LOCUS       sequence-365758-        8508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Aspergillus aculeatus beta-glucosidase (BGL1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8508)
  AUTHORS   Ito Y, Ishigami M, Terai G, Nakamura Y, Hashiba N, Nishi T, Nakazawa
            H, Hasunuma T, Asai K, Umetsu M, Ishii J, Kondo A
  TITLE     A streamlined strain engineering workflow with genome-wide screening
            detects enhanced protein secretion in Komagataella phaffii.
  JOURNAL   Commun Biol. 2022 Jun 8;5(1):561. doi: 10.1038/s42003-022-03475-w.
  PUBMED    35676418
REFERENCE   2  (bases 1 to 8508)
  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: "Commun 
            Biol."; date: "2022-06-8"; volume: "5(1):561. doi"; pages: " 
            10.1038/s42003-022-03475-w"
COMMENT     Sequence Label: pPAP_BGL1
FEATURES             Location/Qualifiers
     source          1..8508
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..42)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(196..784)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(276..295)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(955..1746,1747..1815))
                     /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     1578..1597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1816..1920)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1988..2006
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2044..2066)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2166..2185
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2379..2401
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2393..2410
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2394..2410
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2703..3497
                     /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(2757..2776)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3367..3386
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      complement(4146..4392)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(join(7008..7205,7206..7262))
                     /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="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR"
     promoter        complement(7269..8207)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     primer_bind     complement(8302..8318)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8302..8318)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8315..8337)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8326..8342
                     /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(8350..8356,8357..8374,8375..8380))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8395..8416
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
       61 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      121 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      181 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      241 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      301 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      361 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      421 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      481 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      541 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      601 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      661 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      721 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      781 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      841 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
      901 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
