LOCUS       sequence-363138-        6727 bp    DNA     circular SYN 15-APR-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 6727)
  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 6727)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 1-419
FEATURES             Location/Qualifiers
     source          1..6727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      179..314
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     241..260
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             395..1189
                     /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(449..468)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1059..1078
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1365..1486
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1402..1421)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1456..1475
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1535..1551)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1535..1551)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1548..1570)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1559..1575
                     /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(1583..1589,1590..1607,1608..1613))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1628..1649
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1766..1783)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1937..2525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2017..2036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2696..3487,3488..3556))
                     /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     3319..3338
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3557..3661)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3736..3755)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3927..4306
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4307..4510
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4461..4481
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4507..4531
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5915..5944
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(5990..6009)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6034..6082
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6284..6712
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6371..6390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6581..6602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(join(6721..6727,1..14))
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6726..6727,1..328)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
       61 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
      121 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
      181 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
      241 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
      301 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
      361 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
      421 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
      481 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
      541 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
      601 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
      661 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
      721 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
      781 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
      841 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
      901 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
      961 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     1021 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     1081 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     1141 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     1201 gggttcgcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     1261 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     1321 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     1381 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1441 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     1501 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1561 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1621 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1681 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1741 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1801 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1861 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1921 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2101 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2341 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2521 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2581 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2641 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2701 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2761 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2821 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2881 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2941 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3001 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3061 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3121 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3181 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3241 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3301 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3361 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3421 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3481 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3541 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3601 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3661 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     3721 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     3781 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     3841 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     3901 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     3961 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     4021 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     4081 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     4141 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     4201 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     4261 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     4321 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     4381 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     4441 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     4501 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     4561 cctccataga agacaccggg accgatccag cctccggact gagctcctta aggttaactc
     4621 tagagaattc ggatccatga tggcggccat ggcgacggct cgagtgcgga tggggccgcg
     4681 gtgcgcccag gcgctctggc gcatgccgtg gctgccggtg tttttgtcgt tggcggcggc
     4741 ggcggcggcg gcagcggcgg agcagcaggt cccgctggtg ctgtggtcga gtgaccggga
     4801 cttgtgggct cctgcggccg acactcatga aggccacatc accagcgact tgcagctctc
     4861 tacctactta gatcccgccc tggagctggg tcccaggaat gtgctgctgt tcctgcagga
     4921 caagctgagc attgaggatt tcacagcata tggcggtgtg tttggaaaca agcaggacag
     4981 cgccttttct aacctagaga atgccctgga cctggccccc tcctcactgg tgcttcctgc
     5041 cgtcgactgg tatgcagtca gcactctgac cacttacctg caggagaagc tcggggccag
     5101 ccccttgcat gtggacctgg ccaccctgcg ggagctgaag ctcaatgcca gcctccctgc
     5161 tctgctgctc attcgcctgc cctacacagc cagctctggt ctgatggcac ccagggaagt
     5221 cctcacaggc aacgatgagg tcatcgggca ggtcctgagc acactcaagt ccgaagatgt
     5281 cccatacaca gcggccctca cagcggtccg cccttccagg gtggcccgtg atgtagccgt
     5341 ggtggccgga gggctaggtc gccagctgct acaaaaacag ccagtatcac ctgtgatcca
     5401 tcctcctgtg agttacaatg acaccgctcc ccggatcctg ttctgggccc aaaacttctc
     5461 tgtggcgtac aaggaccagt gggaggacct gactcccctc acctttgggg tgcaggaact
     5521 caacctgact ggctccttct ggaatgactc ctttgccagg ctctcactga cctatgaacg
     5581 actctttggt accacagtga cattcaagtt cattctggcc aaccgcctct acccagtgtc
     5641 tgcccggcac tggtttacca tggagcgcct cgaagtccac agcaatggct ccgtcgccta
     5701 cttcaatgct tcccaggtca cagggcccag catctactcc ttccactgcg agtatgtcag
     5761 cagcctgagc aagaagggta gtctcctcgt ggcccgcacg cagccctctc cctggcagat
     5821 gatgcttcag gacttccaga tccaggcttt caacgtaatg ggggagcagt tctcctacgc
     5881 cagcgactgt gccctcgagc catggaagct tatggaacaa aaactcatct cagaagagga
     5941 tctggatatc taactgactg aaccggtggt ccaaacgggg gaggctaact gaaacacgga
     6001 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     6061 acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     6121 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     6181 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     6241 gccatagcag atctgcgcag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     6301 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     6361 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     6421 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     6481 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     6541 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     6601 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     6661 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     6721 ggaatgt
//
