LOCUS       sequence-251600-        8328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SFA8 in Pichia.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8328)
  AUTHORS   Alcocer MJ, Murtagh GJ, Bailey K, Dumoulin M, Meseguer AS, Parker 
            MJ, Archer DB
  TITLE     The disulphide mapping, folding and characterisation of recombinant 
            Ber e 1, an allergenic protein, and SFA8, two sulphur-rich 2S plant 
            albumins.
  JOURNAL   J Mol Biol. 2002 Nov 15;324(1):165-75.
  PUBMED    12421566
REFERENCE   2  (bases 1 to 8328)
  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: "J Mol 
            Biol."; date: "2002-11-15"; volume: "324(1)"; pages: "165-75"
COMMENT     Sequence Label: pMJA053
FEATURES             Location/Qualifiers
     source          1..8328
                     /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..1201,1202..1213)
                     /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. The EA dipeptides are then removed by 
                     dipeptidyl aminopeptidase A."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKERLK"
     terminator      1632..1878
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(2291..4825)
                     /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(4966..4985)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     misc_feature    5180..5928
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     primer_bind     complement(5964..5986)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6072..6212
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6127..6146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6227..6244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6398..6986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6478..6497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7157..7948,7949..8017))
                     /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     7780..7799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8018..8122)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8190..8208
                     /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 ctcgagaaag
     1201 agaggctgaa gctccctatg gccgaggtag aactgaatcc ggatgctatc agcagatgga
     1261 ggaggcggag atgctcaacc actgcgggat gtacctcatg aaaaatttag gagaaaggag
     1321 ccaagttagt cccagaatga gagaagagga tcacaagcaa ctttgctgca tgcaactgaa
     1381 gaacctagat gagaagtgca tgtgtccggc aatcatgatg atgttgaacg aaccaatgtg
     1441 gataaggatg cgagatcaag tgatgagcat ggctcataac ctgcccatag aatgcaatct
     1501 catgtcccaa ccatgccaaa tgtaataagg gcgaattccc tagggcggcc gcgaattaat
     1561 tcgccttaga catgactgtt cctcagttca agttgggcac ttacgagaag accggtcttg
     1621 ctagattcta atcaagagga tgtcagaatg ccatttgcct gagagatgca ggcttcattt
     1681 ttgatacttt tttatttgta acctatatag tataggattt tttttgtcat tttgtttctt
     1741 ctcgtacgag cttgctcctg atcagcctat ctcgcagctg atgaatatct tgtggtaggg
     1801 gtttgggaaa atcattcgag tttgatgttt ttcttggtat ttcccactcc tcttcagagt
     1861 acagaagatt aagtgagacg ttcgtttgtg caagcttatc gataagcttt aatgcggtag
     1921 tttatcacag ttaaattgct aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct
     1981 catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc ataggcttgg ttatgccggt
     2041 actgccgggc ctcttgcggg atatcgtcca ttccgacagc atcgccagtc actatggcgt
     2101 gctgctagcg ctatatgcgt tgatgcaatt tctatgcgca cccgttctcg gagcactgtc
     2161 cgaccgcttt ggccgccgcc cagtcctgct cgcttcgcta cttggagcca ctatcgacta
     2221 cgcgatcatg gcgaccacac ccgtcctgtg gatctatcga atctaaatgt aagttaaaat
     2281 ctctaaataa ttaaataagt cccagtttct ccatacgaac cttaacagca ttgcggtgag
     2341 catctagacc ttcaacagca gccagatcca tcactgcttg gccaatatgt ttcagtccct
     2401 caggagttac gtcttgtgaa gtgatgaact tctggaaggt tgcagtgtta actccgctgt
     2461 attgacgggc atatccgtac gttggcaaag tgtggttggt accggaggag taatctccac
     2521 aactctctgg agagtaggca ccaacaaaca cagatccagc gtgttgtact tgatcaacat
     2581 aagaagaagc attctcgatt tgcaggatca agtgttcagg agcgtactga ttggacattt
     2641 ccaaagcctg ctcgtaggtt gcaaccgata gggttgtaga gtgtgcaata cacttgcgta
     2701 caatttcaac ccttggcaac tgcacagctt ggttgtgaac agcatcttca attctggcaa
     2761 gctccttgtc tgtcatatcg acagccaaca gaatcacctg ggaatcaata ccatgttcag
     2821 cttgagacag aaggtctgag gcaacgaaat ctggatcagc gtatttatca gcaataacta
     2881 gaacttcaga aggcccagca ggcatgtcaa tactacacag ggctgatgtg tcattttgaa
     2941 ccatcatctt ggcagcagta acgaactggt ttcctggacc aaatattttg tcacacttag
     3001 gaacagtttc tgttccgtaa gccatagcag ctactgcctg ggcgcctcct gctagcacga
     3061 tacacttagc accaaccttg tgggcaacgt agatgacttc tggggtaagg gtaccatcct
     3121 tcttaggtgg agatgcaaaa acaatttctt tgcaaccagc aactttggca ggaacaccca
     3181 gcatcaggga agtggaaggc agaattgcgg ttccaccagg aatatagagg ccaactttct
     3241 caataggtct tgcaaaacga gagcagacta caccagggca agtctcaact tgcaacgtct
     3301 ccgttagttg agcttcatgg aatttcctga cgttatctat agagagatca atggctctct
     3361 taacgttatc tggcaattgc ataagttcct ctgggaaagg agcttctaac acaggtgtct
     3421 tcaaagcgac tccatcaaac ttggcagtta gttctaaaag ggctttgtca ccattttgac
     3481 gaacattgtc gacaattggt ttgactaatt ccataatctg ttccgttttc tggataggac
     3541 gacgaagggc atcttcaatt tcttgtgagg aggccttaga aacgtcaatt ttgcacaatt
