LOCUS       sequence-231108-        4579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Study secretion efficiency of Btl2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4579)
  AUTHORS   Barrero JJ, Casler JC, Valero F, Ferrer P, Glick BS
  TITLE     An improved secretion signal enhances the secretion of model 
            proteins from Pichia pastoris.
  JOURNAL   Microb Cell Fact. 2018 Oct 12;17(1):161. doi: 
            10.1186/s12934-018-1009-5.
  PUBMED    30314480
REFERENCE   2  (bases 1 to 4579)
  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: "Microb Cell
            Fact."; date: "2018-10-12"; volume: "17(1):161. doi"; pages: " 
            10.1186/s12934-018-1009-5"
COMMENT     Sequence Label: pPICZ-pre-Ost1-alphaf(I)-Btl2
FEATURES             Location/Qualifiers
     source          1..4579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             2381..2398
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2402..2648
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2663..3076
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3083..3130
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3149..3523
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      3589..3836
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3605..3623)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3911..4499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3991..4010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
       61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
      121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
      181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
      241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt
      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
      901 caacttgaga agatcaaaaa acaactaatt attcgaaacg atgaggcagg tttggttctc
      961 ttggattgtg ggattgttcc tatgtttttt caacgtgtct tctgctgctc cagtcaacac
     1021 tacaacagaa gatgaaacgg cacaaattcc ggctgaagct gtcatcggtt actcagattt
     1081 agaaggggat ttcgatgttg ctgttttgcc attttccaac agcacaaata acgggttatt
     1141 gtttataaat actactattg ccagcattgc tgctaaagaa gaaggggtat ctctcgagaa
     1201 aagagaggct gaagctgctt ctccaagagc taatgatgct cctattgttt tgttgcatgg
     1261 ttttactggt tggggtagag aagagatgtt gggtttcaaa tattggggtg gtgttagagg
     1321 agatattgaa caatggttga acgataatgg ttacagaact tatactttgg ctgttggtcc
     1381 attgtcttct aactgggata gagcttgtga ggcttacgct caattggttg gtggtactgt
     1441 tgattatggt gctgctcacg ctgctaagca tggtcacgct agatttggta gaacttatcc
     1501 aggtttgttg cctgaattga aaagaggtgg tagagttcat attattgctc actctcaagg
     1561 tggtcaaact gctagaatgt tggtttcttt gttggaaaat ggttctcaag aagagagaga
     1621 gtacgctaag gctcataacg tttctttgtc tccattgttt gagggtggtc atcacttcgt
     1681 tttgtctgtt actactattg ctactcctca cgatggtact actttggtta acatggttga
     1741 tttcactgat agatttttcg atttgcaaaa ggctgttttg aaagctgctg ctgttgcttc
     1801 taacgttcct tacacttctc aagtttacga tttcaagttg gatcaatggg gtttgagaag
     1861 acaacctggt gaatcttttg atcattactt cgagagattg aaaagatccc ctgtttggac
     1921 ttctactgat actgctagat atgatttgtc tattccaggt gctgaaaagt tgaatcaatg
     1981 ggttcaggct tctcctaaca cttactattt gtctttctct actgaaagaa ctcacagagg
     2041 tgctttgact ggtaattact atccagagtt gggtatgaac gctttttctg ctgttgtttg
     2101 tgctcctttc ttgggttctt acagaaatga ggctttgggt attgatgata gatggttgga
     2161 gaacgatggt attgttaata ctgtttctat gaacggtcca aaaagaggtt cttctgatag
     2221 aattgttcct tatgatggta ctttgaagaa aggtgtttgg aatgatatgg gtacttgtaa
     2281 cgttgatcat ttggaagtta ttggtgttga tccaaaccct tcttttgata ttagagcttt
     2341 ctacttgaga ttggctgagc aattggcttc tttgagacca catcaccatc accatcacta
     2401 atcaagagga tgtcagaatg ccatttgcct gagagatgca ggcttcattt ttgatacttt
     2461 tttatttgta acctatatag tataggattt tttttgtcat tttgtttctt ctcgtacgag
     2521 cttgctcctg atcagcctat ctcgcagctg atgaatatct tgtggtaggg gtttgggaaa
     2581 atcattcgag tttgatgttt ttcttggtat ttcccactcc tcttcagagt acagaagatt
     2641 aagtgagacc ttcgtttgtg cggatccccc acacaccata gcttcaaaat gtttctactc
     2701 cttttttact cttccagatt ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc
     2761 caagcacagc atactaaatt tcccctcttt cttcctctag ggtgtcgtta attacccgta
     2821 ctaaaggttt ggaaaagaaa aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc
     2881 aataaaaatt tttatcacgt ttctttttct tgaaaatttt tttttttgat ttttttctct
     2941 ttcgatgacc tcccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag
     3001 tttcattttt cttgttctat tacaactttt tttacttctt gctcattaga aagaaagcat
     3061 agcaatctaa tctaagggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     3121 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct
     3181 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     3241 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
     3301 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     3361 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     3421 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     3481 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacgcggccc
     3541 gacgggtccg aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag
     3601 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
     3661 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     3721 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     3781 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc
     3841 tggagaccaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3901 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3961 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4021 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4081 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4141 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4201 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4261 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4321 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4381 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4441 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4501 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4561 ggattttggt catgagatc
//
