LOCUS       sequence-251673-        8343 bp    DNA     circular SYN 15-APR-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 Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pMJA152
FEATURES             Location/Qualifiers
     source          1..8343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(558..1349,1350..1418))
                     /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     1181..1200
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1419..1523)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1591..1609
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1730..2665
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             join(2677..2733,2734..2931,2932..2943)
                     /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      3376..3622
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(4035..6569)
                     /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(6710..6729)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     misc_feature    6924..7672
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     primer_bind     complement(7708..7730)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    7816..7956
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     7871..7890
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7971..7988)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(8142..8343,1..387))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8222..8241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
       61 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
      121 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
      181 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
      241 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
      301 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
      361 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
      421 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
      481 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
      541 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
      601 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
      661 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
      721 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
      781 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
      841 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
      901 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
      961 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     1021 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     1081 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     1141 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
     1201 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     1261 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     1321 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     1381 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     1441 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     1501 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     1561 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt
     1621 caagaattaa ttctcatgtt tgacagctta tcatcgataa gctgactcat gttggtattg
     1681 tgaaatagac gcagatcggg aacactgaaa aataacagtt attattcgag atctaacatc
     1741 caaagacgaa aggttgaatg aaaccttttt gccatccgac atccacaggt ccattctcac
     1801 acataagtgc caaacgcaac aggaggggat acactagcag cagaccgttg caaacgcagg
     1861 acctccactc ctcttctcct caacacccac ttttgccatc gaaaaaccag cccagttatt
     1921 gggcttgatt ggagctcgct cattccaatt ccttctatta ggctactaac accatgactt
     1981 tattagcctg tctatcctgg cccccctggc gaggttcatg tttgtttatt tccgaatgca
     2041 acaagctccg cattacaccc gaacatcact ccagatgagg gctttctgag tgtggggtca
