LOCUS       sequence-251881-        8343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mutant Ber e 1 in Pichia.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8343)
  TITLE     Alcocer Lab plasmids
REFERENCE   2  (bases 1 to 8343)
  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"
COMMENT     Sequence Label: pMJA157
FEATURES             Location/Qualifiers
     source          1..8343
                     /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..1202,1203..1214)
                     /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
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
     terminator      1647..1893
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(2306..4840)
                     /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(4981..5000)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     misc_feature    5195..5943
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     primer_bind     complement(5979..6001)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6087..6227
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6142..6161
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6242..6259)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6413..7001)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6493..6512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7172..7963,7964..8032))
                     /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     7795..7814
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8033..8137)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8205..8223
                     /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 gagaggctga agctcaggag gaggctcgcg agcagatgca gagacagcag atgctcagcc
     1261 actgccggat gtatatgaga cagcagatgg aggagagcac gtaccagacc atgcccaggc
     1321 ggggaatgga gccgcatatg agcgagtgct gcgagcagct ggaggggatg gacgagagct
     1381 gcagatgcga aggcttaagg atgatgatga ggatgatgca acagaaggag atgcaacccc
     1441 gaggggagca gatgcgaagg atgatgaggc tggccgagaa tatcccttcc cgctgcaacc
     1501 tcagtcccat gagatgcccc atgggtggct ccattgccgg gttctgagaa ttccctaggg
     1561 cggccgcgaa ttaattcgcc ttagacatga ctgttcctca gttcaagttg ggcacttacg
     1621 agaagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag
     1681 atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt
     1741 gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa
     1801 tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc
     1861 actcctcttc agagtacaga agattaagtg agacgttcgt ttgtgcaagc ttatcgataa
     1921 gctttaatgc ggtagtttat cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa
     1981 atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg
     2041 cttggttatg ccggtactgc cgggcctctt gcgggatatc gtccattccg acagcatcgc
     2101 cagtcactat ggcgtgctgc tagcgctata tgcgttgatg caatttctat gcgcacccgt
     2161 tctcggagca ctgtccgacc gctttggccg ccgcccagtc ctgctcgctt cgctacttgg
     2221 agccactatc gactacgcga tcatggcgac cacacccgtc ctgtggatct atcgaatcta
     2281 aatgtaagtt aaaatctcta aataattaaa taagtcccag tttctccata cgaaccttaa
     2341 cagcattgcg gtgagcatct agaccttcaa cagcagccag atccatcact gcttggccaa
     2401 tatgtttcag tccctcagga gttacgtctt gtgaagtgat gaacttctgg aaggttgcag
     2461 tgttaactcc gctgtattga cgggcatatc cgtacgttgg caaagtgtgg ttggtaccgg
     2521 aggagtaatc tccacaactc tctggagagt aggcaccaac aaacacagat ccagcgtgtt
     2581 gtacttgatc aacataagaa gaagcattct cgatttgcag gatcaagtgt tcaggagcgt
     2641 actgattgga catttccaaa gcctgctcgt aggttgcaac cgatagggtt gtagagtgtg
     2701 caatacactt gcgtacaatt tcaacccttg gcaactgcac agcttggttg tgaacagcat
     2761 cttcaattct ggcaagctcc ttgtctgtca tatcgacagc caacagaatc acctgggaat
     2821 caataccatg ttcagcttga gacagaaggt ctgaggcaac gaaatctgga tcagcgtatt
     2881 tatcagcaat aactagaact tcagaaggcc cagcaggcat gtcaatacta cacagggctg
     2941 atgtgtcatt ttgaaccatc atcttggcag cagtaacgaa ctggtttcct ggaccaaata
     3001 ttttgtcaca cttaggaaca gtttctgttc cgtaagccat agcagctact gcctgggcgc
     3061 ctcctgctag cacgatacac ttagcaccaa ccttgtgggc aacgtagatg acttctgggg
     3121 taagggtacc atccttctta ggtggagatg caaaaacaat ttctttgcaa ccagcaactt
     3181 tggcaggaac acccagcatc agggaagtgg aaggcagaat tgcggttcca ccaggaatat
     3241 agaggccaac tttctcaata ggtcttgcaa aacgagagca gactacacca gggcaagtct
     3301 caacttgcaa cgtctccgtt agttgagctt catggaattt cctgacgtta tctatagaga
     3361 gatcaatggc tctcttaacg ttatctggca attgcataag ttcctctggg aaaggagctt
     3421 ctaacacagg tgtcttcaaa gcgactccat caaacttggc agttagttct aaaagggctt
     3481 tgtcaccatt ttgacgaaca ttgtcgacaa ttggtttgac taattccata atctgttccg
     3541 ttttctggat aggacgacga agggcatctt caatttcttg tgaggaggcc ttagaaacgt
     3601 caattttgca caattcaata cgaccttcag aagggacttc tttaggtttg gattcttctt
     3661 taggttgttc cttggtgtat cctggcttgg catctccttt ccttctagtg acctttaggg
     3721 acttcatatc caggtttctc tccacctcgt ccaacgtcac accgtacttg gcacatctaa
     3781 ctaatgcaaa ataaaataag tcagcacatt cccaggctat atcttccttg gatttagctt
