LOCUS       sequence-310206-        4746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 integrative plasmid for stable expression in P. pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4746)
  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 4746)
  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: pPAP003
FEATURES             Location/Qualifiers
     source          1..4746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..89
                     /gene="bla"
                     /label=AmpR promoter
     CDS             90..884
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      1055..1643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1544..1563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1771..1792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1807..1812,1813..1830,1831..1837)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1845..1861
                     /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     1850..1872
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1869..1885
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1869..1885
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1881..2204
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1898..1954
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1955..1972)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1955..1971)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1964..1986)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2180..2199)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            2307..3882
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        2307..2650
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2651..3679
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2651..3679
                     /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      3685..3882
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 ccgaattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
       61 tgcttcaata atattgaaaa aggaagagta tggctaaaat gagaatatca ccggaattga
      121 aaaaactgat cgaaaaatac cgctgcgtaa aagatacgga aggaatgtct cctgctaagg
      181 tatataagct ggtgggagaa aatgaaaacc tatatttaaa aatgacggac agccggtata
      241 aagggaccac ctatgatgtg gaacgggaaa aggacatgat gctatggctg gaaggaaagc
      301 tgcctgttcc aaaggtcctg cactttgaac ggcatgatgg ctggagcaat ctgctcatga
      361 gtgaggccga tggcgtcctt tgctcggaag agtatgaaga tgaacaaagc cctgaaaaga
      421 ttatcgagct gtatgcggag tgcatcaggc tctttcactc catcgacata tcggattgtc
      481 cctatacgaa tagcttagac agccgcttag ccgaattgga ttacttactg aataacgatc
      541 tggccgatgt ggattgcgaa aactgggaag aggacactcc atttaaagat ccgcgcgagc
      601 tgtatgattt tttaaagacg gaaaagcccg aagaggaact tgtcttttcc cacggcgacc
      661 tgggagacag caacatcttt gtgaaagatg gcaaagtaag tggctttatt gatcttggga
      721 gaagcggcag ggcggacaag tggtatgaca ttgccttctg cgtccggtcg ctcagggagg
      781 atatcgggga agaacagtat gtcgagctat tttttgactt actggggatc aagcctgatt
      841 gggagaaaat aaaatattat attttactgg atgaattgtt ttagctgtca gaccaagttt
      901 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
      961 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     1021 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     1081 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     1141 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     1201 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     1261 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     1321 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     1381 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     1441 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     1501 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     1561 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     1621 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     1681 tttgctggcc ttttgctcac atgaacgttg tcttcgcagc tggcacgaca ggtttcccga
     1741 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     1801 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     1861 atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc tgcaggtcga
     1921 ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg
     1981 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     2041 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2101 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2161 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     2221 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2281 gacaagctgt gacgaagaca atacaggaca tggaggccca gaataccctc cttgacagtc
     2341 ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag cccatacatc
     2401 cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga agcaaaaatt
     2461 acggctcctc gctgcagacg tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt
     2521 gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca ctgaggttct
     2581 tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc acatccgaac
     2641 ataaacaacc atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     2701 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     2761 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     2821 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     2881 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     2941 gggtgtcacg ttgcaagacc tccctgaaac cgaactgccc gctgttctgc agccggtcgc
     3001 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     3061 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     3121 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     3181 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     3241 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     3301 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     3361 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     3421 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     3481 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     3541 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     3601 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     3661 tccgagggca aaggaataat cagtactgac aataaaaaga ttcttgtttt caagaacttg
     3721 tcatttgtat agttttttta tattgtagtt gttctatttt aatcaaatgt tagcgtgatt
     3781 tatatttttt ttcgcctcga catcatctgc ccagatgcga agttaagtgc gcagaaagta
     3841 atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac tgggagtggc ttcagcaacg
     3901 ttgtcactga agttggcatc agtatctaca aaccctacat acctcttgaa ggtccagaag
     3961 gtaagcattt gcccgctcca ttcctacccg acatatccac tctacatttt atagttcaag
     4021 gcaccgaaaa agttcgaaac aagaagtttg ttcctgataa caaggatttc tttattggtg
     4081 ggacttcatt tactgtctcc aagaaggaca tttctgctgt cataaccgag atagtctcac
     4141 agttcgagtc tactgatgac caaaagtcag agaaatttac catgaaatct cctcctccgg
     4201 ttgctttagt tgggcataat ctgataggag gcctcaagac actgaagaat gcgggtatca
     4261 ccattcccat acttccaatc ataggatccg gcgcgccgat actcgagaat tatggcttaa
     4321 tcaagtgaat acatcaaagt caaacttaaa atacattctt cgcaggcttg gcttgccaca
     4381 tagttttctt cacaatgctg caaatgacgc ttattatacc cttttggcat cgttgaagct
     4441 tgcacaaacg aacgtcccac ttaatcttct gtactctgaa gaggagtggg aaataccaag
     4501 aaaaacatca aactcgaatg attttcccga acccctacca caagatattc atcagctgcg
     4561 agataggctg atcaggagca agctcgtacg agaagaaaca aaatgacaaa aaaaatccta
     4621 tactatatag gttacaaata aaaaagtatc aaaaatgaag cctgcatctc tcaggcaaat
     4681 ggcattctga catcctctcg ctgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4741 aggccg
//
