LOCUS       sequence-379142-        6728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ExRai-AMPKAR targeted to the mitochondrial outer membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6728)
  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 6728)
  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-Mito-ExRai-AMPKAR
FEATURES             Location/Qualifiers
     source          1..6728
                     /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      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1269..1290
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1396..1417)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1657..1676)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(2281..2299)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2282..2299)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2319..2336)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2325..2549
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2595..3023
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2682..2701)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2892..2913
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3032..3052)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3037..3366
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3217..3352
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3279..3298
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3433..4227
                     /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(3487..3506)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4097..4116
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4401..4522
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4438..4457)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4492..4511
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4571..4587)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4571..4587)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4584..4606)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4595..4611
                     /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(4619..4625,4626..4643,4644..4649))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4664..4685
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4802..4819)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4973..5561)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5053..5072)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5732..6523,6524..6592))
                     /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     6355..6374
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6593..6697)
                     /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 gcttgccacc
      901 atggcaatcc agttgcgttc gctcttcccc ttggcgttgc ccggaatgct ggccctcctt
      961 ggctggtggt ggtttttctc tcgtaaaaaa gcggatccca tgaggagagt ggctaccctg
     1021 gtggatctgg gcggcaccgg cggcagcgag ctcagcttct gcaacgtcta tatcaaggcc
     1081 gacaagcaga agaacggcat caaggcgaac ttcaagatcc gccacaacat cgaggacggc
     1141 ggcgtgcagc tcgcctacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1201 ctgcccgaca accactacct gagcgtgcag tccaaacttt cgaaagaccc caacgagaag
     1261 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1321 gagctgtaca agggcggtac cggagggagc atggtgagca agggcgagga gctgttcacc
     1381 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1441 tccggcgagg gtgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1501 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1561 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1621 gaaggctaca tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1681 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1741 ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttgct gcataagttt
     1801 tctcaagaac agatcggcga aaacattgtg tgcagggtca tttgtaccac gggtcaaatt
     1861 cccatccgag atttgtcagc tgatatttca caagtgctta aggaaaaacg atccataaag
     1921 aaagtttgga catttggtag aaacccagcc tgtgactatc atttaggaaa catttcaaga
     1981 ctgtcaaata agcatttcca aatactacta ggagaagacg gtaacctttt attgaatgac
     2041 atttccacta atgggacctg gttaaatggg caaaaagtcg agaagaacag caatcagtta
     2101 ctgtctcaag gtgatgaaat aaccgttggt gtaggcgtgg aatcagatat tttatctctg
     2161 gtcattttca taaacgacaa atttaagcag tgcctcgagc agaacaaagt tgatcgctag
     2221 gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag agggccctat
     2281 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     2341 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2401 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2461 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2521 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2581 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2641 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2701 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2761 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2821 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2881 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2941 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3001 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3061 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3121 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3181 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3241 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3301 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3361 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     3421 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3481 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3541 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3601 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3661 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3721 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3781 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3841 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3901 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3961 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4021 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4081 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4141 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4201 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4261 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4321 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4381 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4441 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4501 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4561 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4621 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4681 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4741 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4801 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4861 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4921 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4981 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5041 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5101 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5161 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5221 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5281 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5341 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5401 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5461 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5521 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5581 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5641 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5701 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5761 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5821 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5881 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5941 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6001 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6061 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6121 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6181 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6241 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6301 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6361 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6421 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6481 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6541 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6601 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6661 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6721 ctgacgtc
//
