LOCUS       sequence-363131-        5536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To (ectopically) express a mutant of ATP6AP1 lacking the interacting
            domain with PEN2; mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5536)
  AUTHORS   Ma T, Tian X, Zhang B, Li M, Wang Y, Yang C, Wu J, Wei X, Qu Q, Yu 
            Y, Long S, Feng JW, Li C, Zhang C, Xie C, Wu Y, Xu Z, Chen J, Yu Y, 
            Huang X, He Y, Yao L, Zhang L, Zhu M, Wang W, Wang ZC, Zhang M, Bao 
            Y, Jia W, Lin SY, Ye Z, Piao HL, Deng X, Zhang CS, Lin SC
  TITLE     Low-dose metformin targets the lysosomal AMPK pathway through PEN2.
  JOURNAL   Nature. 2022 Mar;603(7899):159-165. doi: 10.1038/s41586-022-04431-8.
            Epub 2022 Feb 23.
  PUBMED    35197629
REFERENCE   2  (bases 1 to 5536)
  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: 
            "10.1038/s41586-022-04431-8"; journalName: "Nature"; date: 
            "2022-03"; volume: "603"; issue: "7899"; pages: "159-165"
COMMENT     Sequence Label: pcDNA3.3-hAP1 420-440
FEATURES             Location/Qualifiers
     source          1..5536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..55)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        40..369
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      220..355
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     282..301
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             436..1230
                     /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(490..509)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1100..1119
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1406..1527
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1443..1462)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1497..1516
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1576..1592)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1576..1592)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1589..1611)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1600..1616
                     /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(1624..1630,1631..1648,1649..1654))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1669..1690
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1807..1824)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1978..2566)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2058..2077)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2737..3528,3529..3597))
                     /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     3360..3379
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3598..3702)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3777..3796)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3968..4347
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4348..4551
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4502..4522
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4548..4572
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             4765..4794
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(4840..4859)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4884..4932
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      join(5134..5536,1..26)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5221..5240)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5431..5452
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
       61 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
      121 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
      181 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
      241 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
      301 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
      361 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
      421 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
      481 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
      541 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
      601 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
      661 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
      721 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
      781 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
      841 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
      901 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
      961 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     1021 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     1081 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     1141 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     1201 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgcg aaatgaccga
     1261 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     1321 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     1381 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     1441 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     1501 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     1561 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     1621 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     1681 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     1741 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     1801 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     1861 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1921 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1981 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2041 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2101 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2161 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2221 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2281 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2341 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2401 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2461 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2521 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2581 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2641 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2701 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2761 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2821 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2881 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     2941 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3001 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3061 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3121 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3181 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3241 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3301 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3361 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     3421 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     3481 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3541 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3601 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3661 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     3721 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     3781 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     3841 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     3901 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
     3961 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     4021 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     4081 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     4141 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     4201 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     4261 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     4321 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     4381 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     4441 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     4501 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg
     4561 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
     4621 gaccgatcca gcctccggac tgagctcctt aaggttaact ctagagaatt cggatccatg
     4681 agcttcttct cccccggcat ctggatgggg ctgctcacct ccctgttcat gctcttcatc
     4741 ttcctcgagc catggaagct tatggaacaa aaactcatct cagaagagga tctggatatc
     4801 taactgactg aaccggtggt ccaaacgggg gaggctaact gaaacacgga aggagacaat
     4861 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acgggtgttg
     4921 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     4981 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     5041 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcag
     5101 atctgcgcag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     5161 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     5221 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5281 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5341 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5401 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5461 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5521 ggttaaaaaa tgagct
//
