LOCUS       sequence-379140-        7910 bp    DNA     circular SYN 14-APR-2025
DEFINITION  ExRai-AMPKAR targeted to the lysosomal membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7910)
  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 7910)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Lyso-ExRai-AMPKAR
FEATURES             Location/Qualifiers
     source          1..7910
                     /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"
     regulatory      905..914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(907..990,991..2157)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFAYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTPHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     primer_bind     2451..2472
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2578..2599)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2839..2858)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(3463..3481)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3464..3481)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3501..3518)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3507..3731
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3777..4205
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3864..3883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4074..4095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4214..4234)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4219..4548
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4399..4534
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4461..4480
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4615..5409
                     /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(4669..4688)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5279..5298
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5583..5704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5620..5639)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5674..5693
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5753..5769)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5753..5769)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5766..5788)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5777..5793
                     /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(5801..5807,5808..5825,5826..5831))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5846..5867
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5984..6001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6155..6743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6235..6254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6914..7705,7706..7774))
                     /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     7537..7556
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7775..7879)
                     /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 cggcgcccgg cagcgcccgg cgacccctgc tgctgctact gctgttgctg
      961 ctgctcggcc tcatgcattg tgcgtcagca gcaatgttta tggtgaaaaa tggcaacggg
     1021 accgcgtgca taatggccaa cttctctgct gccttctcag tgaactacga caccaagagt
     1081 ggccctaaga acatgacctt tgacctgcca tcagatgcca cagtggtgct caaccgcagc
     1141 tcctgtggaa aagagaacac ttctgacccc agtctcgtga ttgcttttgg aagaggacat
     1201 acactcactc tcaatttcac gagaaatgca acacgttaca gcgtccagct catgagtttt
     1261 gcttataact tgtcagacac acaccttttc cccaatgcga gctccaaaga aatcaagact
     1321 gtggaatcta taactgacat cagggcagat atagataaaa aatacagatg tgttagtggc
     1381 acccaggtcc acatgaacaa cgtgaccgta acgccccatg atgccaccat ccaggcgtac
     1441 ctttccaaca gcagcttcag caggggagag acacgctgtg aacaagacag gccttcccca
     1501 accacagcgc cccctgcgcc acccagcccc tcgccctcac ccgtgcccaa gagcccctct
     1561 gtggacaagt acaacgtgag cggcaccaac gggacctgcc tgctggccag catggggctg
     1621 cagctgaacc tcacctatga gaggaaggac aacacgacgg tgacaaggct tctcaacatc
     1681 aaccccaaca agacctcggc cagcgggagc tgcggcgccc acctggtgac tctggagctg
     1741 cacagcgagg gcaccaccgt cctgctcttc cagttcggga tgaatgcaag ttctagccgg
     1801 tttttcctac aaggaatcca gttgaataca attcttcctg acgccagaga ccctgccttt
     1861 aaagctgcca acggctccct gcgagcgctg caggccacag tcggcaattc ctacaagtgc
     1921 aacgcggagg agcacgtccg tgtcacgaag gcgttttcag tcaatatatt caaagtgtgg
     1981 gtccaggctt tcaaggtgga aggtggccag tttggctctg tggaggagtg tctgctggac
     2041 gagaacagca tgctgatccc catcgctgtg ggtggtgccc tggcggggct ggtcctcatc
     2101 gtcctcatcg cctacctcgt cggcaggaag aggagtcacg caggctacca gactatcggt
     2161 ggtggcggtg gtgcggatcc catgaggaga gtggctaccc tggtggatct gggcggcacc
     2221 ggcggcagcg agctcagctt ctgcaacgtc tatatcaagg ccgacaagca gaagaacggc
     2281 atcaaggcga acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgcctac
     2341 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2401 ctgagcgtgc agtccaaact ttcgaaagac cccaacgaga agcgcgatca catggtcctg
     2461 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggcggt
     2521 accggaggga gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     2581 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggtgagggc
     2641 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     2701 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     2761 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta catccaggag
     2821 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     2881 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2941 atcctggggc acaagctgga gtacaacttg ctgcataagt tttctcaaga acagatcggc
     3001 gaaaacattg tgtgcagggt catttgtacc acgggtcaaa ttcccatccg agatttgtca
     3061 gctgatattt cacaagtgct taaggaaaaa cgatccataa agaaagtttg gacatttggt
     3121 agaaacccag cctgtgacta tcatttagga aacatttcaa gactgtcaaa taagcatttc
     3181 caaatactac taggagaaga cggtaacctt ttattgaatg acatttccac taatgggacc
     3241 tggttaaatg ggcaaaaagt cgagaagaac agcaatcagt tactgtctca aggtgatgaa
     3301 ataaccgttg gtgtaggcgt ggaatcagat attttatctc tggtcatttt cataaacgac
     3361 aaatttaagc agtgcctcga gcagaacaaa gttgatcgct aagaattctg cagatatcca
     3421 tcacactggc ggccgctcga gcatgcatct agagggccct attctatagt gtcacctaaa
     3481 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3541 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3601 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3661 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3721 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3841 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3961 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4021 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4081 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4141 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4201 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4261 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4321 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4381 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4441 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4501 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4561 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4621 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4681 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4741 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4801 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4861 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4921 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4981 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5041 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5101 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     5161 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5221 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5281 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5341 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5401 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5461 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5521 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5581 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5641 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5701 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5761 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5821 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5881 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5941 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6001 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6061 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6121 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6181 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6241 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6301 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6361 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6421 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6481 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6541 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6601 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6661 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6721 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6781 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6841 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6901 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6961 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7021 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7081 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7141 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7201 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7261 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7321 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7381 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7441 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7501 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7561 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7621 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7681 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7741 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7801 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7861 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