     2101 aatagtttca tgttccccaa atggcccaaa actgacagtt taaacgctgt cttggaacct
     2161 aatatgacaa aagcgtgatc tcatccaaga tgaactaagt ttggttcgtt gaaatgctaa
     2221 cggccagttg gtcaaaaaga aacttccaaa agtcggcata ccgtttgtct tgtttggtat
     2281 tgattgacga atgctcaaaa ataatctcat taatgcttag cgcagtctct ctatcgcttc
     2341 tgaaccccgg tgcacctgtg ccgaaacgca aatggggaaa cacccgcttt ttggatgatt
     2401 atgcattgtc tccacattgt atgcttccaa gattctggtg ggaatactgc tgatagccta
     2461 acgttcatga tcaaaattta actgttctaa cccctacttg acagcaatat ataaacagaa
     2521 ggaagctgcc ctgtcttaaa cctttttttt tatcatcatt attagcttac tttcataatt
     2581 gcgactggtt ccaattgaca agcttttgat tttaacgact tttaacgaca acttgagaag
     2641 atcaaaaaac aactaattat tcgaaggatc caaacgatga gatttccttc aatttttact
     2701 gcagttttat tcgcagcatc ctccgcatta gctgctccag tcaacactac aacagaagat
     2761 gaaacggcac aaattccggc tgaagctgtc atcggttact cagatttaga aggggatttc
     2821 gatgttgctg ttttgccatt ttccaacagc acaaataacg ggttattgtt tataaatact
     2881 actattgcca gcattgctgc taaagaagaa ggggtatctc tcgagaaaag agaggctgaa
     2941 gctcaggagg agtgtcgcga gcagatgcag agacagcaga tgctcagcca ctgccggatg
     3001 tatatgagac agcagatgga ggagagcacg taccagacca tgcccaggcg gggaatggag
     3061 ccgcatatga gcgagtgctg cgagcagctg gaggggatgg acgaggcatg cagatgcgaa
     3121 ggcttaagga tgatgatgag gatgatgcaa cagaaggaga tgcaaccccg aggggagcag
     3181 atgcgaagga tgatgaggct ggccgagaat atcccttccc gctgcaacct cagtcccatg
     3241 agatgcccca tgggtggctc cattgccggg ttctgagaat tccctagggc ggccgcgaat
     3301 taattcgcct tagacatgac tgttcctcag ttcaagttgg gcacttacga gaagaccggt
     3361 cttgctagat tctaatcaag aggatgtcag aatgccattt gcctgagaga tgcaggcttc
     3421 atttttgata cttttttatt tgtaacctat atagtatagg attttttttg tcattttgtt
     3481 tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt
     3541 aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca
     3601 gagtacagaa gattaagtga gacgttcgtt tgtgcaagct tatcgataag ctttaatgcg
     3661 gtagtttatc acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa tctaacaatg
     3721 cgctcatcgt catcctcggc accgtcaccc tggatgctgt aggcataggc ttggttatgc
     3781 cggtactgcc gggcctcttg cgggatatcg tccattccga cagcatcgcc agtcactatg
     3841 gcgtgctgct agcgctatat gcgttgatgc aatttctatg cgcacccgtt ctcggagcac
     3901 tgtccgaccg ctttggccgc cgcccagtcc tgctcgcttc gctacttgga gccactatcg
     3961 actacgcgat catggcgacc acacccgtcc tgtggatcta tcgaatctaa atgtaagtta
     4021 aaatctctaa ataattaaat aagtcccagt ttctccatac gaaccttaac agcattgcgg
     4081 tgagcatcta gaccttcaac agcagccaga tccatcactg cttggccaat atgtttcagt
     4141 ccctcaggag ttacgtcttg tgaagtgatg aacttctgga aggttgcagt gttaactccg
     4201 ctgtattgac gggcatatcc gtacgttggc aaagtgtggt tggtaccgga ggagtaatct
     4261 ccacaactct ctggagagta ggcaccaaca aacacagatc cagcgtgttg tacttgatca
     4321 acataagaag aagcattctc gatttgcagg atcaagtgtt caggagcgta ctgattggac
     4381 atttccaaag cctgctcgta ggttgcaacc gatagggttg tagagtgtgc aatacacttg
     4441 cgtacaattt caacccttgg caactgcaca gcttggttgt gaacagcatc ttcaattctg
     4501 gcaagctcct tgtctgtcat atcgacagcc aacagaatca cctgggaatc aataccatgt
     4561 tcagcttgag acagaaggtc tgaggcaacg aaatctggat cagcgtattt atcagcaata
     4621 actagaactt cagaaggccc agcaggcatg tcaatactac acagggctga tgtgtcattt
     4681 tgaaccatca tcttggcagc agtaacgaac tggtttcctg gaccaaatat tttgtcacac
     4741 ttaggaacag tttctgttcc gtaagccata gcagctactg cctgggcgcc tcctgctagc
     4801 acgatacact tagcaccaac cttgtgggca acgtagatga cttctggggt aagggtacca
     4861 tccttcttag gtggagatgc aaaaacaatt tctttgcaac cagcaacttt ggcaggaaca
     4921 cccagcatca gggaagtgga aggcagaatt gcggttccac caggaatata gaggccaact
     4981 ttctcaatag gtcttgcaaa acgagagcag actacaccag ggcaagtctc aacttgcaac
     5041 gtctccgtta gttgagcttc atggaatttc ctgacgttat ctatagagag atcaatggct
     5101 ctcttaacgt tatctggcaa ttgcataagt tcctctggga aaggagcttc taacacaggt
