LOCUS       sequence-251677-        8394 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human amyloid Lysozyme in Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8394)
  AUTHORS   Johnson RJ, Christodoulou J, Dumoulin M, Caddy GL, Alcocer MJ, 
            Murtagh GJ, Kumita JR, Larsson G, Robinson CV, Archer DB, Luisi B, 
            Dobson CM
  TITLE     Rationalising lysozyme amyloidosis: insights from the structure and 
            solution dynamics of T70N lysozyme.
  JOURNAL   J Mol Biol. 2005 Sep 30;352(4):823-36. doi: 
            10.1016/j.jmb.2005.07.040.
  PUBMED    16126226
REFERENCE   2  (bases 1 to 8394)
  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.jmb.2005.07"; journalName: "J Mol Biol"; date: 
            "2005-09-30- 30"; volume: "352"; issue: "4"; pages: "823-36"
COMMENT     Sequence Label: pMJA103
FEATURES             Location/Qualifiers
     source          1..8394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..936
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             join(948..1004,1005..1202)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR"
     terminator      1698..1944
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(2357..4891)
                     /codon_start=1
                     /gene="Pichia pastoris HIS4"
                     /product="multifunctional enzyme, required for histidine 
                     biosynthesis"
                     /label=PpHIS4
                     /note="auxotrophic marker for Pichia pastoris"
                     /translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
                     LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
                     VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
                     DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
                     PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
                     TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
                     IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
                     KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
                     LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP
                     GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
                     VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
                     LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
                     AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
                     QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK
                     HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
     primer_bind     complement(5032..5051)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     misc_feature    5246..5994
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     primer_bind     complement(6030..6052)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6138..6278
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6193..6212
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6293..6310)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6464..7052)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6544..6563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7223..8014,8015..8083))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7846..7865
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8084..8188)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8256..8274
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gatctaacat ccaaagacga aaggttgaat gaaacctttt tgccatccga catccacagg
       61 tccattctca cacataagtg ccaaacgcaa caggagggga tacactagca gcagaccgtt
      121 gcaaacgcag gacctccact cctcttctcc tcaacaccca cttttgccat cgaaaaacca
      181 gcccagttat tgggcttgat tggagctcgc tcattccaat tccttctatt aggctactaa
      241 caccatgact ttattagcct gtctatcctg gcccccctgg cgaggttcat gtttgtttat
      301 ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga
      361 gtgtggggtc aaatagtttc atgttcccca aatggcccaa aactgacagt ttaaacgctg
      421 tcttggaacc taatatgaca aaagcgtgat ctcatccaag atgaactaag tttggttcgt
      481 tgaaatgcta acggccagtt ggtcaaaaag aaacttccaa aagtcggcat accgtttgtc
      541 ttgtttggta ttgattgacg aatgctcaaa aataatctca ttaatgctta gcgcagtctc
      601 tctatcgctt ctgaaccccg gtgcacctgt gccgaaacgc aaatggggaa acacccgctt
      661 tttggatgat tatgcattgt ctccacattg tatgcttcca agattctggt gggaatactg
      721 ctgatagcct aacgttcatg atcaaaattt aactgttcta acccctactt gacagcaata
