LOCUS       sequence-310189-        5190 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 5190)
  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 5190)
  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: pPAP002
FEATURES             Location/Qualifiers
     source          1..5190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..89
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(90..158,159..950)
                     /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(308..327)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1162..1750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1651..1670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2403..2424
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2439..2444,2445..2462,2463..2469)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2477..2493
                     /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     2482..2504
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2501..2517
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2501..2517
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2513..2836
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    2530..2586
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2587..2604)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2587..2603)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2596..2618)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2812..2831)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            3158..4733
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        3158..3501
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3502..4530
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3502..4530
                     /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      4536..4733
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 ccgaattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
       61 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
      121 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
      181 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
      241 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
      301 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
      361 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
      421 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
      481 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
      541 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
      601 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
      661 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
      721 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
      781 ataaatctgg agccggtgag cgtggttctc gcggtatcat tgcagcactg gggccagatg
      841 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
      901 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa tttcgcgtat
      961 ccaacggctg cccaggcagc cgtttttatt cctgtcagac caagtttact catatatact
     1021 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1081 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     1321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1741 cctatggaaa aacgccagca acgcggcctt tttacggttc ctgccttttt caatgggtaa
     1801 taactgatat aattaaattg aacggaagac aaaacgtaat cgaactccga atgcggttct
     1861 cctgtaacct taattgtagc atagatcact taaataaact catggcctga catctgtaca
     1921 cgttcttatt ggtcttttag caatcttgaa gtctttctat tgttccggtc ggcattacct
     1981 aataaattcg aatcgagatt gctagtacct gatatcatat gaagtaatca tcacatgcaa
     2041 gttccatgat accctctact aatggaattg aacaaagttt aagcttctcg cacgagtccg
     2101 aatccatact atgcacccct caaagttggg attagtcagg aaagctgagc aattaacttc
     2161 cctcgattgg cctggacttt tcgcttagcc tgccgcaatc ggtaagtttc attatcccag
     2221 cggggtgata gcctctgttg ctcatcaggc caaaatcata tataagctgt agacccagca
     2281 cttcaattac ttgaaattca ccataacact tgctctagtc aagacttaca attaaaaatg
     2341 tgagaccgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     2401 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     2461 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     2521 gattacgcca agcttgcatg cctgcaggtc gactctagag gatccccggg taccgagctc
     2581 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     2641 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     2701 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     2761 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     2821 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     2881 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgacggtct cagctttcga
     2941 tttgtatgtg aaatagctga aattcgaaaa tttcattatg gctgtatcta ctttagcgta
     3001 ttaggcattt gagcattggc ttgaacaatg cgggctgtag tgtgtcacca aagaaaccat
     3061 tcgggttcgg atctggaagt cctcatcacg tgatgccgat ctcgtgtatt ttattttcag
     3121 ataacacctg aggactttta cattgtcttc aatacaggac atggaggccc agaataccct
     3181 ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt gactgtcgcc cgtacattta
     3241 gcccatacat ccccatgtat aatcatttgc atccatacat tttgatggcc gcacggcgcg
     3301 aagcaaaaat tacggctcct cgctgcagac gtgcgagcag ggaaacgctc ccctcacaga
     3361 cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat ataaaaggtt aggatttgcc
     3421 actgaggttc ttctttcata tacttccttt taaaatcttg ctaggataca gttctcacat
     3481 cacatccgaa cataaacaac catgggtaaa aagcctgaac tcaccgcgac gtctgtcgag
     3541 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     3601 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     3661 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     3721 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     3781 cgccgtgcac agggtgtcac gttgcaagac ctccctgaaa ccgaactgcc cgctgttctg
     3841 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     3901 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     3961 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     4021 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     4081 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     4141 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     4201 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     4261 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     4321 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     4381 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     4441 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     4501 cccagcactc gtccgagggc aaaggaataa tcagtactga caataaaaag attcttgttt
     4561 tcaagaactt gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg
     4621 ttagcgtgat ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg
     4681 cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgggagttc
     4741 aacatctttg gataatatca gaatgagaaa gaacagatac gcagtacgtt ttttggtgag
     4801 ctctttgcac ttctttagtt ctttccatca atatcagttg cttatgcact tatgactaat
     4861 attgatgttt aacttcaata tctttaaact tttgttcttc ccgacgttca ttaagaatac
     4921 taatacactt taataattag tttaatattt gtttctatat aatgacattt aattaaaaaa
     4981 gataaaatat aaaaacatca taataactca ccagaggtta agaacaaaaa aacaaattag
     5041 atatctgcta atccaatata gttaaatcaa tctttccttg gtataatggg tatattacat
     5101 atatttcaag gaccgacact cctaccaaat atctaaaatt taccatatta acataacatg
     5161 tatataaacg tcaaatcata atcagcacta
//