     3841 ctgcaagttc atcagcttcc tccctaattt tagcgttcaa caaaacttcg tcgtcaaata
     3901 accgtttggt ataagaacct tctggagcat tgctcttacg atcccacaag gtggcttcca
     3961 tggctctaag accctttgat tggccaaaac aggaagtgcg ttccaagtga cagaaaccaa
     4021 cacctgtttg ttcaaccaca aatttcaagc agtctccatc acaatccaat tcgataccca
     4081 gcaacttttg agttgctcca gatgtagcac ctttatacca caaaccgtga cgacgagatt
     4141 ggtagactcc agtttgtgtc cttatagcct ccggaataga ctttttggac gagtacacca
     4201 ggcccaacga gtaattagaa gagtcagcca ccaaagtagt gaatagacca tcggggcggt
     4261 cagtagtcaa agacgccaac aaaatttcac tgacagggaa ctttttgaca tcttcagaaa
     4321 gttcgtattc agtagtcaat tgccgagcat caataatggg gattatacca gaagcaacag
     4381 tggaagtcac atctaccaac tttgcggtct cagaaaaagc ataaacagtt ctactaccgc
     4441 cattagtgaa acttttcaaa tcgcccagtg gagaagaaaa aggcacagcg atactagcat
     4501 tagcgggcaa ggatgcaact ttatcaacca gggtcctata gataacccta gcgcctggga
     4561 tcatcctttg gacaactctt tctgccaaat ctaggtccaa aatcacttca ttgataccat
     4621 tattgtacaa cttgagcaag ttgtcgatca gctcctcaaa ttggtcctct gtaacggatg
     4681 actcaacttg cacattaact tgaagctcag tcgattgagt gaacttgatc aggttgtgca
     4741 gctggtcagc agcataggga aacacggctt ttcctaccaa actcaaggaa ttatcaaact
     4801 ctgcaacact tgcgtatgca ggtagcaagg gaaatgtcat acttgaagtc ggacagtgag
     4861 tgtagtcttg agaaattctg aagccgtatt tttattatca gtgagtcagt catcaggaga
     4921 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     4981 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     5041 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     5101 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     5161 gcttcctaat gcaggagtcg cataagggag agcgtcgagt atctatgatt ggaagtatgg
     5221 gaatggtgat acccgcattc ttcagtgtct tgaggtctcc tatcagatta tgcccaacta
     5281 aagcaaccgg aggaggagat ttcatggtaa atttctctga cttttggtca tcagtagact
     5341 cgaactgtga gactatctcg gttatgacag cagaaatgtc cttcttggag acagtaaatg
     5401 aagtcccacc aataaagaaa tccttgttat caggaacaaa cttcttgttt cgaacttttt
     5461 cggtgccttg aactataaaa tgtagagtgg atatgtcggg taggaatgga gcgggcaaat
     5521 gcttaccttc tggaccttca agaggtatgt agggtttgta gatactgatg ccaacttcag
     5581 tgacaacgtt gctatttcgt tcaaaccatt ccgaatccag agaaatcaaa gttgtttgtc
     5641 tactattgat ccaagccagt gcggtcttga aactgacaat agtgtgctcg tgttttgagg
     5701 tcatctttgt atgaataaat ctagtctttg atctaaataa tcttgacgag ccagacgata
     5761 ataccaatct aaactcttta aacgttaaag gacaagtatg tctgcctgta ttaaacccca
     5821 aatcagctcg tagtctgatc ctcatcaact tgaggggcac tatcttgttt tagagaaatt
     5881 tgcggagatg cgatatcgag aaaaaggtac gctgatttta aacgtgaaat ttatctcaag
     5941 atctgctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     6001 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     6061 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     6121 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     6181 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     6241 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6301 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6361 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6421 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6481 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6541 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6601 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6661 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6721 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6781 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6841 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6901 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6961 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     7021 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7081 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7141 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7201 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7261 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7321 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7381 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7441 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg
     7501 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7561 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7621 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7681 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7741 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     7801 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7861 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7921 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7981 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     8041 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     8101 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8161 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     8221 ggccctttcg tcttcaagaa ttaattctca tgtttgacag cttatcatcg ataagctgac
     8281 tcatgttggt attgtgaaat agacgcagat cgggaacact gaaaaataac agttattatt
     8341 cga
//