      781 tataaacaga aggaagctgc cctgtcttaa accttttttt ttatcatcat tattagctta
      841 ctttcataat tgcgactggt tccaattgac aagcttttga ttttaacgac ttttaacgac
      901 aacttgagaa gatcaaaaaa caactaatta ttcgaaggat ccaaacgatg agatttcctt
      961 caatttttac tgcagtttta ttcgcagcat cctccgcatt agctgctcca gtcaacacta
     1021 caacagaaga tgaaacggca caaattccgg ctgaagctgt catcggttac tcagatttag
     1081 aaggggattt cgatgttgct gttttgccat tttccaacag cacaaataac gggttattgt
     1141 ttataaatac tactattgcc agcattgctg ctaaagaaga aggggtatct ctcgagaaaa
     1201 gaaaggtttt cgaaagatgt gagctcgcta gaaccttgaa gagattgggt atggacgggt
     1261 accgtggtat ctctttggct aactggatgt gtttggctaa gtgggaatct ggttacaaca
     1321 ctcgagctac caactacaac gctggtgaca ggtcgaccga ctacggtatc ttccaaatta
     1381 attctagata ctggtgtaac cacggtaaga ccccaggcgc cgttaacgct tgtcacttgt
     1441 cttgttctgc tttgctgcag gacaacatcg ctgacgctgt tgcttgtgct aaacgcgttg
     1501 ttagagaccc acaaggtatc agagcttggg ttgcttggag aaatcgatgt caaaacagag
     1561 acgtcagaca atacgttcaa ggttgtggtg tttagtaaga attccctagg gcggccgcga
     1621 attaattcgc cttagacatg actgttcctc agttcaagtt gggcacttac gagaagaccg
     1681 gtcttgctag attctaatca agaggatgtc agaatgccat ttgcctgaga gatgcaggct
     1741 tcatttttga tactttttta tttgtaacct atatagtata ggattttttt tgtcattttg
     1801 tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga atatcttgtg
     1861 gtaggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc cactcctctt
     1921 cagagtacag aagattaagt gagacgttcg tttgtgcaag cttatcgata agctttaatg
     1981 cggtagttta tcacagttaa attgctaacg cagtcaggca ccgtgtatga aatctaacaa
     2041 tgcgctcatc gtcatcctcg gcaccgtcac cctggatgct gtaggcatag gcttggttat
     2101 gccggtactg ccgggcctct tgcgggatat cgtccattcc gacagcatcg ccagtcacta
     2161 tggcgtgctg ctagcgctat atgcgttgat gcaatttcta tgcgcacccg ttctcggagc
     2221 actgtccgac cgctttggcc gccgcccagt cctgctcgct tcgctacttg gagccactat
     2281 cgactacgcg atcatggcga ccacacccgt cctgtggatc tatcgaatct aaatgtaagt
     2341 taaaatctct aaataattaa ataagtccca gtttctccat acgaacctta acagcattgc
     2401 ggtgagcatc tagaccttca acagcagcca gatccatcac tgcttggcca atatgtttca
     2461 gtccctcagg agttacgtct tgtgaagtga tgaacttctg gaaggttgca gtgttaactc
     2521 cgctgtattg acgggcatat ccgtacgttg gcaaagtgtg gttggtaccg gaggagtaat
     2581 ctccacaact ctctggagag taggcaccaa caaacacaga tccagcgtgt tgtacttgat
     2641 caacataaga agaagcattc tcgatttgca ggatcaagtg ttcaggagcg tactgattgg
     2701 acatttccaa agcctgctcg taggttgcaa ccgatagggt tgtagagtgt gcaatacact
     2761 tgcgtacaat ttcaaccctt ggcaactgca cagcttggtt gtgaacagca tcttcaattc
     2821 tggcaagctc cttgtctgtc atatcgacag ccaacagaat cacctgggaa tcaataccat
     2881 gttcagcttg agacagaagg tctgaggcaa cgaaatctgg atcagcgtat ttatcagcaa
     2941 taactagaac ttcagaaggc ccagcaggca tgtcaatact acacagggct gatgtgtcat
     3001 tttgaaccat catcttggca gcagtaacga actggtttcc tggaccaaat attttgtcac
     3061 acttaggaac agtttctgtt ccgtaagcca tagcagctac tgcctgggcg cctcctgcta
     3121 gcacgataca cttagcacca accttgtggg caacgtagat gacttctggg gtaagggtac
     3181 catccttctt aggtggagat gcaaaaacaa tttctttgca accagcaact ttggcaggaa
     3241 cacccagcat cagggaagtg gaaggcagaa ttgcggttcc accaggaata tagaggccaa
     3301 ctttctcaat aggtcttgca aaacgagagc agactacacc agggcaagtc tcaacttgca
     3361 acgtctccgt tagttgagct tcatggaatt tcctgacgtt atctatagag agatcaatgg
     3421 ctctcttaac gttatctggc aattgcataa gttcctctgg gaaaggagct tctaacacag
     3481 gtgtcttcaa agcgactcca tcaaacttgg cagttagttc taaaagggct ttgtcaccat
     3541 tttgacgaac attgtcgaca attggtttga ctaattccat aatctgttcc gttttctgga
     3601 taggacgacg aagggcatct tcaatttctt gtgaggaggc cttagaaacg tcaattttgc
     3661 acaattcaat acgaccttca gaagggactt ctttaggttt ggattcttct ttaggttgtt
     3721 ccttggtgta tcctggcttg gcatctcctt tccttctagt gacctttagg gacttcatat
     3781 ccaggtttct ctccacctcg tccaacgtca caccgtactt ggcacatcta actaatgcaa
     3841 aataaaataa gtcagcacat tcccaggcta tatcttcctt ggatttagct tctgcaagtt
     3901 catcagcttc ctccctaatt ttagcgttca acaaaacttc gtcgtcaaat aaccgtttgg
     3961 tataagaacc ttctggagca ttgctcttac gatcccacaa ggtggcttcc atggctctaa
     4021 gaccctttga ttggccaaaa caggaagtgc gttccaagtg acagaaacca acacctgttt
     4081 gttcaaccac aaatttcaag cagtctccat cacaatccaa ttcgataccc agcaactttt
     4141 gagttgctcc agatgtagca cctttatacc acaaaccgtg acgacgagat tggtagactc
     4201 cagtttgtgt ccttatagcc tccggaatag actttttgga cgagtacacc aggcccaacg
     4261 agtaattaga agagtcagcc accaaagtag tgaatagacc atcggggcgg tcagtagtca