     3601 caatacgacc ttcagaaggg acttctttag gtttggattc ttctttaggt tgttccttgg
     3661 tgtatcctgg cttggcatct cctttccttc tagtgacctt tagggacttc atatccaggt
     3721 ttctctccac ctcgtccaac gtcacaccgt acttggcaca tctaactaat gcaaaataaa
     3781 ataagtcagc acattcccag gctatatctt ccttggattt agcttctgca agttcatcag
     3841 cttcctccct aattttagcg ttcaacaaaa cttcgtcgtc aaataaccgt ttggtataag
     3901 aaccttctgg agcattgctc ttacgatccc acaaggtggc ttccatggct ctaagaccct
     3961 ttgattggcc aaaacaggaa gtgcgttcca agtgacagaa accaacacct gtttgttcaa
     4021 ccacaaattt caagcagtct ccatcacaat ccaattcgat acccagcaac ttttgagttg
     4081 ctccagatgt agcaccttta taccacaaac cgtgacgacg agattggtag actccagttt
     4141 gtgtccttat agcctccgga atagactttt tggacgagta caccaggccc aacgagtaat
     4201 tagaagagtc agccaccaaa gtagtgaata gaccatcggg gcggtcagta gtcaaagacg
     4261 ccaacaaaat ttcactgaca gggaactttt tgacatcttc agaaagttcg tattcagtag
     4321 tcaattgccg agcatcaata atggggatta taccagaagc aacagtggaa gtcacatcta
     4381 ccaactttgc ggtctcagaa aaagcataaa cagttctact accgccatta gtgaaacttt
     4441 tcaaatcgcc cagtggagaa gaaaaaggca cagcgatact agcattagcg ggcaaggatg
     4501 caactttatc aaccagggtc ctatagataa ccctagcgcc tgggatcatc ctttggacaa
     4561 ctctttctgc caaatctagg tccaaaatca cttcattgat accattattg tacaacttga
     4621 gcaagttgtc gatcagctcc tcaaattggt cctctgtaac ggatgactca acttgcacat
     4681 taacttgaag ctcagtcgat tgagtgaact tgatcaggtt gtgcagctgg tcagcagcat
     4741 agggaaacac ggcttttcct accaaactca aggaattatc aaactctgca acacttgcgt
     4801 atgcaggtag caagggaaat gtcatacttg aagtcggaca gtgagtgtag tcttgagaaa
     4861 ttctgaagcc gtatttttat tatcagtgag tcagtcatca ggagatcctc tacgccggac
     4921 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     4981 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     5041 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     5101 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     5161 agtcgcataa gggagagcgt cgagtatcta tgattggaag tatgggaatg gtgatacccg
     5221 cattcttcag tgtcttgagg tctcctatca gattatgccc aactaaagca accggaggag
     5281 gagatttcat ggtaaatttc tctgactttt ggtcatcagt agactcgaac tgtgagacta
     5341 tctcggttat gacagcagaa atgtccttct tggagacagt aaatgaagtc ccaccaataa
     5401 agaaatcctt gttatcagga acaaacttct tgtttcgaac tttttcggtg ccttgaacta
     5461 taaaatgtag agtggatatg tcgggtagga atggagcggg caaatgctta ccttctggac
     5521 cttcaagagg tatgtagggt ttgtagatac tgatgccaac ttcagtgaca acgttgctat
     5581 ttcgttcaaa ccattccgaa tccagagaaa tcaaagttgt ttgtctacta ttgatccaag
     5641 ccagtgcggt cttgaaactg acaatagtgt gctcgtgttt tgaggtcatc tttgtatgaa
     5701 taaatctagt ctttgatcta aataatcttg acgagccaga cgataatacc aatctaaact
     5761 ctttaaacgt taaaggacaa gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc
     5821 tgatcctcat caacttgagg ggcactatct tgttttagag aaatttgcgg agatgcgata
     5881 tcgagaaaaa ggtacgctga ttttaaacgt gaaatttatc tcaagatctg ctgcctcgcg
     5941 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     6001 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     6061 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     6121 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     6181 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     6241 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6301 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6361 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6421 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6481 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6541 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6601 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6661 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6721 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6781 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     6841 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6901 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6961 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7021 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7081 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7141 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     7201 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7261 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7321 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7381 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7441 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     7501 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7561 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7621 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7681 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7741 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     7801 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7861 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7921 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7981 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     8041 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8101 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     8161 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     8221 aagaattaat tctcatgttt gacagcttat catcgataag ctgactcatg ttggtattgt
     8281 gaaatagacg cagatcggga acactgaaaa ataacagtta ttattcga
//
