LOCUS       sequence-234593-        4570 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 4570)
  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 4570)
  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-pro-alphaf-Btl2
FEATURES             Location/Qualifiers
     source          1..4570
                     /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             join(941..997,998..1195,1196..1207)
                     /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="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEA"
     CDS             2372..2389
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2393..2639
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2654..3067
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3074..3121
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3140..3514
                     /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      3580..3827
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3596..3614)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3902..4490)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3982..4001)
                     /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 atgagatttc cttcaatttt
      961 tactgcagtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga
     1021 agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tacttagatt tagaagggga
     1081 tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa
     1141 tactactatt gccagcattg ctgctaaaga agaaggggta tctttggata aaagagaggc
     1201 tgaagctgct tctccaagag ctaatgatgc tcctattgtt ttgttgcatg gttttactgg
     1261 ttggggtaga gaagagatgt tgggtttcaa atattggggt ggtgttagag gagatattga
     1321 acaatggttg aacgataatg gttacagaac ttatactttg gctgttggtc cattgtcttc
     1381 taactgggat agagcttgtg aggcttacgc tcaattggtt ggtggtactg ttgattatgg
     1441 tgctgctcac gctgctaagc atggtcacgc tagatttggt agaacttatc caggtttgtt
     1501 gcctgaattg aaaagaggtg gtagagttca tattattgct cactctcaag gtggtcaaac
     1561 tgctagaatg ttggtttctt tgttggaaaa tggttctcaa gaagagagag agtacgctaa
     1621 ggctcataac gtttctttgt ctccattgtt tgagggtggt catcacttcg ttttgtctgt
     1681 tactactatt gctactcctc acgatggtac tactttggtt aacatggttg atttcactga
     1741 tagatttttc gatttgcaaa aggctgtttt gaaagctgct gctgttgctt ctaacgttcc
     1801 ttacacttct caagtttacg atttcaagtt ggatcaatgg ggtttgagaa gacaacctgg
     1861 tgaatctttt gatcattact tcgagagatt gaaaagatcc cctgtttgga cttctactga
     1921 tactgctaga tatgatttgt ctattccagg tgctgaaaag ttgaatcaat gggttcaggc
     1981 ttctcctaac acttactatt tgtctttctc tactgaaaga actcacagag gtgctttgac
     2041 tggtaattac tatccagagt tgggtatgaa cgctttttct gctgttgttt gtgctccttt
     2101 cttgggttct tacagaaatg aggctttggg tattgatgat agatggttgg agaacgatgg
     2161 tattgttaat actgtttcta tgaacggtcc aaaaagaggt tcttctgata gaattgttcc
     2221 ttatgatggt actttgaaga aaggtgtttg gaatgatatg ggtacttgta acgttgatca
     2281 tttggaagtt attggtgttg atccaaaccc ttcttttgat attagagctt tctacttgag
     2341 attggctgag caattggctt ctttgagacc acatcaccat caccatcact aatcaagagg
     2401 atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt
     2461 aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct
     2521 gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga
     2581 gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac
     2641 cttcgtttgt gcggatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac
     2701 tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag
     2761 catactaaat ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt
     2821 tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat
     2881 ttttatcacg tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac
     2941 ctcccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt
     3001 tcttgttcta ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta
     3061 atctaagggc ggtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     3121 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     3181 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     3241 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     3301 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     3361 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     3421 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     3481 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc cgacgcggcc cgacgggtcc
     3541 gaggcctcgg agatccgtcc cccttttcct ttgtcgatat catgtaatta gttatgtcac
     3601 gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac
     3661 ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta
     3721 tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga
     3781 aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag ctggagacca
     3841 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3901 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3961 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4021 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4081 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4141 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4201 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4261 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4321 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4381 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4441 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4501 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4561 tcatgagatc
//