     4321 aagacgccaa caaaatttca ctgacaggga actttttgac atcttcagaa agttcgtatt
     4381 cagtagtcaa ttgccgagca tcaataatgg ggattatacc agaagcaaca gtggaagtca
     4441 catctaccaa ctttgcggtc tcagaaaaag cataaacagt tctactaccg ccattagtga
     4501 aacttttcaa atcgcccagt ggagaagaaa aaggcacagc gatactagca ttagcgggca
     4561 aggatgcaac tttatcaacc agggtcctat agataaccct agcgcctggg atcatccttt
     4621 ggacaactct ttctgccaaa tctaggtcca aaatcacttc attgatacca ttattgtaca
     4681 acttgagcaa gttgtcgatc agctcctcaa attggtcctc tgtaacggat gactcaactt
     4741 gcacattaac ttgaagctca gtcgattgag tgaacttgat caggttgtgc agctggtcag
     4801 cagcataggg aaacacggct tttcctacca aactcaagga attatcaaac tctgcaacac
     4861 ttgcgtatgc aggtagcaag ggaaatgtca tacttgaagt cggacagtga gtgtagtctt
     4921 gagaaattct gaagccgtat ttttattatc agtgagtcag tcatcaggag atcctctacg
     4981 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     5041 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     5101 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     5161 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     5221 tgcaggagtc gcataaggga gagcgtcgag tatctatgat tggaagtatg ggaatggtga
     5281 tacccgcatt cttcagtgtc ttgaggtctc ctatcagatt atgcccaact aaagcaaccg
     5341 gaggaggaga tttcatggta aatttctctg acttttggtc atcagtagac tcgaactgtg
     5401 agactatctc ggttatgaca gcagaaatgt ccttcttgga gacagtaaat gaagtcccac
     5461 caataaagaa atccttgtta tcaggaacaa acttcttgtt tcgaactttt tcggtgcctt
     5521 gaactataaa atgtagagtg gatatgtcgg gtaggaatgg agcgggcaaa tgcttacctt
     5581 ctggaccttc aagaggtatg tagggtttgt agatactgat gccaacttca gtgacaacgt
     5641 tgctatttcg ttcaaaccat tccgaatcca gagaaatcaa agttgtttgt ctactattga
     5701 tccaagccag tgcggtcttg aaactgacaa tagtgtgctc gtgttttgag gtcatctttg
     5761 tatgaataaa tctagtcttt gatctaaata atcttgacga gccagacgat aataccaatc
     5821 taaactcttt aaacgttaaa ggacaagtat gtctgcctgt attaaacccc aaatcagctc
     5881 gtagtctgat cctcatcaac ttgaggggca ctatcttgtt ttagagaaat ttgcggagat
     5941 gcgatatcga gaaaaaggta cgctgatttt aaacgtgaaa tttatctcaa gatctgctgc
     6001 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     6061 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     6121 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     6181 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     6241 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     6301 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6361 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6421 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6481 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6541 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6601 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6661 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6721 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6781 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6841 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6901 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6961 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7021 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7081 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7141 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7201 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7261 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7321 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7381 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7441 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7501 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     7561 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7621 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7681 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7741 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7801 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
     7861 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7921 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7981 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     8041 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8101 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8161 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     8221 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     8281 gtcttcaaga attaattctc atgtttgaca gcttatcatc gataagctga ctcatgttgg
     8341 tattgtgaaa tagacgcaga tcgggaacac tgaaaaataa cagttattat tcga
//
