5581 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     5641 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     5701 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     5761 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     5821 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     5881 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     5941 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     6001 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     6061 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     6121 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     6181 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     6241 gacccaagct tgcggccgcc accatggtgc ggggttct
//