      961 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1021 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1081 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1141 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1201 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1261 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1321 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1381 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1441 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1501 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1561 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1621 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1681 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1741 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     1801 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1861 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     1921 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     1981 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     2041 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     2101 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     2161 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     2221 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     2281 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     2341 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     2401 gacggccagt gaattctaca gagctttata tcaccttact gaacgctaga gtagacccaa
     2461 ttcccggctc acaccaccct tacatgcaga gctaaccaat aaggtaatta attaacacta
     2521 tatagctcgt ggtgaacact ggcccggagt agtcatacgt gtaggttttt ggcgtgatga
     2581 aaatcaggtg gcgcacgact tttcgtaaag ttcgggaggg agtgctgcaa acggtatata
     2641 aggaccagtt tttctcgcac attatcaatt gctctttagt acaaagataa tatagaaacc
     2701 atatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2761 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2821 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     2881 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     2941 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3001 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3061 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3121 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3181 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3241 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3301 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3361 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3421 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3481 ttgacgagtt cttctgagcg gggaattctc tagatgacgg acaccggcgt ttacatgcga
     3541 aaaaaactaa agtgcgcgca tttcacggcc gaggggggtc ccacttggga ctgagagggg
     3601 gtgggatctg aaatcgagga ggtatcaaga ccccccgttt ctcaactccc taatcaaaaa
     3661 ttacgaagtc ctcgttggaa aggagttaaa ataattaagc ggggtcggac gccataccga
     3721 ggttatcttg caggcatttt actaatattg gaattcggag ctcaacttgc aaccaggcag
     3781 ggtttagcta tgtaatcaat gtaatcaata taataaagca ctaccacatc gaaggtttgg
     3841 gagggaggcc aatagtgtcc cccacagggt gctgatatcg cgattcttgg gtgaggagac
     3901 acatatttca ctcctctcac caaccaacca agcggctcct cgcaagatga tttatccgat
     3961 tatccggaca ctatactccc atccagtttg atgccgattt catcgattgt cctaaataat
     4021 ccttaaatat gtatagaacg gtaccctggg gttacataat ccttatttaa taatccctcc
     4081 cccaccgctt ttcttttttt ttcttcttat tgtctctaga agatctaagc ttgcacaaac
     4141 gaacgtctca cttaatcttc tgtactctga agaggagtgg gaaataccaa gaaaaacatc
     4201 aaactcgaat gattttccca aacccctacc acaagatatt catcagctgc gagataggct
     4261 gatcaggagc aagctcgtac gagaagaaac aaaatgacaa aaaaaatcct atactatata
     4321 ggttacaaat aaaaaagtat caaaaatgaa gcctgcatct ctcaggcaaa tggcattctg
     4381 acatcctctt gattagaatc tagcaagacc ggtcttctcg taagtgccca acttgaactg
     4441 aggaacagtc atgtctaagg ctcgagtcat tgcaccttcg ggagcgccgc gtgaaggggc
     4501 agctgacgcg aagagctacc aacatggacc ttcttcgggt aagaagtgat gacccagtcc
     4561 tgagccgcaa cgtcccagtt agacagatcg cggcgggtca gggtagtcgt ccacaccgtc
     4621 tcctcggagg gcttgagggt gaggcggtcg aatttgcgca acacaacctt gggctcattg
     4681 ggtccaccaa gggaaacata caattgaggc acagcatcac cggcaacacg accagtgttc
     4741 ttgactgtca ccgaaacacg gatcaactca tcgtagaggc gcgggttacc accagagcca
     4801 ccaccggcag gcagaacggg ctgcggagag ccatcagtag caccctcggg cacgtgctcc
     4861 gcggtgtcga ctccatagta cgggtcgcca gatgaggcct tcaggtctgt ggaattaagc
     4921 cagggataga tgaacttgct gattctggtc aatccctcag ggtacacgta gtcagaggca
     4981 ttgccgactt gtccgaaggt gggagcggcg ccagtctcag tggctacttg agcgttggag
     5041 gaagcgttga gaacctggat gtgaaggcca gagtagttga aagtggtgta gctcagacca
     5101 tgtccgaact cgtagatcgg ggtctcattg cgcttgtcga atccgcggta gtcaatgaaa