     5161 gtcttcaaag cgactccatc aaacttggca gttagttcta aaagggcttt gtcaccattt
     5221 tgacgaacat tgtcgacaat tggtttgact aattccataa tctgttccgt tttctggata
     5281 ggacgacgaa gggcatcttc aatttcttgt gaggaggcct tagaaacgtc aattttgcac
     5341 aattcaatac gaccttcaga agggacttct ttaggtttgg attcttcttt aggttgttcc
     5401 ttggtgtatc ctggcttggc atctcctttc cttctagtga cctttaggga cttcatatcc
     5461 aggtttctct ccacctcgtc caacgtcaca ccgtacttgg cacatctaac taatgcaaaa
     5521 taaaataagt cagcacattc ccaggctata tcttccttgg atttagcttc tgcaagttca
     5581 tcagcttcct ccctaatttt agcgttcaac aaaacttcgt cgtcaaataa ccgtttggta
     5641 taagaacctt ctggagcatt gctcttacga tcccacaagg tggcttccat ggctctaaga
     5701 ccctttgatt ggccaaaaca ggaagtgcgt tccaagtgac agaaaccaac acctgtttgt
     5761 tcaaccacaa atttcaagca gtctccatca caatccaatt cgatacccag caacttttga
     5821 gttgctccag atgtagcacc tttataccac aaaccgtgac gacgagattg gtagactcca
     5881 gtttgtgtcc ttatagcctc cggaatagac tttttggacg agtacaccag gcccaacgag
     5941 taattagaag agtcagccac caaagtagtg aatagaccat cggggcggtc agtagtcaaa
     6001 gacgccaaca aaatttcact gacagggaac tttttgacat cttcagaaag ttcgtattca
     6061 gtagtcaatt gccgagcatc aataatgggg attataccag aagcaacagt ggaagtcaca
     6121 tctaccaact ttgcggtctc agaaaaagca taaacagttc tactaccgcc attagtgaaa
     6181 cttttcaaat cgcccagtgg agaagaaaaa ggcacagcga tactagcatt agcgggcaag
     6241 gatgcaactt tatcaaccag ggtcctatag ataaccctag cgcctgggat catcctttgg
     6301 acaactcttt ctgccaaatc taggtccaaa atcacttcat tgataccatt attgtacaac
     6361 ttgagcaagt tgtcgatcag ctcctcaaat tggtcctctg taacggatga ctcaacttgc
     6421 acattaactt gaagctcagt cgattgagtg aacttgatca ggttgtgcag ctggtcagca
     6481 gcatagggaa acacggcttt tcctaccaaa ctcaaggaat tatcaaactc tgcaacactt
     6541 gcgtatgcag gtagcaaggg aaatgtcata cttgaagtcg gacagtgagt gtagtcttga
     6601 gaaattctga agccgtattt ttattatcag tgagtcagtc atcaggagat cctctacgcc
     6661 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
     6721 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
     6781 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
     6841 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
     6901 caggagtcgc ataagggaga gcgtcgagta tctatgattg gaagtatggg aatggtgata
     6961 cccgcattct tcagtgtctt gaggtctcct atcagattat gcccaactaa agcaaccgga
     7021 ggaggagatt tcatggtaaa tttctctgac ttttggtcat cagtagactc gaactgtgag
     7081 actatctcgg ttatgacagc agaaatgtcc ttcttggaga cagtaaatga agtcccacca
     7141 ataaagaaat ccttgttatc aggaacaaac ttcttgtttc gaactttttc ggtgccttga
     7201 actataaaat gtagagtgga tatgtcgggt aggaatggag cgggcaaatg cttaccttct
     7261 ggaccttcaa gaggtatgta gggtttgtag atactgatgc caacttcagt gacaacgttg
     7321 ctatttcgtt caaaccattc cgaatccaga gaaatcaaag ttgtttgtct actattgatc
     7381 caagccagtg cggtcttgaa actgacaata gtgtgctcgt gttttgaggt catctttgta
     7441 tgaataaatc tagtctttga tctaaataat cttgacgagc cagacgataa taccaatcta
     7501 aactctttaa acgttaaagg acaagtatgt ctgcctgtat taaaccccaa atcagctcgt
     7561 agtctgatcc tcatcaactt gaggggcact atcttgtttt agagaaattt gcggagatgc
     7621 gatatcgaga aaaaggtacg ctgattttaa acgtgaaatt tatctcaaga tctgctgcct
     7681 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     7741 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     7801 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     7861 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     7921 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     7981 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     8041 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     8101 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     8161 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     8221 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     8281 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     8341 tca
//
