LOCUS       sequence-310188-        5183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 episomal expression plasmid for P. pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5183)
  AUTHORS   Pullmann P, Knorrscheidt A, Munch J, Palme PR, Hoehenwarter W, 
            Marillonnet S, Alcalde M, Westermann B, Weissenborn MJ
  TITLE     A modular two yeast species secretion system for the production and 
            preparative application of unspecific peroxygenases.
  JOURNAL   Commun Biol. 2021 May 12;4(1):562. doi: 10.1038/s42003-021-02076-3.
  PUBMED    33980981
REFERENCE   2  (bases 1 to 5183)
  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: "2021-05-12"; volume: "4(1):562. doi"; pages: " 
            10.1038/s42003-021-02076-3"
COMMENT     Sequence Label: pPAP001
FEATURES             Location/Qualifiers
     source          1..5183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gene            233..1808
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        233..576
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             577..1605
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             577..1605
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      1611..1808
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        2283..2354
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2355..2423,2424..3215)
                     /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     complement(2573..2592)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3427..4015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3916..3935
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4108..4584
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     protein_bind    4661..4682
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4697..4702,4703..4720,4721..4727)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4735..4751
                     /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)."
     primer_bind     4740..4762
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4759..4775
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4759..4775
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4771..5094
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    4788..4844
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4845..4862)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4845..4861)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4854..4876)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5070..5089)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggtctcagct ttcgatttgt atgtgaaata gctgaaattc gaaaatttca ttatggctgt
       61 atctacttta gcgtattagg catttgagca ttggcttgaa caatgcgggc tgtagtgtgt
      121 caccaaagaa accattcggg ttcggatctg gaagtcctca tcacgtgatg ccgatctcgt
      181 gtattttatt ttcagataac acctgaggac ttttacattg tcttcaatac aggacatgga
      241 ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg
      301 tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca tacattttga
      361 tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cagacgtgcg agcagggaaa
      421 cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa
      481 aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg
      541 atacagttct cacatcacat ccgaacataa acaaccatgg gtaaaaagcc tgaactcacc
      601 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
      661 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
      721 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
      781 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
      841 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctccc tgaaaccgaa
      901 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
      961 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     1021 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     1081 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     1141 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     1201 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     1261 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     1321 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     1381 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     1441 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     1501 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     1561 agtggaaacc gacgccccag cactcgtccg agggcaaagg aataatcagt actgacaata
     1621 aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc
     1681 tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag
     1741 atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg
     1801 ctatactggg agttcaacat ctttggataa tatcagaatg agaaagaaca gatacgcagt
     1861 acgttttttg gtgagctctt tgcacttctt tagttctttc catcaatatc agttgcttat
     1921 gcacttatga ctaatattga tgtttaactt caatatcttt aaacttttgt tcttcccgac
     1981 gttcattaag aatactaata cactttaata attagtttaa tatttgtttc tatataatga
     2041 catttaatta aaaaagataa aatataaaaa catcataata actcaccaga ggttaagaac
     2101 aaaaaaacaa attagatatc tgctaatcca atatagttaa atcaatcttt ccttggtata
     2161 atgggtatat tacatatatt tcaaggaccg acactcctac caaatatcta aaatttacca
     2221 tattaacata acatgtatat aaacgtcaaa tcataatcag cactaccgaa tttttctaaa
     2281 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2341 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2401 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2461 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2521 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2581 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2641 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2701 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2761 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2821 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2881 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     2941 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3001 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3061 gtgagcgtgg ttctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3121 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3181 ctgagatagg tgcctcactg attaagcatt ggtaatttcg cgtatccaac ggctgcccag
     3241 gcagccgttt ttattcctgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3301 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3361 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3421 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3481 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3541 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3601 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3661 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3721 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3781 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3841 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3901 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3961 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4021 cagcaacgcg gcctttttac ggttcctgcc tttttcaatg ggtaataact gatataatta
     4081 aattgaacgg aagacaaaac ggatcctttt ttgtagaaat gtcttggtgt cctcgtccaa
     4141 tcaggtagcc atctctgaaa tatctggctc cgttgcaact ccgaacgacc tgctggcaac
     4201 gtaaaattct ccggggtaaa acttaaatgt ggagtaatgg aaccagaaac gtctcttccc
     4261 ttctctctcc ttccaccgcc cgttaccgtc cctaggaaat tttactctgc tggagagctt
     4321 cttctacggc ccccttgcag caatgctctt cccagcatta cgttgcgggt aaaacggagg
     4381 tcgtgtaccc gacctagcag cccagggatg gaaaagtccc ggccgtcgct ggcaataata
     4441 gcgggcggac gcatgtcatg agattattgg aaaccaccag aatcgaatat aaaaggcgaa
     4501 cacctttccc aattttggtt tctcctgacc caaagacttt aaatttaatt tatttgtccc
     4561 tatttcaatc aattgaacaa ctatcaaaac acaaaatgtg agaccgcagc tggcacgaca
     4621 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     4681 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     4741 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc
     4801 tgcaggtcga ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta
     4861 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4921 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4981 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5041 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5101 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5161 tccgcttaca gacaagctgt gac
//