     5161 acaccttccg agaaatcatc ttggggagct ccgttgccgt tgttgagctc acggacaagg
     5221 taatccccgt acgcctccct cgtcttgccc caggtgaatg gagatttggc gcccgggttg
     5281 acgcggccgt agagcacgtc agccaaggag ttgccagact cctggccagg caagcccgcc
     5341 cagaggatgg cagtaacgtt ggggtggtca taccactcgt caaccaaaac aggtccaacg
     5401 gagtggatga caacgatggt gttgttgcag ttgtttgcag cagccttgat gaggttgtcg
     5461 ccgttcttcc agagggtgag gttgttgcgg tcgccctcgt ttccgtccac ggagatatag
     5521 ccctctcccg cgtccgagtt gacaaataca agagagacac tggcttgttt agcgagggtc
     5581 tccacctggc tcagcgccca gttgtccgtg atggcgtaga cgctgccctt atgcttgagc
     5641 acctcggctt gaatagcctg ctcaggggtc acgagatatg ggaattcggc agtgccgcta
     5701 ccccaagcca tagcaagagt accgttgtca cagccacggt cagagcagcc attggcaccg
     5761 tacgagttgg atccagcatc ttcacccagg atcgcaactt tgcgctcctt tccggtcagc
     5821 ggcagggcat tgttgttctt cagtagaaca gtactgtctg ctcccaactt gcgaataacc
     5881 tcgctgtggt tgcgctgcac attgacaaag tgattgacct tctcataggg cccttcctgg
     5941 gggtagaaat acttgaagcc gtattcatcg cgagtccagg agctgaagtt aggcggctgg
     6001 tacaggcggt cgcggccaac cttgtagtag gcagccatga tacggacagc catgtcgtca
     6061 acgcgccact gcgggacggt accgttgagc acagcaatgg tcaggttggt accccagaaa
     6121 ctagtggcag aatcgaaggt gatatcgcca ggcattgaca tatccaagcc ggccaaagca
     6181 gagccaacac cactgtggtg agcaccccag tcagacatca caaagccctg gaagccgagc
     6241 tcggccttca gaagcttgtt cagagtgtaa ctgttctggc aaccgtagct gttgttgatc
     6301 tggttgtagg aacacatgat ggcgccaacg ccggcgcgaa cggcatccgc gaagggccag
     6361 aggtacattt catgaatggt cttgtcatca acgttagagc tgatcgtgtc ggagatattg
     6421 aatccgtagc ccgcagcctc tgcgacctgg cggaaatgct cttgctcatt gagaatgtaa
     6481 tgcttggctg tcgccacgac accagcgtct tgaataccct taatcgtctc cgcaaagagc
     6541 acaccagtaa gagccgggtc tggagagaaa ccttcccagt tgcgacctcc atcagggctc
     6601 ctgccgaggg gacccgcggc cggtcccaat tgaacatcaa ttcctttgtc actgaactct
     6661 tgacccatag cctgaccacg tagataagca aggttcttgt cccatgtcgc agcaacgttg
     6721 acaccagcag ggaaagccga attgtagtca ctatcacgaa ttcccaaggg actgtcctga
     6781 agacacatgc caccgatgtt cagtcttggg acaccaccag tctgaccgac gcacttctcc
     6841 agctcccatc cagttccggt ggtcaggttg accttctcat ccagagtcat ctgggataca
     6901 atggccactg cacgctggta ggcttccgcc cactctccct ggccattggc ccacggagag
     6961 gggtagaaag gaggagagaa cgccagttca tcgaattcgt cgacgtctct tttatccaaa
     7021 gatacccctt cttctttagc agcaatgctg gcaatagtag tatttataaa caataacccg
     7081 ttatttgtgc tgttggaaaa tggcaaaaca gcaacatcga aatccccttc taaatctaag
     7141 taaccgatga cagcttcagc cggaatttgt gccgtttcat cttctgttgt agtgttgact
     7201 ggagcagcta atgcggagga tgctgcgaat aaaactgcag taaaaattga aggaaatctc
     7261 atactagtcg tttcgaataa ttagttgttt tttgatcttc tcaagttgtc gttaaaagtc
     7321 gttaaaatca aaagcttgtc aattggaacc agtcgcaatt atgaaagtaa gctaataatg
     7381 atgataaaaa aaaaggttta agacagggca gcttccttct gtttatatat tgctgtcaag
     7441 taggggttag aacagttaaa ttttgatcat gaacgttagg ctatcagcag tattcccacc
     7501 agaatcttgg aagcatacaa tgtggagaca atgcataatc atccaaaaag cgggtgtttc
     7561 cccatttgcg tttcggcaca ggtgcaccgg ggttcagaag cgatagagag actgcgctaa
     7621 gcattaatga gattattttt gagcattcgt caatcaatac caaacaagac aaacggtatg
     7681 ccgacttttg gaagtttctt tttgaccaac tggccgttag catttcaacg aaccaaactt
     7741 agttcatctt ggatgagatc acgcttttgt catattaggt tccaagacag cgtttaaact
     7801 gtcagttttg ggccatttgg ggaacatgaa actatttgac cccacactca gaaagccctc
     7861 atctggagtg atgttcgggt gtaatgcgga gcttgttgca ttcggaaata aacaaacatg
     7921 aacctcgcca ggggggccag gatagacagg ctaataaagt catggtgtta gtagcctaat
     7981 agaaggaatt ggaatgagcg agctccaatc aagcccaata actgggctgg tttttcgatg
     8041 gcaaaagtgg gtgttgagga gaagaggagt ggaggtcctg cgtttgcaac ggtctgctgc
     8101 tagtgtatcc cctcctgttg cgtttggcac ttatgtgtga gaatggacct gtggatgtcg
     8161 gatggcaaaa aggtttcatt caacctttcg tctttggatg ttagatctcg aataataact
     8221 gttatttttc agtgttcccg atctgcgtct atttcacaat accaacatgg gatccgcggc
     8281 cgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     8341 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     8401 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     8461 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggttt
//
